UNIVERSIDAD DE SALAMANCA

FACULTAD DE BIOLOGÍA

DEPARTAMENTO DE BIOLOGÍA ANIMAL, PARASITOLOGÍA,

ECOLOGÍA, EDAFOLOGÍA Y QUÍMICA AGRÍCOLA

TESIS DOCTORAL

El papel de la plasmina en la supervivencia de

Dirofilaria immitis y en la patología vascular del
hospedador durante la dirofilariosis cardiopulmonar

Tesis Doctoral presentada por el Licenciado D. Javier González Miguel

para optar a la Mención de Doctor Europeo por la Universidad de Salamanca.

Salamanca, 2015.
 UNIVERSIDAD DE SALAMANCA
FACULTAD DE BIOLOGÍA

DEPARTAMENTO DE BIOLOGÍA ANIMAL, PARASITOLOGÍA,

ECOLOGÍA, EDAFOLOGÍA Y QUÍMICA AGRÍCOLA

TESIS DOCTORAL

El papel de la plasmina en la supervivencia de

Dirofilaria immitis y en la patología vascular del
hospedador durante la dirofilariosis cardiopulmonar

Fdo.: D. Javier González Miguel

Salamanca, 2015
 DEPARTAMENTO DE BIOLOGÍA ANIMAL,
PARASITOLOGÍA, ECOLOGÍA, EDAFOLOGÍA
Y QUÍMICA AGRÍCOLA
ÁREA DE PARASITOLOGÍA

D. Fernando Simón Martín, Catedrático de Parasitología de la Universidad de

Salamanca y D. Rodrigo Morchón García, Profesor Ayudante Doctor adscrito al Área
de Parasitología de la Universidad de Salamanca,

CERTIFICAN:

Que la Tesis Doctoral titulada “El papel de la plasmina en la supervivencia de

Dirofilaria immitis y en la patología vascular del hospedador durante la
dirofilariosis cardiopulmonar” ha sido realizado bajo su dirección, por D. Javier
González Miguel, con D.N.I. 72098244-Y, licenciado en Biología por la Universidad
de Salamanca, en el Área de Parasitología del Departamento de Biología Animal,
Parasitología, Ecología, Edafología y Química Agrícola de la Universidad de
Salamanca. Dicha Tesis Doctoral reúne las condiciones necesarias para ser defendida y
optar a la Mención de Doctor Europeo por la Universidad de Salamanca.

Y para que así conste, a los efectos legales, expiden y firman el presente
certificado en Salamanca, a 25 de Mayo de 2015.

Fdo.: Fernando Simón Martín Fdo.: Rodrigo Morchón García

 DEPARTAMENTO DE BIOLOGÍA ANIMAL,
PARASITOLOGÍA, ECOLOGÍA, EDAFOLOGÍA
Y QUÍMICA AGRÍCOLA
ÁREA DE PARASITOLOGÍA

La presente Tesis Doctoral está elaborada en el formato de compendio de

artículos/publicaciones según la normativa aprobada por la Comisión de Doctorado y
Posgrado de la Universidad de Salamanca el 15 de febrero de 2013 y consta de las
siguientes publicaciones:

1. Excretory/secretory antigens from Dirofilaria immitis adult worms interact

with the host fibrinolytic system involving the vascular endothelium
Javier González-Miguela, Rodrigo Morchóna, Isabel Melladoa, Elena Carretónb,
José Alberto Montoya-Alonsob, Fernando Simóna
a
Laboratorio de Parasitología, Facultad de Farmacia, Universidad de Salamanca,
b
37007 Salamanca, España; Medicina Interna, Facultad de Veterinaria,
Universidad de Las Palmas de Gran Canaria, 35413 Arucas, Las Palmas,
España.
Molecular & Biochemical Parasitology 181 (2012) 134-140.
Factor de impacto (2012): 2,734. doi:10.1016/j.molbiopara.2011.10.010

2. Surface associated antigens of Dirofilaria immitis adult worms activate the

host fibrinolytic system
Javier González-Miguela, Rodrigo Morchóna, Elena Carretónb, José Alberto
Montoya-Alonsob, Fernando Simóna
a
Laboratorio de Parasitología, Facultad de Farmacia, Universidad de Salamanca,
b
37007 Salamanca, España; Medicina Interna, Facultad de Veterinaria,
Universidad de Las Palmas de Gran Canaria, 35413 Arucas, Las Palmas,
España.
Veterinary Parasitology 196 (2013) 235-240
Factor de impacto (2013): 2,545. doi:10.1016/j.vetpar.2013.01.028
3. Surface-displayed glyceraldehyde 3-phosphate dehydrogenase and galectin
from Dirofilaria immitis enhance the activation of the fibrinolytic system of
the host
Javier González-Miguela, Rodrigo Morchóna, Mar Siles-Lucasb, Ana Oleagab,
Fernando Simóna
a
Laboratorio de Parasitología, Facultad de Farmacia, Universidad de Salamanca,
b
37007 Salamanca, España; Laboratorio de Parasitología, IRNASA, CSIC,
37008 Salamanca, España.
Acta Tropica 145 (2015) 8-16.
Factor de impacto (2014): 2,519. doi:10.1016/j.actatropica.2015.01.010

4. Can the activation of plasminogen/plasmin system of the host by metabolic

products of Dirofilaria immitis participate in heartworm disease
endarteritis?
Javier González-Miguela, Rodrigo Morchóna, Elena Carretónb, José Alberto
Montoya-Alonsob, Fernando Simóna
a
Laboratorio de Parasitología, Facultad de Farmacia, Universidad de Salamanca,
b
37007 Salamanca, España; Medicina Interna, Facultad de Veterinaria,
Universidad de Las Palmas de Gran Canaria, 35413 Arucas, Las Palmas,
España.
Parasites & Vectors (2015) 8: 194.
Factor de impacto (2014): 3,25. doi:10.1186/s13071-015-0799-0

5. Fibrinolysis and Proliferative Endarteritis: Two Related Processes in

Chronic Infections? The Model of the Blood-Borne Pathogen Dirofilaria
immitis
Javier González-Miguela, Rodrigo Morchóna, Mar Siles-Lucasb, Fernando
Simóna
a
Laboratorio de Parasitología, Facultad de Farmacia, Universidad de Salamanca,
b
37007 Salamanca, España; Laboratorio de Parasitología, IRNASA, CSIC,
37008 Salamanca, España.
PLoS ONE (2015) 10(4):e0124445.
Factor de impacto (2014): 3,534. doi:10.1371/journal.pone.0124445
 DEPARTAMENTO DE BIOLOGÍA ANIMAL,
PARASITOLOGÍA, ECOLOGÍA, EDAFOLOGÍA
Y QUÍMICA AGRÍCOLA
ÁREA DE PARASITOLOGÍA

D. Fernando Simón Martín, Catedrático de Parasitología de la Universidad de

Salamanca y D. Rodrigo Morchón García, Profesor Ayudante Doctor adscrito al Área
de Parasitología de la Universidad de Salamanca,

AUTORIZAN:

Que la Tesis Doctoral titulada “El papel de la plasmina en la supervivencia de

Dirofilaria immitis y en la patología vascular del hospedador durante la
dirofilariosis cardiopulmonar” sea presentada en la modalidad de compendio de
artículos/publicaciones (Comisión de Doctorado y Posgrado, 15 de febrero de 2013).

Y para que así conste, a los efectos legales, expiden y firman el presente
certificado en Salamanca, a 25 de Mayo de 2015.

Fdo.: Fernando Simón Martín Fdo.: Rodrigo Morchón García

 Javier González Miguel ha sido beneficiario de una Beca adscrita a Contrato
Art. 83 LOU suscrito con la empresa Chemical Ibérica Productos Veterinarios S. L.
durante 8 meses (enero-septiembre 2008), de una Beca de postgrado del Programa
Nacional de Formación de Profesorado Universitario (FPU) del Ministerio de
Ciencia e Innovación (AP2009-3479) durante 4 años (julio 2009-julio 2013) y
actualmente se encuentra contratado como Personal Docente Investigador a través de
un Proyecto Art. 83 LOU por la Universidad de Salamanca.

Este trabajo ha sido financiado en parte, por la Agencia de Desarrollo

Económico de Castilla y León (cofinanciado con fondos FEDER), Junta de Castilla y
León (grant SA090/A09), España.
 “La ciencia es respecto del alma lo que es la luz respecto de los ojos,
y si las raíces son amargas, los frutos son muy dulces”
Aristóteles

“Heavenly Fruits” – Vladimir Kush

Esta Tesis Doctoral está profundamente dedicada a todas aquellas personas que
me han prestado su aliento y han brindado su apoyo científico, económico, logístico y
psicoterapéutico para que este árbol se muestre sano y lustroso; y de forma muy especial
a Alberto, Antonia, Fernando, Rodrigo y Fernando. Mil gracias.
ÍNDICE DE CONTENIDOS
 ÍNDICE DE CONTENIDOS

INTRODUCCIÓN 12

REVISIÓN BIBLIOGRÁFICA 15
1. Sistemática y filogenia 16
2. Biología y ciclo de D. immitis 16
2.1. Desarrollo de D.immitis en los vectores 17
2.2. Desarrollo de D.immitis en los hospedadores definitivos 18
2.3. Composición proteica de D. immitis 19
2.4. La bacteria simbionte intracelular Wolbachia 22
3. Importancia epidemiológica de la dirofilariosis 23
3.1. Distribución de la dirofilariosis cardiopulmonar canina 25
3.1.1. América 25
3.1.2. Europa 25
3.1.3. África 26
3.1.4. Asia y Australia 26
4. Las relaciones parásito/hospedador en la dirofilariosis 27
4.1. Tejidos del hospedador afectados por la patología vascular. La pared
arterial 27
4.1.1. El endotelio vascular 29
4.1.2. El músculo liso perivascular 29
4.1.3. La matriz extracelular 30
4.2. Características clínicas de la dirofilariosis cardiopulmonar 31
4.2.1. Daños vasculares 31
4.2.2. Desarrollo posterior de la enfermedad 34
4.2.3. Patología aguda 35
4.2.4. Sintomatología 36
4.3. Mecanismos patogénicos 38
4.4. Respuesta inmune 41
4.5. Mecanismos de evasión y supervivencia 42
5. El sistema fibrinolítico 44
5.1. Componentes del sistema fibrinolítico 45
5.1.1. Sistema Plasminógeno – Plasmina 45
5.1.2. Activadores del plasminógeno 46
 ÍNDICE DE CONTENIDOS

5.1.3. Inhibidores de la fibrinolisis 48

5.1.4. Receptores del plasminógeno 50
5.2. Interacción de los patógenos con el sistema fibrinolítico de sus
hospedadores 51
5.3. Fisiopatología de la fibrinolisis 55
6. Diagnóstico y manejo de la dirofilariosis 57
6.1. Diagnóstico de la dirofilariosis cardiopulmonar canina 57
6.2. Tratamiento y prevención 59
Bibliografía 62

HIPÓTESIS Y OBJETIVOS 84

PRIMER CAPÍTULO 86
Excretory/secretory antigens from Dirofilaria immitis adult worms interact
with the host fibrinolytic system involving the vascular endothelium

SEGUNDO CAPÍTULO 106

Surface associated antigens of Dirofilaria immitis adult worms activate the
host fibrinolytic system

TERCER CAPÍTULO 120

Surface-displayed glyceraldehyde 3-phosphate dehydrogenase and galectin
from Dirofilaria immitis enhance the activation of the fibrinolytic system of
the host

CUARTO CAPÍTULO 147

Can the activation of plasminogen/plasmin system of the host by metabolic
products of Dirofilaria immitis participate in heartworm disease endarteritis?

QUINTO CAPÍTULO 167

Fibrinolysis and proliferative endarteritis: two related processes in chronic
infections? The model of the blood-borne pathogen Dirofilaria immitis

CONCLUSIONES 201

ABSTRACT 203
INTRODUCCIÓN
 INTRODUCCIÓN

La dirofilariosis cardiopulmonar causada por Dirofilaria immitis es una

parasitosis de transmisión vectorial que afecta a las poblaciones de cánidos y félidos
domésticos y silvestres de todo el mundo. Además, D. immitis puede transmitirse al
hombre; de hecho, casos de dirofilariosis humana se detectan con frecuencia creciente,
por lo que a la indudable importancia de la dirofilariosis desde el punto de vista
veterinario, hay que añadir el interés médico de las infecciones zoonósicas.

D. immitis se ha adaptado en diversos grados a sus distintos hospedadores,

estableciendo con ellos un amplio espectro de relaciones. Si bien existen precisas
descripciones de las consecuencias patológicas de la infección tanto en perros y gatos
como en humanos, disponemos de mucha menos información sobre las moléculas y
células, tanto del parásito como de sus hospedadores, implicadas en esas relaciones, así
como de los mecanismos en los que intervienen.

Al igual que las filarias tropicales, los vermes adultos de D. immitis presentan
una gran longevidad, pese a vivir en un entorno tan hostil como la sangre. Este hecho,
necesariamente, debe estar asociado a mecanismos de supervivencia desarrollados por
el parásito, que le permitan modificar en beneficio propio su entorno inmediato,
constituido por la propia sangre y la pared arterial. Además de los mecanismos
antiinflamatorios previamente estudiados por nuestro equipo, en la actualidad, la
activación del sistema fibrinolítico de los hospedadores por parte de los organismos
invasores es considerada un mecanismo de supervivencia clave en las relaciones que se
establecen entre los patógenos sanguíneos y sus hospedadores, ya que no solo
contribuye a mantener la hemostasia, hecho fundamental para los patógenos hemáticos,
sino que también se ha relacionado con su capacidad invasiva.

En la dirofilariosis crónica, el aparente equilibrio que se prolonga durante largos

períodos de tiempo, beneficioso tanto para Dirofilaria como para el hospedador, puede
alterarse por la influencia de diversos factores derivados tanto del parásito (presencia
física de los vermes, acción de sus productos metabólicos y la muerte de algunos de
ellos), como del hospedador (respuesta inmune y conformación del sistema vascular),
dando comienzo a la patología vascular, cuyos eventos principales son la aparición de
endarteritis proliferativa y la formación de coágulos. Aunque se han propuesto diversos
factores desencadenantes de la formación de vellosidades en la pared arterial durante la

13
 INTRODUCCIÓN

dirofilariosis animal, no existen hasta el momento estudios experimentales orientados a

la identificación de los mecanismos moleculares implicados. Recientemente,
investigaciones realizadas en patología vascular humana han relacionado la
sobreproducción de plasmina, molécula clave del sistema fibrinolítico, con la
proliferación y migración de las células de la pared arterial, así como con la destrucción
de la matriz extracelular. Dichos procesos presentan una evidente similitud con los que
conducen a la aparición de endarteritis proliferativa en la dirofilariosis cardiopulmonar.

Considerando todos estos hechos, en la presente Tesis Doctoral se han

investigado las moléculas de D. immitis implicadas en la activación del sistema
fibrinolítico con la consecuente producción de plasmina y el posible papel de esta
enzima en el desarrollo de la endarteritis proliferativa durante la dirofilariosis
cardiopulmonar.

14
REVISIÓN BIBLIOGRÁFICA
 Revisión bibliográfica

1. SISTEMÁTICA Y FILOGENIA

Dirofilaria immitis (Leidy, 1856) es un nematodo filaroideo incluido dentro de la

familia Onchocercidae. Esta familia comprende a su vez dos subfamilias, la subfamilia
Onchocercinae, con los géneros Onchocerca, Brugia, Wuchereria y Mansonella; y la
subfamilia Dirofilariinae, con los géneros Dirofilaria y Loa (Marquardt et al., 2000; Bain,
2002). No obstante, esta clasificación basada en caracteres morfológicos ha sido
cuestionada desde el punto de vista molecular. Xie et al. (1994) propusieron una relación
más cercana entre los géneros Loa y Mansonella por una parte, y entre Onchocerca y
Dirofilaria por otra, comparando la región espaciadora del ARN ribosomal 5S. Estos
resultados fueron indirectamente corroborados gracias al estudio filogenético de las
bacterias endosimbiontes del género Wolbachia presentes en las filarias (Sironi et al.,
1995; Bandi et al., 1998), y más recientemente mediante análisis filogenéticos basados
en las secuencias mitocondriales correspondientes a la citocromo oxidasa I y al ARN
ribosomal 12S de las propias filarias (Casiraghi et al., 2001; Huang et al., 2009).

2. BIOLOGÍA Y CICLO DE D. IMMITIS

Los vermes adultos de D. immitis son largos, delgados, de aspecto filiforme y con
un marcado dimorfismo sexual. Las hembras miden 250-300 mm de longitud por 1-1,3
mm de diámetro, mientras que los machos, más pequeños y con su extremo posterior
enrollado en espiral, miden 120-200 mm de longitud por 0,7-0,9 mm de diámetro
(Manfredi et al., 2007). Poseen un ciclo biológico indirecto que implica a un hospedador
definitivo vertebrado y a un vector (Figura 1). Su escasa especificidad de hospedador
determina que D. immitis pueda parasitar numerosas especies de mamíferos (Barriga,
1982). De todas ellas, los perros y los cánidos silvestres sirven de reservorios de la
enfermedad, al ser los hospedadores a los que mejor se ha adaptado el parásito. Pese a
que se trata de un hospedador menos adecuado para su desarrollo, el gato puede albergar
la fase adulta del parásito, mientras que el hombre también puede verse afectado, aunque
de manera accidental y sin ninguna implicación para la transmisión (McCall et al., 2008;
Simón y Genchi, 2000). En el hospedador humano, los vermes inmaduros de D. immitis
pueden alcanzar una rama de la arteria pulmonar, donde estimulan una reacción
inflamatoria y tromboembólica que destruye los vermes y causa nódulos pulmonares

16
 Revisión bibliográfica

(Simón et al., 2005). La poca especificidad de D. immitis también se ve reflejada a nivel

de su hospedador intermediario. Al menos 70 especies de mosquitos culícidos son
consideradas como vectores potenciales del parásito, aunque solo se ha demostrado la
capacidad vectorial real en algunas de ellas, pertenecientes a los géneros Culex, Aedes,
Culiseta, Mansonia y Coquilletidia dentro de la subfamilia Culicinae y al género
Anopheles dentro de la subfamilia Anophelinae (Cancrini y Kramer, 2001; Cancrini et
al., 2006).

Figura 1. Ciclo biológico de D. immitis. L3, larvas de 3er estadio; mf, microfilarias.

2.1. Desarrollo de D. immitis en los vectores

Cuando las hembras de mosquitos susceptibles se alimentan sobre reservorios

infectados, ingieren microfilarias (larvas de primer estadio) presentes en la sangre
periférica de estos. Aproximadamente 24 horas después, las microfilarias, que miden 290-
330 µm de longitud por 5-7 µm de diámetro (Venco et al., 2011), pasan del tubo digestivo
del vector a los tubos de Malpighi, donde comienzan el desarrollo a larvas infectantes.
Dicho proceso dura entre 10 y 15 días, dependiendo de diferentes condiciones
ambientales, principalmente de la temperatura y comprende dos mudas. La primera da
lugar a las larvas de segundo estadio (L2) y se produce aproximadamente entre los días

17
 Revisión bibliográfica

8º y 10º después de la infección. La siguiente, que tiene como resultado la larva de tercer
estadio (L3) y que implica un importante aumento de tamaño, tiene lugar unos 3 días
después. Las L3 completan su desarrollo emigrando hacia la región cefálica del vector.
Una vez allí, se acumulan en las piezas bucales y son inoculadas en el tejido subcutáneo
de un nuevo hospedador en la siguiente toma de sangre (Manfredi et al., 2007).

2.2. Desarrollo de D. immitis en los hospedadores definitivos

Los mosquitos parasitados depositan una gota de hemolinfa con las L3 durante la
toma de sangre. Estas penetran en la piel por sus propios medios mudando a larvas de
cuarto estadio (L4) entre 3 y 12 días post-
infección, con un considerable aumento de
tamaño asociado. La siguiente muda dará lugar a
los vermes adultos inmaduros (50-70 días post-
infección), los cuales alcanzan su localización
definitiva en las arterias pulmonares y el
ventrículo derecho del corazón entre los días 70º
y 85º después de la infección (Figura 2). La
fecundación comienza a partir de los 120 días
post-infección, cuando se completa el desarrollo
de los vermes adultos, alcanzando estos la Figura 2. Vermes adultos (macho y
madurez sexual (Manfredi et al., 2007). Una vez hembra) de D. immitis en el corazón de un
perro con dirofilariosis (Simón et al.,
realizada la fecundación, las hembras liberan 2012).
microfilarias al torrente circulatorio. Estas aparecen en la circulación periférica entre los
6 meses y medio y 7 meses post-infección. La microfilaremia aumenta durante los 10
meses siguientes y se mantiene constante durante varios años, desapareciendo después
progresivamente. La longevidad de las microfilarias puede alcanzar los dos años,
mientras que los vermes adultos pueden vivir 7 años o más en su localización definitiva
(McCall et al., 2008). Por otra parte, algunos perros infectados no presentan microfilarias
en sangre (infecciones amicrofilarémicas), situación que puede deberse a distintos
factores como el envejecimiento de las hembras, infecciones por vermes de un solo sexo
y/o la respuesta inmune del hospedador (Simón y Genchi, 2000).

18
 Revisión bibliográfica

2.3. Composición proteica de D. immitis

El conocimiento de la composición proteica de un patógeno es esencial para poder

definir las relaciones que establece con su hospedador a nivel molecular. No obstante,
esta información es aún escasa en el caso de D. immitis. La base de datos de secuencias
proteicas del National Center for Biotechnology Information (NCBI) contiene 277
resultados para D. immitis, un número escaso en comparación con el de otros nematodos
en los que se han desarrollado muchos más estudios moleculares (Caenorhabditis
elegans: 85050 resultados; Brugia malayi: 34705 resultados).

Durante la década de los 80 del pasado siglo se llevaron a cabo los primeros
trabajos de identificación proteica en D. immitis, con el objetivo de buscar moléculas que
pudieran ser empleadas en el diagnóstico inmunológico de la dirofilariosis
cardiopulmonar. Se caracterizaron tanto bioquímica como inmunológicamente
numerosos antígenos circulantes en suero de perros infectados naturalmente (Ehrenberg
et al., 1987), así como otros antígenos presentes en la superficie de larvas L3 (Philipp y
Davis, 1986; Ibrahim et al., 1989) y vermes adultos (Scott et al., 1988).

En la década siguiente, la mejora de las técnicas de biología molecular permitió

un aumento considerable de los estudios de identificación y caracterización proteica en
D. immitis. Muchos de ellos fueron realizados por el grupo de investigación liderado por
los doctores Glenn R. Frank y Robert B. Grieve (Heska Corporation, EEUU). Entre los
más relevantes se encuentran la identificación y clonación de un grupo de antígenos que
han servido para el diagnóstico de la dirofilariosis cardiopulmonar, como P20, P22L y
P22U (Frank y Grieve, 1991; Frank et al., 1996; Frank et al., 1999) o un inhibidor de la
aspartil proteasa (Dit33) (Frank et al., 1998). Además, llevaron a cabo la clonación de
una proteína similar a la glutatión peroxidasa (Di29) excretada por vermes adultos (Tripp
et al., 1998), así como la identificación de un gran número de antígenos de L3 y L4 con
características proteolíticas (Richer et al., 1992) o posibles aplicaciones
inmunoprofilácticas (Grieve et al., 1992). Otros trabajos realizados en esta década
permitieron comparar los repertorios antigénicos de las L2, L3 y L4 (Scott et al., 1990) o
la identificación y clonación molecular de la proteína de choque térmico p27, una
peroxirredoxina, una paramiosina, un factor quimiotáctico de neutrófilos o la ciclofilina
Dicyp-3, entre otras (Limberger y McReynolds, 1990; Owhashi et al., 1993; Lillibridge
et al., 1996; Hong et al., 1998; Zipfel et al., 1998).

19
 Revisión bibliográfica

A partir del año 2000, frente a la producción de proteínas recombinantes de forma

individual, algunas con importantes funciones antioxidantes (Chandrashekar et al., 2000),
relacionadas con la muda (Crossgrove et al., 2002), la estructura (Harris y Fuhrman,
2002), la proliferación y diferenciación (Shea et al., 2004) o como posibles dianas
terapéuticas (Yates y Wolstenholme, 2004), las nuevas técnicas de identificación masiva
han permitido aumentar notablemente el conocimiento sobre las proteínas de D. immitis.
Mediante la combinación de electroforesis bidimensional y espectrometría de masas,
nuestro grupo ha llevado a cabo los primeros estudios de proteómica en los extractos
antigénicos de D. immitis (Oleaga et al., 2009; González-Miguel et al., 2010a y b) (Figura
3). Partiendo de un extracto proteico soluble de vermes adultos y con el objetivo de
conocer qué antígenos son reconocidos por el sistema inmune de perros, gatos o humanos
infectados con D. immitis, se identificaron 39 proteínas inmunógenas del parásito (Tabla
1). Estas proteínas pertenecen principalmente a cuatro grupos funcionales que incluyen
enzimas metabólicas, enzimas detoxificantes o con potencial redox, moléculas
relacionadas con respuesta al estrés y proteínas estructurales. Esto sugiere la importancia
que tiene para el parásito la generación de energía mediante procesos metabólicos tales
como la glicolisis anaerobia, o los mecanismos defensivos mediante el empleo de un
repertorio de enzimas relacionadas con la detoxificación o la respuesta al estrés. Destaca
también el gran número de proteínas que han sido previamente estudiadas en otros
parásitos por su papel como receptores del plasminógeno, como la actina, la enolasa, la
fructosa-bifosfato aldolasa (FBAL) o la gliceraldehído-3-fosfato deshidrogenasa
(GAPDH) (González-Miguel et al., 2010b). Bastantes de las proteínas identificadas están
representadas por diversas isoformas, algunas de las cuales no son reconocidas por el
sistema inmune de los hospedadores, lo que parece indicar que D. immitis posee sistemas
bioquímicos redundantes que hacen más difícil su interferencia por el sistema inmune del
hospedador (Simón et al. 2012).

20
 Revisión bibliográfica

Figura 3. Electroforesis bidimensional representativa de 40 µg de un extracto de proteínas somáticas de

vermes adultos de D. immitis. Geles de pH 5-8 y 7-10 y 12% de acrilamida teñidos con nitrato de plata. Los
spots identificados por espectrometría de masas están numerados (Oleaga et al., 2009; González-Miguel et
al., 2010a y b).

Más recientemente, la publicación de una base de datos sobre el transcriptoma de

D. immitis (Fu et al., 2012), los nuevos datos disponibles sobre el genoma del parásito
(Godel et al., 2012), así como la
aparición de técnicas más efectivas
de identificación están permitiendo
completar el mapa proteico del
parásito. Geary et al. (2012)
identificaron 110 proteínas en el
secretoma de D. immitis mediante la
combinación de cromatografía en
fase líquida con la espectrometría de
Figura 4. Comparación de la abundancia relativa de los
grupos funcionales asignados a las proteínas de D. immitis
masas (LC-MS). Aplicando una
identificadas por LC-MS en el compartimento antigénico metodología similar nuestro grupo
somático del parásito (Morchón et al., 2014).
identificó un total de 108 y 16
proteínas, respectivamente, en los compartimentos somático y de superficie del parásito
(Morchón et al., 2014). Atendiendo a las funciones de estas proteínas, de nuevo aquellas
enzimas relacionadas con el metabolismo energético, los procesos redox, las proteínas
con funciones estructurales o respuesta al estrés fueron las más representadas (Figura 4).

21
 Revisión bibliográfica

Número Código de
Proteína Especie Proceso biológico
de spot acceso (NCBI)

1-4 AAF32254 Proteína de choque térmico Hsp 70 Wuchereria bancrofti Respuesta al estrés
5-7 CAA34719 Actina Caenorhabditis elegans Motilidad celular
8 P30162 Actina-1 Onchocerca volvulus Motilidad celular
9 CAE11787 Disulfuro isomerasa Brugia malayi Homeostasis redox
10-12 AAC24752 Precursor de transglutaminasa Dirofilaria immitis Homeostasis redox
13,14 AAV33247 Fosfoglicerato mutasa Onchocerca volvulus Glicolisis
15-17 A8NLA3 Oxidorreductasa dependiente de FAD Brugia malayi Transporte electrónico
18,19 EDP29666 Glucosa fosfato isomerasa Brugia malayi Glicolisis
20 EDP37909 Inhibidor α de la disociación del Rab Brugia malayi Transporte proteico
GDP
21 P30163 Actina-2 Caenorhabditis elegans Motilidad celular
22 1D4X_A Cadena A de la Mg-Atp actina Caenorhabditis elegans Motilidad celular
23-28 XP_001896281 Enolasa Brugia malayi Glicolisis
29-32 XP_001670614 Proteína hipotética CBG05397 Caenorhabditis briggsae -
33-39 AAB52600 Fructosa-bifosfato aldolasa Onchocerca volvulus Glicolisis
40-42 XP_001891892 Fosfoglicerato quinasa Brugia malayi Glicolisis
43-45 XP_001900957 Fumarasa Brugia malayi Metabolismo aeróbico
46-49 XP_001899850 Gliceraldehído-3- Brugia malayi Glicolisis
fosfato deshidrogenasa
50 XP_001900208 Lactato deshidrogenasa Brugia malayi Glicolisis anaerobia
51 XP_001897743 Aldo/ceto oxidorreductasa Brugia malayi Procesos redox
52-54 XP_001899521 Proteína de desorganización muscular 1 Brugia malayi Adhesión celular
55 AAZ42332 Subunidad β de la proteína G Caenorhabditis remanei Transducción de señales
56-59 Q27384 Precursor del inhibidor de pepsina Dit33 Dirofilaria immitis -
60 NP_508842 Actina-4 Caenorhabditis elegans Motilidad celular
61 AAB08736 Proteína pequeña de choque térmico Dirofilaria immitis Respuesta al estrés
p27
62 P52033 Precursor de la Glutatión peroxidasa Dirofilaria immitis Respuesta al estrés
Di29 oxidativo
63-68 AAF37720 Galectina Dirofilaria immitis Respuesta inmune
69-72 XP_001897269 Triosafosfato isomerasa Brugia malayi Glicolisis
73 CAA73325 Glutatión transferasa Brugia malayi Detoxificación
74-75 AAD11968 P22U Dirofilaria immitis -
76-77 AAC38831 Tiorredoxina peroxidasa Dirofilaria immitis Homeostasis redox
78-81 CAA61152 Proteína pequeña de choque térmico Brugia pahangi Respuesta al estrés
82 AA799423 Peptidil-prolil isomerasa Taenia solium Plegamiento proteico
83-88 CAA48632 Proteína OV25-1 Onchocerca volvulus Respuesta al estrés
89,90 BAA96354 Proteína de unión a Dirofilaria immitis Transducción de señales
fosfatidiletanolamina
91 XP_001899662 Precursor del antígeno OV-16 Brugia malayi Transducción de señales
92,93 AAC47233 Ciclofilina Ovcyp-2 Onchocerca volvulus Plegamiento proteico
94 XP_001902628 Bmcyp-2 Brugia malayi Plegamiento proteico
95,96 BAA02004 Precursor del factor quimiotáctico de Dirofilaria immitis -
neutrófilos Di-NCF
97,98 XP_001901495 Nucleósido-difosfato quinasa Brugia malayi Metabolismo de
nucleótidos

Tabla 1. Proteínas inmunógenas de D. immitis identificadas por MALDI-TOF MS (Oleaga et al., 2009;
González-Miguel et al., 2010a y b). La numeración de spots hace referencia a su localización en el mapa
proteómico de D. immitis ilustrado en la figura 3. Se incluye el nombre de la proteína, especie en la que ha
sido identificada, número de acceso a la información disponible en la base datos del NCBI, y proceso
biológico asignado a cada proteína según las bases de datos Gen Ontology (http://www.geneontology.org)
y Swiss-Prot/Uniprot (http://beta.uniprot.org).

2.4. La bacteria simbionte intracelular Wolbachia

D. immitis alberga bacterias endosimbiontes del género Wolbachia (Kozek et al.,

2007). Estas bacterias fueron descubiertas en los años 70 mediante el empleo de la

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 Revisión bibliográfica

microscopía electrónica (McLaren et al., 1975; Vincent et al., 1975; Kozek y Figueroa,
1977) y “redescubiertas”, dos décadas después, en vermes adultos de D. immitis
permitiendo demostrar su pertenencia al género Wolbachia, orden Rickettsiales (alfa 2
proteobacterias), mediante el empleo de técnicas de biología molecular (Sironi et al.,
1995). Wolbachia se ha encontrado en hexápodos, crustáceos y quelicerados, en los que
causa alteraciones reproductivas. No obstante, con los nematodos de la familia
Onchocercidae ha establecido una relación simbiótica obligatoria al ser su presencia
crucial para la embriogénesis y la muda de las filarias, mientras que estas proporcionan
aminoácidos para el crecimiento de las bacterias (Lamb et al., 2004; Foster et al., 2005;
Fenn y Blaxter, 2006).

Wolbachia se transmite por vía materna representando un componente estable de

la estructura de D. immitis, al encontrarse en todas sus fases evolutivas. Es especialmente
abundante en las larvas que se desarrollan en los hospedadores vertebrados (L3 y L4), en
los cordones hipodérmicos de los adultos de ambos sexos y en los órganos genitales de
las hembras (McGarry et al., 2004). Recientemente, ha sido detectada también en las
gónadas somáticas o en la pared intestinal de nuevas especies de filarias de la familia
Onchocercidae, lo que sugiere que la relación entre las bacterias y las filarias es más
compleja y diversa de lo que se suponía hasta el momento (Ferri et al., 2011).

3. IMPORTANCIA EPIDEMIOLÓGICA DE LA
DIROFILARIOSIS

La dirofilariosis es una enfermedad cosmopolita que afecta a poblaciones caninas

y felinas, tanto domésticas como silvestres de áreas templadas y tropicales de todo el
mundo. Además, en los últimos años la dirofilariosis está experimentando una expansión
hacia áreas con climas más fríos, introduciéndose en países donde no se había denunciado
la transmisión, a la vez que aumentan las prevalencias en las zonas históricamente
endémicas. Diversos factores, tanto dependientes del comportamiento humano en
relación con las mascotas, como climáticos, pueden influir en la expansión de la
enfermedad. Entre ellos, el movimiento sin control de animales infectados, la
introducción de nuevas especies de mosquitos capaces de actuar como vectores o el
cambio climático, causado por el calentamiento global y el desarrollo de la actividad

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 Revisión bibliográfica

humana en nuevas áreas. Todo ello provoca que la dirofilariosis cardiopulmonar sea
considerada actualmente un problema veterinario de primera magnitud y una enfermedad
emergente en algunas áreas (Genchi et al., 2009; Morchón et al., 2012a).

El conocimiento de la distribución de la infección canina es fundamental desde el

punto de vista epidemiológico, ya que la transmisión de Dirofilaria en un área dada
depende de la existencia de perros parasitados que actúen como reservorios de la
enfermedad (Figura 5) (Simón et al., 2012). En comparación con los numerosos estudios
llevados a cabo en perros, la información disponible sobre la distribución de la
dirofilariosis felina es bastante escasa. No obstante, las infecciones felinas por D. immitis
tienden a detectarse en las mismas áreas que la dirofilariosis canina, si bien con
prevalencias entre el 5 y el 20% de las que presentan los perros en las mismas áreas
(Newcombe y Ryan, 2002). Con respecto a la dirofilariosis humana, puesto que algunos
vectores de la enfermedad se alimentan indistintamente de reservorios animales o de
humanos, en áreas donde la dirofilariosis canina es endémica, la dirofilariosis pulmonar
humana existe y puede suponer un problema desde el punto de vista médico. Además,
debido tanto a su carácter asintomático, como a su localización en órganos internos, la
revisión retrospectiva de los casos publicados no proporciona una distribución real de la
enfermedad. En los últimos años, la mejora en las herramientas diagnósticas, junto con el
creciente interés de la comunidad científica ha permitido aumentar notablemente el
número de casos denunciados.

Figura 5. Distribución geográfica de los casos de dirofilariosis cardiopulmonar canina denunciados en el

mundo.

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 Revisión bibliográfica

3.1. Distribución de la dirofilariosis cardiopulmonar canina

3.1.1. América

La dirofilariosis canina ha sido descrita en perros de prácticamente todo el

continente americano a excepción de Chile donde no fue encontrada, y en la Guayana
Francesa y Uruguay donde no se han realizado estudios (Labarthe y Guerrero, 2005;
Guerrero et al., 2006; Lee et al., 2010). Las prevalencias varían dependiendo de la
climatología: Canadá (0,24%) (Slocombe y Villeneuve, 1993), Costa del Golfo de
Estados Unidos (48,8%) (Levy et al., 2011), Estado de Amazonas en Brasil (57,6%)
(Soares et al., 2014), Cuba (63,2%) (Duménigo et al., 1988) o zonas rurales del norte de
Argentina (74%) (Vezzani et al., 2006).

En Estados Unidos, los mapas de incidencia realizados por la American

Heartworm Society desde el año 2001 han permitido conocer, además, la dinámica de la
distribución. Se ha observado que la dirofilariosis se ha extendido desde los estados de
las costas Atlántica y del Golfo hasta los más occidentales, encontrándose actualmente en
todos e incrementando su prevalencia en la mayor parte de ellos, aunque la transmisión
en Alaska no ha sido bien documentada (Nelson et al., 2005; Guerrero et al., 2006; Lee
et al., 2010).

3.1.2. Europa

La presencia del parásito en Europa ha sido históricamente denunciada en países

de la cuenca mediterránea y del sur, como Grecia, Italia, Francia, España y Portugal en
los que la enfermedad es endémica. En Grecia se han denunciado prevalencias en torno
al 10-15% por todo el territorio (Haralampidis, 2003). En Italia D. immitis presenta
prevalencias superiores al 50% en el área hiperendémica del valle del río Po, al norte del
país (Genchi et al., 2001), mientras que en Francia se localiza principalmente en el sur, a
lo largo de la costa Mediterránea (Doby et al., 1986; Guerrero et al., 1992). En España la
distribución de la dirofilariosis canina se reparte zonalmente por todo el territorio, aunque
es especialmente prevalente en zonas asociadas a regadíos como la ribera del Tormes en
Salamanca (33,3%), delta del río Ebro (35,8) o Huelva (36,7%). También se han
observado altas prevalencias en las islas, donde la influencia marina proporciona la
humedad adecuada para las poblaciones de mosquitos, como la isla de Gran Canaria
(23,87%) o Ibiza (39%) (Guerrero et al., 1989; Montoya et al., 2007; Simón et al., 2014).

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 Revisión bibliográfica

En Portugal se han denunciado prevalencias medias en torno al 15-20% a lo largo de todo

el país, encontrándose la más alta en la isla de Madeira (30%) (Araujo, 1996; Genchi et
al., 2001; Vieira et al., 2014a y b).

Por otra parte, un gran número de estudios llevados a cabo en la última década
demuestra que la dirofilariosis canina se ha expandido hacia países del centro y norte del
continente, donde anteriormente no se conocía o donde solo se habían encontrado casos
esporádicos (Morchón et al., 2012a; Simón et al., 2012). Esto ha permitido denunciar
recientemente la presencia de D. immitis en países como Alemania, Ucrania o Rusia
(Kartashev et al., 2011; Hamel et al., 2013; Genchi et al., 2014).

3.1.3. África

La escasez de estudios epidemiológicos realizados en el continente africano junto

con la variedad de los métodos de diagnóstico empleados y el número de especies de
filarias existentes, no permiten conocer la distribución real de la dirofilariosis canina en
África (Simón et al., 2012). Datos revisados por Genchi et al. (2001) han permitido
reportar la presencia del parásito en Marruecos, Túnez, Egipto, Tanzania, Kenia,
Mozambique, Malawi, Senegal, Angola, Gabón, Nigeria y Sierra Leona. Más
recientemente se ha descrito la enfermedad en perros de zonas urbanas en Ghana (Clarke
et al., 2014).

3.1.4. Asia y Australia

Un número mayor de estudios, aunque realizados de manera esporádica, ha

permitido localizar la presencia de D. immitis en muchas regiones de Asia y Australia,
con prevalencias variables. En Irán la dirofilariosis canina se reparte por todo el territorio,
alcanzando prevalencias del 51,4% en las zonas más húmedas del norte (Khedri et al.,
2014) y así mismo, en la India se localiza principalmente en el noreste del país
(Chakravarty y Chaudhuri, 1983; Patnaik, 1989). En la parte más oriental del continente
se han denunciado prevalencias elevadas del parásito en China (21,6%) (Sun et al., 2012),
Taiwan (57%) (Wu y Fan, 2003), Japón (59%) (Tanaka et al., 1985), Corea del Sur
(69,5%) (Song et al., 2003) y Malasia (70%) (Lok, 1988). En Australia, D. immitis es
endémica a lo largo de las áreas costeras del norte y oeste, así como en la parte oriental
de los estados de Queensland, Nueva Gales del Sur y Victoria (Kendall et al., 1991;
Bidgood y Collins, 1996).

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 Revisión bibliográfica

4. LAS RELACIONES PARÁSITO/HOSPEDADOR EN LA

DIROFILARIOSIS

El conocimiento de los mecanismos que rigen las interacciones entre los parásitos
y sus hospedadores, así como la implicación de los tejidos del hospedador y sus
alteraciones patológicas es fundamental para establecer las pautas adecuadas de manejo
y control de las parasitosis. Estas relaciones resultan especialmente complejas en la
dirofilariosis como consecuencia de dos factores: la capacidad de D. immitis para infectar
distintos hospedadores en los que el parásito muestra diversos grados de adaptación y
desarrollo; y la presencia de la bacteria simbionte Wolbachia que afecta a la inmunidad
desarrollada por el hospedador, a la patogenia y consecuentemente al cuadro clínico de la
enfermedad (Simón et al., 2009).

Pese a que la patología en general y la patología vascular en particular de la

dirofilariosis cardiopulmonar han sido ampliamente descritas, los estudios sobre las
relaciones que establece D. immitis con sus hospedadores a nivel molecular (mecanismos
patogénicos, respuesta inmune y estrategias de supervivencia) son relativamente escasos.

4.1. Tejidos del hospedador afectados por la patología vascular. La

pared arterial

Las arterias pulmonares lobares constituyen la localización definitiva primaria de

los vermes adultos de D. immitis. Allí los parásitos pueden sobrevivir durante largos
períodos de tiempo (más de 7 años), en números que pueden variar entre uno y más de
250 individuos. La pared arterial es, por tanto, el tejido del hospedador con el que el
parásito interacciona de manera inmediata, estableciendo un amplio intercambio
molecular y causando las primeras y algunas de las más graves alteraciones patológicas
de la dirofilariosis cardiopulmonar.

La pared de las arterias pulmonares, cuya estructura ha sido recientemente

revisada en profundidad por Townsley (2012), presenta, al igual que la de los demás vasos
sanguíneos, tres capas concéntricas (Figura 6). La túnica íntima está formada por un
revestimiento de células endoteliales dispuestas longitudinalmente en contacto directo

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 Revisión bibliográfica

con la sangre, la membrana basal y una región de tejido conectivo subendotelial donde se
pueden encontrar células musculares lisas de
manera esporádica. A continuación y separada
de la íntima por una lámina elástica interna se
encuentra la túnica media. Es la capa de mayor
grosor y está compuesta principalmente por
células musculares lisas que se disponen
concéntricamente y están embebidas en una
matriz extracelular rica en colágenos, elastina,
fibrilina y proteoglicanos. Finalmente la
túnica adventicia es la capa más alejada del
lumen vascular y consta principalmente de
fibroblastos dispuestos longitudinalmente
Figura 6. Imagen representativa de la
dentro de una matriz que contiene colágeno y disposición en capas de una arteria. Tomada de
Encyclopædia Britannica Online.
elastina separada del medio externo por una
<http://global.britannica.com/EBchecked/medi
lámina elástica (Figura 7). a/95216>

Pese a esta descripción de la pared arterial constituida por tres capas

estructuralmente distintas, la evidencia indica que los componentes celulares y
extracelulares de estas capas están ampliamente interconectados, de tal manera que los
límites que se han marcado tradicionalmente son inciertos. Esto es particularmente
evidente si se considera que dichas interconexiones juegan un papel clave en la capacidad
funcional de la pared arterial. Así, a lo largo de gran parte de la red arterial pulmonar, la
lámina elástica interna se compone de membranas fenestradas que permiten el desarrollo
de proyecciones endoteliales hacia la túnica media facilitando la comunicación entre el
endotelio y el músculo liso perivascular. Estas interacciones célula-célula se consideran
esenciales para la integración local de la vasoconstricción y de la vasodilatación, así como
para la coordinación de las respuestas vasculares en la red de vasos interconectados
(Martinez-Lemus, 2012; Townsley, 2012).

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 Revisión bibliográfica

Figura 7. Corte histológico

mostrando una arteria pulmonar
normal canina. La túnica íntima,
media y adventicia muestran un
grosor y estructura celular normal.
Tinción: hematoxilina-eosina.
Aumento: 10X (Quinn y Williams,
2011).

4.1.1. El endotelio vascular

El endotelio es una monocapa continua formada por células unidas a la vez entre
sí y a la membrana basal subyacente. Las células endoteliales son células polarizadas, con
un dominio apical en contacto con la sangre y uno distal basal en contacto con el
subendotelio (Dejana et al., 1995). El endotelio vascular está considerado el principal
órgano de regulación de numerosas funciones vasculares teniendo, además, un papel
clave en la regulación de la homeostasis al constituir la interfase entre la sangre y los
tejidos. Cualquier alteración tanto en la composición de la sangre como en el flujo
sanguíneo puede convertir un endotelio sano con características antitrombóticas,
antiinflamatorias y vasodilatadoras en un tejido donde predomine la coagulación, la
inflamación y la vasoconstricción (Michiels, 2003).

4.1.2. El músculo liso perivascular

El músculo liso perivascular constituye el componente mayoritario de la túnica

media y se presenta generalmente en forma de haces de células musculares lisas de
apariencia fusiforme y con un núcleo alargado. Estas se encuentran rodeadas de una
lámina externa e interconectadas por uniones en hendidura. La principal función de las
células musculares lisas dentro de la túnica media es la de controlar el diámetro vascular
mediante los procesos de contracción y relajación celular. Para ello se disponen
concéntrica y perpendicularmente al eje longitudinal del vaso (Martinez-Lemus, 2012;
Townsley, 2012). Además, estas células juegan un papel fundamental en otros procesos
fisiológicos como el mantenimiento del tono vascular, reparación, cicatrización y
desarrollo arterial, así como en las principales enfermedades vasculares, incluyendo la

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 Revisión bibliográfica

formación de la placa de ateroma (Schwartz, 1997). Las células musculares lisas

vasculares tienen la capacidad de modular su fenotipo o estado de diferenciación. Por
ello, en arterias adultas intactas, las células musculares son normalmente quiescentes y
presentan un estado diferenciado conocido como fenotipo contráctil, el cual es esencial
para la estabilidad hemodinámica. En contraste, la formación de lesiones derivadas de
aterosclerosis o estenosis implica la diferenciación de las células musculares lisas a un
estado conocido como fenotipo sintético. Este proceso incluye una reorganización
estructural visible dentro de las células que permite aumentar su capacidad para proliferar,
migrar hacia la túnica íntima y secretar componentes de la matriz extracelular (Dupont et
al., 2005).

4.1.3. La matriz extracelular

La matriz extracelular constituye uno de los mayores componentes de los vasos

sanguíneos, suponiendo más de la mitad de la masa de la pared de arterias y venas
(Hungerford y Little, 1999). Pese a estar formada principalmente por colágenos y
elastinas, otros componentes como la fibronectina, microfibrillas (principalmente
fibrilinas) y abundante material soluble o amorfo como proteoglicanos y glicoproteínas
pequeñas ricas en leucina están presentes entre los espacios extracelulares de la pared de
los vasos, siendo cruciales para su integridad (Bou-Gharios et al., 2004).

La matriz extracelular define las propiedades mecánicas críticas para una correcta
función del sistema vascular. Además, el adecuado balance entre su producción y
degradación es crucial para el mantenimiento de la estructura del tejido, así como para su
desarrollo y reparación (Wagenseil y Mecham, 2009). Los diferentes tipos celulares de
los vasos sanguíneos son los encargados de la síntesis de los componentes de la matriz
extracelular. Se asume que las células endoteliales son las principales responsables de la
síntesis y deposición de los componentes de la matriz extracelular de la túnica íntima
(Davis y Senger, 2005). En la túnica media el material extracelular, incluido el colágeno
y las fibras elásticas es producido principalmente por las células del músculo liso durante
el desarrollo, mientras que en la túnica adventicia, el colágeno es sintetizado y secretado
por los fibroblastos, como en otros tejidos conectivos (Bou-Gharios et al., 2004;
Wagenseil y Mecham, 2009).

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La degradación de la matriz extracelular es realizada por un grupo de enzimas

conocidas como metaloproteasas de matriz que incluye colagenasas, gelatinasas,
estromelisinas, metaloelastasas, así como cualquier otra proteína capaz de degradar
específicamente algún componente de la matriz extracelular (Galis y Khatri, 2002). Estas
enzimas, cuya síntesis es realizada por las células endoteliales, musculares lisas y por los
fibroblastos, son capaces de degradar completamente la matriz extracelular y, por tanto,
sus mecanismos de acción han de ser estrictamente regulados (Bou-Gharios et al., 2004).
Además, puesto que otras muchas moléculas que no forman parte de la matriz extracelular
son sustratos potenciales de las metaloproteasas, su actividad puede afectar a procesos
tan importantes como la migración celular, diferenciación, crecimiento, procesos
inflamatorios, neovascularización o apoptosis (Nagase et al., 2006).

4.2. Características clínicas de la dirofilariosis cardiopulmonar

La dirofilariosis cardiopulmonar está descrita en el perro, principal hospedador de

D. immitis, como una enfermedad grave, que potencialmente puede causar la muerte del
hospedador. Presenta un desarrollo generalmente crónico muy complejo, afectando
progresivamente al sistema vascular, al parénquima pulmonar y en sus últimas fases, a
las cámaras derechas del corazón (Furlanello et al., 1998; Venco, 2007; McCall et al.,
2008). Los daños provocados en el hospedador se atribuyen principalmente a los vermes
adultos. Estos, localizados en las arterias pulmonares, inducen las primeras lesiones en
las paredes de los vasos, hecho clave para la evolución de la enfermedad y para el
desarrollo posterior de la patología pulmonar y cardíaca (Simón et al., 2012) (Figura 8).

4.2.1. Daños vasculares

Con la llegada de los vermes a las arterias pulmonares comienzan los primeros
daños en el tejido que está en contacto con los parásitos, el endotelio. Como respuesta al
trauma mecánico, se producen cambios anatómicos en la pared arterial, aumentando el
tamaño tanto de las células endoteliales como el de los espacios intracelulares, a la vez
que se desorientan los ejes longitudinales de las células (Venco y Vezzoni, 2001). La
desorganización del endotelio facilita la infiltración de células inflamatorias,
principalmente neutrófilos, hacia los espacios perivasculares. Por otra parte, la exposición
del subendotelio favorece la activación plaquetaria. Todo ello desemboca en un proceso

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de endarteritis proliferativa, fenómeno de hiperplasia característico de la dirofilariosis

cardiopulmonar (Figura 9).

Figura 8. Progresión de la patología de la dirofilariosis cardiopulmonar. La enfermedad tiene

habitualmente un curso crónico afectando inicialmente al sistema vascular, para luego extenderse al tejido
pulmonar y a las cámaras derechas del corazón. La muerte simultánea de un gran número de vermes adultos
puede desencadenar la forma aguda de la enfermedad (Simón et al., 2012).

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Figura 9. Imágenes representativas de la presencia de endarteritis proliferativa en la arteria pulmonar de

un perro con dirofilariosis. Se puede observar el endotelio engrosado con presencia de vellosidades y
coloración purpúrea del mismo (Carretón et al., 2012).

Figura 10. Endarteritis proliferativa. (A) Sección de la arteria pulmonar de un gato con dirofilariosis donde
se pueden observar numerosas microvellosidades debido al engrosamiento de la túnica íntima y a la
hiperplasia de las células endoteliales. (B) Arteria pulmonar de un gato con dirofilariosis donde se aprecia
un verme de D. immitis rodeado en parte por un trombo. Se observa una gran proliferación vellosa con
infiltración de células inflamatorias. Tinción: hematoxilina-eosina (McCracken y Patton, 1993).

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La endarteritis proliferativa está causada por la proliferación y migración de

células de la pared arterial hacia el lumen vascular, por lo que la pared de la arteria deja
de ser lisa y blanca y desarrolla microvellosidades intravasculares, que dan a la superficie
de la arteria pulmonar un aspecto rugoso y una tonalidad púrpura (Venco, 2007; Carretón
et al., 2012). Se ha descrito que este proceso va acompañado de proliferación y migración,
tanto de células endoteliales como del músculo liso perivascular (Adcock, 1961; Atwell
et al., 1986; Hidaka et al., 2004; Kawabata et al., 2008), así como de degradación de la
matriz extracelular (Wang et al., 2005) (Figura 10).

Además, se pierde la capacidad de contracción/relajación de la pared arterial, las

arterias lesionadas pierden elasticidad, se vuelven tortuosas y aunque sufren dilatación,
la luz disminuye debido al engrosamiento provocado por la formación de
microvellosidades. Esto puede provocar oclusión de las arteriolas más estrechas por
embolización (Kaiser et al., 1989; Carretón et al., 2012).

4.2.2. Desarrollo posterior de la enfermedad

La patología pulmonar aparece de manera secundaria, provocada por los cambios

vasculares. El aumento de la permeabilidad vascular, la disminución del calibre de los
vasos y las obstrucciones que alteran el flujo sanguíneo conducen a un aumento de la
presión en la arteria pulmonar, produciéndose hipertensión pulmonar (Venco y Vezzoni,
2001; Venco et al., 2011). Esta, aunque suele ser moderada, puede triplicarse en la arteria
pulmonar durante el ejercicio debido a la pérdida de elasticidad de la pared arterial
(Kittleson, 1998). Por otra parte, la salida de líquido y antígenos parasitarios desde los
vasos dañados hacia el parénquima pulmonar perivascular provoca edema e inflamación
(Figura 11A). La presencia de eosinófilos y neutrófilos conforma infiltrados intersticiales
y alveolares que provocan una fibrosis irreversible y esta a su vez, la disminución del área
de intercambio gaseoso y el aumento de la resistencia vascular pulmonar (Rawlings,
1986). Además, la aparición de enfisema y de hipertrofia de la musculatura bronquial
asociada también ha sido descrita (Dillon et al., 1995; Venco et al., 2011).

En animales sometidos a ejercicio físico y con una gran carga parasitaria, el estado
de hipertensión pulmonar provoca una dilatación del ventrículo derecho acompañada de
hipertrofia compensatoria para mantener la alta presión de perfusión y mover la sangre a
los pulmones (Venco, 2007; Wang et al., 2005) (Figura 11B). La persistencia de la

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hipertensión pulmonar, junto con el aumento del ritmo cardíaco producido por el ejercicio
o por fenómenos tromboembólicos, puede generar una dilatación irreversible de la parte
derecha del corazón, determinando una insuficiencia cardíaca congestiva. Diferentes
complicaciones como edema miocárdico, fibrosis, isquemia o insuficiencia valvular
como consecuencia del agrandamiento de las cámaras atrio-ventriculares o por la
presencia de los vermes en el interior del corazón pueden agravar finalmente la situación
(Venco y Vezzoni, 2001; Venco, 2007). Además de la patología cardiopulmonar, la
dirofilariosis puede causar alteraciones en órganos y tejidos tan dispares como el riñón,
el hígado, el cerebro, la cámara anterior del ojo o la cavidad peritoneal, debido entre otras
cosas a localizaciones ectópicas o aberrantes del parásito (McCall et al., 2008).

A B

Figura 11. (A) Edema pulmonar en un perro con dirofilariosis (Carretón et al., 2012). (B) Cardiomegalia
con dilatación de las cámaras cardíacas derechas del corazón en un perro con dirofilariosis. Se puede
observar la presencia de vermes adultos de D. immitis saliendo a través del tronco de la arteria pulmonar
(Carretón et al., 2012).

4.2.3. Patología aguda

De forma paralela al desarrollo crónico de la enfermedad descrito hasta el

momento, pueden producirse procesos agudos que suponen un riesgo inmediato para la
vida de los animales que los padecen (Venco, 2007). Aparecen cuando se produce la
muerte súbita y simultánea de muchos vermes adultos, de manera natural o como
consecuencia de un tratamiento filaricida. Los parásitos vivos tienen capacidad para
controlar la formación de trombos, pero cuando mueren, la liberación masiva de
productos antigénicos al torrente circulatorio produce una trombosis masiva y una
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exacerbación de las reacciones inflamatorias en el endotelio vascular (Venco y Vezzoni,

2001) (Figura 12). Los vermes muertos son arrastrados distalmente hacia las arterias más
finas, mientras que los fragmentos del parásito se calcifican y son parcialmente
incorporados a la pared de la arteria, con la posterior cicatrización y formación de gran
cantidad de tejido conectivo fibroso. Todo ello provoca un grave deterioro del flujo
sanguíneo, hipoxia e inflamación granulomatosa de la pared arterial con un crecimiento
exagerado de las vellosidades y un aumento de la permeabilidad, con la formación de
edemas perivasculares (Carretón et al., 2012).

Por último, dentro de la forma

aguda de la enfermedad, destaca el
síndrome de la vena cava. Esta
variante clínica suele presentarse en
animales de pequeño tamaño y con
una alta carga parasitaria, se presenta
de improviso y su pronóstico es de
reservado a grave, presentando una
alta mortalidad. Consiste en la
acumulación masiva de vermes
Figura 12. Tromboembolismo (flecha amarilla)
adultos en el atrio ventricular derecho. localizado en la arteria pulmonar de un perro que murió
de dirofilariosis cardiopulmonar (Cortesía de L. Venco,
Las turbulencias que aparecen en el
Ospedale Veterinario “Città di Pavia”, Pavia, Italia).
torrente sanguíneo inducen daños
mecánicos en la pared de los eritrocitos provocando hemólisis. Además, la masa de
parásitos se opone al retorno venoso al corazón, lo que provoca un estado de shock
cardiocirculatorio (Kitagawa et al., 1987; Furlanello et al., 1998; Carretón et al., 2012).

4.2.4. Sintomatología

En la dirofilariosis cardiopulmonar canina, los síntomas, en general, van

apareciendo progresivamente en consonancia con el desarrollo crónico habitual de la
enfermedad. La mayor parte de los perros infectados no presenta síntomas durante meses
o años, y estos dependen de la carga parasitaria, la reactividad individual y el ejercicio
físico al que son sometidos (Dillon et al., 1995). Cuando aparecen los síntomas, estos
incluyen tos, disnea y taquipnea, intolerancia al ejercicio, pérdida de peso, síncope,
hemoptisis o epistaxis y ascitis (Figura 13). Además, aparecen sonidos pulmonares en los

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lóbulos caudales, arritmias y ruidos cardíacos por insuficiencia de la tricúspide. Cuando

se desarrolla la insuficiencia cardíaca congestiva derecha se presenta, además, pulso
venoso yugular e ingurgitación de las yugulares, hepatomegalia, edema pulmonar y
derrame pleural (Carretón et al., 2012).

En relación con la variante aguda de la enfermedad y ante la aparición de

tromboembolismos tras la muerte masiva de vermes adultos de D. immitis, los perros
pueden mostrar disnea y hemoptisis potencialmente fatales. La muerte súbita es rara pero
puede sobrevenir como consecuencia de una insuficiencia respiratoria, caquecsia o
fenómenos tromboembólicos graves (Venco y Vezzoni, 2001). El síndrome de vena cava
se manifiesta con signos de colapso cardiovascular, disnea, soplo cardíaco y
hemoglobinuria debida a hemólisis mecánica como consecuencia de las turbulencias
causadas por la masa de vermes al dificultar el flujo sanguíneo (Venco et al., 2011).

Figura 13. Ascitis en un perro con dirofilariosis (Cortesía de J. A. Montoya-Alonso, Facultad de

Veterinaria, Universidad de Las Palmas de Gran Canaria, Las Palmas de Gran Canaria, España).

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4.3. Mecanismos patogénicos

Históricamente, la patogénesis de la dirofilariosis ha sido atribuida a la presencia

física de los vermes adultos en las arterias pulmonares de los hospedadores. Sin embargo,
la complejidad del cuadro clínico de la dirofilariosis cardiopulmonar no puede explicarse
sin otros factores relacionados con el parásito y sus moléculas excretadas. La idea de
explicar los mecanismos patogénicos de la dirofilariosis a través de la alteración de la
función fisiológica del endotelio causada por productos excretados por las filarias, fue
postulada en los años 80 y 90 del pasado siglo por un equipo multidisciplinar de la
Universidad de Michigan (EEUU). Estos investigadores propusieron que los vermes
adultos de D. immitis contribuyen a la alteración de la capacidad de relajación de las
arterias pulmonares. Dicho proceso estaría mediado por la liberación de productos
parasitarios que cambian el comportamiento de las células endoteliales vasculares, pero
no del músculo liso asociado. Identificaron al óxido nítrico (NO), los productos de la
actividad de una ciclooxigenasa (que no fueron especificados), la prostaglandina D2 y la
histamina como las moléculas responsables de dicha alteración (Kaiser et al., 1989 y
1992; Mupanomunda et al., 1997; Kaiser y Williams, 1998).

En el mismo sentido, no se conocen con exactitud los mecanismos patogénicos

que provocan la formación de microvellosidades. Se ha postulado que el daño mecánico
causado por la sola presencia de los vermes adultos en las arterias pulmonares podría
participar en el proceso patológico (Atwell et al., 1985). También se ha señalado que la
pared arterial podría responder a sustancias activas o factores de crecimiento secretados
por las plaquetas que invaden el espacio perivascular, como consecuencia del daño en la
superficie arterial. Otros dos mecanismos que podrían contribuir a la progresión de la
endarteritis proliferativa son, en primer lugar, las reacciones antígeno-anticuerpo que
activan el complemento, alterando la función endotelial y aumentando la permeabilidad
vascular, y, en segundo lugar, el aumento de la lipoproteína de baja densidad (LDL) en el
plasma, lo que contribuiría a estimular la proliferación de las células del músculo liso
hacia la íntima (Furlanello et al., 1998; Venco, 2007; Venco et al., 2011). No obstante,
puesto que la gravedad de la proliferación vellosa está directamente relacionada con la
duración de la infección y la carga parasitaria (Venco, 2007), es posible que productos
excretados por el parásito puedan influir en el proceso.

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Otra gran parte de los hallazgos realizados sobre los mecanismos patogénicos de
la dirofilariosis cardiopulmonar se han centrado en la inmunopatología de la enfermedad
(Figura 14). Los datos obtenidos indican que los hospedadores infectados o inmunizados
con D. immitis desarrollan una respuesta inmune dual Th1/Th2 (Marcos-Atxutegui et al.,
2003; Kramer et al., 2005). La respuesta de tipo Th1, proinflamatoria, es estimulada por
la bacteria simbionte Wolbachia e induce la expresión del ARNm de la óxido nítrico
sintasa inducible (iNOS), del interferón gamma (IFN-γ), así como la producción de NO
y de anticuerpos IgG2a. La respuesta de tipo Th2, antiinflamatoria, está dirigida
preferentemente contra los antígenos de D. immitis (Marcos-Atxutegui et al., 2003;
Morchón et al., 2007a y b). Esta polarización de la respuesta inmune se ha observado
también en infecciones naturales. Una respuesta de tipo Th2, caracterizada por una intensa
expresión del ARNm de las citoquinas IL-4 e IL-10 y la producción de IgG, se ha descrito
en infecciones caninas microfilarémicas. Una respuesta de tipo Th1, caracterizada por la
ausencia de expresión de IL-10 y una intensa expresión de iNOS y la producción de IgG2,
predomina en las infecciones caninas amicrofilarémicas. Estos datos sugieren que las
microfilarias circulantes pueden estimular una respuesta de tipo Th2 ineficiente,
permitiendo la supervivencia a largo plazo de los vermes adultos (Morchón et al., 2007b).

El descubrimiento de que Wolbachia es clave para el desarrollo de las reacciones

inflamatorias en la dirofilariosis ha supuesto un gran avance en este aspecto. Se ha
demostrado que la mayor parte de los pacientes con dirofilariosis pulmonar presenta una
respuesta exclusiva de IgG1 (Th1) contra la proteína mayoritaria de la cubierta de
Wolbachia (WSP) (Simón et al., 2003; Simón et al., 2007). Otros trabajos indican que la
WSP activa la quimiotaxis de neutrófilos (Bazzocchi et al., 2003) y que es capaz de
inhibir su apoptosis in vitro, lo que podría contribuir a prolongar la reacción inflamatoria
(Bazzocchi et al., 2007). Además, se ha demostrado en otras filariosis que Wolbachia
interacciona con los macrófagos a través de la familia de receptores Toll-like (TLR2,
TLR4 o TLR6) (Brattig et al., 2004; Turner et al., 2009). Por otra parte, cultivos de células
endoteliales estimulados con WSP mostraron un aumento significativo en la expresión de
los eicosanoides proinflamatorios, tromboxano B2 y leucotrieno B4, así como en la de las
enzimas responsables de su síntesis (ciclooxigenasa-2 y 5-lipooxigenasa). Ambos
eicosanoides alcanzaron niveles máximos en infecciones felinas experimentales a los 180
días post-infección, momento en el que comienzan las reacciones inflamatorias más
graves (Morchón et al., 2007c). Además, el tromboxano B2 presenta elevados niveles

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tanto en infecciones crónicas caninas y felinas, como en pacientes humanos con

dirofilariosis pulmonar, lo que se correlaciona, en todos los casos, con elevados títulos de
anticuerpos IgG anti-WSP, indicando una intensa liberación de Wolbachia por la
destrucción de los vermes (Morchón et al., 2006, 2007c y 2009). Finalmente, la expresión
de iNOS y NOS endotelial (eNOS), así como la de otras moléculas relacionadas tanto con
la adhesión y transmigración de leucocitos (VCAM, ICAM y PECAM), como con la
proliferación celular (E-cadherina y VEGF), también se vieron aumentadas en cultivos
de células endoteliales estimulados con WSP (Morchón et al., 2008). No obstante, el
hecho de que muchas de estas moléculas también se vean activadas en cultivos similares
estimulados con antígenos somáticos parasitarios (Simón et al., 2008) o que filarias que
carecen de Wolbachia también induzcan reacciones inflamatorias, parece sugerir que
además de las bacterias, las propias filarias participan también en la generación de la
patología inflamatoria de la dirofilariosis cardiopulmonar (Simón et al., 2012).

Figura 14. Mecanismos inmunopatogénicos en la dirofilariosis cardiopulmonar mediados por una

respuesta inmune dual Th1/Th2. (A) La respuesta de tipo Th2, antiinflamatoria, es estimulada por los
antígenos de D. immitis y la presencia de microfilarias. Se incrementa la expresión de IL-4 e IL-10, así
como los niveles de anticuerpos relacionados con la respuesta Th2, IgG (en perros) o IgE (en humanos). (B)
La respuesta de tipo Th1, proinflamatoria, es estimulada por las bacterias Wolbachia liberadas a partir de
vermes muertos. La proteína mayoritaria de la superficie de Wolbachia (WSP) estimula la producción de
IFN-γ, interacciona con macrófagos (probablemente a través de receptores TLR) e inhibe su apoptosis.
Wolbachia estimula además la producción de anticuerpos típicos de la respuesta Th1 y la expresión de
mediadores proinflamatorios en células endoteliales vasculares. En estas mismas células los niveles de
expresión de moléculas de adhesión/transmigración (VCAM, PECAM, ICAM, E-cadherina y VEGF)
también se ven aumentados. Algunos de estos estímulos también son producidos por un extracto de
antígenos somáticos de D. immitis (DiSA) (Simón et al., 2012).

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4.4. Respuesta inmune

El sistema inmune es una sofisticada y compleja herramienta que ha evolucionado

para destruir a los patógenos invasores (Sorci et al., 2013). Sin embargo, frente a la fuerte
respuesta inmune desarrollada por los hospedadores parasitados, los helmintos en
particular son capaces de persistir en el hospedador, produciendo generalmente
infecciones de tipo crónico (Moreau y Chauvin, 2010). En este sentido, el desarrollo de
las larvas infectantes hasta vermes adultos en la dirofilariosis cardiopulmonar y el carácter
crónico en la mayor parte de la infecciones, sugiere una escasa eficiencia de la respuesta
inmune y/o una habilidad del parásito para evadir los mecanismos de control del
hospedador. No obstante, está generalmente aceptado que el hospedador es capaz de
controlar la carga parasitaria manteniéndola dentro de unos límites compatibles con su
propia supervivencia, destruyendo una parte de las larvas adquiridas por las reinfecciones
(Simón et al., 2001 y 2012).

En la dirofilariosis los datos experimentales parecen demostrar que la respuesta

inmune está influenciada por la presencia de microfilarias y por el estatus clínico de los
hospedadores infectados (Simón et al., 2012). En perros con dirofilariosis, la
microfilaremia se ha relacionado con niveles significativamente superiores de anticuerpos
IgG anti-D. immitis y anti-Wolbachia en comparación con lo que ocurre en infecciones
amicrofilarémicas (Grieve et al., 1979; Morchón et al., 2007b; Marcos-Atxutegi et al.,
2004). En el mismo sentido, una mayor respuesta de anticuerpos IgG anti-Wolbachia fue
encontrada en la orina de perros microfilarémicos con glomerulonefritis asociada
(Morchón et al., 2012b). Por otra parte, comparando ambos tipos de anticuerpos en perros
amicrofilarémicos, la respuesta es mayor en aquellos con tromboembolismos pulmonares
masivos que en los que no presentan sintomatología (Kramer et al., 2005; Simón et al.,
2007).

En estudios llevados a cabo con gatos infectados experimentalmente se ha

observado una respuesta IgG moderada y de corta duración solo en los 2 primeros meses
post-infección contra los antígenos de L3 y una respuesta IgG intensa entre los 2 y los 6
meses post-infección contra los antígenos de los vermes adultos (Bazzocchi et al., 2000;
Prieto et al., 2001 y 2002). Esta respuesta desciende en gatos tratados con ivermectina,
mientras que las IgG anti-Wolbachia se incrementan, probablemente como consecuencia

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de la muerte de las larvas y la liberación de Wolbachia al organismo del hospedador

(Prieto et al., 2002).

En las infecciones humanas, pacientes diagnosticados de dirofilariosis pulmonar

presentan una respuesta de IgG o IgM frente a los complejos antigénicos somático y
excretor, además de niveles elevados de IgG anti-Wolbachia (Simón et al., 1991, Simón
et al., 2003), mientras que en seropositivos asintomáticos (sin nódulos pulmonares)
predominan las IgE y los anticuerpos anti-Wolbachia se mantienen en niveles más bajos
(Sato et al., 1985; Espinoza et al., 1993; Simón et al., 2003).

4.5. Mecanismos de evasión y supervivencia

Pese a la grave patología que produce, D. immitis es capaz de sobrevivir durante

años en el sistema vascular de su hospedador definitivo asegurando su reproducción,
interaccionando con su entorno inmediato a través de los antígenos de la interfase
parásito/hospedador. Por ello, la larga esperanza de vida de D. immitis puede ser
considerada como un reflejo de la evolución de estrategias altamente efectivas
relacionadas con sus mecanismos de evasión y supervivencia.

Los pocos datos disponibles muestran que cada fase evolutiva de D. immitis ha
desarrollado diferentes estrategias para evadir la respuesta inmune del hospedador (Simón
et al., 2001) (Figura 15). Se ha demostrado que las L3 eliminan entre el 10 y el 20% de
su contenido antigénico de superficie (constituido principalmente por dos moléculas de 6
y 35 kDa), no siendo repuesto posteriormente. Esto permite ofrecer un bajo perfil
antigénico difícil de detectar, en un estadio de corta duración pero de vital importancia
para el establecimiento de la infección en el hospedador definitivo (Ibrahim et al., 1989).
Por su parte, los vermes adultos expresan en su superficie glicolípidos no inmunógenos,
y son capaces de retener plaquetas y adsorber albúmina, IgG y la fracción C3 del
complemento como mecanismo de enmascaramiento y así evitar el reconocimiento de los
antígenos cuticulares (Scott et al., 1988; Bilge et al., 1989; Kadispasaoglu y Bilge, 1989).
Además, se han detectado proteasas con capacidad para hidrolizar anticuerpos IgG en la
epicutícula de las microfilarias (Tamashiro et al., 1987). En cuanto a los productos
secretados por D. immitis, se ha demostrado que la estimulación de cultivos de células
endoteliales vasculares con los antígenos excretores/secretores del parásito produce un
aumento significativo en la producción de prostaglandina E2 (PGE2), así como un

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significativo descenso en la transmigración de monocitos (Morchón et al., 2010). La

PGE2 es un eicosanoide derivado del ácido araquidónico que produce efectos
inmunosupresores y antiinflamatorios (Liu y Weller, 1990), por lo que altos niveles de
esta molécula pueden relacionarse con la supervivencia del parásito, lo que ha sido
postulado también en otras especies de filarias (Liu, et al., 1990; Brattig et al., 2006).

Finalmente, como se ha señalado con anterioridad, los estudios proteómicos están

generando abundante información sobre la composición proteica de D. immitis. El
análisis mediante esta tecnología de los diferentes compartimentos antigénicos del
parásito parece indicar que D. immitis posee un gran número de enzimas cuyas funciones
están directamente relacionadas con su supervivencia. Diferentes isoformas de proteínas
de choque térmico, peroxirredoxinas, moléculas antioxidantes y detoxificantes y enzimas
implicadas en la generación de energía están ampliamente representadas en el repertorio
antigénico del parásito (Oleaga et al., 2009; González-Miguel et al., 2010a y b; Geary et
al., 2012; Morchón et al., 2014).

Figura 15. Mecanismos de evasión y supervivencia descritos en D. immitis. (A) Las larvas infectantes L3
evaden la respuesta inmune del hospedador liberando grandes cantidades de dos polipéptidos de superficie
de 6 y 35 KDa. (B) En localización vascular, los vermes adultos pueden enmascarar su superficie mediante
la adsorción de moléculas del hospedador. Además, poseen glicolípidos no inmunógenos y un gran número
de isoformas de proteínas de choque térmico, peroxirredoxinas, antioxidantes y moléculas detoxificantes.
En el endotelio sus productos de excreción/secreción estimulan la expresión del eicosanoide
antiinflamatorio PGE2 y la disminución en la transmigración de monocitos. Las microfilarias poseen
además proteasas de superficie con capacidad para digerir anticuerpos del hospedador (Simón et al., 2012).

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5. EL SISTEMA FIBRINOLÍTICO

La coagulación sanguínea es un complejo evento enzimático que culmina con la

formación de una proteína insoluble de aspecto filamentoso llamada fibrina (Figura 16).
Junto con las plaquetas, la fibrina conforma un tapón que evita el sangrado cuando se
produce un daño vascular. El conjunto de mecanismos encargados de lisar el coágulo de
fibrina una vez formado y, por tanto, de restaurar el estado fisiológico normal que permita
una correcta permeabilidad vascular, constituye el sistema fibrinolítico (Mosher, 1990;
Gaffney y Longstaff, 1994).

Como se ha descrito anteriormente,

D. immitis provoca un daño vascular tanto
por procesos mecánicos como enzimáticos,
lo que sin duda estaría relacionado con una
intensa actividad de la cascada de la
coagulación. No obstante, D. immitis
sobrevive durante largos períodos en el
sistema vascular de hospedadores
inmunocompetentes sin verse afectado,
aparentemente, por este proceso. Por otra
Figura 16. Glóbulos rojos atrapados en una parte, cuando mueren los vermes adultos
malla de filamentos de fibrina. Tomado de
pueden producirse los graves
Encyclopædia Britannica Online.
http://global.britannica.com/EBchecked/topic/6 tromboembolismos que caracterizan la
9202/bleeding-and-blood-clotting#ref64585
patología aguda y que comprometen de
inmediato la vida del hospedador. Por tanto, es razonable asumir que los vermes adultos
interaccionan en vida con su entorno intravascular mediante mecanismos de
supervivencia, ya que dichos fenómenos tromboembólicos, además de producir graves
consecuencias para la vida del animal parasitado, pueden ser nocivos para el propio
parásito. Por ello, es probable que, como ocurre en otros patógenos sanguíneos, D. immitis
interaccione de algún modo con el sistema fibrinolítico de su hospedador.

Se revisan a continuación las principales características bioquímicas y funcionales

de los componentes más importantes del sistema fibrinolítico, la naturaleza de su
interacción con diferentes grupos de patógenos, así como sus implicaciones
fisiopatológicas.

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5.1. Componentes del sistema fibrinolítico

5.1.1. Sistema Plasminógeno - Plasmina

El funcionamiento del sistema fibrinolítico tiene como base la conversión de un

proenzima, el plasminógeno, en su enzima proteolíticamente activa, la plasmina, la cual
es capaz de degradar fibrina y, así, eliminar el coágulo previamente formado (Collen y
Lijnen, 1991) (Figura 17).

Figura 17. Esquema simplificado del sistema fibrinolítico. PAI-1, inhibidor del activador del
plasminógeno-1; PDFs, productos de la degradación de la fibrina; TAFIa, inhibidor de la fibrinolisis
activable por trombina activado; tPA, activador tisular del plasminógeno; uPA, activador del plasminógeno
de tipo uroquinasa; uPAR, receptor del activador del plasminógeno de tipo uroquinasa.

El plasminógeno es una glicoproteína plasmática de 92 kDa sintetizada en el

hígado y formada por 791 aminoácidos enlazados por 24 puentes disulfuro, 16 de los
cuales dan lugar a 5 regiones homólogas de triple lazo llamadas kringles (Cesarman-Maus
y Hajjar, 2005) (Figura 18). Al tratarse de un zimógeno, su conversión a plasmina
depende de la escisión, por diferentes tipos de activadores, de un enlace peptídico Arg-
Val en la posición 560-561 (Holvoet et al., 1985). Esto libera las cadenas pesada y ligera
de la molécula de plasmina quedando unidas por dos puentes disulfuro. La cadena pesada
de 65 kDa presenta los 5 dominios kringle y la cadena ligera de 25 kDa contiene el sitio
activo de His602, Asp645 y Ser740, lo que conforma la característica tríada catalítica de las

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proteasas de serina (Lähteenmäki et al., 2001). La plasmina como serina proteasa activa
es capaz de degradar eficientemente el coágulo formado, generando productos solubles
de degradación y logrando que la fibrina exponga sus residuos de lisina carboxi-
terminales. Estos residuos son sitios de unión tanto para los dominios kringle 1 y 4 del
plasminógeno como para los de sus activadores, lo que se traduce en un incremento
significativo del proceso fibrinolítico (Cesarman-Maus y Hajjar, 2005). Además de con
la fibrina, la plasmina puede realizar su actividad proteolítica sobre otros muchos
sustratos, jugando un papel importante en procesos como la invasión celular, la
quimiotaxis o la remodelación de los tejidos (Syrovets y Simmet, 2003).

Figura 18. Estructura del plasminógeno. Los dominios kringle que contienen los sitios de unión a lisina
están marcados como K1-K5. Se indican los aminoácidos que conforman la tríada catalítica (His602, Asp645
y Ser740) y el sitio de escisión para los activadores del plasminógeno entre los aminoácidos Arg560 y Val561.
Las líneas discontinuas señalan puentes disulfuro (Lähteenmäki et al., 2001). PA, activadores del
plasminógeno.

El plasminógeno está presente en grandes cantidades tanto a nivel vascular como

tisular. Debido al papel principal que cumple esta molécula, no solo en el sistema
fibrinolítico, sino también en otros procesos celulares, es de suma importancia que esta
gran reserva de actividad proteolítica se halle fuertemente regulada. Esto se logra gracias
a la actividad de diferentes activadores, inhibidores y receptores.

5.1.2. Activadores del plasminógeno

Dos son los activadores fisiológicos del plasminógeno identificados hasta el

momento, el activador tisular del plasminógeno (tPA) y el activador del plasminógeno de

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tipo uroquinasa (uPA). La activación del plasminógeno mediada por tPA está relacionada
principalmente con la disolución del coágulo de fibrina a nivel vascular, mientras que
uPA se une a su receptor celular específico (uPAR) potenciando la activación del
plasminógeno unido a células (Lijnen, 2006). Ambos son capaces de reconocer y escindir
el puente peptídico Arg560-Val561 del plasminógeno facilitando su conversión a plasmina.

Activador tisular del plasminógeno (tPA)

El tPA es una proteasa de serinas de 72 kDa sintetizada principalmente por las

células endoteliales. Esta glicoproteína contiene cinco dominios estructurales entre los
cuales se incluyen un finger tipo fibronectina, un dominio homólogo del factor de
crecimiento epidérmico, dos estructuras kringle homólogas a las que contiene el
plasminógeno y el dominio de proteasa de serinas (Pennica et al., 1983). El tPA es
sintetizado como un polipéptido de cadena sencilla el cual, a diferencia de la mayoría de
los precursores monocatenarios de la familia de las proteasas de serinas, es
enzimáticamente activo. Por otra parte, su actividad en fase fluida puede aumentar
mediante la hidrólisis del puente péptidico Arg275-Ile276 por parte de la plasmina y su
consiguiente transformación en proteasa de doble cadena (Tate et al., 1987). El principal
papel del tPA es degradar la fibrina en los vasos sanguíneos. Pese a que la eficiencia
catalítica del tPA es intrínsecamente baja, esta puede aumentar más de 100 veces en
presencia de la propia fibrina. Esta estimulación ocurre a través de la formación de un
complejo ternario mediante la unión del tPA con el plasminógeno y la fibrina. La fibrina
no actúa simplemente como sustrato, sino también como cofactor estimulando su propia
degradación. En un primer momento el tPA de cadena sencilla inicia la activación del
plasminógeno en contacto con la red de fibrina intacta. A continuación, cuando la fibrina
se encuentra parcialmente degradada por la acción de la plasmina, esta muestra nuevos
sitios de unión al plasminógeno mediante la generación proteolítica de residuos carboxi-
terminales de lisina. Estos residuos interaccionan con los dominios kringle presentes en
el plasminógeno y en el tPA, aumentando notablemente el proceso fibrinolítico (Gebbink,
2011). Además de su papel principal en la disolución de coágulos sanguíneos, estudios
recientes muestran al tPA como un importante modulador del sistema nervioso central,
jugando un importante papel en los procesos de memoria, estrés, degeneración neuronal
y Alzheimer (Zorio et al., 2008; Rijken y Lijnen, 2009).

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Activador del plasminógeno de tipo uroquinasa (uPA)

El uPA es secretado como una proteína inactiva de cadena sencilla por una gran
variedad de tipos celulares que incluyen células endoteliales vasculares, musculares lisas,
monocitos y macrófagos, fibroblastos, células epiteliales, así como células tumorales
malignas de diferente origen (Fuhrman, 2012). Se trata de una glicoproteína de 55 kDa
de masa molecular, constituida por un dominio homólogo al factor de crecimiento
epidérmico, una estructura kringle homóloga a la que contiene el plasminógeno y una
tríada catalítica clásica del tipo de las proteasas de serina (Cesarman-Maus y Hajjar,
2005). Diferentes moléculas como la plasmina o la calicreína pueden llevar a cabo su
activación mediante la rotura del puente peptídico Lys158-Ile159. El resultado es una
proteasa de doble cadena unida por puente disulfuro cuya afinidad por el plasminógeno
aumenta aproximadamente 300 veces. Además, en esta forma puede unirse a su receptor
de membrana (uPAR), lo cual está considerado como un paso crítico en la función de
uPA, permitiendo dicha unión focalizar la proteólisis llevada a cabo por uPA en el espacio
pericelular inmediato (Nicholl et al., 2006). Pese a que su afinidad por la fibrina es mucho
más baja que la que posee el tPA, uPA puede ser un activador efectivo del plasminógeno
tanto en presencia como en ausencia de fibrina (Cesarman-Maus y Hajjar, 2005), lo que
favorece la implicación del sistema uPA/uPAR en otros procesos como migración celular,
diferenciación, proliferación y degradación de matrices. Por ello, uPA está considerada
como una proteína crucial tanto en el crecimiento de la neoíntima como en el remodelado
vascular, pudiendo ser otras moléculas, como las metaloproteasas de matriz o factores de
crecimiento, susceptibles a la actividad de su dominio proteolítico (Nicholl et al., 2006;
Fuhrman, 2012).

5.1.3. Inhibidores de la fibrinolisis

Inhibidores de los activadores del plasminógeno (PAIs)

El inhibidor del activador del plasminógeno-1 (PAI-1) está considerado como el

principal regulador de la fibrinolisis in vivo (Loskutoff, 1991). Se trata de una
glicoproteína de 47 KDa perteneciente a la superfamilia de las serpinas o inhibidores de
proteasas de serina (Schleef et al., 1989). Después de iniciada la formación del trombo
como resultado de un daño vascular, PAI-1 se libera principalmente a partir del endotelio
y de las plaquetas, aunque puede ser sintetizado por otros tipos celulares. En condiciones

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fisiológicas normales, la liberación de PAI-1 atenúa la activación del plasminógeno

actuando a nivel de sus activadores tPA y uPA, contribuyendo así a la estabilización del
trombo para el mantenimiento apropiado de la cicatrización de la herida y de la
permeabilidad vascular (Iwaki et al., 2012). Con un menor grado de importancia, el
inhibidor del activador del plasminógeno-2 (PAI-2) está relacionado con el
mantenimiento de la hemostasia durante el embarazo y el parto, mientras que el inhibidor
del activador del plasminógeno-3 (PAI-3) se encuentra en ciertos fluidos humanos, como
los fluidos seminales y foliculares, presentando una baja actividad en plasma (Zorio et
al., 2008).

Inhibidor de la fibrinolisis activable por trombina (TAFI)

La generación de residuos de lisina carboxi-terminales en la fibrina parcialmente

degradada provoca una acumulación 30 veces mayor de plasminógeno en la superficie
del coágulo y por tanto un aumento de la lisis en la segunda fase de la fibrinolisis. El
TAFI es una glicoproteína de 60 KDa producida por el hígado, que tras su activación por
trombina, tripsina o plasmina puede funcionar como una carboxipeptidasa eliminando los
residuos de lisina carboxi-terminales de la red de fibrina. Al actuar solamente en la
segunda fase de la lisis del coágulo, TAFI es considerado más bien un atenuador de la
fibrinolisis (Nesheim et al., 1997; Sakharov et al., 1997; Rijken y Lijnen, 2009).

Inhibidores de la plasmina

La fibrinolisis también puede ser regulada a nivel de la plasmina por la serpina

alfa 2-antiplasmina (Lijnen, 2006). Esta glicoproteína plasmática de 67 kDa es sintetizada
en el hígado y realiza su función antifibrinolítica a tres niveles: 1) mediante la formación
de un complejo con la plasmina, 2) inhibiendo la fijación del plasminógeno al coágulo de
fibrina y 3) mediante su entrecruzamiento con la fibrina a través del factor XIIIa, lo que
hace a esta última más resistente a la plasmina (Carpenter y Mathew, 2008). En menor
medida, la plasmina puede ser también inhibida mediante la formación de complejos no
covalentes con la alfa 2-macroglobulina (Cesarman-Maus y Hajjar, 2005).

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5.1.4. Receptores del plasminógeno

Otro de los puntos clave en la regulación de la fibrinolisis es la llevada a cabo por

los receptores celulares del plasminógeno. La unión del plasminógeno a la superficie
celular permite, no solo dirigir la actividad proteolítica de la plasmina a aquellos sitios
donde es requerida, sino también aumentar significativamente dicha actividad enzimática
a nivel pericelular. Esto se debe a la existencia de residuos de lisina en sus extremos
carboxi-terminales que permiten la unión a residuos kringle, lo que constituye la principal
característica de los receptores fisiológicos del plasminógeno. Todo ello favorece, por
una parte, la localización conjunta y simultánea del plasminógeno y sus activadores (tPA,
uPA), y por otra la protección de la plasmina de nueva generación de la acción inhibitoria
de la alfa 2-antiplasmina (Rijken y Lijnen, 2009; Madureira et al., 2011). Una gran
variedad de células puede expresar un elevado número de receptores del plasminógeno.
Entre ellos, los mejor caracterizados incluyen la α-enolasa, complejo glicoprotéico IIb-
IIIa, antígeno de nefritis de Heymann, integrina αMβ2 y anexina A2 (Cesarman-Maus y
Hajjar, 2005).

Figura 19. El tetrámero de anexina A2 se expresa en la superficie celular de numerosas células funcionando
como receptor del plasminógeno. Consta de dos moléculas de anexina A2 unidas por un dímero de la
proteína S100A10, el cual puede unir plasminógeno y tPA a través de sus residuos de lisina carboxi-
terminales favoreciendo su localización conjunta en la membrana celular. Esto puede favorecer la acción
del complejo uPA/uPAR con la consecuente generación de plasmina a nivel pericelular (Madureira et al.,
2011).

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La anexina A2 ha sido propuesta como el principal correceptor del plasminógeno

y del tPA en las células endoteliales vasculares (Hajjar et al., 1994). Lleva a cabo su
acción cuando se expresa en la superficie celular como un tetrámero compuesto de dos
moléculas de anexina A2 unidas por un dímero de la proteína S100A10. La anexina A2
contiene sitios de unión a fosfolípidos con capacidad para anclarse a la superficie de la
membrana celular, mientras que los residuos de lisina carboxi-terminales de S100A10
son los encargados de unir y hacer coincidir al plasminógeno con el tPA. El complejo de
membrana uPA/uPAR también puede verse beneficiado de esta fijación del plasminógeno
produciéndose, en cualquier caso, la localización conjunta del plasminógeno y sus
activadores y la consecuente generación de plasmina a nivel pericelular (Flood y Hajjar,
2011; Madureira et al., 2011; Luo y Hajjar, 2013) (Figura 19).

5.2. Interacción de los patógenos con el sistema fibrinolítico de sus

hospedadores

La plasmina es una proteasa de serinas de amplio espectro con una gran actividad
proteolítica. Entre sus sustratos más comunes encontramos la fibrina, pero también
diferentes componentes de la matriz extracelular y del tejido conectivo. El reclutamiento
de esta enzima por parte de cualquier patógeno sanguíneo significaría una ventaja
evolutiva, ya que no solo supondría un mecanismo efectivo para evitar su posible
inmovilización por la red de coágulos de fibrina, sino también una ayuda para su
diseminación y establecimiento en el hospedador mediante la degradación de los
componentes de la matriz extracelular (Sun, 2006; Bhattacharya et al., 2012). Otras
funciones como la degradación de inmunoglobulinas y de moléculas del complemento, la
activación de metaloproteasas, la estimulación de la adherencia y de la invasión, así como
la degradación de proteínas para la nutrición, han sido atribuidas a la interacción entre los
patógenos y el sistema fibrinolítico (Kitt y Leigh, 1997; Yavlovich et al., 2004; Yavlovich
y Rottem, 2007; Gong et al., 2008; Chung et al., 2011; Siemens et al., 2011; Bergmann
et al., 2013).

La utilización del sistema fibrinolítico del hospedador como medio para obtener
un beneficio ha sido ampliamente estudiada en organismos bacterianos desde hace un par
de décadas (Lottenberg et al., 1994) (Figura 20). Un gran número de trabajos han
permitido relacionar el empleo de la función proteolítica de la plasmina por parte de

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diferentes especies de bacterias patógenas con el control de la hemostasia o con

mecanismos para la mejora de su diseminación y de la evasión de los sistemas de
vigilancia de la respuesta innata (Degen et al., 2007). Esta interacción es posible gracias
al reclutamiento del plasminógeno en la superficie bacteriana mediante la expresión de
proteínas que pueden actuar como receptores y a su transformación en plasmina mediante
la acción de activadores del plasminógeno (Bhattacharya et al., 2012).

Figura 20. Imágenes captadas mediante microscopía electrónica de barrido de emisión de campo donde se
puede apreciar la degradación de coágulos de fibrina provocada por Streptococcus pneumoniae. (A) Matriz
de fibrina formada por gruesos haces formados a su vez por varias fibrillas de fibrina retorcidas sobre sí
mismas. (B) Matriz de fibrina en degradación tras ser incubada con neumococos recubiertos con
plasminógeno y su posterior activación a plasmina mediante uPA (Bergmann y Hammerschmidt, 2007).

La capacidad para expresar receptores de plasminógeno ha sido demostrada en un

gran número de especies de bacterias (Sanderson-Smith et al., 2012). Estos receptores
permiten inmovilizar el plasminógeno facilitando su conversión a plasmina por parte de
los activadores, a la vez que evitan la función inhibitoria de la alfa 2-antiplasmina. Entre
los mejor caracterizados se encuentran la enolasa y la GAPDH identificadas en los
estreptococos de los grupos A y C. Estas enzimas, típicamente glicolíticas y con funciones
básicas para el mantenimiento celular, pueden ser expresadas en la superficie bacteriana
y llevar a cabo multitud de funciones (Lähteenmäki et al., 2001; Bhattacharya et al.,
2012). Además, la secreción de receptores bacterianos de plasminógeno a través de
vesículas de membrana externa también ha sido descrita, lo que permitiría la proteólisis

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externa en el entorno pericelular como mecanismo para la nutrición y la mejora en la

diseminación (Bergmann y Hammerschmidt, 2007; Toledo et al., 2012).

Tres son los principales activadores del plasminógeno que han sido descritos
como proteínas secretadas o unidas a membrana en diferentes grupos bacterianos: la
estreptoquinasa y la estafiloquinasa, producidas respectivamente por diferentes especies
de los generos Streptococcus y Staphylococcus, y la proteasa Pla identificada en Yersinia
pestis. Los dos primeros activan el plasminógeno mediante la formación de complejos
catalíticamente activos tanto con el plasminógeno como con la plasmina, mientras que
Pla es una aspartil proteasa que genera plasmina mediante su acción proteolítica (Degen
et al., 2007).

Más recientemente, la capacidad para interaccionar con el sistema fibrinolítico del

hospedador ha sido estudiada en organismos eucariotas causantes de enfermedades
parasitarias (Figuera et al., 2013b). Un número limitado de trabajos ha permitido
demostrar esta interacción en diferentes especies de protozoos y helmintos parásitos
analizando antígenos excretados o de superficie. Las proteínas con capacidad para fijar
plasminógeno identificadas hasta el momento en organismos parásitos aparecen en la
tabla 2. La mayor parte de los estudios se han llevado a cabo con proteínas de las que su
función como receptores del plasminógeno ya era conocida en especies de bacterias
(enolasa, GAPDH, actina, FBAL…), mientras que otras proteínas como la LACK o la
SMP-1 han sido relacionadas por primera vez con el sistema fibrinolítico en organismos
parásitos (Gómez-Arreaza et al., 2011; Figuera et al., 2013a y b).

Al igual que en los trabajos llevados a cabo con bacterias, la interacción entre los
parásitos y el sistema fibrinolítico del hospedador ha sido relacionada siempre con
funciones a priori beneficiosas para el agente patógeno. Así, se ha vinculado la fijación
de plasminógeno con la virulencia de los parásitos y con su éxito tanto en el proceso de
infección como en su establecimiento en el hospedador. Esto ha sido recientemente
sugerido en tripanosomas (Avilán et al., 2011). Trypanosoma cruzi puede fijar
plasminógeno tanto en el hospedador vertebrado como en el vector y utilizarlo para
atravesar tejidos (Almeida et al., 2004; Rojas et al., 2008). Además, se ha postulado que
los parásitos del género Leishmania pueden interactuar con el plasminógeno en el
momento de su inoculación en el hospedador definitivo como promastigotes, o como
amastigotes cuando son liberados por los macrófagos para infectar otras células. La

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generación de plasmina podría ayudar a los parásitos a reducir la matriz de fibrina

asociada a la reacción inflamatoria y favorecer el encuentro entre Leishmania y nuevos
macrófagos (Maldonado et al., 2006). Funciones relacionadas con la interacción célula-
célula para la adherencia y penetración han sido propuestas en la utilización del
plasminógeno por parte de los ooquinetos de Plasmodium falciparum y P. berghei. Tras
su activación a plasmina podría ser utilizado para ayudar a la degradación de las proteínas
de superficie de las células epiteliales del insecto vector para permitir la invasión
parasitaria (Ghosh et al., 2011). Por su parte, se ha relacionado la fijación de
plasminógeno por Trichomonas vaginalis con su penetración en la membrana basal lo
que permitiría su asociación con la fibronectina y laminina. Esto garantizaría el acceso
del parásito a factores de crecimiento y nutrientes contribuyendo al éxito de la infección
(Mundodi et al., 2008). En el caso de los helmintos, funciones similares se han propuesto
para O. volvulus, cuyas microfilarias podrían utilizar la actividad proteolítica de la
plasmina unida a su superficie para facilitar su migración a través de los tejidos del
hospedador (Jolodar et al., 2003). Además, en Schistosoma bovis, helminto parásito con
localización intravascular, se ha relacionado la fijación de plasminógeno con una
regulación del sistema hemostático por parte del parásito para evitar la formación de
coágulos (Ramajo-Hernández et al., 2007; de la Torre-Escudero et al., 2010).

La mayor parte de los receptores de plasminógeno identificados tanto en bacterias

como en parásitos comparten mecanismos similares de unión con los receptores
fisiológicos de sus hospedadores (Miles et al., 2005; Figuera et al., 2013b). Se ha
demostrado la participación de los residuos de lisina en la interacción de estos receptores
con el plasminógeno mediante la realización de ensayos competitivos con análogos de
este aminoácido, como el ácido ε-amino caproico. Los residuos de lisina que participan
en la fijación del plasminógeno han sido identificados en los extremos carboxi-terminales
de los receptores, pero también formando parte de motivos internos como el
“FYDKERKVY” descrito en la enolasa de S. pneumoniae (Bergmann et al., 2003) y
encontrado posteriormente con ligeras modificaciones en las enolasas de otras bacterias,
hongos o parásitos (Figuera et al., 2013b).

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Proteína Especie Referencia

Enolasa Leishmania mexicana Vanegas et al., 2007; Figuera et al., 2013a

Trichomonas vaginalis Mundodi et al., 2008
Plasmodium falciparum Ghosh et al., 2011
Plasmodium berghei Ghosh et al., 2011
Fasciola hepatica Bernal et al., 2004
Echinostoma caproni Marcilla et al., 2007
Schistosoma bovis Ramajo-Hernández et al., 2007; de la Torre-
Escudero et al., 2010
Schistosoma japonicum Yang et al., 2010
Clonorchis sinensis Wang et al., 2011
Taenia pisiformis Zhang et al., 2015
Onchocerca volvulus Jolodar et al., 2003
GAPDH Trichomonas vaginalis Lama et al., 2009
Schistosoma bovis Ramajo-Hernández et al., 2007
Clonorchis sinensis Hu et al., 2014
Onchocerca volvulus Erttmann et al., 2005
LACK Leishmania mexicana Gómez-Arreaza et al., 2011
SMP-1 Leishmania mexicana Figuera et al., 2013a
Anexina B30 Clonorchis sinensis He et al., 2014
Actina Schistosoma bovis Ramajo-Hernández et al., 2007
FBAL Schistosoma bovis Ramajo-Hernández et al., 2007
ATP: guanidino quinasa Schistosoma bovis Ramajo-Hernández et al., 2007
Fosfoglicerato mutasa Schistosoma bovis Ramajo-Hernández et al., 2007
Triosafosfato isomerasa Schistosoma bovis Ramajo-Hernández et al., 2007
Adenilato quinasa Schistosoma bovis Ramajo-Hernández et al., 2007
Proteína hipotética AAW24823 Schistosoma bovis Ramajo-Hernández et al., 2007
Proteína hipotética AAP06049 Schistosoma bovis Ramajo-Hernández et al., 2007

Tabla 2. Proteínas fijadoras de plasminógeno identificadas en organismos parásitos. FBAL, fructosa-

bifosfato aldolasa; GAPDH, gliceraldehído-3-fosfato deshidrogenasa.

5.3. Fisiopatología de la fibrinolisis

El gran número de sustratos sobre los que pueden realizar su función los
componentes con actividad proteolítica del sistema fibrinolítico, requiere una estricta
regulación del sistema para evitar una proteólisis indiscriminada (Draxler y Medcalf,
2015). En caso contrario podría producirse un escenario patológico, debido a que la
plasmina y los activadores del plasminógeno están implicados en procesos como la
proliferación, migración, inflamación y degradación de la matriz extracelular (Figura 21).
Esto ha permitido relacionar la sobreactivación del sistema fibrinolítico con situaciones
patológicas tan importantes como el crecimiento de la placa arterial, la aterosclerosis
crónica, síndromes coronarios agudos, restenosis, la remodelación vascular e incluso con
el cáncer, provocando que la plasmina sea considerada actualmente como una diana
terapéutica potencialmente interesante desde diversos puntos de vista (Nicholl et al.,
2006; Zorio et al., 2008).

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 Revisión bibliográfica

Un gran número de trabajos llevados a cabo en investigación cardiovascular

relacionada con humanos, algunos realizados mediante la utilización de ratones
deficientes en componentes del sistema fibrinolítico, han permitido relacionar la
activación del plasminógeno con la proliferación y migración de células vasculares
humanas, así como con la degradación de matrices extracitoplasmáticas (Nicholl et al.,
2005 y 2006; Yang et al., 2005; Roth et al., 2006; Hayashi et al., 2009). Del mismo modo
se ha podido medir un incremento en los niveles de tPA, uPA y/o plasmina en diferentes
situaciones patológicas, que tienen en común alguno o todos los mecanismos
anteriormente citados (Syrovets y Simmet, 2004; Nicholl et al., 2006; Draxler y Medcalf,
2015). Incluso a altas concentraciones, se ha descrito que la plasmina puede provocar
desorganización tanto del endotelio como del músculo liso perivascular e iniciación de la
apoptosis celular si la estimulación persiste (Rossignol et al., 2006; Ho-Tin-Noe et al.,
2009; Doeuvre et al., 2010).

Figura 21. Esquema de la fisiopatología de la fibrinolisis.

Por otra parte, la plasmina puede unirse a una gran variedad de células, incluyendo
monocitos, macrófagos, células dendríticas y otras, a través de receptores de baja afinidad
provocando agregación de neutrófilos, degranulación plaquetaria y liberación de ácido
araquidónico desde las células endoteliales. Esto indica una relación directa entre la
plasmina y la activación proinflamatoria a gran escala, incluyendo la liberación de
mediadores lipídicos y especies reactivas del oxígeno, quimiotaxis y expresión de
citoquinas, así como la inducción de la expresión de otros genes proinflamatorios

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(Syrovets y Simmet, 2004). La plasmina juega, por tanto, un papel central en la respuesta
inmune celular, ayudando a la eliminación de organismos infecciosos. No obstante, su
activación excesiva en enfermedades autoinmunes o inflamatorias de tipo crónico puede
exacerbar la estimulación de células inflamatorias y por tanto, la patogénesis de la
enfermedad (Syrovets et al., 2012).

Todos estos datos han permitido evidenciar la implicación de la activación

fibrinolítica en enfermedades tan graves como el cáncer. Se ha demostrado que la
sobreexpresión constitutiva del gen de uPA es una característica de la progresión maligna,
lo que conduce a altos niveles de uroquinasa unida al receptor y por tanto, a una excesiva
activación del plasminógeno. La plasmina unida a membrana, protegida de los
inhibidores circulantes, es capaz de degradar las proteínas de la matriz extracelular tales
como laminina y fibronectina, así como activar varias metaloproteasas de la matriz, lo
que contribuye aún más a la degradación de la matriz extracelular. La plasmina generada
en la superficie de las células tumorales es considerada, por lo tanto, como un evento
clave en la invasión tumoral y la metástasis (Schmitt et al., 1997; Andreasen et al., 2000;
Syrovets y Simmet, 2004).

6. DIAGNÓSTICO Y MANEJO DE LA DIROFILARIOSIS

El diagnóstico de la dirofilariosis es un aspecto fundamental dentro del cuadro

general del manejo de la parasitosis. Dadas las implicaciones que tiene la enfermedad,
tanto en su aspecto clínico veterinario y humano, como en los estudios epidemiológicos,
el diagnóstico constituye el primer paso en el control de la zoonosis. Por otra parte, las
complicaciones derivadas de la muerte masiva de los vermes adultos de D. immitis en el
circuito vascular precisa de un número suficiente de técnicas diagnósticas que permita
valorar la gravedad de cada situación y la intensidad de la parasitación, para seleccionar
la pauta correcta de tratamiento que evite o disminuya el riesgo de tromboembolismos en
el animal afectado (Knight, 1995).

6.1. Diagnóstico de la dirofilariosis cardiopulmonar canina

En el perro, los métodos diagnósticos se basan en la identificación de antígenos

circulantes del parásito adulto en suero y en la detección de microfilarias en muestras de

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sangre. Existen test comerciales basados en métodos inmunocromatográficos capaces de

detectar proteínas secretadas por el tejido ovárico de las hembras adultas de D. immitis.
Este diagnóstico presenta una alta especificidad al no observarse reacciones cruzadas con
otras filarias caninas, como D. repens o Acanthocheilonema reconditum. Su sensibilidad
también es grande pese a que pueden producirse falsos negativos en infecciones
prepatentes (de menos de 5 meses), cuando las infecciones están producidas por un
número muy escaso de vermes, o en infecciones causadas solo por machos (McCall,
1992). La detección de microfilarias está considerado como un método complementario
pero necesario, ya que se estima que un 30% de las infecciones caninas por D. immitis en
áreas endémicas son amicrofilarémicas (Rawlings et al., 1982). Este método se realiza
habitualmente por examen microscópico después de proceder a la concentración de las
microfilarias mediante el test de Knott o similares (Venco et al., 2011) (Figura 22).
Debido a la existencia de otras especies
de filarias que pueden aparecer en sangre
periférica, esta técnica debe de ir
acompañada de la determinación
específica de las microfilarias mediante
diferenciación morfológica (Carretón et
al., 2012), tinción histoquímica de las
zonas anatómicas con actividad fosfatasa
(Chalifoux y Hunt, 1971; Peribáñez et al.,
Figura 22. Observación de microfilarias mediante el 2001) o mediante amplificación del ADN
test de Knott. En la imagen puede verse una
microfilaria de D. immitis teñida con azul de por la reacción en cadena de la polimerasa
metileno (40X) (Carretón et al., 2012).
(Favia et al., 1996). En cualquier caso, la
positividad de una muestra a los test de microfilarias y de antígenos indicaría sin duda
una infección microfilarémica por D. immitis. La forma amicrofilarémica de la infección
se revela por un test negativo de microfilarias acompañado de un test positivo de
antígenos, mientras que la negatividad en el test de antígenos y positividad en el de
microfilarias, mostraría una infección por una especie distinta a D. immitis (Simón et al.,
2012).

Además de estos test, existen otros complementarios, como la radiografía torácica,

la ecocardiografía o la electrocardiografía, que pueden aportar datos interesantes sobre la
situación clínica de cada paciente y resultar de mucho interés a la hora de establecer un

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 Revisión bibliográfica

correcto protocolo de tratamiento. El examen radiográfico de tórax no permite estimar la

carga parasitaria (Venco et al., 2003), pero puede ser de utilidad en estadios avanzados
de la enfermedad, poniendo de manifiesto ensanchamientos de las arterias pulmonares,
anomalías del patrón pulmonar y en los casos más graves, cardiomegalia derecha (Venco
et al., 2011). La ecocardiografía, en cambio, sí permite realizar una estimación
aproximada del número y localización de las filarias, lo que le aporta cierto valor
diagnóstico. Los vermes de D. immitis aparecen como dos líneas paralelas hiperecógenas,
en el lumen de las cámaras del corazón y vasos pulmonares (Atkins et al., 1996; Venco
et al., 1998). La ecocardiografía asociada a impulsos Doppler permite además evaluar
con precisión la gravedad de la hipertensión pulmonar, por lo que esta prueba es altamente
recomendable cuando las características clínicas y radiológicas sugieren una infección
grave (Venco et al., 2011). En perros que se encuentran en la fase terminal de la
enfermedad, el análisis electrocardiográfico permite revelar alteraciones tanto del eje
como del ritmo eléctrico del corazón (Venco et al., 2011).

Recientemente, se ha investigado la utilización de moléculas liberadas a la sangre

como consecuencia del daño celular en los vasos y el corazón, de una perfusión
inadecuada o de la lisis de trombos, como biomarcadores tempranos en la dirofilariosis
cardiopulmonar. Esto permitiría realizar una mejor evaluación del estado clínico del perro
infectado, establecer un pronóstico y monitorizar el tratamiento elegido. Los resultados
iniciales sugieren la posibilidad de usar la troponina I cardíaca y la mioglobina como
marcadores de daño cardíaco y el dímero-D como herramienta de apoyo en el diagnóstico
de tromboembolismos pulmonares (Carretón, 2013).

6.2. Tratamiento y prevención

Las reacciones secundarias resultantes de la destrucción masiva de los vermes

adultos de D. immitis en el circuito sanguíneo complican en gran medida el tratamiento
de la dirofilariosis cardiopulmonar, desaconsejándose en ciertos pacientes. En cualquier
caso, se debe evaluar la situación de cada animal, considerando factores tales como el
número de parásitos, edad y tamaño del perro, gravedad de la enfermedad pulmonar y el
tipo de restricción de la actividad física al cual puede ser sometido el animal antes de
establecer un correcto tratamiento (Venco et al., 2004). En función de estos factores la
dirofilariosis se clasifica actualmente en dos categorías o niveles de gravedad (McCall et

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al., 2008). El primer nivel comprende los perros con bajo riesgo de complicaciones
tromboembólicas e incluye los animales asintomáticos con baja carga parasitaria,
ausencia de lesiones de la vasculatura o parénquima pulmonar, que presenten radiografías
torácicas normales, bajo nivel de antígenos circulantes, ausencia de parásitos en
ecocardiografía, sin enfermedades concomitantes y con posibilidad de limitar su actividad
física durante el tratamiento. En el grupo de alto riesgo de complicaciones
tromboembólicas se incluyen los perros que cumplan, al menos, una de las siguientes
condiciones: síntomas relacionados con la enfermedad (tos, lipotimias y ascitis),
radiografías torácicas anormales compatibles con la dirofilariosis, elevado nivel de
antígenos circulantes, visualización de los parásitos mediante ecocardiografía,
enfermedades concomitantes o imposibilidad de limitar la actividad física del animal
(Venco et al., 2011).

El tratamiento adulticida debe realizarse exclusivamente con clorhidrato de

melarsomina. El protocolo clásico consiste en administrar dos inyecciones por vía
intramuscular separadas por un intervalo de 24 horas en dosis de 2,5 mg/kg. No obstante,
la American Heartworm Society recomienda un protocolo diferido añadiendo una tercera
inyección con una dosis similar de melarsomina, al menos un mes antes de estas dos. Este
protocolo resulta más eficaz al eliminar los vermes adultos de forma escalonada [50% de
los adultos (90% machos y 10% hembras)] con la primera inyección y el resto con la
segunda y tercera, y más seguro al dar suficiente tiempo a los pulmones para recuperarse
del tromboembolismo causado por la muerte de los vermes en la primera inyección. Hay
que tener en cuenta que el tromboembolismo es una consecuencia inevitable del
tratamiento adulticida. Por ello, es esencial que este vaya acompañado de la restricción
del ejercicio durante un mes desde la administración del fármaco adulticida para
minimizar las complicaciones derivadas de la muerte de los parásitos. Además, para
controlar los síntomas del tromboembolismo pulmonar se puede administrar prednisona
a 0,5 mg/kg/12 h la primera semana y 0,5 mg/kg/24 h durante la segunda semana, seguido
de 0,5 mg/kg/48 h durante 1 ó 2 semanas (Carretón et al., 2012; Simón et al., 2012).
Puesto que la melarsomina no puede eliminar filarias menores de 4 meses de edad, el
tratamiento debe comenzar con la administración de lactonas macrocíclicas a dosis
preventivas durante los dos o tres meses previos para eliminar las larvas migratorias y las
microfilarias. Por otra parte, la liberación masiva de Wolbachia durante el tratamiento
adulticida y su implicación en la patogénesis de la enfermedad debe ser controlada. Por

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ello, el tratamiento con tetraciclinas del tipo doxiciclina a dosis de 10 mg/kg/12 h durante
4 semanas antes de la administración del adulticida es recomendable, ya que elimina un
90% de la población de Wolbachia y provoca un debilitamiento y disminución en la
fertilidad de las filarias adultas (Venco et al., 2011; Carretón et al., 2012; Simón et al.,
2012).

En perros en los que no es recomendable la aplicación de una terapia causal, puede

llevarse a cabo un tratamiento sintomático que incluye la administración de diversos
fármacos (corticosteroides, digoxina, opiáceos…) y/o restricción de la actividad física por
confinamiento en jaula (Dillon et al., 1995). La terapia quirúrgica puede aplicarse en
perros con síndrome de vena cava, empleando Flexible Alligator Forceps©, introducidos
a través de la vena yugular. Este instrumento permite extraer los vermes adultos de D.
immitis localizados en el ventrículo derecho y arteria pulmonar con un riesgo de
mortalidad intraoperatoria muy bajo y con una tasa de supervivencia y curación
directamente proporcional al porcentaje de vermes extraídos (Ishihara et al., 1990)
(Figura 23).

Teniendo en cuenta la gravedad de la enfermedad, la dificultad a la hora de

clasificar a los perros infectados y los riesgos tromboembólicos derivados de la terapia
elegida, la profilaxis constituye una alternativa de fundamental importancia. El
tratamiento profiláctico de elección se basa en la administración mensual de lactonas
macrocíclicas como ivermectina,
milbemicina oxima, moxidectina o
selamectina (McCall et al., 1986;
McTier et al., 1992). Se recomienda que
los cachorros de zonas endémicas
comiencen la profilaxis cuanto antes,
nunca más tarde de los dos meses de
edad, iniciándose esta un mes antes del
comienzo del período de transmisión y
finalizando un mes después de que este Figura 23. Verme adulto de D. immitis extraído de la
cavidad cardíaca derecha de un gato con dirofilariosis
termine (Atkins, 2011; American
a través de la vena yugular empleando Flexible
Heartworm Society, 2012). Alligator Forceps© (Venco y Vezzoni, 2001).

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Yang, J., Qiu, C., Xia, Y., Yao, L., Fu, Z., Yuan, C., Feng, X., Lin, J., 2010. Molecular
cloning and functional characterization of Schistosoma japonicum enolase which
is highly expressed at the schistosomulum stage. Parasitol. Res. 107, 667-677.

Yang, Z., Eton, D., Zheng, F., Livingstone, A.S., Yu, H., 2005. Effect of tissue
plasminogen activator on vascular smooth muscle cells. J. Vasc. Surg. 42, 532-
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Yates, D.M., Wolstenholme, A.J., 2004. An ivermectin-sensitive glutamate-gated

chloride channel subunit from Dirofilaria immitis. Int. J. Parasitol. 34, 1075-1081.

Yavlovich, A., Katzenell, A., Tarshis, M., Higazi, A.A., Rottem, S., 2004. Mycoplasma
fermentans binds to and invades HeLa cells: involvement of plasminogen and
urokinase. Infect. Immun. 72, 5004-5011.

Yavlovich, A., Rottem, S., 2007. Binding of host extracellular matrix proteins to
Mycoplasma fermentans and its effect on adherence to, and invasion of HeLa
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Zhang, S., Guo, A., Zhu, X., You, Y., Hou, J., Wang, Q., Luo, X., Cai, X., 2015.
Identification and functional characterization of alpha-enolase
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HIPÓTESIS Y OBJETIVOS
 HIPÓTESIS Y OBJETIVOS

Uno de los hechos clave de la dirofilariosis cardiopulmonar es el desarrollo de

endarteritis proliferativa, un proceso hiperplásico de la pared arterial, de gran
importancia para el desarrollo posterior de la patología pulmonar y cardíaca. Pueden
producirse, además, graves tromboembolismos causados por la muerte de los vermes
adultos de Dirofilaria immitis, que ocurren, de forma natural o como consecuencia de
un tratamiento filaricida. Puesto que los mecanismos de estas alteraciones no son bien
conocidos, es necesario estudiarlos, ya que su entendimiento puede facilitar el manejo
de estas situaciones por los clínicos veterinarios, contribuyendo a mejorar la calidad de
vida de los animales afectados. Dada la capacidad de supervivencia de D. immitis en sus
hospedadores y el hecho de que los tromboembolismos aparecen cuando mueren los
vermes, nuestra primera hipótesis fue que el parásito controla y modifica el hábitat
sanguíneo para facilitar su supervivencia a través de moléculas presentes en sus
productos antigénicos, generando un estado neto antitrombótico mediante la utilización
de productos profibrinolíticos.

Por otra parte, la supuesta activación del sistema fibrinolítico a largo plazo por
parte del parásito podría generar una sobreproducción de plasmina, producto final de
esta ruta. Este fenómeno ha sido relacionado en otros contextos con situaciones
patológicas graves que incluyen proliferación y migración de las células de la pared
arterial, así como destrucción de la matriz extracelular. Dada la aparente similitud entre
estos procesos patológicos y los que se producen durante el desarrollo de la endarteritis
proliferativa en la dirofilariosis cardiopulmonar, nuestra segunda hipótesis fue que la
sobreactivación de la ruta fibrinolítica por parte de D. immitis estaría directamente
relacionada con la aparición de dichos procesos patológicos en la pared vascular de los
animales afectados. Para demostrar ambas hipótesis propusimos los siguientes objetivos
dentro de la presente Tesis Doctoral:

1. Analizar la interacción de los antígenos de D. immitis con el sistema fibrinolítico

de su hospedador en relación con los mecanismos de supervivencia a nivel
vascular.
2. Estudiar si la activación del sistema fibrinolítico por parte del parásito tiene
influencia en los procesos patológicos descritos en el desarrollo de la endarteritis
proliferativa en la dirofilariosis cardiopulmonar.

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“Excretory/secretory antigens from Dirofilaria immitis adult worms interact
with the host fibrinolytic system involving the vascular endothelium”
 PRIMER CAPÍTULO

Los antígenos excretores/secretores de los vermes adultos de Dirofilaria immitis

interaccionan con el sistema fibrinolítico del hospedador implicando al endotelio
vascular

Javier González-Miguel, Rodrigo Morchón, Isabel Mellado, Elena Carretón, José

Alberto Montoya-Alonso, Fernando Simón

Molecular & Biochemical Parasitology 181 (2012) 134-140.

Factor de impacto (2012): 2,734.

Resumen

Dirofilaria immitis es el agente causal de la dirofilariosis cardiopulmonar canina

y felina. El parásito puede sobrevivir durante largos períodos de tiempo (7 años o más)
en el sistema circulatorio de los reservorios inmunocompetentes, produciendo
generalmente una enfermedad vascular inflamatoria crónica. Además, la muerte
simultánea de grupos de vermes adultos puede desencadenar una patología aguda
caracterizada por la exacerbación de las reacciones inflamatorias y la aparición de
tromboembolismos graves. En el contexto de las relaciones D. immitis/hospedador, el
objetivo de este trabajo fue investigar la interacción entre los antígenos
excretores/secretores de los vermes adultos de D. immitis (DiES) y el sistema
fibrinolítico del hospedador. Mediante el empleo de un enzimoinmunoensayo
demostramos que el extracto DiES es capaz de fijar plasminógeno y generar plasmina,
aunque este hecho requiere la presencia del activador tisular del plasminógeno (tPA).
Por otra parte, establecemos que el extracto DiES aumenta la expresión de tPA en
cultivos de células endoteliales vasculares. Adicionalmente, 10 proteínas fijadoras de
plasminógeno del extracto DiES fueron identificadas por espectrometría de masas
(HSP60, actina-1/3, actina, actina 4, transglutaminasa, GAPDH, Ov87, LOAG_14743,
galectina y P22U). Los datos sugieren que los antígenos del extracto DiES interaccionan
con el entorno del parásito regulando la activación del sistema fibrinolítico del
hospedador e implicando al endotelio vascular en el proceso.

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Abstract
Dirofilaria immitis is the causative agent of canine and feline heartworm
disease. The parasite can survive for long periods of time (7 years or more) in the
circulatory system of immunocompetent reservoirs, producing usually a chronic
inflammatory vascular disease. In addition, the simultaneous death of groups of adult
worms can trigger an acute disease characterized by the exacerbation of inflammatory
reactions and the emergence of serious thromboembolic events. In the context of the D.
immitis/host relationships, the aim of this study was to investigate the interaction
between the excretory/secretory antigens from D. immitis adult worms (DiES) and the
fibrinolytic system of the host. Using an enzyme-linked immunosorbent assay we
showed that DiES extract is able to bind plasminogen and generate plasmin, although
this fact requires the presence of the tissue plasminogen activator (t-PA). Moreover, we
established that DiES extract enhances t-PA expression in cultured vascular endothelial
cells. Additionally, 10 plasminogen-binding proteins from DiES extract were identified
by mass spectrometry (HSP60, actin-1/3, actin, actin 4, transglutaminase, GAPDH,
Ov87, LOAG_14743, galectin and P22U). The data suggest that DiES antigens interact
with the environment of the parasite regulating the activation of the fibrinolytic system
of the host with involvement of the vascular endothelium in the process.

Keywords: Dirofilaria immitis, excretory/secretory antigens, plasminogen binding, t-

PA, endothelial cells, mass spectrometry.

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Highlights
> Excretory/secretory antigens from D. immitis adult worms (DiES) bind plasminogen.
> Plasminogen is activated by this extract and plasmin is generated.
> DiES extract stimulate t-PA expression in vascular endothelial cell cultures.
> We identify 10 plasminogen-binding proteins of DiES extract.

Graphical Abstract

1. Introduction
Heartworm disease (HD) is a serious and potentially fatal disease caused by the
filaroid nematode Dirofilaria immitis that affects dogs and cats all over the world [1].
The adult worms lodge in the pulmonary arteries and the right ventricle of infected hosts
where they can live for years [2], causing a chronic inflammatory pathology. Initially
the damages affect the arteries (endarteritis and perivascular inflammation), spreading
later to the lung parenchyma and the right heart chambers [3]. In addition, when groups
of worms die naturally or as a consequence of filaricide treatment, very serious
alterations occur, with the exacerbation of inflammatory reactions and the formation of
massive thromboembolisms [4] that put the life of the infected animals in immediate
risk.

Since D. immitis can survive in the long term in the vascular system of
immunocompetent hosts, it is reasonable to assume that adult worms interact with their
intravascular environment, modulating the immune response and the associated
pathology by means of the action of their metabolic products [excretory/secretory (ES)
antigens], as it occurs in other parasitic infections [5,6]. A key point of vantage in blood
parasites is the hemostasis, which is closely associated to fibrinolysis, inflammatory
reactions and angiogenesis [7]. During the fibrinolysis, plasminogen binds to specific
receptors together with activators of the process from which the most important is the t-
PA that is mainly synthesized and secreted by endothelial cells. This binding determines
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the transformation of plasminogen into serin protease plasmin, which is the enzyme that
lyses the fibrin [8], degrading it into soluble products including the D-dimer [9].
Moreover, the activation of the plasminogen–plasmin system plays a key role in the
degradation of extracellular matrices [10] that has been related to cell invasion and
intra-organic migration of different pathogens [11,12]. Plasminogen and t-PA bind to
receptors present on cells in the fibrin clots, to annexin-A2 of vascular endothelial cells
and integrin αMβ2 of leukocytes [8]. Activation of plasminogen by binding to
molecules secreted by bacteria with which it forms complexes has also been described
[13]. Related to parasites, several molecules associated to the surface of protozoa and
helminths [14-19] as well as molecules of the ES products [12,20], that bind
plasminogen, have been identified. Additionally, it has been described that such
interactions are mediated by carboxyl-terminal lysine residues of the plasminogen
receptors [21].

We have previously observed some facts that suggest the interaction of D.

immitis with its vascular environment: (i) DiES promote vasodilation, stimulating the
expression of prostaglandin E2 by vascular endothelial cells, also limiting the
transmigration of monocytes to the perivascular tissue [22]. (ii) In another study we
found that serum levels of D-dimer are significantly higher in the 47% of dogs with HD
analyzed than in the healthy dogs used as controls, indicating the presence of
thromboembolisms and their degradation [23].

Although D. immitis is able to survive in the circulatory system of its hosts for
years, there are no data on the interaction of adult worms with the fibrinolytic system of
their hosts. The aim of this study was to demonstrate that the DiES antigens can bind
plasminogen, generate plasmin, and stimulate the increase of t-PA synthesis by vascular
endothelial cells. Additionally, we identified some plasminogen binding proteins of
DiES extract using immunoproteomic techniques and mass spectrometry (MS).

2. Materials and Methods

2.1. Collection of ES extract of proteins from D. immitis adult worms
DiES were prepared as previously described [22] with minor modifications and
stored at −80 °C. In brief, live worms (25) obtained from a naturally infected dog were
washed in sterile phosphate-buffered saline solution (PBS) pH 7.2 and incubated for

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24 h in 50 ml of Eagle's minimum essential medium (EMEM) supplemented with

50 U/ml penicillin and 50 μg/ml streptomycin at 37 °C. A cocktail of protease inhibitors
was added to the medium following the methodology described by Maizels et al. [24].
The medium was dialyzed against water for 24 h and filtered through an Amicon YC05
membrane (Millipore). The protein concentration of DiES was measured by DC protein
assay commercial kit (Bio-Rad). DiES was tested for the presence of endotoxin
contamination using a quantitative Limulus amebocyte lysate test (BioWhittaker). The
endotoxin quantity was under the sensitivity level of cell stimulation (<0.4 U/mg
protein).

2.2. Plasminogen binding assay

To determine whether plasminogen would bind components of the DiES extract,
an enzyme-linked immunosorbent assay (ELISA) was performed. Multiwell microplates
(Costar) were coated with 1 μg/well of DiES extract diluted in carbonate buffer, pH 9.6,
overnight at 4 °C. The wells were blocked with 1% BSA in PBS and incubated
successively with increasing amounts (from 0 μg to 3 μg) of human plasminogen (Acris
Antibodies), with a sheep anti-human plasminogen IgG (Acris Antibodies) at 1:2000
dilution and then with a peroxidase-conjugated donkey anti-sheep IgG (Sigma) at
1:4000 dilution. All incubations were performed for 1 h at 37 °C and between each step
washed three times with PBS wash buffer (PBS containing 0.05% Tween20). Ortho-
phenylene-diamine was used as a chromogen. Optical densities (OD) were measured at
492 nm in an Easy Reader (Bio-Rad). In parallel, competition assays were performed by
including 50 mM of the lysine analogue ɛ-aminocaproic acid (ɛACA) during
plasminogen incubation. Some wells coated with BSA only were used as negative
controls.

2.3. Plasminogen activation assay

Plasminogen activation assay was performed in a test volume of 100 μl by
measuring the amidolytic activity of generated plasmin [15]. In each well 2 μg of
human plasminogen (Acris Antibodies) were incubated in PBS with 3 μg of the
chromogenic substrate S-2251 (Sigma) in the presence of 1 μg of DiES. Activation of
plasminogen was initiated by addition of 15 ng of t-PA (Sigma). In parallel, plasmin
generation was also measured in the absence of t-PA. Plates were incubated at 37 °C for

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2 h and the hydrolysis of the chromogenic substrate was monitored by measuring

absorbance at 405 nm every 30 min. Each sample was analyzed in triplicate.

2.4. Cell culture and stimulation of endothelial cells

Vascular endothelial cells HAAE-1 from ATCC (LGC Promochem) were grown
and treated as previously described [22]. In brief, endothelial cells were grown in Ham's
F12k medium (ATCC) supplemented with 2 mM L-glutamine, 10% fetal bovine serum
(FBS) (ATCC), 50 U/ml penicillin, 50 μg/ml streptomycin, 0.1 mg/ml heparin (Sigma)
and 0.03 mg/ml endothelial cell growth supplement (ECGS) (Sigma). Plates were
precoated with 0.1% pig gelatine (Sigma). Cells were cultured at 37 °C in a humidified
atmosphere in the presence of 5% CO2–95% air. Medium was changed every 3 days.
Endothelial cells (106 cells/plate) were plated on 100 mm culture plates and grown for
4 days to obtain confluent cultures and treated with 1 μg/ml of DiES for 24 h. Non-
stimulated cells were used as controls under the same conditions.

2.5. Two-dimensional electrophoresis (2-DE) of DiES extract

The 2-DE of DiES was performed as described before by us for the somatic
antigen of adult worms of D. immitis [25]. Briefly, DiES extract was purified with the
ReadyPrep 2-D Cleanup Kit (Bio-Rad) and resuspended in rehydration buffer 2-D (7 M
urea, 2 M thiourea, 4% 3-[(3-cholamidopropyl) dimethylammonio]-1-propanesulfonate
(CHAPS)). The samples were divided into 125 μl aliquots (containing 60 μg of protein)
and stored at −20 °C until use. When they were used DiES aliquots were supplemented
with ampholytes and DTT, incubated and centrifuged to remove all particulate material,
and then applied to 7-cm IPG strips (Bio-Rad) with linear pH ranges of 3–10, 5–8 and
7–10, using a Protean IEF Cell (Bio-Rad) for isoelectric focusing (IEF). After IEF,
strips were reduced and alkylated, and second dimension separation was done in 12%
acrylamide gels. Gels were then silver stained with the PlusOne Silver Staining Kit,
Protein (GE Healthcare) or transferred to nitrocellulose membranes for their
immunoblot analysis. The 2-D images were scanned with the GS-800 Densitometer
(Bio-Rad) and analyzed with the Quantity One Software v.4.6.5 (Bio-Rad).

2.6. Immunoblot assays

To determine which proteins of DiES extract bind plasminogen, they were
electrotransferred from 2D gels to nitrocellulose membranes at 20 V for 30 min using a

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Trans-Blot SD Semi-Dry Transfer cell (Bio-Rad). Blots were blocked with 2% BSA in
PBS wash buffer, for 1 h at room temperature. DiES membranes were incubated
overnight at 4 °C with 10 μg/ml of human plasminogen. Then, the blots were incubated
with a sheep anti-human plasminogen IgG (Acris Antibodies) at 1:1000 dilution and
with a peroxidase-conjugated donkey anti-sheep IgG (Sigma) at 1:2000 dilution, each
incubation for 90 min. All incubations were performed at 37 °C with shaking and
between each step washed three times with washing buffer for 5 min per wash. Protein
bands were revealed with 4-chloro naphthol. Negative controls were also used in which
the plasminogen had been omitted. In addition, competition assays were performed by
including 50 mM ɛACA during plasminogen incubation. Membranes were digitized
with the scanner GS-800 Densitometer (Bio-Rad) using the Quantity One Software
v.4.6.5 (Bio-Rad). Matching of 2-D gels with the homologous Western blot to identify
plasminogen-binding proteins, the assignment of molecular weights (MW) and
isoelectric points (pI) of each protein were analyzed using the PDQuest Software v.8.0.1
(Bio-Rad). All assays were performed in triplicate to assess the reproducibility of the
spot pattern.

Western blot analysis for the t-PA expression was performed as previously
described [22]. Treated and non-treated vascular endothelial cells were lysed in ice-cold
lysis buffer. Protein samples (20 μg) were separated by SDS–PAGE under reducing
conditions and blotted onto polyvinylidine difluoride membranes. Membranes were
blocked before incubation with the primary antibody rabbit anti-t-PA (Santa Cruz
Biotechnology) at 1:1000. After incubation with HRP-conjugated anti-rabbit secondary
antibody at 1:20,000 dilution, bands were visualized by a luminol-based detection
system with p-iodophenol enhancement. Anti-α-tubulin antibody (Oncogene Research
Products) was used to confirm loading of comparable amount of protein in each lane.
Protein expression was quantified by densitometry using Scion Image Software.

2.7. MS and protein identification

In gel digestion of proteins and MS analysis were done as described before by us
[25]. The spots containing plasminogen-binding proteins were excised manually from
the gels and sent to the Unit of Proteomics of the Centro Nacional de Investigaciones
Cardiovasculares (Madrid, Spain) for MS analysis. For peptide mass fingerprinting and
the acquisition of LIFT TOF/TOF spectra, an aliquot of the digestion of each spot was

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deposited onto a 600 μm AnchorChip MALDI probe (Bruker-Daltonics). Peptide mass

fingerprint spectra were measured on a Bruker Ultraflex TOF/TOF MALDI mass
spectrometer (Bruker-Daltonics) [26] in positive-ion reflector mode. The measured
tryptic peptide masses were transferred through the MS BioTools program (Bruker-
Daltonics) as inputs to search the National Centre for Biotechnology Information non-
redundant database (NCBInr) using Mascot software (Matrix Science). When necessary,
MS/MS data from the LIFT TOF/TOF spectra were combined with MS peptide mass
fingerprint (PMF) data for database searches.

2.8. Statistical analysis

The results from the plasminogen binding assay, plasminogen activation assay
and Western blots for the t-PA expression were analyzed with the Student's t-test. The
results were expressed as the mean ± SD of at least 3 independent experiments. In all
experiments, a significant difference was defined as a p-value of <0.05 for a confidence
level of 95%.

3. Results
3.1. Proteins of DiES extract bind plasminogen
The binding level of plasminogen to DiES extract was studied by ELISA. This
test showed that the DiES extract binds plasminogen and that this binding is directly
proportional to the amount of plasminogen (Fig. 1). The negative control consisting of
wells coated only with BSA showed some non-specific binding activity, but always in a
value significative lower than that shown by the DiES extract (p < 0.05). To determine
whether or not lysine residues are involved in binding, a competition experiment
including 50 mM ɛACA was carried out. In this case the binding between DiES extract
and plasminogen was inhibited about 70%, resulting in slightly higher optical densities
than the negative control (Fig. 1).

3.2. Plasminogen is activated by proteins of DiES extract and plasmin is generated

The ability to activate plasminogen by DiES extract and to generate plasmin was
assessed by measuring the amidolytic activity of plasmin generated in the presence of
the antigenic extract and plasminogen. This effect was measured in the presence or
absence of a physiological activator of the process, t-PA, to observe the ability of the
DiES extract proteins of activating plasminogen on their own. Negative controls

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replacing DiES for BSA or t-PA were also used. As shown in Fig. 2, the generation of
plasmin by t-PA is enhanced by DiES reaching optical density values significative
higher (p < 0.05) than the negative controls in the presence of t-PA. However, DiES
extract is unable to generate plasmin without t-PA resulting in optical density values
identical to the negative control.

Figure 1. Plasminogen binding to 1 μg of DiES extract of D. immitis measured over a range of

plasminogen amounts per cavity using a microtiter plate method. (■) Incubation with increasing amounts
of plasminogen, 0–3 μg. (●) Competition assay with 50 mM ɛACA included during plasminogen
incubation. (▴) Negative control consisted of wells coated only with BSA. Each point is the mean of
three replicates ± SD. The asterisk (*) designates significant (p < 0.05) differences.

Figure 2. Plasminogen activation and plasmin generation by DiES extract of D. immitis. (□) 15 ng of t-
PA was added to mixtures containing 2 μg of human plasminogen and 3 μg of the chromogenic substrate
S-2251 (Sigma) in the presence or absence of 1 μg of DiES (or BSA as negative control) in a test volume
of 100 μl. (■) No t-PA were added to reaction mixtures. Each point is the mean of three replicates ± SD.
The asterisk (*) designates significant (p < 0.05) differences.

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3.3. DiES extract stimulate t-PA expression in vascular endothelial cell cultures
D. immitis is an intravascular parasite for which we previously observed
interactions of DiES antigens with the vascular endothelium. Additionally, the DiES
extract activates the transformation of plasminogen to plasmin in a t-PA-dependent
manner. Thus, the objective of this experiment was to determine whether or not DiES
enhances the synthesis of t-PA in vascular endothelial cells (HAAE-1). Proteins from
DiES-treated vascular endothelial cell extracts were separated by SDS–PAGE and
analyzed by Western blotting using anti-t-PA antibody. As shown in Fig. 3, DiES
induced a significative increase in t-PA (p < 0.05) protein expression after 24 h of
stimulation.

Figure 3. Effect of DiES on the expression of t-PA in human vascular endothelial cells. Protein extracts
from lysed DiES treated or untreated confluent cell cultures were analyzed by Western blot for t-PA. α-
tubulin served as a protein control. Results were expressed as the mean ± SEM of at least 3 independent
experiments. The asterisk (*) designates significant (p < 0.05) differences from control cells. (□)
Stimulated endothelial cells. (■) Non-treated control cells. AU, arbitrary units.

3.4. Two-dimensional analysis of DiES extract

To obtain an overall view of all the proteins of the DiES, this extract were first
electrofocused using 3–10 linear immobilized pH gradient strips. Silver nitrate staining
of these 2-D gels revealed about 570 spots in the excretome of D. immitis with pIs
between 5 and 9.8, and a broad range of MWs (10–150 kDa). Only 24 spots were
observed with pI < 5 (not shown).

In order to improve spot resolution and detection, once the spot MW and pI
ranges were determined, the DiES extract were electrofocused in 5–8 and 7–10 IPG
strips. With these new conditions, silver staining revealed a total of 636 spots, most of

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them (594) located between pH 5 and 8. The remaining 96 spots had pIs between 8 and
9.8 (Fig. 4A and B).

Figure 4. Representative 2-DE of 60 μg of the DiES extract from adult D. immitis worms. The gels were
in the 5–8 and 7–10 pH ranges, 12% polyacrylamide and silver-stained (A and B). Plasminogen-binding
spots revealed on ligand blots from gels A and B (C and D). Reference molecular masses are indicated on
the left. The plasminogen-binding spots analyzed by MS are circled and numbered.

3.5. Identification of plasminogen-binding proteins

To identify plasminogen-binding proteins, ligand blotting of 2D gels of 5–8 and
7–10 pH with plasminogen was performed, after electrotransfering them to
nitrocellulose membranes.

As shown in Fig. 4C and D, 81 plasminogen-binding spots were revealed. This

represents a binding rate of 12.73% of total spots revealed in the excretome of D.
immitis. Most of them (n = 60) were resolved in a narrow range of MWs and pIs

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(between 37 and 150 KDa, and 5.2 and 7.2, respectively). In the control blots, in which
plasminogen incubation was omitted, the anti-plasminogen antibody did not reveal any
spots (not shown). In the competition experiments, the inclusion of 50 mM ɛACA
inhibited plasminogen binding to DiES demonstrating the specificity of the reaction
(Fig. S1).

MW Sequence
Spot pI Mascot
Accesion code Protein definition Species (kDa) coverage
number theor/exp score
theor/exp (%)

23 ACY25666 Chaperonin-like Brugia malayi 61.4/67.1 5.7/5.6 11 130

protein HSP60
27 AF121264_1 Chaperonin protein Onchocerca 64.5/67.0 5.7/5.8 17 145
HSP60 volvulus
28 AF121264_1 Chaperonin protein Onchocerca 64.5/65.2 5.7/5.8 16 130
HSP60 volvulus
31 ACT1_CAEEL Actin-1/3 Caenorhabditis 42.1/65.2 5.3/5.9 11 41
elegans
32 XP_001894819 Actin Brugia malayi 42.1/43.3 5.3/5.8 4 64

33 NP_508842 ACTin family Caenorhabditis 37.5/39.4 5.4/6.0 17 142

member (act-4) elegans
37 AAC24752 Transglutaminase Dirofilaria 57.6/61.0 5.7/6.3 19 91
precursor immitis
66 XP_001899850 Glyceraldehyde 3- Brugia malayi 32.1/40.8 8.5/7.5 20 207
phosphate
dehydrogenase
67 XP_001899850 Glyceraldehyde 3- Brugia malayi 32.1/40.7 8.5/7.8 25 292
phosphate
dehydrogenase
69 AAD00843 Ov87 Onchocerca 36.7/36.4 8.9/8.2 24 161
volvulus
71 AAD00843 Ov87 Onchocerca 36.7/36.5 8.9/9.0 16 157
volvulus
72 XP_003150284 Hypothetical Loa loa 13.3/33.7 6.7/6.3 11 94
protein
LOAG_14743
73 AAF37720 Galectin Dirofilaria 32.2/30.1 6.0/6.6 11 118
immitis
78 AAD11968 P22U Dirofilaria 24.4/22.0 8.9/9.2 66 499
immitis
79 AAD11968 P22U Dirofilaria 24.4/22.0 8.9/9.4 62 458
immitis
80 AAD11968 P22U Dirofilaria 24.4/22.0 8.9/9.6 62 489
immitis
81 AAD11968 P22U Dirofilaria 24.4/22.0 8.9/9.8 54 201
immitis

Table 1. Plasminogen-binding protein spots of DiES extract identified by MALDI-TOF MS. Exp,
experimental; theo, theoretical.

The matching of spots revealed by ligand-blotting with their homologous spots

in the silver-stained 2-D gels allowed to select a total of 81 plasminogen-binding spots
of D. immitis, which were manually excised from 2-D gels and submitted to analysis by

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MS. Table 1 shows the identity of these proteins and their MWs and pIs (theoretical and
experimental), the NCBI accession number, the sequence coverage and the Mascot
score. Seventeen of 81 spots were identified (21%) and corresponded to 10 different
proteins. The proteins identified were chaperonine protein HSP60, actin-1/3, actin, actin
4, transglutaminase precursor, glyceraldehyde 3-phosphate dehydrogenase (GAPDH),
Ov87, hypothetical protein LOAG_14743, galectin and P22U. Between 1 and 4
isoforms of each protein were identified. Most proteins were identified by their
similarity to homologous proteins from other species of filarial worms. Thus of the 17
spots identified 9 corresponded to other filarial proteins (Brugia malayi, Onchocerca
volvulus and Loa loa), while 6 spots corresponded to 3 proteins of D. immitis deposited
in databases (transglutaminase precursor, galectin and P22U). The 2 remaining spots
corresponded to proteins from the nematode Caenorhabditis elegans.

4. Discussion
D. immitis infections are typically characterized by the persistence of adult
worms in vascular location for years in which they are exposed to multiple aggression
mechanisms from the host and where their presence causes severe and sometimes fatal
pathological changes. One of these mechanisms is the generation of thromboembolisms.
This process is physiologically regulated by the fibrinolytic system which is able to lyse
fibrin clots. Its activation by molecules of D. immitis could have beneficial effects for
the survival of the parasite in the circulatory system.

In this study we demonstrate in an in vitro system that DiES antigens bind

plasminogen. This binding is dependent on the presence of lysine residues, as it is
inhibited by ɛ-aminocaproic acid. We also demonstrate that DiES extract activates
plasminogen and generate plasmin in a t-PA-dependent manner. All this is consistent
with experimental studies carried out in bacteria, protozoa and helminths [17,19,27,28].

We also demonstrated that DiES antigens significantly stimulate the basal

production of t-PA by vascular endothelial cells cultured in vitro. This is consistent also
with the key role of vascular endothelium in the regulation of haemostasis [7] and with
the intravascular niche of D. immitis. We have previously shown that DiES antigens
interact with the vascular endothelium, stimulating vasodilatation and reducing
leukocyte transmigration, which highlights the importance of the endothelium in the

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activation of mechanisms that likely promote parasite survival, also limiting the damage
to the host [22].

On the other hand, plasmin produced by plasminogen activation is also involved

in the lysis of extra-cytoplasmic matrices [29], which is interpreted as a mechanism
related to cell invasion and intra-organic migration of different parasites [12,14]. As
many molecules involved in the binding of plasminogen are multifunctional, we cannot
rule out that though the activation of the fibrinolytic system has predictable beneficial
effects, both for the parasite and for the host, some of the molecules involved in the
process can exert other activities, contributing to the generation of damage on the host.
A consequence of the arrival of the D. immitis adult worms into the pulmonary arteries
is the appearance of proliferative endarteritis caused by the proliferation and migration
of smooth muscle cells of the vascular wall into the lumen [3]. It has been shown that
the over-expression of t-PA in damaged endothelium induces proliferation and
migration of smooth muscle cells in humans [30]. Since we have observed an over-
expression of t-PA by vascular endothelial cells stimulated by DiES, it will be necessary
to study in the future if the proliferative endarteritis is associated with over-expression
of t-PA by endothelial cells and therefore the activation of the fibrinolytic system of the
host by D. immitis adult worms.

The proteomic analysis of DiES extract also allowed us to identify 17

plasminogen-binding spots by MS, which corresponded to 10 proteins. Their
identification was possible by the existence of a significant amount of available
information on the filarial worms. Of the proteins identified in the DiES, HSP 60,
different proteins of the family of actins and GAPDH are among the best characterized
binding-plasminogen molecules. The HSP 60 is a binding protein belonging to the
family of heat shock proteins. Its plasminogen-binding activity has been demonstrated
in bacteria [31,32], in which this activity has been associated with disruption of the
extracellular matrix of tissues and invasion [32].

Three proteins of the actin family binding plasminogen (actin-1/3, actin and
actin-4) have been identified in the DiES extract. The interaction between actin and
plasminogen is well known, as well as the fact that specific binding occurs through
lysine residues which stimulate the tPA-dependent plasmin generation [33]. In addition,

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its function as plasminogen receptor has been demonstrated on the surface of

endothelial cells [34] and in the tegument of Schistosoma bovis [17]. The glycolytic
enzyme GAPDH is a multifunctional molecule whose plasminogen-binding activity has
been observed in different pathogens such as bacteria and fungi [35,36], blood
helminths such as S. bovis [17] and tissue helminths like O. volvulus [16].

The other proteins identified in our study (transglutaminase precursor, Ov87,

galectin, P22U and LOAG_14743) have been related to plasminogen binding for the
first time. However, there is evidence that some of them are associated with nearby or
related fibrinolysis processes. It has been demonstrated that galectin-1, which belongs to
the family of galectins (like galectin and Ov87 proteins identified here) acts as receptor
of t-PA and is the responsible for the increase of catalytic activity that occurs in the
pancreatic cancer [37]. Moreover, the LOAG_14743 is a hypothetical protein of the
annexin family. Within this family, annexin-A2 is one of the best-characterized
plasminogen receptors on endothelial cells [38].

To conclude, we have demonstrated that DiES antigens in vitro activate the

plasminogen binding and plasmin production, involving the vascular endothelium in the
regulation of this process through the stimulus of the expression of t-PA by vascular
endothelial cells. These facts demonstrate the interaction of D. immitis with its vascular
environment through his metabolic products, promoting mechanisms for its own
survival. Ten plasminogen binding molecules of the DiES extract have been identified
by proteomic analysis and MS, suggesting that D. immitis adult worms use different
molecules to maintain the balance of the vascular environment. Future studies are
needed to obtain a complete understanding of this process during HD and to elucidate if
molecules of the plasminogen binding process are involved in other mechanisms related
to the occurrence of pathological changes in the pulmonary arteries.

Acknowledgements
This research was supported by Agencia de Desarrollo Económico de Castilla y
León (cofinanced with FEDER funds), Junta de Castilla y León (grant SA090/A09),
Spain.

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Supplementary material

Supplemental Figure S1. Competition assay of DiES-plasminogen immunoblot analysis. The assay was
performed by including 50 mM εACA during plasminogen incubation. The membranes were in the 5-8
and 7-10 pH ranges. Reference molecular masses are indicated on the left. As shown in Figure S1 the
inclusion of 50 mM εACA inhibit plasminogen binding to DiES.

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“Surface associated antigens of Dirofilaria immitis
adult worms activate the host fibrinolytic system”
 SEGUNDO CAPÍTULO

Los antígenos asociados a la superficie de los vermes adultos de Dirofilaria immitis

activan el sistema fibrinolítico del hospedador

Javier González-Miguel, Rodrigo Morchón, Elena Carretón, José Alberto Montoya-

Alonso, Fernando Simón

Veterinary Parasitology 196 (2013) 235-240.

Factor de impacto (2013): 2,545.

Resumen

La dirofilariosis cardiopulmonar (Dirofilaria immitis) se caracteriza por eventos

aparentemente contradictorios, como la supervivencia a largo plazo de los vermes
adultos en el sistema circulatorio de los hospedadores infectados y el desarrollo de
procesos potencialmente mortales como la aparición de tromboembolismos. Por lo
tanto, mecanismos desarrollados por los parásitos como la activación del sistema
fibrinolítico, son un punto clave para la supervivencia tanto de los vermes como del
hospedador. El objetivo de este trabajo fue investigar la interacción entre los antígenos
asociados a la superficie de los vermes adultos de D. immitis (DiSAA) y el sistema
fibrinolítico del hospedador. Se demostró que el extracto DiSAA es capaz de fijar
plasminógeno y generar plasmina, ocurriendo esto último de un modo dependiente del
activador tisular del plasminógeno (tPA). Adicionalmente, 11 proteínas fijadoras de
plasminógeno del extracto DiSAA fueron identificadas mediante proteómica y
espectrometría de masas (MS): (actina-5C, actina-1, enolasa, fructosa-bifosfato
aldolasa, GAPDH, dominio proteico MSP, MSP 2, lectina de unión a beta-
galactosidasa, galectina, proteína contenedora del dominio inmunoglobulina I y
ciclofilina Ovcyp-2). Debido a que en un trabajo previo hemos demostrado la
interacción positiva entre los antígenos excretores/secretores de D. immitis (DiES) y el
sistema fibrinolítico del hospedador y a que muchas de las moléculas identificadas aquí
son compartidas por ambos compartimentos antigénicos, se propone que DiSAA
coopera en la activación del sistema fibrinolítico promoviendo la lisis de los coágulos
de fibrina.

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Abstract
Cardiopulmonary dirofilariosis (Dirofilaria immitis) is characterized by apparent
contradictory events, like the long-term survival of adult worms in the circulatory system
of the infected hosts and the development of life-threatening events like
thromboembolisms and others. Thus parasite mechanisms, like the activation of
fibrinolytic system, are key to the survival of both the worms and the host. The aim of
this study was to investigate the interaction between D. immitis adult worms surface-
associated antigens (DiSAA) and the fibrinolytic system of the host. We demonstrate that
DiSAA extract is able to bind plasminogen and generate plasmin, with the latter occurring
in a tissue plasminogen activator (t-PA) dependent manner. Additionally, 11
plasminogen-binding proteins from DiSAA extract were identified by proteomics and
mass spectrometry (MS) (actin-5C, actin-1, enolase, fructose-bisphosphate aldolase,
GAPDH, MSP domain protein, MSP 2, beta-galactosidase-binding-lectin, galectin,
immunoglobulin I-set domain-containing protein and cyclophilin Ovcyp-2). Because in a
previous work we have shown the positive interaction between the excretory/secretory
antigens of D. immitis (DiES) and the host fibrinolytic system and many of the molecules
identified here are shared by both antigens, we hypothesize that DiSAA cooperate in host
fibrinolytic system activation promoting the fibrin clot lysis.

Key words: Dirofilaria immitis; surface associated antigens; fibrinolysis; plasminogen

binding.

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1. Introduction
Dirofilaria immitis is the causative agent of canine and feline cardiopulmonary
dirofilariosis and human pulmonary dirofilariosis. It is a vector-borne transmitted disease
with a cosmopolitan distribution (Genchi et al., 2001). D. immitis adult worms can survive
for years (7 or more) in the pulmonary arteries and right ventricle of dogs (Quiroz-
Romero, 1984), causing a chronic vascular disease mainly associated with inflammatory
reactions. Moreover, the simultaneous death of groups of adult worms can trigger an acute
pathology characterized by the exacerbation of the inflammatory reactions and the
occurrence of serious thromboembolisms (Venco, 2007) that poses an immediate risk for
the life of the affected hosts.

The fibrinolytic system activity is based on the conversion of plasminogen into

plasmin, the enzyme responsible of fibrin clots lysis (Cesarman-Maus and Hajjar, 2005).
This process is regulated by the t-PA, mainly synthesized by vascular endothelial cells. It
has been recently demonstrated that the metabolic products excreted by D. immitis “in
vitro” stimulate the host fibrinolytic system. Furthermore, this stimulation causes an over-
expression of t-PA in vascular endothelial cells (González-Miguel et al., 2012),
suggesting the existence of regulatory mechanisms of the thromboembolisms by this
parasite in its intravascular habitat. On the other hand, parasite-surface molecules have
been widely linked to key roles related to the host/parasite relationships (Fetterer and
Rhoads, 1993) and some of them have been related to the plasminogen-binding activity
in protozoa (Almeida et al., 2004, Vanegas et al., 2007 and Mundodi et al., 2008) and
helminth parasites (Jolodar et al., 2003, Erttmann et al., 2005 and Ramajo-Hernández et
al., 2007). In this work we demonstrate that different surface-associated molecules of D.
immitis adult worms bind plasminogen and generate plasmin, activating the host
fibrinolytic system.

2. Materials and Methods

2.1. Collection of surface associated antigens from D. immitis adult worms (DiSAA)
DiSAA extract was obtained following the methodology described by
Wedrychowicz et al. (1994) with minor modifications. In brief, live worms (7) obtained
from a naturally infected dog were washed and then incubated in saline solution
containing 0.25% CTAB with a cocktail of protease inhibitors (Maizels et al., 1991) at
37 °C for 4 h. The worms were separated from detergent and extracted proteins were

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precipitated with sodium acetate 0.002 M with nine volumes of 96% ethanol, at −20 °C
for 48 h followed by centrifugation (10,000 × g, 10 min). The resulting pellets were re-
suspended in PBS pH 7.2 and stored at −80 °C until use. Previously, protein concentration
of DiSAA was measured by DC protein assay commercial kit (Bio-Rad).

2.2. Plasminogen binding assay

To determine whether the plasminogen would bind surface components of D.
immitis an ELISA test was carried out as described previously (González-Miguel et al.,
2012). In brief, multiwell microplates (Costar) were coated with 1 μg/well of DiSAA
extract, blocked and then incubated with increasing amounts (from 0 μg to 3 μg) of human
plasminogen (acris antibodies). After incubation with the corresponding antibodies and
with a chromogen, the optical density was measured at 492 nm in an easy reader (Bio-
Rad). In parallel, competition assays were performed by including 50 mM of the lysine
analogue ɛ-aminocaproic acid (ɛACA) during plasminogen incubation.

2.3. Plasminogen activation assay

Plasminogen activation assay was performed in a test volume of 100 μl by
measuring the amidolytic activity of generated plasmin (González-Miguel et al., 2012).
In each well 2 μg of human plasminogen (acris antibodies) were incubated in PBS with
3 μg of the chromogenic substrate S-2251 (Sigma) in the presence of 1 μg of DiSAA
extract. Activation of plasminogen was initiated by addition of 15 ng of t-PA (Sigma). In
parallel, plasmin generation was also measured in the absence of t-PA. Plates were
incubated at 37 °C for 2 h and the hydrolysis of the chromogenic substrate was monitored
by measuring absorbance at 405 nm every 30 min. Each sample was analyzed in triplicate.

2.4. Two-dimensional electrophoresis (2-DE) of DiSAA extract and immunoblot

assay
The 2-DE of DiSAA extract was performed as described before by us (González-
Miguel et al., 2012). Briefly, DiSAA extract aliquots were supplemented with
ampholytes, incubated and centrifugated, and then applied to 7 cm IPG strips (Bio-Rad)
with linear pH ranges of 3–10, 5–8 and 7–10, using a Protean IEF Cell (Bio-Rad) for
isoelectric focusing (IEF). After IEF, strips were reduced and alkilated, and second
dimension was done in 12% acrylamide gels. Gels were then silver stained with the
PlusOne silver staining kit, protein (GE Healthcare).

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To determine which proteins of DiSAA extract bind plasminogen an immunoblot

was performed (González-Miguel et al., 2012). The 2-D gels were transferred to
nitrocellulose membranes which were blocked and then incubated overnight at 4 °C with
10 μg/ml of human plasminogen. After incubating the membranes with the corresponding
antibodies, proteins were revealed with 4-chloro naphthol.

The 2-D gels and membranes were scanned and analyzed with the quantity one
software v.4.6.5 (Bio-Rad). Matching of 2-D gels with the homologous Western blot to
identify plasminogen-binding proteins was analyzed using the PDQuest Software v.8.0.1
(Bio-Rad).

2.6. MS and protein identification

In gel digestion of proteins and MS analysis were done as described before by us
(González-Miguel et al., 2012). The spots containing plasminogen-binding proteins were
excised manually from the gels and sent to the unit of Proteomics of the Centro de
Investigación Príncipe Felipe (Valencia, Spain) for MS analysis. For peptide mass
fingerprinting and the acquisition of LIFT TOF/TOF spectra, an aliquot of the digestion
of each spot was deposited onto a 600 μm AnchorChip MALDI probe (Bruker-Daltonics).
Peptide mass fingerprint spectra were measured on a Bruker Ultraflex TOF/TOF MALDI
mass spectrometer (Bruker-Daltonics) in positive-ion reflector mode. The measured
tryptic peptide masses were transferred through the MS BioTools software (Bruker-
Daltonics) as inputs to search the National Centre for Biotechnology Information non-
redundant database (NCBInr) using Mascot software (Matrix Science). When necessary,
MS/MS data from the LIFT TOF/TOF spectra were combined with MS peptide mass
fingerprint data for database searches.

2.7. Statistical analysis

The results from the plasminogen binding assay and plasminogen activation assay
were analyzed with the Student's t-test. The results were expressed as the mean ± SD of
at least 3 independent experiments. In all experiments, a significant difference was
defined as a p-value < 0.05 for a confidence level of 95%.

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Results
3.1. Proteins of DiSAA extract bind plasminogen
The binding capacity of plasminogen to DiSAA extract was measured by ELISA.
The test showed that DiSAA binds plasminogen obtaining optical densities statistically
higher (p < 0.05) than those of the control wells (coated only with BSA) (Fig. 1). This
binding is also directly proportional to the amount of plasminogen. The competition assay
showed that the inclusion of 50 mM ɛACA inhibits the plasminogen-binding (Fig. 1),
demonstrating that this union is dependent on lysine residues.

Figure 1. Plasminogen binding to 1 μg of DiSAA extract of D. immitis measured over a range of

plasminogen amounts using a microtiter plate method. (■) Incubation with increasing amounts of
plasminogen, 0–3 μg. (●) Competition assay with 50 mM ɛACA included during plasminogen incubation.
(▴) Negative control consisted of wells coated only with BSA. Each point is the mean of three replicates
±SD. The asterisk (*) designates significant (p < 0.05) differences.

3.2. The plasminogen-binding activity of DiSAA extract generates plasmin

The ability to activate plasminogen by DiSAA extract and to generate plasmin
was assessed by measuring the amidolytic activity of plasmin generated in the presence
of the antigenic extract and plasminogen. This effect was measured in the presence or
absence of t-PA, to observe the ability of the DiSAA extract proteins of activating
plasminogen on their own. Negative controls replacing DiSAA by BSA or t-PA were also
used. As shown in Fig. 2, the generation of plasmin by t-PA is enhanced by DiSAA
reaching optical density values significative higher (p < 0.05) than the negative controls.

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Furthermore this effect is inhibited by 50 mM ɛACA, indicating the involvement of lysine

residues in the process. However, DiSAA extract is unable to generate plasmin without
t-PA resulting in optical density values identical to the negative control.

Figure 2. Plasminogen activation and plasmin generation by DiSAA extract of D. immitis. (□) 15 ng of t-
PA was added to mixtures containing 2 μg of human plasminogen, 3 μg of S-2251 (Sigma) and 1 μg of
DiSAA extract (or BSA as negative control) in the presence or absence of 50 mM of ɛACA in a test volume
of 100 μl. (■) No t-PA was added to reaction mixtures. Each point is the mean of three replicates ±SD. The
asterisk (*) designates significant (p < 0.05) differences.

3.3. Two-dimensional analysis of DiSAA extract

To obtain an overall view of all the proteins of the DiSAA, this extract were first
electrofocused using 3–10 linear immobilized pH gradient strips. Silver nitrate staining
of these 2-D gels revealed about 315 spots in the D. immitis surface proteome, many
sparsely settled, with isoelectric points (pIs) between 5 and 9.7, and a broad range of
molecular weights (MWs) (10–145 kDa). Only 4 spots were observed with pI‹5 (not
shown). In order to improve spot resolution and detection, once the spot MW and pI
ranges were determined, the DiSAA extract were electrofocused in 5–8 and 7–10 IPG
strips. With these new conditions, silver staining revealed a total of 347 spots, most of
them (318) located between pIs 5 and 8. The remaining 29 spots had pIs between 8 and
9.8 (Fig. 3A and B).

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Figure 3. Representative 2-DE of 60 μg of the DiSAA extract from adult D. immitis worms. The gels were
in the 5–8 and 7–10 pH ranges, 12% polyacrylamide and silver-stained (A and B). Plasminogen-binding
spots revealed on ligand blots from gels A and B (C and D). Reference molecular masses are indicated on
the left. The plasminogen-binding spots analyzed by MS are circled and numbered.

3.4. Identification of plasminogen-binding proteins

To identify plasminogen-binding proteins, a ligand blotting with plasminogen of
2D gels of 5–8 and 7–10 pH was performed. As shown in Fig. 3C and D, 61 plasminogen-
binding spots were revealed (17.58% for total spots revealed in the surface proteome).
Most of them (n = 42) were resolved in a narrow range of MWs and pIs (between 40 and
100 kDa, and 5.4 and 8, respectively). In the control blots, in which plasminogen
incubation was omitted, the anti-plasminogen antibody did not reveal any spots (not
shown).

The matching of spots revealed by ligand-blotting with their homologous in the

silver-stained 2-D gels allowed us to select a total of 53 plasminogen-binding spots of D.
immitis, which were manually excised from 2-D gels and submitted to analysis by MS.
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Table 1 shows the identity of these proteins and their MWs and pIs (theoretical and
experimental), the number of access to similar information available in the NCBI
database, the sequence coverage and the Mascot score. Sixteen of 53 spots were identified
(30.18%) and corresponded to 11 different proteins. Between 1 and 4 isoforms of each
protein were identified. Most proteins were identified by their similarity to homologous
proteins from other species of filarial worms. Thus of the 16 spots identified 14
corresponded to other filarial proteins (Brugia malayi, Onchocerca volvulus and Loa loa).
The 2 remaining spots corresponded to a protein from the nematode Trichinella spiralis
(actin-5C) and to a protein from the free-living protist parasite Acanthamoeba castellanii
(actin-1).

MW
Spot pI Queries Mascot
Accesion code Protein definition Species (kDa)
number theor/exp matched score
theor/exp

17 EFV54220 Actin-5C Trichinella 41.8/54.1 5.3/5.7 4 154

spiralis
18 P02578 Actin-1 Acanthamoeba 41.7/52.8 5.4/5.8 6 190
castellanii
20 XP_001896281 Enolase Brugia malayi 47.5/59.6 6.0/6.3 6 248
22 Q7YZX3 Enolase Onchocerca 47.1/55.5 6.0/6.6 8 64
volvulus
26 AAB52600 Fructose- Onchocerca 39.2/40.5 7.7/7.7 8 247
bisphosphate volvulus
aldolase
42 AAB52600 Fructose- Onchocerca 39.2/39.0 7.7/7.9 2 59
bisphosphate volvulus
aldolase
28 P48812 GAPDH Brugia malayi 36.1/40.0 7.7/7.2 13 128
30 P48812 GAPDH Brugia malayi 36.1/39.8 7.7/7.4 17 259
32 P48812 GAPDH Brugia malayi 36.1/37.4 7.7/7.8 11 84
46 P48812 GAPDH Brugia malayi 36.1/36.0 7.7/8.0 9 71
35 XP_001900868 MSP domain protein Brugia malayi 18.1/40.1 5.5/6.0 2 64
with Glu-rich
domain
60 P13263 Major sperm protein Onchocerca 14.3/15.7 7.8/8.8 18 265
2 volvulus
37 AAA20541 Beta-galactosidase- Onchocerca 32.0/33.6 6.0/6.6 20 143
binding-lectin volvulus
38 XP_001900812 Galectin Brugia malayi 31.8/30.3 6.4/7.8 3 68
39 XP_003139445 Immunoglobulin I- Loa loa 22.5/24.2 6.6/7.6 8 307
set domain-
containing protein
58 AAC47233 Cyclophilin Ovcyp- Onchocerca 18.5/20.9 8.3/9.4 1 71
2 volvulus

Table 1. Plasminogen-binding protein spots of DiSAA extract identified by MALDI-TOF MS. GAPDH,
Glyceraldehyde 3-phosphate dehydrogenase; Exp, experimental; theo, theoretical.

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4. Discussion
Cardiopulmonary dirofilariosis is caused by the long-term presence of D. immitis
adult worms in the pulmonary arteries and right ventricle of their definitive host. In this
location, worms are exposed to the effector mechanisms of the host immune system.
Moreover, the death of the adult worms triggers some pathological processes that
immediately threat the host's life, the thromboembolisms being one of the most serious.
However, D. immitis can survive through different mechanisms of both immune evasion
and modulation of its vascular environment (Simón et al., 2012). In the live worms there
are two antigenic compartments participating in the parasite/host relationships, the
excreted metabolic products (DiES antigens) and the surface-associated antigens, many
of which are also excreted (Smith, 1991), taking part in the excretory/secretory antigens.
In this study we demonstrate that the DiSAA bind plasminogen and stimulate plasmin
generation. Surface-associated antigens have also been related to the plasminogen
recruitment in both bacterial (Bergmann and Hammerschmidt, 2007) and parasitic
infections (Jolodar et al., 2003 and Ghosh and Jacobs-Lorena, 2011). It has also been
postulated that plasmin produced by plasminogen activation plays a key role in the
degradation of extracellular matrices, migration through the tissues (Bergmann and
Hammerschmidt, 2007) and evasion of the immune response (Barthel et al., 2012). For
the conversion of plasminogen into plasmin t-PA is necessary, being mainly synthesized
and secreted by the vascular endothelium (Cesarman-Maus and Hajjar, 2005). Given that
it has been previously shown that the DiES causes an over-expression of t-PA in cultured
vascular endothelial cells (González-Miguel et al., 2012), it is possible that a combined
action of both compartments (DiES and DiSAA) could exist. Thus D. immitis worms are
not only capable of activating the fibrinolytic system in order to avoid clot formation in
the systemic level by action of the DiES, but also in their immediate environment by the
action of the DiSAA. This combined action would be of great importance as a parasite
survival mechanism.

The combination of proteomic, immunomic and MS techniques allowed us to

identify a total of 16 spots of the DiSAA extract that corresponded to 11 plasminogen-
binding proteins. These were identified using the available information of filarial proteins
in databases. Among them, various enzymes from the group of actins (Dudani et al.,
2005 and Ramajo-Hernández et al., 2007), enolase (Jolodar et al., 2003, Marcilla et al.,
2007 and Mundodi et al., 2008), fructose-bisphosphate aldolase (Ramajo-Hernández et
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al., 2007 and de la Paz Santangelo et al., 2011) and GAPDH (Erttmann et al.,
2005 and Ramajo-Hernández et al., 2007) have been extensively studied due to their
interaction with the fibrinolytic system in different types of pathogens. Furthermore, 3
isoforms of actin, 2 of GAPDH and the galectin were identified as plasminogen-binding
proteins on the DiES antigens (González-Miguel et al., 2012). The other proteins
identified in our study (MSP domain protein, major sperm protein 2, beta-galactosidase-
binding-lectin, immunoglobulin I-set domain-containing protein and cyclophilin Ovcyp-
2) have been identified as plasminogen binding proteins for the first time. These proteins
are involved in different biological processes such as structural activity (MSP domain
protein and MSP 2), carbohydrate binding (beta-galactosidase-binding-lectin) or protein
folding (cyclophilin Ovcyp-2). Further studies are needed to know the real effect of the
identified plasminogen-binding proteins on the survival mechanisms of D. immitis.

In summary, we have demonstrated the “in vitro” interaction between the surface-
associated antigens of D. immitis and the host fibrinolytic system, supplementing the
already demonstrated fibrinolytic activity of the excretory/secretory antigens of this
species.

Acknowledgements
This research was supported by Agencia de Desarrollo Económico de Castilla y
León (cofinanced with FEDER funds), Junta de Castilla y León (grant SA090/A09),
Spain.

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“Surface-displayed glyceraldehyde 3-phosphate dehydrogenase and galectin from
Dirofilaria immitis enhance the activation of the fibrinolytic system of the host”
 TERCER CAPÍTULO

La gliceraldehído-3-fosfato deshidrogenasa y la galectina asociadas a la superficie

de Dirofilaria immitis potencian la activación del sistema fibrinolítico del hospedador

Javier González-Miguel, Rodrigo Morchón, Mar Siles-Lucas, Ana Oleaga, Fernando

Simón

Acta Tropica 145 (2015) 8-16.

Factor de impacto (2014): 2,519.

Resumen

La dirofilariosis cardiopulmonar es una enfermedad cosmopolita causada por

Dirofilaria immitis, un parásito filaroideo cuyos vermes adultos viven durante años en el
sistema vascular de su hospedador. Estudios previos han demostrado que D. immitis
puede utilizar sus antígenos excretores/secretores (ES) y de superficie para potenciar la
fibrinolisis, lo que podría limitar la formación de coágulos en su entorno. Además, varias
isoformas de la gliceraldehído-3-fosfato deshidrogenasa (GAPDH) y la galectina (GAL)
fueron identificadas en ambos extractos antigénicos como proteínas fijadoras de
plasminógeno. El objetivo de este trabajo es estudiar la interacción de la GAPDH y la
GAL de D. immitis con el sistema fibrinolítico del hospedador. Este estudio incluye la
clonación, secuenciación y expresión de las formas recombinantes de la GAPDH y la
GAL de D. immitis (rDiGAPDH y rDiGAL) y el análisis de sus capacidades como
proteínas fijadoras de plasminógeno. Los resultados indican que rDiGAPDH y rDiGAL
son capaces de fijar plasminógeno y estimular la generación de plasmina mediada por el
activador tisular del plasminógeno (tPA). Esta interacción necesita la implicación de
residuos de lisina, muchos de los cuales se encuentran localizados externamente en ambas
proteínas como se demuestra con el modelado molecular de sus estructuras secundarias.
Además, mostramos que rDiGAPDH y rDiGAL aumentan la expresión del activador del
plasminógeno de tipo uroquinasa (uPA) en cultivos de células endoteliales caninas y que
ambas proteínas se expresan en la superficie de D. immitis en contacto directo con la
sangre del hospedador. Estos datos sugieren que D. immitis podría utilizar la GAPDH y
la GAL asociadas a la superficie como receptores fisiológicos del plasminógeno para
modificar el equilibrio fibrinolítico hacia la generación de plasmina, lo que podría

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constituir un mecanismo de supervivencia para evitar la formación de coágulos en su

hábitat intravascular.

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Abstract
Cardiopulmonary dirofilariosis is a cosmopolitan disease caused by Dirofilaria
immitis, a filaroid parasite whose adult worms live for years in the vascular system of its
host. Previous studies have shown that D. immitis can use their excretory/secretory (ES)
and surface antigens to enhance fibrinolysis, which could limit the formation of clots in
its surrounding environment. Moreover, several isoforms of the glyceraldehyde 3-
phosphate dehydrogenase (GAPDH) and galectin (GAL) were identified in both
antigenic extracts as plasminogen-binding proteins. The aim of this work is to study the
interaction of the GAPDH and GAL of D. immitis with the fibrinolytic system of the
host. This study includes the cloning, sequencing and expression of the recombinant
forms of the GAPDH and GAL of D. immitis (rDiGAPDH and rDiGAL) and the
analysis of their capacity as plasminogen-binding proteins. The results indicate that
rDiGAPDH and rDiGAL are able to bind plasminogen and stimulate plasmin generation
by tissue plasminogen activator (tPA). This interaction needs the involvement of lysine
residues, many of which are located externally in both proteins as have been shown by
the molecular modeling of their secondary structures. In addition, we show that
rDiGAPDH and rDiGAL enhance the expression of the urokinase-type plasminogen
activator (uPA) on canine endothelial cells in culture and that both proteins are
expressed on the surface of D. immitis in close contact with the blood of the host. These
data suggest that D. immitis could use the associated surface GAPDH and GAL as
physiological plasminogen receptors to shift the fibrinolytic balance towards the

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generation of plasmin, which might constitute a survival mechanism to avoid the clot
formation in its intravascular habitat.

Key words: Dirofilaria immitis; glyceraldehyde 3-phosphate dehydrogenase; galectin;

plasminogen; fibrinolysis.

Abbreviations
ES, excretory/secretory; GAPDH, glyceraldehyde 3-phosphate dehydrogenase;
GAL, galectin; rDiGAPDH, recombinant form of the GAPDH of D. immitis; rDiGAL,
recombinant form of the GAL of D. immitis; tPA, tissue plasminogen activator; uPA,
urokinase-type plasminogen activator; OP, optical density; εACA, lysine analogue ε-
aminocaproic acid; CnAOEC, canine aortic endothelial cells; DiES, excretory/secretory
antigens from D. immitis adult worms.

Highlights
The Dirofilaria immitis GAPDH and GAL were cloned and sequenced.
rDiGAPDH and rDiGAL bind plasminogen and generate plasmin activating
fibrinolysis.
Plasminogen activation by rDiGAPDH and rDiGAL requires tPA and lysine
residues.
rDiGAPDH and rDiGAL enhance the expression of uPA in canine endothelial cells.
rDiGAPDH and rDiGAL are located exposed to the host on the surface of D.
immitis.

Graphical Abstract

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1. Introduction
Fibrinolysis is one of the main anticlotting mechanisms of the hemostatic
system. Its key molecule is plasminogen, an abundant component of blood and
zymogen of serine protease plasmin, enzyme responsible for degrading fibrin clots. The
conversion of plasminogen into plasmin is regulated by binding to receptors via its five
kringle domains, which have affinity for lysine residues and plasminogen activators
(tPA and uPA) (Cesarman-Maus and Hajjar, 2005).

In order to maintain and propagate in the circulatory system, many bloodborne

pathogens not only require adaptations to evade the activity of the host immune system,
but also need to prevent blood clotting through interaction with the fibrinolytic system
(Mebius et al., 2013). Cardiopulmonary dirofilariosis is a chronic and potentially fatal
parasitic disease that affects dogs and cats around the world (Genchi et al., 2001). It is
characterized by the presence of D. immitis adult worms in the pulmonary arteries and
right ventricle of the infected hosts, where they can live for years causing a chronic
inflammatory pathology (Venco, 2007). In previous studies, we have demonstrated the
ability of D. immitis to bind plasminogen, enhancing plasmin generation by tPA by
using two antigenic compartments (ES and surface) in an in vitro system. We have also
observed that the ES antigens are able to induce an overexpression of the fibrinolytic
activator tPA in vascular endothelial cells in culture. Additionally, we have respectively
identified a total of 10 and 11 plasminogen-binding proteins in the ES and surface
extracts of the parasite, which included different isoforms of GAPDH and GAL
(González-Miguel et al., 2012 and González-Miguel et al., 2013).

GAPDH has historically been regarded as a “housekeeping” protein. However,

its involvement in numerous cellular processes in addition to glycolysis has been
recently demonstrated. These include DNA repair, tRNA export, membrane fusion and
transport, cytoskeletal dynamics and cell death (Tristan et al., 2011). Moreover its
relationship with the fibrinolytic system has been widely studied being identified as
plasminogen-binding protein in bacteria (Bhattacharya et al., 2012), fungi (Crowe et al.,
2003) and parasites (Erttmann et al., 2005, Ramajo-Hernández et al., 2007 and Lama et
al., 2009). GAPDH is one of the most studied plasminogen receptors in parasites
together with enolase, which has been reported as plasminogen-binding protein for the

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related filaria Onchocerca volvulus (Jolodar et al., 2003) or Schistosoma bovis (Ramajo-
Hernández et al., 2007 and de la Torre-Escudero et al., 2010) among others.

Galectins are β-galactoside-binding proteins characterized by its high level of

evolutionary conservation, having been identified in many species from nematodes to
mammals. Galectins have a wide range of biological functions in different processes
including homeostasis, apoptosis, and vascular embryogenesis and in pathological
conditions such as pre-eclampsia, inflammation, diabetes, atherosclerosis and cancer
(Astorgues-Xerri et al., 2014). Related to filarial worms, onchocercal molting L3
strongly express GAL, being this protein proposed as good target for protective
responses (Joseph et al., 2000). The interaction between this molecule and plasminogen
has not yet demonstrated. However, the link between GAL-1 expression and cancer cell
invasion with the demonstration of a direct interaction between tPA and GAL-1 in
pancreatic cancer cells and stromal fibroblasts surrounding the tumor has been recently
shown. This interaction enhanced tPA proteolytic activity and increased cell migration
and invasion (Roda et al., 2009).

The aim of this study was to perform the molecular and functional
characterization of the D. immitis GAPDH and GAL showing their capabilities as
plasminogen-binding proteins, their relationships with the endothelium-dependent
components of the fibrinolytic system and confirming their presence on the surface of
the parasite.

2. Materials and methods

2.1. Parasite material
Adult worms of D. immitis were obtained from hearts of infected dogs from
Gran Canary (Canary Islands, Spain) through the jugular vein using Flexible Alligator
Forceps.

2.2. RNA isolation, RT-PCR, and cloning of GAPDH and GAL cDNA
Total RNA from adult worms was extracted using the NucleoSpin RNA II kit
(Macherey-Nagel) according to the manufacturer's instructions. First-strand cDNA was
synthesized from D. immtis adult worms RNA using the first-strand cDNA synthesis kit
(Roche) as recommended by the manufacturer. The cDNA sequence of the D. immitis
GAPDH and GAL were amplified using the following primers:

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GAPDHFwd (5´-ATGAGCAAACCAAAGATTGGAATC)
GAPDHRev (5´-TTATCTGCTGGCGATGTAAGAG)
GALFwd (5´-ATGCACCACAACGAATATGAAACGAATTAC)
GALRev (5´-CTAGTGCATTTGAATACCGCTCACTTC)

The primers from GAPDH were designed on the consensus sequence resulting
after the alignment of GAPDH cDNA sequences from O. volvulus and Brugia malayi
(GenBank accession numbers U96177.1 and U18137.1 respectively). The primers from
GAL were designed on the sequence of GAL cDNA sequences from D. immitis
(GenBank accession number AF237485.1). PCR was performed in 1 cycle at 94 °C for
5 m, 35 cycles at 94 °C for 1 m, 46 °C for 46 s and 72 °C for 1 min 30 s, and 1 cycle at
72 °C for 5 m. The PCR products were electrophoresed in an agarose gel and the bands
were purified from the gel using the StrataPrep DNA Gel Extraction kit (Stratagene) as
recommended by the manufacturers. The GAPDH and GAL PCR products were cloned
into the pSC-A vector using the StrataClone PCR Cloning kit (Stratagene) following the
manufacturer's instructions. Both clones were purified with the Machery-Nagel
NucleoSpin Plasmid kit.

2.3. Expression and purification of the rDiGADPH and rDiGAL

PCR products containing the whole rDiGADPH and rDiGAL coding sequences
were cloned into the TOPO vector (Gateway System, Invitrogen) following the
manufacturer's instructions. The recombinant plasmids were transformed into the
Escherichia coli XL1B. Transformed cells were grown in LB-agar plates with
ampicillin (100 μg/ml) overnight at 37 °C. Three colonies for each molecule were
grown in liquid LB plus ampicillin overnight at 37 °C in agitation, and cells were
harvested for plasmid extraction. Extracted plasmids were digested with EcoRI to check
the insert presence. The TOPO vectors containing the fragments of interest were used
for a ligation reaction with the pDEST7 vector (Gateway System, Invitrogen) following
the manufacturer's instructions. Ligation reaction was transformed into XL1B cells and
grown in LB-agar plates with ampicillin (100 μg/ml) overnight at 37 °C. Three colonies
for each molecule were grown in liquid LB plus ampicillin overnight at 37 °C in
agitation, and cells were harvested for plasmid extraction. Extracted plasmids were
digested with EcoRI to check the insert presence. Vectors containing the inserts of
interest were sequenced with the T7 primer (Sequencing Service of the Salamanca

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University) to check for the correct reading frame. The vectors containing the molecule
of interest in reading frame were used to transform BL-21 expression cells. These were
grown in liquid LB plus ampicillin (100 μg/ml) overnight at 37 °C in agitation. Cultures
were diluted 1:20 in fresh medium and further growth until reaching an optical density
(OD) of 0.5 at 600 nm. Then, expression of the recombinant proteins was induced by
adding L-arabinose at a final concentration of 0.2% and further growing at 37 °C for 4 h
in agitation. The induced cells were harvested and sonicated in a buffer containing
50 mM Na2PO4, 300 mM NaCl and 10 mM imidazole, pH 8 for rDiGADPH and 8 M
urea, 100 mM NaH2PO4 and 10 mM Tris–Cl, pH 7.9 for rDiGAL. After a 20 min
centrifugation step at 10,000 × g, the supernatant was applied to a HIS-Select® Nickel
Affinity Gel (Sigma) for affinity purification of the histidine-tagged rDiGADPH and
rDiGAL, according to the manufacturer's instructions. Urea was eliminated for rDiGAL
by washing the column with wash buffer (100 mM NaH2PO4, and 10 mM Tris–Cl pH
6.3) containing decreasing concentrations of urea (from 6 M to 0 M). Then, the
recombinant proteins were eluted with elution buffer (50 mM NaH2PO4, 300 mM NaCl
and 250 mM imidazole, pH 7.9). The eluted rDiGAPDH and rDiGAL were dialyzed in
PBS for 24 h at 4 °C and stored at −80 °C until use. The purity and yield of each protein
obtained after purification were assessed in 12% polyacrylamide gels using Coomasie
blue staining. The densitometry was calculated with the PDQUEST program (Bio-Rad).

2.4. Bioinformatic analyses

The deduced amino-acid sequence of rDiGAPDH and rDiGAL were analyzed
using the following bioinformatic tools: BLAST searching of the homologous
sequences in the NCBI and Swissprot/Uniprot databases (http://www.ncbi.nlm.nih.gov/,
http://www.uniprot.org/); analysis of conserved domains with SMART
(http://smart.embl-heidelberg.de); theoretical isoelectric point (pI) and the molecular
weight (MW) calculations (http://www.expasy.org/tools/pi_tool.html); prediction of
transmembrane domains with the TMHMM Server v. 2.0
(http://www.cbs.dtu.dk/services/TMHMM-2.0); prediction of signal peptides with
SignalP 3.0 (Bendtsen et al., 2004) (http://www.cbs.dtu.dk/services/SignalP); search for
glycosyl–phosphatidyl anchors in the sequence with the big-PI Predictor (Eisenhaber et
al., 2000) (http://mendel.imp.ac.at/sat/gpi/gpi_server.html); multiple sequence
alignment with ClustalW 2.1 (http://www.ebi.ac.uk/Tools/msa/clustalw2/) and
prediction of the secondary structures and three-dimensional modeling with the Swiss-
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Model server (Arnold et al., 2006; http://swissmodel.expasy.org/) using as templates the

X-ray crystal structure of a GAPDH from B. malayi (code pdb: 4K9D) for DiGAPDH
(identity of 96.76%) and the crystal structure of Toxascaris leonine GAL (code pdb:
4HL0) for DiGAL (identity of 90.11%). The 3-D models were visualized with the
RasMol software v. 2.7.5.2.

2.5. Plasminogen binding assays

To determine whether the rDiGAPDH and rDiGAL would bind plasminogen an
ELISA test was carried out as described previously (González-Miguel et al., 2012). In
brief, multiwell microplates (Costar) were coated with 0.5 μg/well of each protein,
blocked and then incubated with increasing concentrations (from 0 to 30 μg/ml) of
human plasminogen (Acris antibodies). After incubation with the corresponding
antibodies and with a chromogen, the OD was measured at 492 nm in an easy reader
(Bio-Rad). In parallel, competition assays were performed by including 50 mM of the
lysine analogue ϵ-aminocaproic acid (ϵACA) during plasminogen incubation.

2.6. Plasminogen activation assays

Plasminogen activation assay was performed in a test volume of 100 μl by
measuring the amidolytic activity of generated plasmin (González-Miguel et al., 2012).
In each well 2 μg of human plasminogen (Acris antibodies) were incubated in PBS with
3 μg of the chromogenic substrate D-Val-Leu-Lys 4-nitroanilide dihydrochloride
(Sigma) in the presence of 1 μg of rDiGAPDH or rDiGAL. Activation of plasminogen
was initiated by addition of 15 ng of tPA (Sigma). In parallel, plasmin generation was
also measured in the absence of tPA. Plates were incubated at 37 °C for 2 h and the
hydrolysis of the chromogenic substrate was monitored by measuring absorbance at
405 nm every 30 min. Each sample was analyzed in triplicate.

2.7. Cell culture and stimulation of endothelial cells

Canine aortic endothelial cells (CnAOEC) (Cell Applications, Inc.) were grown
in canine endothelial growth mediums (Cell Applications, Inc.). Plates were precoated
with an attachment factor solution (Cell Applications, Inc.) and cells were cultured at
37 °C in a humidified atmosphere in the presence of 5% carbon dioxide and 95% air.
Medium was changed every 3 days. Endothelial cells (106 cells/plate) were plated on
100 mm culture plates and grown for 4 days to obtain confluent cultures and treated

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with 1 μg/ml of rDiGAPDH or rDiGAL for 24 h. Non-stimulated cells were used as

controls under the same conditions.

2.8. Cell lysates and Western blot analyses

Western blot analysis was performed as previously described (Morchón et al.,
2010) with slightly modifications. Treated and control CnAOEC were lysed in ice-cold
lysis buffer (20 mM Tris–HCl (pH 7.5), 140 mM NaCl, 10 mM
ethylendiaminetetraacetic acid, 10% glycerol, 1% Igepal CA-630, aprotinin, pepstanin,
pepstatin, and leupeptin at 1 μg/ml each, 1 mM phenylmethylsulfonyl fluoride, and
1 mM sodium orthovanadate). Protein samples (10 μg) were separated by SDS–PAGE
under reducing conditions and blotted onto polyvinylidine difluoride membranes.
Membranes were blocked before incubation with the primary antibodies anti-tPA and
anti-uPA (Santa Cruz Biotechnology, Inc.) according to the manufacturer's
recommendations. After incubation with HRP-conjugated secondary antibodies, bands
were visualized by a luminol-based detection system with p-iodophenol enhancement.
Anti-α-tubulin antibody (Oncogene Research Products) was used to confirm loading of
comparable amount of protein in each lane. Protein expression was quantified by
densitometry using Scion Image Software (Scion).

2.9. Generation of an anti-rDiGAPDH and anti-rDiGAL antisera

Antisera against D. immitis rGAPDH and rGAL were generated by subcutaneous
immunization of four New Zealand female rabbits with three doses of each protein in
0.2% saponin solution. First dose of 1 mg at the beginning of the experiment, plus two
doses of 500 μg 7 and 10 days later. Rabbits were bled 20 days after the last dose. Sera
were collected, serially diluted and titred by ELISA. The reactivity and specificity of the
sera were also assessed by Western blot on rDiGAPDH, rDiGAL or on ES extract from
D. immitis adult worms (DiES) containing the corresponding native proteins. In brief,
recombinant proteins (2 μg each) and DiES (10 μg) were electrophoresed on 12% SDS–
PAGE gels, electrotransferred onto nitrocellulose membranes, blocked with 2% BSA
and incubated with the antisera against rDiGAPDH, rDiGAL or with a negative control
serum at 1/500 dilution. After washing, immunoblots were incubated with 1/2000
diluted peroxidase-labeled anti-rabbit IgG and revealed with 4-chloro naphthol. Images
were digitized with the scanner GS-800 Densitometer (Bio-Rad) using the Quantity One
Software v.4.6.5 (Bio-Rad).

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2.10. Immunolocalization of proteins in D. immitis adult worms

Immunolocalization assays were carried out in microtome-cut 5 μm sections
after dehydrate and embed in paraffin D. immitis adult worms. The sections were placed
on microscope slides, deparaffinized in xylene, rehydrated and blocked with 1% BSA in
PBS. Then, sections were firstly incubated with the anti-rDiGAPDH or anti-rDiGAL
rabbit antisera or with a negative serum (rabbit preinmune serum), all of them diluted
1/50 in blocking buffer. Secondly, samples were incubated with an anti-rabbit IgG
antibody conjugated to Alexa Fluor 594 (Invitrogen) diluted 1/400 in blocking buffer
containing phalloidin-Alexa Fluor 488 (Invitrogen) diluted 1/200, which binds to actin
microfilaments. All incubations were carried out for 1 h at 37 °C in a humid chamber
and between each step, three washes of 5 min with PBS containing 0.05% Tween-20
were performed. Finally, the samples were washed four times, mounted in antifade
reagent (Prolong Gold, Invitrogen) and analyzed with a Leica TCS-NT confocal
microscope.

2.11. Statistical analysis

The results from the plasminogen-binding assay, plasminogen activation assay
and Western blots for the tPA and uPA expression were analyzed with the Student's t-
test. The results were expressed as the mean ± SD of at least three independent
experiments. In all experiments, a significant difference was defined as a p-value of
<0.05 for a confidence level of 95%.

3. Results
3.1. Amplification, cloning, sequencing, and expression of D. immitis GAPDH and
GAL
Amplification of D. immitis GAPDH and GAL cDNA by RT-PCR resulted in a
PCR product of around 1000 and 850 bp respectively. These were cloned into the pSC-
A vector and fully sequenced. BLAST analysis of the sequence demonstrated its
identity as glyceraldehyde 3-phosphate dehydrogenase and galectin. The GAPDH new
sequence was deposited in the Gen-Bank under accession number JQ780095.1. The full
D. immitis GAPDH and GAL cDNA contained 1020 and 846 nucleotides, encoded
proteins of 339 and 280 amino acids, with a theoretical molecular weight of 36,179 and
32,085 Da, and pI of 7.11 and 6.08 respectively.

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The bioinformatics analyses of the deduced amino acid sequences did not reveal
a signal peptide, transmembrane helices or glycosyl-phosphatidyl inositol anchors. The
percentage identity between DiGAPDH and homologous sequences from other
organisms (O. volvulus, S. bovis, Candida albicans, Streptococcus pyogenes, Bacillus
anthracis and Trichomonas vaginalis) whose GAPDH had been previously related with
plasminogen-binding activities was analyzed using multiple sequence alignment with
the ClustalW program (Fig. 1). The analysis revealed a range of identities between the
94.99% of the filaria O. volvulus and the 43.07% of the protozoa T. vaginalis.
Additionally, seven conserved lysine residues in all the sequences were found and
highlighted. Amino-acid conservation of DiGAPDH and DiGAL was analyzed by
alignment with homologous sequences from other parasites (Figs. S1 and S2).
DiGAPDH and DiGAL revealed a strong identity with the homologous sequences from
other filarial nematodes (B. malayi, O. volvulus, Loa loa and Wuchereria bancrofti)
ranging from 97.46% and 94.99% in the case of DiGAPDH (Fig. S1) and from 96.79%
and 94.81% in the case of DiGAL (Fig. S2). These sequences also showed high
identities in the alignment with proteins from other non-filarial parasitic helminths.
DiGAPDH revealed identities of 87.61%, 76.33% and 73.96% with the GAPDH from
Ascaris suum, S. mansoni and Fasciola hepatica (Fig. S1); and DiGAL revealed
identities of 88.13 and 82.73 with GAL from A. suum and Haemonchus contortus (Fig.
S2). Conserved lysine residues of DiGAL were also highlighted. In silico three-
dimensional modeling of the molecules predicted the 3D structures showing in the case
of DiGAPDH a homo-tetramer with 15 α-helices and 4 β-sheets (Fig. 2A). Molecular
modeling of DiGAL showing a monomer with the presence of 2 α-helices and 26 β-
sheets (Fig. 2B). Conserved lysine residues were highlighted and were visualized on the
outside of the proteins.

The D. immitis GAPDH and GAL cDNA were cloned into the expression vector
TOPO/pDEST. After induction of expression in E. coli, the hexahistidine-tagged
recombinant proteins were purified under denaturing conditions using nickel affinity
chromatography. The purified recombinant proteins rDiGAPDH and rDiGAL had
molecular weights of 38.6 kDa and 34.6 kDa in polyacrylamide gel.

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Figure 1. Alignment of the D. immitis GAPDH sequence (AFL46382) with the GAPDH from O. volvulus
(CAA70607), S. bovis (ACC78613), C. albicans (AAC49800), S. pyogenes (AAK33348), B. anthracis
(AIF58743) and T. vaginalis (AAA30325), which have previously been associated with plasminogen-
binding activities. The percentage of sequence identity between D. immitis sequence and the others is
indicated. The amino acids conserved in all the sequences are labeled with asterisks, and conservative and
semiconservative substitutions are labeled with two and one point, respectively. Conserved lysine
residues are shaded in yellow.

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Figure 2. Molecular modeling of D. immitis GAPDH (A) and GAL (B). The secondary structure of the
proteins were predicted with the Swiss-Model web server (http://swissmodel.expasy.org/) by analogy
with the X-ray crystallography available models. The three-dimensional models of the molecules were
visualized with the RasMol application v. 2.7.5.2. Conserved lysine residues of proteins were highlighted
as red balls.

3.2. rDiGAPDH and rDiGAL bind plasminogen

The binding level of plasminogen to rDiGAPDH and rDiGAL was assessed by
ELISA (Fig. 3). Analyses showed that both recombinant proteins bind plasminogen and
that this binding is directly proportional to the amount of plasminogen. Comparing the
results obtained by both recombinant proteins, rDiGAPDH showed higher plasminogen-
binding capacity than rDiGAL (Fig. 3). The negative control consisting of wells coated
only with BSA showed some non-specific binding activity, but always with values
significantly lowers than those obtained by rDiGAPDH and rDiGAL (p < 0.05). To
determine whether or not lysine residues are involved in binding, a competition
experiment including 50 mM ϵACA was carried out. In this case the binding was
inhibited about 90% in the case of rDiGAPDH and approximately 70% in the case of
rDiGAL, resulting in slightly higher optical densities than the negative control (Fig. 3).

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Figure 3. Plasminogen binding to 0.5 µg of rDiGAPDH (A) or rDiGAL (B) measured over a range of
plasminogen concentrations using a microtiter plate method. (■) Incubation with increasing
concentrations of plasminogen, 0–30 µg/ml. (▲) Competition assay with 50 mM εACA included during
plasminogen incubation. (●) Negative control consisted of wells coated only with BSA. Each point is the
mean of three replicates ± SD. The asterisk (*) designates significant (p < 0.05) differences.

3.3. rDiGAPDH and rDiGAL enhance the activation of plasminogen by tPA

In order to assess the ability of rDiGAPDH and rDiGAL to activate plasminogen
and generate plasmin on their own, the amidolytic activity of plasmin generated in the
presence or absence of tPA was measured. Negative controls replacing each
recombinant protein for BSA or tPA were also used. Fig. 4 shows the capacity of
rDiGAPDH and rDiGAL to stimulate plasmin generation by tPA obtaining optical
densities significantly higher than the negative controls (p < 0.05). Both proteins
obtained similar results and plasminogen-activation did not occur in the absence of tPA.
Furthermore this effect is inhibited by 50 mM ϵACA, indicating the involvement of
lysine residues in the process.

3.4. rDiGAPDH and rDiGAL enhance the expression of uPA and not of tPA in
canine endothelial cells
To study the possible effect of rDiGAPDH and rDiGAL on the expression of the
main activators of fibrinolysis (tPA and uPA), the parasitic proteins were employed to
stimulate CnAOEC in culture. Proteins from rDiGAPDH/rDiGAL-treated or untreated
vascular endothelial cell extracts were separated by SDS–PAGE and analyzed by
Western blotting using anti-tPA or anti-uPA antibodies. As shown in Fig. 5, both
proteins induced a significant increase in uPA protein expression after 24 h of

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stimulation (p < 0.01), being this increase higher in the case of rDiGAPDH stimulation.
None of the proteins led to significant differences in the protein expression of tPA.

Figure 4. Plasminogen activation and plasmin generation by rDiGAPDH (A) and rDiGAL (B). (□) 15 ng
of tPA was added to mixtures containing 2 µg of human plasminogen, 3 µg of D-Val-Leu-Lys 4-
nitroanilide dihydrochloride (Sigma) and 1 µg of each recombinant protein (or BSA as negative control)
in the presence or absence of 50 mM of εACA in a test volume of 100 µl. (■) No tPA was added to
reaction mixtures. Optical densities measured at 405 nm after 120 min of incubation. Each point is the
mean of three replicates ± SD. The asterisk (*) designates significant (p < 0.05) differences.

Figure 5. Effect of rDiGAPDH and rDiGAL on the expression of tPA and uPA in canine vascular
endothelial cells. Protein extracts from lysed rDiGAPDH or rDiGAL treated or untreated confluent cell
cultures were analyzed by Western blot for tPA and uPA. α-tubulin served as a protein control. Results
were expressed as the mean ± SD of at least 3 independent experiments. The asterisk (*) designates
significant (p<0.05) differences from control cells. (■) Stimulated endothelial cells with 1µg/ml of
rDiGAPDH or rDiGAL. (□) Non-treated control cells.

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3.5. Immunolocalization of DiGAPDH and DiGAL

In a first step, antisera against rDiGAPDH and rDiGAL were generated. The
reactivity and specificity of these antisera were tested in ELISA and Western blot prior
to their use in the immunolocalization studies. The antibody titers of these antisera were
higher than 1/500, with an OD of 1.12 and 1.07, respectively, while a negative serum
showed an OD of 0.12 and 0.16 at the same dilution. The anti-rDiGAPDH and anti-
rDiGAL antisera reacted strongly and specifically with the recombinant proteins and
with the native GAPDH and GAL proteins in the DiES extract in the Western blot
analyses. The negative sera showed no reactivity with any of the proteins tested (data
not shown).

The anatomical localization of DiGAPDH and DiGAL was carried out in

histological sections of D. immitis adult worms by immunofluorescence using the rabbit
polyclonal antisera previously generated. As shown in Fig. 6, all sections showed green
fluorescence throughout the soma of the parasite, as a result of the binding of
phalloidin-Alexa Fluor 488, actin ligand which serves as a positive control of the
technique. Sections incubated with the anti-rDiGAPDH or anti-rDiGAL antisera
showed, in addition, specific reactivity (in red) against the parasitic GAPDH and GAL,
respectively. Both proteins are located scattered throughout all the soma, being
especially abundant in the cuticle (reflected by an orange color in the overlay of
Phalloidin-Alexa Fluor 488 + Alexa Fluor 594 images). Sections incubated with a rabbit
negative serum showed no specific red fluorescence from recombinant proteins.

4. Discussion
Two recent in vitro studies demonstrated the participation of the ES and surface
antigens of D. immitis in the activation of the fibrinolytic system. In addition, some of
the antigens responsible for this enhancement were identified (González-Miguel et al.,
2012 and González-Miguel et al., 2013). Taking into account these previous data and
the importance of the anticlotting mechanisms for D. immitis, a parasite that survives for
years in the pulmonary arteries of its host, the objective of this work was to investigate
the participation of the D. immitis GAPDH and GAL in the fibrinolytic system
activation using recombinant forms of both proteins. This involves knowing whether or
not these proteins are able to bind plasminogen, stimulate plasmin generation, interact

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with the expression of the main fibrinolytic activators and express in an antigenic
compartment of the parasite in contact with the blood of the host.

Figure 6. Immunolocalization of DiGAPDH and DiGAL in sections from D. immitis adult worms.
Images of parasite sections incubated with phalloidin-Alexa Fluor 488 (in green, specific binding to
Actin) plus the negative or the anti-rDiGAPDH or anti-rDiGAL rabbit sera and an anti-rabbit IgG-Alexa
Fluor 594 (in red). Corresponding transmitted light images are also adressed. Magnification 4X.

Two peptide sequences of 339 and 280 amino acids were respectively obtained
by cloning and sequencing of the D. immitis GAPDH and GAL cDNAs. The subsequent
bioinformatics analysis have highlighted the high degree of evolutionary conservation
of these proteins, both in the structural characteristics of their 3D models, and in the
multiple sequence alignments carried out with homologous proteins from other
helminth parasite species. On the other hand, none of the two proteins showed structural

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motifs for their transport or expression on the cell surface (signal peptide,
transmembrane motifs or GPI anchors). However, both proteins have been identified by
immunoproteomic techniques in the ES and surface extracts of D. immitis (González-
Miguel et al., 2012 and González-Miguel et al., 2013). This may be related to
unconventional mechanisms of protein transport, as for example with the association of
these proteins with exosome-like secretion vesicles. This has been postulated as an
extracellular transport mechanism both for glycolytic enzymes from several groups of
parasites (Gómez-Arreaza et al., 2014) and for galectins (Nickel, 2003).

Both the rDiGAPDH and rDiGAL showed ability to bind plasminogen (higher in
the case of rDiGAPDH) and to stimulate plasmin generation in an in vitro system.
Plasminogen activation occurred only in the presence of the tPA, as observed previously
using the ES and surface antigens from D. immitis (González-Miguel et al.,
2012 and González-Miguel et al., 2013) or GAPDH from different bacteria, fungi or
parasites species (reviewed by Figuera et al., 2013). In addition, to our knowledge, this
is the first time that GAL is proposed as a physiological receptor of plasminogen.
Competition assays with the ϵ-ACA acid revealed the involvement of lysine residues
from both proteins in the binding of plasminogen. The interaction between plasminogen
and their receptors has been related to the presence of carboxyl-terminal lysine residues
(Plow et al., 1995). DiGAPDH alignment with homologous sequences from other
organisms that have been related to plasminogen-binding activities (Figuera et al., 2013)
shows that the carboxyl-terminal domain of these proteins are not highly conserved, and
that in some cases domains lack lysines (see Fig. 1). However, there are highly
conserved internal lysine residues in the amino acid sequences of DiGAPDH and
DiGAL. Therefore, it is possible that conserved internal lysine residues are involved in
the binding of plasminogen to these proteins as it has been postulated for the enolase of
Streptococcus pneumoniae (Ehinger et al., 2004). In addition, after viewing the spatial
location of the conserved internal lysine residues of the DiGAPDH and DiGAL in their
3D models, these residues seem to be located externally in these molecules, which
would facilitate the accessibility of plasminogen.

rDiGAPDH and rDiGAL did not cause a stimulation of basal tPA production in
canine endothelial cell cultures. However, it has been shown that whole D. immitis ES
antigens are able to produce an overexpression of this fibrinolytic activator in human

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endothelial cells in culture (González-Miguel et al., 2012). This suggests that other
molecules of D. immitis are responsible for this process. On the other hand, rDiGAPDH
and rDiGAL produced a significant stimulation of the basal uPA production in canine
endothelial cells in culture. To our knowledge, this is the first time that the relationship
between a parasitic antigen and the overproduction of an activator of fibrinolysis in an
in vitro system has been demonstrated. This could have particular relevance since uPA,
in addition to its role as activator of fibrinolysis, plays a key role in tissue remodeling
inducing proliferation and cell migration (Nicholl et al., 2005a), and high levels in its
expression are related to cardiovascular disease (Fuhrman, 2012).

In order to ascertain whether the ability of rDiGAPDH and rDiGAL to bind

plasminogen may have relevance in vivo, it is necessary that these proteins are
expressed and/or located in tissues of the parasite in close contact with the host blood
(Hawley et al., 2000). Immunofluorescence study indicates that DiGAPDH and DiGAL
are especially abundant on the surface of D. immitis, in addition to have an intracellular
localization which is expected for a glycolytic enzyme and a lectin. In the case of the
GAPDH, this is consistent with the results of a recent study in which it was identified as
one of the five most abundant proteins in the cuticle of D. immitis (Morchón et al.,
2014).

The ability of these molecules to enhance the activation of fibrinolysis could be

an important survival mechanism for D. immitis in its intravascular environment.
However, overstimulation of the plasminogen/plasmin system has been related to
cellular invasion and intra-organic migration in different pathogens (Jong et al.,
2003 and Bernal et al., 2004). In addition, the overproduction of plasmin has been
linked with the proliferation and migration of vascular cells and with the degradation of
extracellular matrices in humans (Nicholl et al., 2005b; Yang et al., 2005; Roth et al.,
2006 and Hayashi et al., 2009). This suggests a possible involvement of the over-
activation of the fibrinolytic system by D. immitis antigens in the long-term
development of the pathogenic mechanisms that occur during cardiopulmonary
dirofilariosis.

In conclusion, we have shown that D. immitis GAPDH and GAL are able to bind
plasminogen and enhance plasmin generation by tPA with the involvement of lysine

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residues. In addition, these proteins stimulate the expression of the fibrinolytic activator
uPA on canine endothelial cells in culture and they are expressed on the surface of the
worms. Therefore, DiGAPDH and DiGAL could be used by D. immitis to stimulate the
activation of the fibrinolytic system through the plasminogen binding to its surface or
excreted molecules, as a mechanism to avoid blood clot formation in its close
environment.

Acknowledgements
This research was supported by Agencia de Desarrollo Económico de Castilla y
León (cofinanced with FEDER funds), Junta de Castilla y León (grant SA090/A09),
Spain.

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Supplementary material

D.immitis MSKPKIGINGFGRIGRLVLRAAVEKNTVDVVAVNDPFINIDYMVYMFKYDSTHGRFKGNV 60
L.loa MSKPKIGINGFGRIGRLVLRAAVEKDTVDVVAVNDPFINIDYMVYMFKYDSTHGRFKGNV 60
W.bancrofti ----------FGRIGRLVLRAAVEKDTVDVVAVNDPFINIDYMVYMFKYDSTHGRFKGSV 50
B.malayi MSKPKVGINGFGRIGRLVLRAAVEKDTVDVVAVNDPFINIDYMVYMFKYDSTHGRFKGSV 60
O.volvulus MSKPKIGINGFGRIGRLVLRAAVEKDTVEVVAVNDPFINIDYMVYMFKYDSTHGRFKGHV 60
A.suum MSKPKVGINGFGRIGRLVLRAAVEKDTVEVVAVNDPFINIDYMVYMFKYDSTHGRFKGDV 60
S.mansoni MSRAKVGINGFGRIGRLVLRAAFLKNTVDVVSVNDPFIDLEYMVYMIKRDSTHGTFPGEV 60
F.hepatica MSKPKVGINGFGRIGRLVLRAAVERGTVEVVAVNDPFIDLNYMVYMFKYDSTHGTFKGDV 60
************. :.**:**:******:::*****:* ***** * * *

D.immitis SAEGGKLVVTN-GQTTHHISVHNSKDPAEIPWGVDGAEYVVESTGVFTTTEKASAHLKGG 119

L.loa SAEGGKLVVTN-GKRTHHISVHNSKDPAEIPWGVDGAEYVVESTGVFTTTEKASAHLKGG 119
W.bancrofti SAEGGKLVVTN-GKTTHHISVHNSKDPAEIPWGVDGAEYVVESTGVFTTTDKASAHLKGG 109
B.malayi SAEGGKLIVTN-GKTTHHISVHNSKDPAEIPWGVDGAEYVVESTGVFTTTDKASAHLKGG 119
O.volvulus SAEGGKLIVTN-GKTTHQIAVHNSKDPAEIPWGVEGAEYVVESTGVFTHTEKASAHLKGG 119
A.suum HAEGGKLVVSTPGKATHHITVHNSKDPSEIAWSADGAEYVVESTGVFTTVEKASAHLKGG 120
S.mansoni STENGKLKVNG-----KLISVHCERDPANIPWDKDGAEYVVESTGVFTTIDKAQAHIKNN 115
F.hepatica SVHDKKLCING-----MRISVYNERSPETIPWDKDSAEYVVESTGVFTTMEKAQAHLKNN 115
... ** :. *:*: .:.* *.*. :.************ :**.**:*..

D.immitis -AKKVIISAPSADAPMFVMGVNNETYDKANNHIISNASCTTNCLAPLAKVIHDKFGIIEG 178

L.loa -AKKVVISAPSADAPMFVMGVNNDKYDKANNHIISNASCTTNCLAPLAKVIHDKFGIIEG 178
W.bancrofti -AKKVIISAPSADAPMFVMGVNNDKYDKANNHIISNASCTTNCLAPLAKVIHDKFGIIEG 168
B.malayi -AKKVIISAPSADAPMFVMGVNNDMYDKANNHIISNASCTTNCLAPLAKVIHDKFGIIEG 178
O.volvulus -AKKVIISAPSADAPMFVMGVNNDKYDKANNHIISNASCTTNCLAPLAKVIHDKFGIIEG 178
A.suum -AKKVIISAPSADAPMFVMGVNEDKYDGANNHVISNASCTTNCLAPLAKVINDKFGIVEG 179
S.mansoni RAKKVIISAPSADAPMFVVGVNENSYEKSMS-VVSNASCTTNCLAPLAKVIHDKFEIVEG 174
F.hepatica RAKKVIISAPSPDAPMFVVGVNHDKYDKCMN-VVSNASCTTNCLSPLAKVLHENFGIEEG 174
****:*****.******:***.: *: . . ::**********:*****::::* * **

D.immitis LMTTVHATTATQKTVDGPSGKLWRDGRGAGQNIIPASTGAAKAVGKVIPDLNGKLTGMAF 238

L.loa LMTTVHATTATQKTVDGPSGKLWRDGRGAAQNIIPASTGAAKAVGKVIPDLNGKLTGMAF 238
W.bancrofti LMTTVHATTATQKTVDGPSGKLWRDGRGAGQNIIPASTGAAKAVGKVIPDLNGKLTGMAF 228
B.malayi LMTTVHATTATQKTVDGPSGKLWRDGRGAGQNIIPASTGAAKAVGKVIPGLNGKLTGMAN 238
O.volvulus LMTTVHATTATQKTVDGPSGKLWRDGRGAGQNIIPASTGAAKAVGKVIPDLNGKLTGMAS 238
A.suum LMTTVHATTATQKTVDGPSGKQWRDGRGAGQNIIPASTGAAKAVGKVIPQLNGKLTGMAF 239
S.mansoni LMTTVHSFTATQKVVDGPSSKLWRDGRGAMQNIIPASTGAAKAVGKVIPALNGKLTGMAF 234
F.hepatica LMTTIHSYTATQRVVDGPSVKLWRDGRGAAQNIIPASTGAAKAVGKVIPDLNGKLTGMAF 234
****:*: ****:.***** * ******* ******************* *********

D.immitis RVPTPDVSVVDLTCRLQKGATMDEIKAAVKEAAAGPMKGILEYTEDQVVSSDFIGDAHSS 298

L.loa RVPTPDVSVVDLTCRLQKGATMDEIKAAVKEAANGPMKGILDYTEDQVVSTDFVGDTHSS 298
W.bancrofti RVPTPDVS---------------------------------------------------- 236
B.malayi RVPTPDVSVVDLTCRLQKGATMDEIKAAVKEAANGPMKGILEYTEDQVVSTDFTGDTHSS 298
O.volvulus RVPTPDVSVVDLTCRLQKGASMDEIKAAVKEAAAGPMKGILEYTEDQVVSSDFVGDPHSS 298
A.suum RVPTPDVSVVDLTARLEKAATMDEIKAAIKEAAAGPMKGILGYTEDSVVSTDFVGDSHSS 299
S.mansoni RVPTPDVSVVDLTCRLGKGASYEEIKAAVKAAASGPLKGILEYTEDEVVSSDFVGSTSSS 294
F.hepatica RVPTPDVSVVDLTCKLSRPTTYDQIKAALKAAAEGPMKGILEYTEDQVVSMDFRGCSASS 294
********
Identity (%)
D.immitis IFDALACISLNPNFVKLIAWYDNEYGYSNRVVDLISYIASR--- 339 -
L.loa IFDALACISLNPNFVKLIAWYDNEYGYSNRVVDLISYIASR--- 339 97.46
W.bancrofti -------------------------------------------- 96.46
B.malayi IFDSLACISLNPNFVKLIAWYDNEYGYSNRVVDLISYIASR--- 339 95.58
O.volvulus IFDALACISLNPNFVKLIAWYDNEYGYSNRVVDLISYNASK--- 339 94.99
A.suum IFDANACISLNPHFVKLISWYDNEFGYSCRVVDLIAYISKH--- 340 87.61
S.mansoni IFDAKAGISLNNNFVKLVSWYDNEFGYSCRVVDLITHMHKVDHA 338 76.33
F.hepatica IFDAGAGLALNDTFVKIVAWYDNEYGYSCRVIDLINHMHTIDHL 338 73.96

Figure S1. Alignment of the D. immitis GAPDH sequence (AFL46382) with the GAPDH from L. loa
(EJD75047), W. bancrofti (EJW73626), B. malayi (P48812), O. volvulus (O01360), A. suum
(BAB68543), S. mansoni (P20287) and F. hepatica (AIE44418). The percentage of sequence identity
between D. immitis sequence and the others is indicated. The amino acids conserved in all the sequences
are labelled with asterisks, and conservative and semiconservative substitutions are labelled with two and
one point, respectively.

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D.immitis MHHNEYETNYPVPYRSKLTESFEPGQTLLVKGKTAEDSVRFTINLHNTSADFSGNDVPLH 60
B.malayi -MPNEYETIYPVPYRSKLTESFEPGQTLLVKGKTAEDSVRFTINLHNTSADFSGNDVPLH 59
O.volvulus -MTNEYETNYPVPYRSKLTESFEPGQTLLVKGKTAEDSVRFTINLHNTSADFSGNDVPLH 59
L.loa -MANEYETNYPVPYRSKLTETFDPGQTLLVKGKTAEDSVRFTVNLHNTSADFSGNDVPLH 59
W.bancrofti ------------------------------------------------------------
A.suum ---MATETSYPVPYRSKLTEPFEPGQTLIIKGKTAEDSVRFTINLHNTSADFSGNDVPLH 57
H.contortus ---MAFETNYPIPYRSKLTEPFEPGQTLTVKGKTGEDSVRFTINLHNSSADFSGNDVPLH 57

D.immitis ISVRFDEGKIVFNTFSKGEWGKEERKSNPYKKGDDIDIRIRAHDSKYTIYVDQKEVKEYE 120

B.malayi ISVRFDEGKIVFNTFSKGEWGKEERKSNPYKKGDDIDIRIRAHDSKYTIYVEQKEVKEYE 119
O.volvulus ISVRFDEGKIVFNTFSKGEWGKEERKSNPYKKGDDIDIRIRAHDSKYTIYVDQKEVKEYE 119
L.loa ISVRFDEGKIVFNTFSKGEWGKEERKSNPYKKGDDIDIRIRAHDSKYTIYVDQKEVKEYE 119
W.bancrofti ---------IVFNTFSKGEWGKEERKSNPYKKGDDIDIRIRAHDSKYTIYVDQKEVKEYE 51
A.suum ISVRFDEGKIVFNTFSKGEWGKEERKSNPYKKGDDIDIRIRAHDSKFSISVDQKEVKEYE 117
H.contortus VSVRFDEGKIVCNSFAKGEWGKEERKSNPYKKGDDIDIRIRAHDSKFQIFVDQKELKEYE 117
** *:*:******************************: * *:***:****

D.immitis HRVPLSS-TRFSIDGDILVTYIHWGGKYYPVPYESGLSGEGLVPGKSLLIFATPEKKGKR 179

B.malayi HRVPLSSVTHFSIDGDVLVTYIHWGGKYYPVPYESGLSGESLMPGKSLLIFATPEKKGKR 179
O.volvulus HRVPLSAVTHFSIDGDVLVTYIHWGGKYYPVPYESGLSGEGLVPGKSLLIFATPEKKGKR 179
L.loa HRVPLSSVTHFSIDGDVLVTYIHWGGKYYPVPYESGLSGEGLVPGKSLLIFATPEKKGKR 179
W.bancrofti HRVPLSSVTHFSIDGDVLITYIHWGGKYYPVPYESGLSGDGLVPGKSLLIFATPEKKGKR 111
A.suum HRVPLSSVTHFSVDGDILITYIHWGGKYYPVPYESGLAGDGLAPGKSLLIFATPEKKGKR 177
H.contortus HRLPLSSVTHFSIDGDVLITYIHWGGKYYPVPYESGLAGEGLAPGKSIFLYGMPEKKGKR 177
**:***: *:**:***:*:******************:*:.* ****::::. *******

D.immitis FHINLLKKNGDIALHFSPRFDEKAIVRNSLIAGEWGNEEREGKMILEKGIGFDLEIKNEE 239

B.malayi FHINLLKKNGDIALHFNPRFDEKAIVRNSLIAGEWGNEEREGKMILEKGIGFDLEIKNEE 239
O.volvulus FHINLLKKNGDIALHFNPRFDEKAIVRNSLIAGEWGNEEREGKMILEKGIGFDLEIKNEE 239
L.loa FHINLLKKNGDIALHFNPRFDEKAIVRNSLIAGEWGNEEREGKMILEKGIGFDLEIKNEE 239
W.bancrofti FHINLLKKNGDIALHFNPRFDEKV------------------------------------ 135
A.suum FHINLLKKNGDIALHFNPRFDEKAIVRNSLISGEWGNEEREGKNPLEKGIGCDLEFRNEE 237
H.contortus FHINLLKKNGDIALHFNPRFDEKAVVRNSLISNEWGNEEREGKMPFEKAVGFDLEIKNEE 237
****************.******.
Identity (%)
D.immitis YAFQIFINSERYATYAHRLDPHEINGLQIGGDLEVSGIQMH 280 -
B.malayi YAFQIFINSERYATYAHRLDPHEINGLQIGGDLKVSGIQMH 280 96.79
O.volvulus YAFQIFINGERYATYAHRLDPREINGLQIGGDLEVSGIQMH 280 96.07
L.loa YAFQIFINSERYATYAHRVDPHEINGLQIGGDLEVSGIQMR 280 96.07
W.bancrofti ----------------------------------------- 94.81
A.suum YAFQIYVDGERFATYAHRLDPHDINGLQIGGDVEVTGIQMV 278 88.13
H.contortus YAFQIMVNGERFASYAHRLEPHELNGLQIGGDVEITGIQLH 278 82.73

Figure S2. Alignment of the D. immitis galectin sequence (AAF37720) with the galectins from B. malayi
(Q9NGY0), O. volvulus (Q25597), L. loa (EFO26688), W. bancrofti (EJW74751), A. suum (F1KZZ8)
and H. contortus (AAD11972). The percentage of sequence identity between D. immitis sequence and the
others is indicated. The amino acids conserved in all the sequences are labelled with asterisks, and
conservative and semiconservative substitutions are labelled with two and one point, respectively.
Conserved lysine residues are shaded in yellow.

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“Can the activation of plasminogen/plasmin system of the host by metabolic
products of Dirofilaria immitis participate in heartworm disease endarteritis?”
 CUARTO CAPÍTULO

¿Puede la activación del sistema plasminógeno/plasmina del hospedador por los

productos metabólicos de Dirofilaria immitis participar en la endarteritis de la
dirofilariosis cardiopulmonar?

Javier González-Miguel, Rodrigo Morchón, Elena Carretón, José Alberto Montoya-

Alonso, Fernando Simón

Parasites & Vectors (2015) 8:194.

Factor de impacto (2014): 3,25.

Resumen

Antecedentes: La endarteritis proliferativa es uno de los mecanismos

patológicos clave en la dirofilariosis cardiopulmonar, una parasitosis cosmopolita
causada por Dirofilaria immitis y que afecta a perros y gatos de todo el mundo. Se ha
demostrado que los antígenos excretores/secretores de los vermes adultos de D. immitis
(DiES) fijan plasminógeno (PLG) y activan la fibrinolisis, lo que puede suponer un
mecanismo de supervivencia para el parásito en su entorno intravascular. Sin embargo,
la sobreproducción de plasmina (producto final de la ruta) ha sido relacionada con
procesos patológicos similares a los descritos en la endarteritis proliferativa. El objetivo
de este trabajo es relacionar la aparición de esta condición patológica con la activación
del sistema PLG/plasmina del hospedador por DiES.

Métodos: La proliferación celular a través de la técnica del cristal violeta, la

migración celular mediante un ensayo de cicatrización de heridas y la destrucción de la
matriz extracelular mediante la medición de la degradación del colágeno y los niveles
de metaloproteasas de matriz, fueron estudiados en un modelo in vitro utilizando células
endoteliales y musculares lisas vasculares de perro. Estas células fueron tratadas con
una mezcla de DiES + PLG. Células sin tratar, células estimuladas solamente con DiES
o con PLG, o con una mezcla de DiES + PLG + εACA (un inhibidor de la conversión
PLG-plasmina) fueron empleadas como controles. Además, el efecto de DiES en la
expresión de los activadores fibrinolíticos tPA y uPA, el inhibidor PAI-1 y el receptor
de PLG Anexina A2 fue analizado en ambos tipos de cultivos por western blot.

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Resultados: La plasmina generada por la unión de DiES + PLG produjo un

aumento significativo en la proliferación y migración celular de las células endoteliales
y del músculo liso, así como un aumento en la destrucción de la matriz extracelular
basada en una degradación mayor del colágeno de tipo I y en un nivel incrementado de
la metaloproteasa de matriz-2. DiES también induce un aumento en la expresión de tPA
y uPA en los cultivos de células endoteliales, así como una disminución en la expresión
de PAI-1 en ambos tipos de células.

Conclusiones: Nuestro estudio muestra una interrelación entre la plasmina

causada por la activación de la fibrinolisis por los productos metabólicos de D. immitis
y la aparición de procesos patológicos similares a los descritos en la aparición de la
endarteritis proliferativa en la dirofilariosis cardiopulmonar.

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Abstract
Background
Proliferative endarteritis is one of the key pathological mechanisms of
cardiopulmonary dirofilariosis, a cosmopolitan parasitosis caused by Dirofilaria immitis
affecting dogs and cats around the world. It has been shown that the excretory/secretory
antigens from D. immitis adult worms (DiES) bind plasminogen (PLG) and activate
fibrinolysis, which can lead to a survival mechanism for the parasite in its intravascular
environment. However, overproduction of plasmin (final product of the route) has been
related to pathological processes similar to those described in proliferative endarteritis.
The aim of this study is to relate the appearance of this pathological condition with the
activation of the PLG/plasmin system of the host by DiES.

Methods
Cell proliferation through the crystal violet technique, cell migration by wound
healing assay and degradation of the extracellular matrix by measuring collagen
degradation and levels of matrix metalloproteinases were studied in an “in vitro” model
using canine vascular endothelial and smooth muscle cells. These cells were treated with
a mixture of DiES + PLG. Untreated cells, cells only stimulated with DiES or with PLG,
or with a mixture of DiES + PLG + εACA (an inhibitor of the PLG-plasmin conversion)
were employed as controls. In addition, the effect of DiES on the expression of the
fibrinolytic activators tPA and uPA, the inhibitor PAI-1 and the PLG receptor Annexin
A2 was analyzed in both types of cultures by western blot.

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Results
Plasmin generated by DiES + PLG binding produced a significant increase in the
cell proliferation and migration of the endothelial and smooth muscle cells, as well as an
increase in the destruction of the extracellular matrix based on a further degradation of
Type I Collagen and an increased level of matrix metalloproteinase-2. DiES also induce
an increase in the expression of tPA and uPA in endothelial cells in culture, as well as a
decrease in the expression of PAI-1 in both types of cells.

Conclusions
Our study reports an interrelationship between plasmin caused by fibrinolysis
activation by metabolic products of D. immitis and the appearance of pathological events
similar to those described in the emergence of proliferative endarteritis in the
cardiopulmonary dirofilariosis.

Key words: Dirofilaria immitis; excretory/secretory antigens; fibrinolysis; plasmin;

endothelial cells; smooth muscle cells; proliferative endarteritis.

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1. Background
Dirofilaria immitis is the filaroid nematode that causes the canine and feline
cardiopulmonary dirofilariosis, a vector-borne parasitosis with cosmopolitan distribution.
D. immitis is also responsible for the human pulmonary dirofilariosis, a clinical entity
characterized by the formation of benign lung nodules that may be confused with
carcinomas in radiology [1].

The dog acts as definitive host and reservoir of the parasite. The adult worms
lodge in its pulmonary artery and right ventricle of the heart causing a chronic
inflammatory pathology at vascular level [2]. One of the key factors of the disease is the
appearance of proliferative endarteritis, which has resulted in the formation of
intravascular microvilli. This process has previously been described as being
accompanied by an increase and migration of the arterial wall cells [3-6], as well as the
destruction of the extracellular matrix (ECM) [7]. These changes cause disorganization
of the endothelium and reduction of the vascular lumen in the pulmonary arteries, with
the consequent extension of pathology to the pulmonary parenchyma [8]. On the other
hand, the simultaneous death of groups of adult worms can trigger an acute disease
characterized by the exacerbation of the inflammatory reactions and the emergence of
serious thromboembolic events threatening the life of the affected hosts [9]. However, D.
immitis possesses the ability to regulate these pathological mechanisms and survive for
long periods (over 7 years) in their intravascular environment. Recently, it has been
shown that both excretory/secretory and surface-associated antigens of D. immitis interact
with the fibrinolytic system of the host binding PLG and generating plasmin [10,11].

PLG is a glycoprotein predominantly released by the liver into the blood

circulation. After its activation PLG becomes a serine protease (plasmin), whose main
target are the fibrin clots. Under physiological conditions, this process is strictly regulated
at the vascular level for the complex formed by receptors that bind PLG through
carboxyterminal lysine residues (Annexin A2, among others) and activators [tissue
plasminogen activator (tPA) and urokinase-type plasminogen activator (uPA)], whose
activity is inhibited primarily by the plasminogen activator inhibitor-1 (PAI-1) [12,13].

The activation of the fibrinolytic system by D. immitis antigens and the

consequent maintenance of haemostasis, a priori beneficial for both the parasite and host,

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could have pathological consequences. An over-activation of the PLG/plasmin system

has been related to cell invasion and intra-organic migration of different pathogens
[14,15]. In addition, in human cardiovascular research the overproduction of plasmin has
also been linked with the proliferation and migration of human vascular cells and with
the degradation of extracitoplasmatic matrices [16-19]. These mechanisms have
similarities with those that cause the formation of microvilli in the cardiopulmonary
dirofilariosis, although their molecular aspects have not been conveniently studied to date
in this parasitosis.

The objective of this work is to demonstrate that the overproduction of plasmin

by the antigens of D. immitis may relate to the mechanisms described in the formation of
microvilli at the vascular level in cardiopulmonary dirofilariosis. For this purpose, cell
proliferation and migration, degradation of the ECM and expression of some components
of the fibrinolytic system were studied in canine vascular endothelial and smooth muscle
cells in culture stimulated with the parasitic antigens and PLG.

2. Methods
2.1. Cell culture
Canine endothelial cells (CnAOEC) and canine smooth muscle cells (CnAOSMC)
from Cell Applications, INC were respectively grown in canine endothelial and canine
smooth muscle cell growth mediums (Cell Applications, INC). CnAOEC plates were
precoated with an attachment factor solution (Cell Applications, INC). Cells were
cultured at 37°C in a humidified atmosphere in the presence of 5% carbon dioxide and
95% air. Medium was changed every 3 days. Expansion was carried out by trypsinizing
the cells, (Trypsin/EDTA, Cell Applications, INC), and re-plating them when the
proliferating cells had reached a sufficient density. Passaging was performed at ratios of
1:6 (CnAOEC) or 1:3 (CnAOSMC). Cell counts were performed using the equipment
Countess® Automated Cell Counter (Invitrogen) following the manufacturer’s
instructions.

2.2. Reagents and stimulation of CnAOEC and CnAOSMC

DiES were prepared as previously described [12] with minor modifications and
stored at −80°C. In brief, live worms (25) obtained from a naturally infected dog were
washed in sterile phosphate-buffered saline solution (PBS) pH 7.2 and incubated for 24 h

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in 50 ml of Eagle’s minimum essential medium (EMEM) supplemented with 50 U/ml

penicillin and 50 μg/ml streptomycin at 37°C. A cocktail of protease inhibitors was added
to the medium following the methodology described by Maizels et al. [20]. The medium
was dialyzed against water for 24 h and filtered through an Amicon YC05 membrane
(Millipore). The protein concentration of DiES was measured by DC protein assay
commercial kit (Bio-Rad). DiES extract was tested for the presence of endotoxin
contamination using a quantitative Limulus amebocyte lysate test (BioWhittaker). The
endotoxin quantity was under the sensitivity level of cell stimulation (<0.4 U/mg protein).

For stimulations, CnAOEC and CnAOSMC were grown for 4 days to obtain
confluent cultures and were treated with 1 μg/ml of DiES [21], 10 μg/ml of PLG (Acris
Antibodies) [17] or with a mixture of both treatments. Untreated cells and cells treated
with DiES + PLG + 50 mM of the lysine analogue ε-aminocaproic acid (εACA) as an
inhibitor of PLG activation were used as control cells under the same conditions.

2.3. Cell proliferation assay

Cells were plated on 24-well plates to a density of 104 CnAOEC/well or 1.5 × 104
CnAOSMC/well and allowed to attach overnight. After stimulation cell proliferation was
analyzed by crystal violet nuclei staining over 10 days to determine the number of viable
cells, as previously described [22]. Briefly, every two days, cells were rinsed with PBS,
fixed with 4% formaldehyde for 10 min and stained with 0.2% crystal violet for 30 min
at room temperature. After several rinses with PBS, the cells were allowed to dry
overnight and crystal violet bound to cells was extracted by incubation with 2 ml/well of
10% acetic acid. The absorbance of the samples was then measured at 595 nm and
transformed to “number of viable cells” using a curve that correlated absorbance and
number of endothelial or smooth muscle cells previously determined.

2.4. Cell migration assay

Cell migration was assessed by quantifying the percentage of wound closure in
the wound-healing assay [23]. In brief, CnAOEC and CnAOSMC were cultured in 60 mm
plates (3 × 105 cells/plate) and allowed to attach overnight before wound creation.
Confluent monolayers were wounded using a sterile pipette tip and the medium was
exchanged for fresh medium before cell stimulation to remove cellular debris. The extent
of wound closure of the treated and control cells was monitored over a time course by

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microscopy and determined along 8 hours by calculating the migrated distance/total

wound distance.

2.5. Collagen degradation assay

The concentration of collagen in the supernatants was analyzed by ELISA.
Treated and control cells were cultured with medium for 48 hours. Then the culture
supernatants were collected, filtered and added to multi-well plates (Costar). After
incubation overnight at 4°C, wells were blocked with 1% BSA in PBS and incubated with
a rabbit anti-Type I Collagen antibody (1:2500) (Acris Antibodies), followed by a
peroxidase-conjugated goat anti-rabbit IgG (Sigma) at 1:500 dilution. All incubations
were performed for 1 h at 37°C and between each step washed three times with PBS wash
buffer (PBS containing 0.05% Tween20). Ortho-phenylene-diamine was used as a
chromogen. Optical densities (OD) were measured at 492 nm in an Easy Reader (Bio-
Rad).

2.6. Matrix metalloproteinases (MMPs) levels assays

The levels of the MMP-2 and MMP-9 metalloproteinases in the culture media of
the different experimental groups were analyzed by gelatin zymography according to the
methodology described by Marangoni et al. [24]. Media samples employed in the
collagen degradation assays were electrophoresed on a 10% polyacrylamide gel
copolymerized with 1% gelatin (Sigma) together with a MMP marker (Cosmobio) as a
positive control. The gels were washed for 1 hour in 2.5% Triton X-100 and incubated
for 20 hours at 37°C in agitation in an enzymatic activation buffer ph 7.5 (50 mM Tris;
200 mM NaCl; 5 mM CaCl2; 0,2% Brij-35). The gels were finally stained with
Coomassie blue. The positivity was assessed as appearance of clear bands on a dark
background with molecular weights of 72 kDa (MMP-2) and 92 kDa (MMP-9). The
levels of the MMPs were calculated after measuring the density of the existing bands,
which is directly proportional to the amount of gelatin degraded into the gel.

2.7. Cell lysates and Western blot analyses

Western blot analyses were performed as previously described [21] with slight
modifications. CnAOEC and CnAOSMC previously treated with 1 μg/ml of DiES for
24 h were lysed in ice-cold lysis buffer (20 mM Tris–HCl (pH 7.5), 140 mM NaCl, 10 mM
ethylendiaminetetraacetic acid, 10% glycerol, 1% Igepal CA-630, aprotinin, pepstatin,

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and leupeptin at 1 μg/ml each, 1 mM phenylmethylsulfonyl fluoride, and 1 mM sodium

orthovanadate). Non-stimulated cells were used as controls under the same conditions.
Protein samples (10 μg) were separated by SDS-PAGE under reducing conditions and
blotted onto polyvinylidine difluoride membranes. Membranes were blocked before
incubation with primary antibodies: anti-tPA, anti-uPA, anti-Annexin A2 and anti-PAI-1
(Santa Cruz Biotechnology Inc) according to the manufacturer’s recommendations. After
incubation with HRP-conjugated secondary antibodies, bands were visualized by a
luminol-based detection system with p-iodophenol enhancement. Anti-α-tubulin antibody
(Oncogene Research Products) was used to confirm loading of comparable amount of
protein in each lane. Protein expression was quantified by densitometry using Scion
Image Software (Scion).

2.8. Statistical analysis

Cell proliferation and migration, collagen degradation and MMP levels
significance measurements (comparisons between groups) were performed by ANOVA
and corrected for repeated measurements when appropriate. If ANOVA revealed overall
significant differences, individual means were evaluated post hoc using Bonferroni’s
procedure. The results from the Western blots for the tPA, uPA, annexin A2 and PAI-1
expression were analyzed with the Student’s t-test. All the results were expressed as the
mean ± SD of three experiments performed with duplicates. In all experiments, a
significant difference was defined as a p-value of <0.05 for a confidence level of 95%.

3. Results
3.1. DiES produce proliferation of CnAOEC and CnAOSMC via PLG/plasmin
system
The effect of DiES and PLG on the proliferation of endothelial and smooth muscle
cells was quantified using the crystal violet technique in a period of 10 days (Figure 1).
Both cultures showed typical curves of cell growth in all experimental groups with a
progressive growth between days 0 and 8 post-treatment, experiencing cell death and an
evident decrease of viable cells between days 8 and 10 post-treatment. Crystal violet
staining showed an increase significantly greater in the number of viable cells in
CnAOEC and CnAOSMC in culture stimulated with DiES + PLG than that showed by
other experimental groups on day 8 post-treatment (p < 0.05), indicating that this
treatment stimulates the proliferation of CnAOEC and CnAOSMC.

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Figure 1. Cell proliferation assay performed by the crystal violet technique measuring cell viability over a
10 days period. The experiment was carried out in canine endothelial (A) and smooth muscle cells (B)
untreated ( ) or treated with 1 µg/ml of DiES + 10 µg/ml of PLG ( ), 1 µg/ml of DiES ( ), 10 µg/ml of
PLG ( ), or with 1 µg/ml of DiES + 10 µg/ml of PLG + 50 mM of the εACA ( ).Results are expressed
as number of viable cells (x 10,000). Each point is the mean ± SD from three independent experiments. The
asterisk (*) designates significant (p < 0.05) differences between DiES + PLG treatment and control groups.

3.2. DiES produce migration of CnAOEC and CnAOSMC via PLG/plasmin system
Wound Healing assay was performed to assess migration of endothelial
(Figure 2A) and smooth muscle cells (Figure 2B). The quantification was carried out by
measuring the distance of migration in comparison with negative control (untreated cells)
up to 8 hours post-treatment. In both CnAOEC and CnAOSMC cultures a significant
increase of cell migration after stimulation with DiES + PLG with respect to the other
experimental groups (p < 0.05) occurred, this increase was most pronounced in cultured
endothelial cells.

3.3. DiES produce ECM degradation of CnAOEC and CnAOSMC via PLG/plasmin
system
To examine ECM degradation, Type I Collagen in the culture supernatant of
treated and untreated CnAOEC and CnAOSMC were measured by ELISA (Figure 3). A
lower concentration of Type I Collagen and therefore a further degradation of the secreted
collagen by the cells was observed in the CnAOEC and CnAOSMC stimulated with
DiES + PLG than that obtained by the control cells (p < 0.05). In addition, the same
culture media from treated and untreated cells was analyzed with gelatin zymography for
MMP-2 and MMP-9 levels (Figure 4). Density of the bands was measured by the Quantity
One Software (Bio-Rad). The results show a significantly higher level of MMP-2 in the

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CnAOEC and CnAOSMC treated with DiES + PLG than that obtained in the control cells
(p < 0.05). No significant differences in the MMP-9 levels were observed (Figure 4).

Figure 2. Cell migration by Wound-Healing assay. Confluent cell cultures were wounded post-treatment
and migration distances were measured at 8 hours. The experiment was carried out in canine endothelial
(A) and smooth muscle cells (B) untreated ( ) or treated with 1 µg/ml of DiES + 10 µg/ml of PLG ( ), 1
µg/ml of DiES ( ), 10 µg/ml of PLG ( ), or with 1 µg/ml of DiES + 10 µg/ml of PLG + 50 mM of the
εACA ( ).The results were expressed as percentage of the migration ability of the negative control cells
(100%). Each point is the mean ± SD from three independent experiments. The asterisk (*) designates
significant (p < 0.05) differences between DiES + PLG treatment and control groups.

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Figure 3. Type I Collagen degradation assay measured in culture supernatants from canine endothelial (A)
and smooth muscle cells (B) untreated ( ) or treated with 1 µg/ml of DiES + 10 µg/ml of PLG ( ), 1
µg/ml of DiES ( ), 10 µg/ml of PLG ( ), or with 1 µg/ml of DiES + 10 µg/ml of PLG + 50 mM of the
εACA ( ).The results were expressed as percentage of the Type I Collagen concentration in the culture
supernatant from negative control cells (100%). Each point is the mean ± SD from three independent
experiments. The asterisk (*) designates significant (p < 0.05) differences between DiES + PLG treatment
and control groups.

Figure 4. Representative zymography of culture supernatants from canine endothelial (A) and smooth
muscle cells (B) untreated ( ) or treated with 1 µg/ml of DiES + 10 µg/ml of PLG ( ), 1 µg/ml of DiES
( ), 10 µg/ml of PLG ( ), or with 1 µg/ml of DiES + 10 µg/ml of PLG + 50 mM of the εACA ( ). Note
the gelatinolytic bands associated with MMP-2 (72 KDa) and MMP-9 (92 KDa) levels. The results are
expressed as percentage of the MMPs levels in the culture supernatant from negative control cells (100%).
Each point is the mean ± SD from three independent experiments. The asterisk (*) designates significant
(p < 0.05) differences between DiES + PLG treatment and control groups.

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3.4. Effect of DiES on the fibrinolytic system components (tPA, uPA, Annexin II and
PAI-1) expression in CnAOEC and CnAOSMC
To determine the effect of DiES on some of the main components of the
fibrinolytic system, proteins from DiES-treated or untreated CnAOEC and CnAOSMC
extracts were separated by SDS–PAGE and analyzed by Western blotting using anti-tPA,
anti-uPA, anti-Annexin A2 and anti-PAI-1 antibodies. DiES induced a marked increase
in the expression of the main fibrinolytic activators tPA and uPA in cultured endothelial
cells (p<0.05) (Figure 5A and B), as well as a slight decrease in the expression of the
main fibrinolytic inhibitor PAI-1 in both types of cultures (p<0.05) (Figure 5D).
Significant differences in the expression of tPA and uPA in CnAOSMC (Figure 5A and
B) and in the expression of Annexin A2 in both cell types between DiES-treated or
untreated cultures were not found (Figure 5C).

Figure 5. Effect of DiES on the expression of tPA (A), uPA (B), annexin A2 (C) and PAI-1 (D) in canine
vascular endothelial (EC) and smooth muscle cells (SMC). Protein extracts from lysed DiES untreated or
treated confluent cell cultures were analyzed by Western blot for tPA, uPA annexin A2 and PAI-1. α-tubulin
served as a protein control. Results were expressed as the mean ± SD of at least 3 independent experiments.
The asterisk (*) designates significant (p < 0.05) differences from control cells. (■) Non-treated control
cells. (■) Stimulated endothelial or smooth muscle cells with 1µg/ml of DiES.

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4. Discussion
Plasmin is the serine protease responsible for initiating the fibrinolytic process
through the lysis of the fibrin clots. Apart from its pivotal role in the maintenance of
haemostasis, plasmin possesses exceptionally broad specificity for target substrates
playing important physiological and pathological roles in tissue remodeling, cell
migration, wound healing, angiogenesis, inflammation and degradation of ECM [25]. In
addition, plasmin is also upregulated in chronic inflammatory diseases, including
atherosclerosis and arthritis [26]. The cardiopulmonary dirofilariosis is a chronic disease
with an important inflammatory component at the vascular level in which the interaction
between the excretory/secretory and surface antigens of its aetiologic agent (D. immitis)
and the overproduction of plasmin has been recently shown [10,11]. The objective of this
work was to establish a relationship between this interaction and the mechanisms
responsible for the formation of intravascular microvilli in the cardiopulmonary
dirofilariosis. Similar lesions have been previously explained through mechanical damage
caused by the sole presence of the adult worms in the pulmonary arteries [27]. However,
the pathogenic mechanisms causing the proliferative endarteritis at the molecular level
have not been described in cardiopulmonary dirofilariosis.

Firstly, we have developed an “in vitro” model of canine endothelial and smooth
muscle cells to study the relationship between the excretory/secretory antigens of the
parasite and the different cell types of the canine arterial wall. Our data show that
stimulation of both types of cultures with DiES + PLG cause the proliferation and
migration of CnAOEC and CnAOSMC. Cells treated separately with DiES or PLG did
not generate growth curves or migration distances significantly different from non-treated
cells. In this sense, Morchón et al. [22] observed that the stimulation of human endothelial
cells in culture with somatic antigens of D. immitis (DiSA) did not alter cell proliferation.
Finally, the inhibition of both effects by including the 50 mM εACA in the stimulations
demonstrates the participation of plasmin generated by DiES + PLG binding on the
proliferation and migration of CnAOEC and CnAOSMC.

Two facts reveal the involvement of plasmin generated by DiES + PLG binding

in the degradation of the ECM. First, this treatment for 48 h causes a significant increase
in the degradation of the Type I Collagen in the culture media of CnAOEC and
CnAOSMC. This molecule represents the main component of the ECM of elastic arteries.

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Its alteration has been associated with vascular disease and its degradation products with
the proliferation and migration of smooth muscle cells in remodelling arteries [28]. These
results are consistent with that observed in vivo by Wang et al. [7] who reported a
significantly lower amount of collagen in heartworm-infected dogs than in clinically
normal dogs. The degradation of the ECM is also related to the increase in the levels of
MMPs. Our results show a significantly higher level of MMP-2 or gelatinase A in the
culture media of CnAOEC and CnAOSMC treated with DiES + PLG for 48 h, than that
obtained from the other control groups. The MMP-2 can digest a large number of the
ECM molecules including Type I, II, III, IV, V and XI collagens, laminin, aggrecan core
protein, etc. [29]. In addition, the pathophysiological study of the action of gelatinases
shows that an increase in its activity can be responsible for tissue remodeling,
hypertrophy, angiogenesis and chronic inflammation [30].

All these data are consistent with previous studies, which relate the over-activation
of the PLG/plasmin system with different processes of cell proliferation and migration,
as well as the degradation of the ECM [16-19]. However, none of them linked these
pathogenic processes with the plasmin generated through the interaction of a blood-borne
pathogen with its host fibrinolytic system. To complete the knowledge of this interaction
we analyze the participation of DiES in the basal production of the main components of
the fibrinolytic system secreted by the cells of the arterial wall. Our results show that
DiES increases the expression of the fibrinolytic activators tPA and uPA in CnAOEC
during the first 24 h of stimulation, an effect that is not observed in CnAOSMC cultures.
These data are consistent with those obtained previously where an increase in the basal
production of tPA in human endothelial cells in culture was demonstrated [10]. In
addition, the presence of tPA is required for the activation of PLG in various parasites,
including D. immitis [31]. The increase in the expression of uPA could have special
significance since, in addition to its function as an activator of fibrinolysis, it plays a key
role in tissue remodeling inducing cell proliferation and migration [32], and high levels
in its expression are related to cardiovascular disease [33]. DiES causes a significant
reduction in the production of PAI-1 in CnAOEC and CnAOSMC in culture. PAI-1 is a
member of the serine protease inhibitor (SERPIN) superfamily and is the primary
physiological inhibitor of the tPA and uPA activity [34]. Finally, DiES does not cause
significant changes in the expression of Annexin A2 PLG receptor. However, it has

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recently been shown that D. immitis is able to excrete numerous antigens which can carry
out similar functions as PLG-binding proteins [10].

Conclusions
In conclusion, the data obtained in this study suggest that metabolic products of
D. immitis may tip the fibrinolytic balance towards the generation of plasmin, and that
this fact is related to the appearance of pathological phenomena similar to those described
in the formation of intravascular microvilli in cardiopulmonary dirofilariosis.

List of abbreviations
DiES, excretory/secretory antigens from D. immitis adult worms; PLG,
plasminogen; ECM, extracellular matrix; tPA, tissue plasminogen activator; uPA,
urokinase-type plasminogen activator; PAI-1, plasminogen activator inhibitor-1;
CnAOEC, canine aortic endothelial cells; CnAOSMC, canine aortic smooth muscle cells;
εACA, lysine analogue ε-aminocaproic acid; OP, optical density; MMPs, matrix
metalloproteinases.

Competing interests
The authors declare that they have no competing interests.

Authors' contributions
JGM and FS conceived and designed the experiments; JGM and RM performed
the experiments; JGM, RM, EC, JAMA and FS analyzed the data; JGM and FS wrote the
manuscript. All the authors have read and approved the final version of the manuscript.

Acknowledgements
This research was supported by Agencia de Desarrollo Económico de Castilla y
León (cofinanced with FEDER funds), Junta de Castilla y León (grant SA090/A09),
Spain.

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“Fibrinolysis and proliferative endarteritis: two related processes in chronic
infections? The model of the blood-borne pathogen Dirofilaria immitis”
 QUINTO CAPÍTULO

Fibrinolisis y endarteritis proliferativa: ¿Dos procesos relacionados en las

infecciones crónicas? El modelo del patógeno sanguíneo Dirofilaria immitis

Javier González-Miguel, Rodrigo Morchón, Mar Siles-Lucas, Fernando Simón

PLoS ONE (2015) 10(4):e0124445.

Factor de impacto (2014): 3,534.

Resumen

La interacción entre los patógenos sanguíneos y la fibrinolisis es uno de los

mecanismos más importantes que intervienen en la invasión y en el establecimiento de
los agentes infecciosos en sus hospedadores. Sin embargo, la sobreproducción de
plasmina (producto final de la ruta) ha sido relacionada en otros contextos con la
proliferación y migración de las células de la pared arterial y con la degradación de la
matriz extracelular. Recientemente hemos identificado antígenos con capacidad para
activar la fibrinolisis pertenecientes a Dirofilaria immitis, un parásito sanguíneo cuyo
proceso patológico clave (la endarteritis proliferativa) se produce por mecanismos
similares a los indicados anteriormente. El objetivo de este trabajo es estudiar cómo dos
de estos antígenos [la actina (ACT) y la fructosa-bifosfato aldolasa (FBAL)] altamente
conservados en los patógenos, activan la fibrinolisis y establecer una relación entre esta
activación y el desarrollo de la endarteritis proliferativa durante la dirofilariosis
cardiopulmonar. Se demostró que ambas proteínas fijan plasminógeno, potencian la
generación de plasmina, estimulan la expresión de los activadores fibrinolíticos tPA y
uPA en cultivos de células endoteliales y que se encuentran en la superficie del verme en
contacto con la sangre del hospedador. Técnicas de enzimoinmunoensayo, western blot e
inmunofluorescencia fueron utilizadas para este propósito. Adicionalmente, la
implicación de residuos de lisina en esta interacción se analizó mediante bioinformática.
La participación de la plasmina generada por la unión de la ACT/FBAL y el
plasminógeno en la proliferación y migración celular, así como en la degradación de la
matriz extracelular se demostró en un modelo in vitro de cultivos de células endoteliales
y del músculo liso. Los resultados obtenidos indican que la ACT y la FBAL de D. immitis
activan la fibrinolisis, lo que podría ser utilizado por el parásito como un mecanismo de

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supervivencia para evitar la formación de coágulos. Sin embargo, la sobreproducción a

largo plazo de plasmina puede desencadenar procesos patológicos similares a los
descritos en la aparición de la endarteritis proliferativa. Procesos similares podrían ocurrir
en otros patógenos sanguíneos debido al alto grado de conservación evolutiva de estos
antígenos.

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Abstract
The interaction between blood-borne pathogens and fibrinolysis is one of the most
important mechanisms that mediate invasion and the establishment of infectious agents
in their hosts. However, overproduction of plasmin (final product of the route) has been
related in other contexts to proliferation and migration of the arterial wall cells and
degradation of the extracellular matrix. We have recently identified fibrinolysis-
activating antigens from Dirofilaria immitis, a blood-borne parasite whose key
pathological event (proliferative endarteritis) is produced by similar mechanisms to those
indicated above. The objective of this work is to study how two of this antigens [actin
(ACT) and fructose-bisphosphate aldolase (FBAL)] highly conserved in pathogens,
activate fibrinolysis and to establish a relationship between this activation and the
development of proliferative endarteritis during cardiopulmonary dirofilariasis. We
demonstrate that both proteins bind plasminogen, enhance plasmin generation, stimulate
the expression of the fibrinolytic activators tPA and uPA in endothelial cell cultures and
are located on the surface of the worm in contact with the host’s blood. ELISA, western
blot and immunofluorescence techniques were employed for this purpose. Additionally,
the implication of lysine residues in this interaction was analyzed by bioinformatics. The
involvement of plasmin generated by the ACT/FBAL and plasminogen binding in cell
proliferation and migration, and degradation of the extracellular matrix were shown in an
“in vitro” model of endothelial and smooth muscle cells in culture. The obtained results
indicate that ACT and FBAL from D. immitis activate fibrinolysis, which could be used
by the parasite like a survival mechanism to avoid the clot formation. However, long-
term overproduction of plasmin can trigger pathological events similar to those described
in the emergence of proliferative endarteritis. Due to the high degree of evolutionary

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conservation of these antigens, similar processes may occur in other blood-borne

pathogens.

1. Introduction
The interaction between pathogens and their hosts at molecular level is the key
point that mediates invasion and establishment of the infection. One of these events is the
prevention of blood clotting through interaction with the hemostatic system, which is used
by many blood-borne pathogens as a survival mechanism [1]. The fibrinolytic system,
one of the main anticlotting mechanisms of the hemostatic system, is composed by
plasminogen (PLG), an abundant component of blood and zymogen of serine protease
plasmin, enzyme responsible for degrading fibrin clots. The conversion of PLG into
plasmin is regulated by binding to receptors via its five kringle domains, which have
affinity for lysine residues and PLG activators [tissue plasminogen activator (tPA) and
urokinase-type plasminogen activator (uPA)] [2]. On the other hand, plasmin is believed
to play an important role in a number of physiological and pathophysiological processes
such as tissue remodeling, wound healing, angiogenesis or inflammation [3].
Overproduction of plasmin has been also linked with the proliferation and migration of
human vascular cells and with the degradation of the extracellular matrix (ECM) [4–6].
In addition, plasmin is also upregulated in chronic inflammatory diseases, including
atherosclerosis and arthritis [7].

Activation of fibrinolysis by pathogen antigens has been widely studied in bacteria

where the expression of fibrinolytic receptors is considered an effective system for
invasion and dissemination [8]. In addition, the ability to interact with the fibrinolytic
system has been recently found in many eukaryotic pathogens causing parasitic
infections, such as Leishmania mexicana, Plasmodium falciparum, Fasciola hepatica,
Schistosoma bovis or Onchocerca volvulus [9]. Dirofilaria immitis is a zoonotic filaria
responsible for canine and feline cardiopulmonary dirofilariasis and human pulmonary
dirofilariasis [10]. These are chronic pathological processes that occur in the pulmonary
arteries, where D. immitis adult worms survive for long periods (over 7 years) causing an
inflammatory and thrombotic disease. Its key factor is the appearance of proliferative
endarteritis, which results in the formation of intravascular microvilli. It has been
described that this process is accompanied by an increase and migration of the cells of
the arterial wall [11–14], as well as the destruction of the ECM [15]. These changes cause

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disorganization of the endothelium and reduction of the vascular lumen in the pulmonary
arteries, with the consequent extension of pathology to the pulmonary parenchyma [16].

In previous research, we have studied the interaction between the

excretory/secretory (ES) and surface antigens of D. immitis and the fibrinolytic system of
the host. The ability of these antigenic complexes to bind PLG, generate plasmin in a
tPA-dependent manner and to induce an overexpression of the fibrinolytic activator tPA
in vascular endothelial cells in culture was demonstrated. Additionally, we have
respectively identified a total of 10 and 11 PLG-binding proteins in the ES and surface
extracts of the parasite, which included different isoforms of highly conserved proteins
like ACT and FBAL [17,18].

Despite the fact that the pathogenic mechanisms described in the formation of
intravascular microvilli during cardiopulmonary dirofilariasis are similar to those
associated with the pathophysiology of plasmin, a relationship between plasmin over-
production during Dirofilaria infection and their pathological implications has not yet
been established. The aim of the present work is to study the participation of two highly
conserved proteins of D. immitis (ACT and FBAL) in the activation of the fibrinolytic
system of the host and to establish its relationship with the proliferative endarteritis
pathogenic mechanisms in the cardiopulmonary dirofilariasis.

2. Materials and methods

2.1. Parasite material
Adult worms of D. immitis were obtained from hearts of naturally infected dogs
from Gran Canary (Canary Island, Spain) through the jugular vein using Flexible
Alligator Forceps. The surgeries were carried out by veterinarians at the Veterinary
Medicine Service of Las Palmas de Gran Canaria University (Canary Island, Spain) as
part of a routine practice for treating animals. The pet owners were adequately informed
and gave their verbal consent to the use of the samples in the study.

2.2. RNA isolation, RT-PCR, and cloning of DiACT and DiFBAL cDNA
RNA isolation, RT-PCR and cloning of the cDNA from the selected proteins were
carried out as described in detail previously [19]. Total RNA from adult worms was
extracted using the NucleoSpin RNA II kit (Macherey-Nagel) according to the
manufacturer’s instructions. Then, first-strand cDNA was synthesized from D. immtis

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adults worms RNA using the first-strand cDNA synthesis kit (Roche) as recommended
by the manufacturer. The cDNA sequence of the D. immitis actin (DiACT) and fructose-
bisphosphate aldolase (DiFBAL) were amplified using the following primers:

ACTFwd (5´-ATGTGTGACGACGACGTTGCGG)
ACTRev (5´-CTAGAAACATTTGCGATGAACAATTG)
FBALFwd (5´-ATGACCTCTTACTCACAGTTTCTG)
FBALRev (5´-TTAGTATGCATGATTAGCAATGTAG)

The primers from DiACT were designed on the consensus sequence resulting after
the alignment of ACT cDNA sequences from Loa loa and Brugia malayi (GenBank
accession numbers XM_003146804.1 and XM_001894784.1 respectively). The primers
from DiFBAL were designed on the consensus sequence resulting after the alignment of
FBAL cDNA sequences from O. volvulus, B. malayi and L. loa (GenBank accession
numbers AF155220.1, XM_001894495.1 and XM_003138767.1 respectively). PCR
amplifications were performed in 1 cycle at 94°C for 5 min, 35 cycles at 94°C for 1 min,
46°C for 46 sec, and 72°C for 1 min 30 sec and 1 cycle at 72°C for 5 min. The PCR
products were finally electrophoresed in an agarose gel and the bands were purified using
the StrataPrep DNA Gel Extraction kit (Stratagene). The DiACT and DiFBAL PCR
products were cloned into the pSC-A vector using the StrataClone PCR Cloning kit
(Stratagene) following the manufacturer’s instructions. Both clones were purified with
the Machery-Nagel NucleoSpin Plasmid kit.

2.3. Expression and purification of the rDiACT and rDiFBAL proteins

PCR products containing the whole DiACT and DiFBAL coding sequences were
cloned into the TOPO vector (Invitrogen) following the manufacturer’s instructions. The
recombinant plasmids were transformed into the Escherichia coli XL1B. Transformed
cells were grown in LB-agar plates with ampicillin (100 μg/ml) overnight at 37°C. Three
colonies for each molecule were grown in liquid LB plus ampicillin overnight at 37°C in
agitation, and cells were harvested for plasmid extraction. Extracted plasmids were
digested with EcoRI to check the insert presence. Transformed vectors were used for a
ligation reaction with the pDEST vector (Invitrogen). Ligation reaction was used for
transformant selection as above-mentioned. Vectors containing the inserts of interest
were used to transform BL-21 cells. These were grown in liquid LB plus ampicillin (100

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μg/ml) overnight at 37°C in agitation. Cultures were diluted 1:10 in fresh medium and
further grown until reaching an optical density (OD) of 0.5. Then, expression of the
recombinant protein was induced by 0.2% L-arabinose 20% at 37°C for 4 h in agitation.
The induced cells were harvested and sonicated in a buffer containing 8M urea, 100mM
NaH2PO4 and 10mM Tris-Cl, pH 7.9. After a 20 min centrifugation step at 10.000 x g,
the supernatant was applied to a HIS-Select Nickel Affinity Gel (Sigma) for affinity
purification of the histidine-tagged rDiACT and rDiFBAL, according to the
manufacturer’s instructions. Urea was eliminated by washing the column with wash
buffer (100mM NaH2PO4, and 10mM Tris-Cl pH 6.3) containing decreasing
concentrations of urea (from 6M to 0M). Then, the recombinant proteins were eluted with
elution buffer (50mM NaH2PO4, 300mM NaCl and 250mM imidazole, pH 7.9). The
eluted rDiACT and rDiFBAL were dialysed in PBS for 24 h at 4°C and stored at −80°C
until use. The purity and yield of each protein obtained after purification were assessed
in 12% polyacrylamide gels using Coomasie blue staining.

2.4. Bioinformatic analyses

The deduced amino-acid sequence of rDiACT and rDiFBAL were analysed using
the following bioinformatic tools: BLAST searching of the homologous sequences in the
NCBI and Swissprot/Uniprot databases (http://www.ncbi.nlm.nih.gov/,
http://www.uniprot.org/); analysis of conserved domains with SMART
(http://smart.embl-heidelberg.de); theoretical isoelectric point (pI) and the molecular
weight (MW) calculations (http://www.expasy.org/tools/pi_tool.html); prediction of
transmembrane domains with the TMHMM Server v. 2.0
(http://www.cbs.dtu.dk/services/TMHMM-2.0); prediction of signal peptides with
SignalP 3.0 [20] (http://www.cbs.dtu.dk/services/SignalP); search for glycosyl-
phosphatidyl anchors in the sequence with the big-PI Predictor [21]
(http://mendel.imp.ac.at/sat/gpi/gpi_server.html); multiple sequence alignment with
ClustalW 2.1 (http://www.ebi.ac.uk/Tools/msa/clustalw2/) and prediction of the
secondary structures and three-dimensional modelling with the Swiss-Model server [22]
(http://swissmodel.expasy.org/). The 3-D models were visualized with the RasMol
software v.2.7.5.2.

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2.5. PLG binding assays

The ability of rDiACT and rDiFBAL to act as plasminogen-binding proteins was
assessed by ELISA. Following the methodology described by González-Miguel et al.
(2012) multiwell microplates (Costar) were coated with 0.5 μg/well of each recombinant
protein. Then, plates were blocked and incubated with increasing amounts (from 0 μg to
3 μg) of human PLG (Acris antibodies). After incubation with the corresponding
antibodies and with a chromogen, binding was revealed by measuring OD at 492 nm in
an Easy Reader (Bio-Rad). Between each incubation, plates were washed three times with
PBS containing 0.05% Tween20 (PBST). In order to study the involvement of lysine
residues in this binding, competition assays were performed by including 50 mM of the
lysine analogue ε-aminocaproic acid (εACA) during PLG incubation [17].

2.6. PLG activation assays

The participation of rDiACT and rDiFBAL in plasmin generation was studied
following the methodology previously described [17]. In brief, 2 μg of human PLG (Acris
antibodies) were incubated in PBS with 3 μg of the chromogenic substrate D-Val-Leu-
Lys 4-nitroanilide dihydrochloride (Sigma) in the presence of 1 μg of each recombinant
protein in a final volume of 100 μl. Activation of PLG was initiated by addition of 15 ng
of t-PA (Sigma), however, plasmin generation in the absence of tPA was also analyzed.
After incubation of the plates (1 h at 37°C) the hydrolysis of the chromogenic substrate,
which is directly proportional to the amidolytic activity of generated plasmin was
revealed by measuring absorbance at 405 nm. Each sample was analyzed in triplicate.

2.7. Generation of an anti-rDiFBAL antiserum

Antiserum against rDiFBAL was generated by subcutaneous immunization of two
New Zealand female rabbits with 3 doses of protein in 0.2% saponin solution. First dose
of 1 mg at the beginning of the experiment, plus 2 doses of 500 μg 7 and 10 days later.
Rabbits were sacrificed by an intravenous overdose of pentobarbital and bled 20 days
after the last dose. Serum was collected, serially diluted and titred by ELISA. The
reactivity and specificity of the serum was also assessed by Western blot. Animal
procedures for this purpose complied with the Spanish (Real Decreto RD53/2013) and
the European Union (European Directive 2010/63/EU) guidelines on animal
experimentation for the protection and humane use of laboratory animals, and were
conducted at the accredited Animal Experimentation Facility (Servicio de

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Experimentación Animal) of the University of Salamanca (Register number:

PAE/SA/001). Procedures were approved by the Ethics Committee of the University of
Salamanca and all efforts were made to minimize suffering. These included good practice
for housing and care minimizing discomfort, distress and pain in animals.

2.8. Immunolocalization of DiACT and DiFBAL in D. immitis adult worms

Confocal microscopy studies were carried out on adult worm sections. D. immitis
adult worms were fixed in 10% buffered formalin and embedded in paraffin. For
immunofluorescence, 6 μm-thick sections were placed on polylisinated slides,
deparaffinised in xylene (2×8 min each), and rehydrated. Sections were then blocked with
1% BSA in PBST for 1 h at 37°C, after which they were incubated with the anti-rDiFBAL
rabbit serum diluted 1:50 in blocking buffer for 1 h at 37°C. Samples were washed three
times with PBST and incubated at 4°C overnight with an anti-rabbit IgG antibody
conjugated to Alexa Fluor 594 (Molecular Probes) diluted 1:400 in blocking buffer
containing phalloidin-Alexa Fluor 488 (Molecular Probes) diluted 1:200, which
specifically binds to ACT microfilaments. The samples were then washed four times and
mounted in an antifade reagent (Prolong Gold, Molecular Probes). Negative controls were
carried out by serum from a non-immunized rabbit.

2.9. Cell culture

Canine endothelial cells (CnAOEC) and canine smooth muscle cells (CnAOSMC)
from Cell Applications, INC were respectively grown in canine endothelial and canine
smooth muscle cell growth mediums (Cell Applications, INC). CnAOEC plates were
precoated with an attachment factor solution (Cell Applications, INC). Cells were
cultured at 37°C in a humidified atmosphere in the presence of 5% carbon dioxide and
95% air. Medium was changed every 3 days. Expansion was done by trypsinizing the
cells (Trypsin/EDTA, Cell Applications, INC) and replating them when the proliferating
cells had reached a sufficient density. Passaging was done at ratios of 1:6 (CnAOEC) or
1:3 (CnAOSMC). Cell counts were performed using the equipment Countess Automated
Cell Counter (Invitrogen) following the manufacturer's instructions.

2.10. Reagents and stimulation of CnAOEC and CnAOSMC

For stimulations, CnAOEC and CnAOSMC were grown for 4 days to obtain
confluent cultures and were treated with 1 μg/ml of rDiACT or rDiFBAL, 10 μg/ml of

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PLG (Acris Antibodies) [4] or with a mixture of both treatments (rDiACT + PLG or
rDiFBAL + PLG). Untreated cells and cells treated with rDiACT/rDiFBAL + PLG + 50
mM of εACA as an inhibitor of PLG activation were used as control cells under the same
conditions.

2.11. Cell lysates and Western blot analyses

Western blot analyses were performed as described in detailed previously [23]
with slightly modifications. CnAOEC and CnAOSMC previously treated with 1μg/ml of
rDiACT or rDiFBAL for 24 h were lysed in ice-cold lysis buffer (20mM Tris—HCl (pH
7.5), 140mM NaCl, 10mM ethylendiaminetetraacetic acid, 10% glycerol, 1% Igepal CA-
630, aprotinin, pepstatin, and leupeptin at 1μg/ml each, 1mM phenylmethylsulfonyl
fluoride, and 1mM sodium orthovanadate). Non-stimulated cells were used as controls
under the same conditions. Protein samples (10 μg) were separated by SDS-PAGE under
reducing conditions and electrotransferred onto polyvinylidine difluoride membranes.
Then, membranes were blocked before incubation with the following primary rabbit
polyclonal antibodies: anti-tPA and anti-uPA (Santa Cruz Biotechnology Inc) according
to the manufacturer's recommendations. After incubation with HRP-conjugated anti-
rabbit secondary antibodies, bands were visualized by a luminol-based detection system
with p-iodophenol enhancement. Anti-α-tubulin antibody (Oncogene Research Products)
was used as control to confirm loading of comparable amount of protein in each lane.
Protein expression was quantified by densitometry using the PDQuest Software v.8.0.1
(Bio-Rad).

2.12. Cell proliferation assay

Cells were plated on 24-well plates to a density of 104 CnAOEC/well or 1.5 x 104
CnAOSMC/well and allowed to attach overnight. After stimulations cell proliferation
was analyzed by crystal violet nuclei staining over 10 days determining the number of
viable cells as previously described [24]. Briefly, every two days, cells were rinsed with
PBS, fixed with 4% formaldehyde for 10 min and stained with 0.2% crystal violet for 30
min at room temperature. After several rinses with PBS, the cells were allowed to dry
overnight and crystal violet bound to cells was extracted by incubation with 2 ml/well of
10% acetic acid. The absorbance of the samples was then measured at 595 nm and
transformed to “number of viable cells” using a curve that correlated absorbance and
number of endothelial or smooth muscle cells previously determined.

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2.13. Cell migration assay

Cell migration was assessed by quantifying the percentage of wound closure in
the wound-healing assay [25]. In brief, CnAOEC or CnAOSMC were cultured in 60 mm
plates (3 x 105 cells/plate) and allowed to attach overnight before wound creation.
Confluent monolayers were wounded using a sterile pipette tip and the medium was
exchanged for fresh medium before cell stimulation to remove cellular debris. The extent
of wound closure of the treated and control cells was monitored over a time course by
microscopy and determined along 8 hours by calculating the migrated distance/total
wound distance.

2.14. Collagen degradation assay

The concentration of collagen in the supernatants was analyzed by ELISA.
Treated and control cells were cultured with medium for 48 hours. Then the culture
supernatants were collected, filtered and added to multi-well plates (Costar). After
incubation overnight at 4°C, wells were blocked with 1% BSA in PBS and incubated with
a rabbit anti-Type I Collagen antibody (1:2500) (Acris Antibodies), and then with a
peroxidase-conjugated goat anti-rabbit IgG (Sigma) at 1:500 dilution. All incubations
were performed for 1 h at 37°C and between each step washed three times with PBST.
Ortho-phenylene-diamine was used as a chromogen. ODs were measured at 492 nm in an
Easy Reader (Bio-Rad).

2.15. Matrix Metalloproteinase (MMP) levels assays

The levels of MMP-2 and MMP-9 metalloproteinases in the culture media of the
different experimental groups was analyzed by gelatin zymography [26] according to the
methodology described by Marangoni et al. (2011) [27]. Media samples employed in the
collagen degradation assays were electrophoresed on a 10% polyacrylamide gel
copolymerized with 1% gelatin (Sigma) together with a MMP marker (Cosmobio) as a
positive control. The gels were washed for 1 hour in 2.5% Triton X-100 and incubated
for 20 hours at 37°C in agitation in an enzymatic activation buffer ph 7.5 (50 mM Tris;
200 mM NaCl; 5 mM CaCl2; 0,2% Brij-35). The gels were finally stained with Coomassie
blue. The positivity was assessed as appearance of clear bands on a dark background with
molecular weights of 72 kDa (MMP-2) and 92 kDa (MMP-9). The levels of MMPs were
calculated after measuring the intensity of the existing bands, which is directly
proportional to the amount of gelatin degraded into the gel [28].

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2.16. Statistical analysis

The results from the PLG binding assay, PLG activation assay and Western blots
for the tPA and uPA expression were analyzed with the Student’s t-test. Cell proliferation
and migration, collagen degradation and MMP levels significance measurements
(comparisons between groups) were performed by ANOVA and corrected for repeated
measurements when appropriate. If ANOVA revealed overall significant differences,
individual means were evaluated post hoc using Bonferroni’s procedure. All the results
were expressed as the mean ± SD of three experiments performed with duplicates. In all
experiments, a significant difference was defined as a p-value of <0.05 for a confidence
level of 95%.

3. Results
3.1. Amplification, cloning, sequencing, and expression of D. immitis ACT and FBAL
Amplification of D. immitis ACT and FBAL cDNA by RT-PCR respectively
resulted in PCR products of around 1100 and 1000 bp. After their cloning into the pSC-
A vector, fragments were fully sequenced and their identities demonstrated as actin and
fructose bisphosphate aldolase by BLAST analysis. The new sequences were respectively
deposited in the Gen-Bank under accession numbers JQ780093.1 and JQ780094.1. The
full cDNA respectively contained 1131 and 1092 nucleotides, encoded a protein of 376
and 363 amino acids, with a theoretical molecular weight of 41.820 and 39.423 Da, and
pI of 5.29 and 7.65.

The bioinformatics analyses of the deduced amino acid sequences did not reveal
a signal peptide, transmembrane helices or glycosyl-phosphatidyl inositol anchors. The
percentage identity between DiACT/DiFBAL and homologous sequences from other
organisms was analyzed using multiple sequence alignment with the ClustalW program
(Figs 1 and 2). The analysis revealed that DiACT and DiFBAL are highly conserved
proteins. Thus, in the alignment of these sequences with homologous proteins from other
filarial nematodes (Wuchereria bancrofti, O. volvulus, B. malayi and L. loa) DiACT and
DiFBAL respectively obtained identities of 100% and range of identities between 94.21
and 96.69%. These identities also obtained high values when DiACT and DiFBAL were
aligned with homologous proteins from other parasitic helminthes (Figs 1 and 2).
Aditionally, a PLG-binding domain to actin within amino acids 56 to 70
(GDEAQSKRGILTLKY) and 19 and 12 conserved lysine residues in the DiACT and

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DiFBAL alignment were respectively found and highlighted. In silico three-dimensional

modelling of the molecules predicted the 3D structures showing in the case of DiACT a
monomer with 20 α-helices and 19 β-sheets (Fig 3A). Molecular modelling of DiFBAL
showing a homo-tetramer with the presence of 14 α-helices and 13 β-sheets (Fig 3B). The
PLG-binding domain (GDEAQSKRGILTLKY) and the conserved lysine residues were
highlighted and were visualized on the outside of the proteins.

The D. immitis ACT and FBAL cDNA were cloned into the expression vector
TOPO/pDEST. After induction of expression in E. coli, the recombinant proteins were
purified under denaturing conditions using nickel affinity chromatography. The purified
rDiACT and rDiFBAL respectively had molecular weights of 43.6 kDa and 41.6 kDa in
polyacrylamide gel.

3.2. rDiACT and rDiFBAL bind PLG

An ELISA was carried out to determine the binding level of PLG to rDiACT and
rDiFBAL (Fig 4). Analyses showed that both recombinant proteins bind PLG in a similar
way and that this binding is directly proportional to the amount of PLG (Fig 4). Wells
coated only with BSA, used as negative control, showed some non-specific binding
activity, but always with values significantly lowers than those obtained by rDiACT and
rDiFBAL (p<0.05). The addition of 50 mM εACA respectively resulted in the inhibition
of about 70% and 90% of the binding level of rDiACT and rDiFBAL, demonstrating the
involvement of lysine residues in the process (Fig 4).

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Fig. 1. Multiple sequence alignment of DiACT with homologous proteins. Alignment of the D. immitis
ACT sequence (I3WTW3) with the ACT from W. bancrofti (EJD75047), O. volvulus (EJW73626), B.
malayi (P48812), L. loa (O01360), Ascaris suum (BAB68543), Homo sapiens (P20287) and S. bovis
(AIE44418). The percentage of sequence identity between D. immitis sequence and the others is indicated.
The amino acids conserved in all the sequences are labelled with asterisks, and conservative and
semiconservative substitutions are respectively labelled with two and one point. Conserved lysine residues
are shaded in yellow. The PLG-binding domain (GDEAQSKRGILTLKY) are shaded in grey.

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Fig. 2. Multiple sequence alignment of DiFBAL with homologous proteins. Alignment of the D. immitis
FBAL sequence (I3WTW4) with the FBAL from O. volvulus (Q9U9R9), L. loa (J0DRR2), B. malayi
(A8P3ES), W. bancrofti (J9EVQ4), A. suum (U1M5S0), Haemonchus contortus (R4H2V1) and S.
japonicum (C1LB95). The percentage of sequence identity between D. immitis sequence and the others is
indicated. The amino acids conserved in all the sequences are labelled with asterisks, and conservative and
semiconservative substitutions are respectively labelled with two and one point. Conserved lysine residues
are shaded in yellow.

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Fig. 3. Molecular modelling of D. immitis ACT and FBAL. The secondary structure of the proteins was
predicted with the Swiss-Model web server (http://swissmodel.expasy.org/) by analogy with the X-ray
crystallography available models. The three-dimensional models of DiACT (A) and DiFBAL (B) were
visualized with the RasMol application v. 2.7.5.2. Conserved lysine residues of proteins were highlighted
as red balls. The PLG-binding domain (GDEAQSKRGILTLKY) is highlighted in yellow.

Fig. 4. PLG binding assay of rDiACT and rDiFBAL. PLG binding to 0.5 μg of rDiACT (A) or rDiFBAL
(B) analyzed over a range of PLG amounts using a microtiter plate method. (■) Incubation with increasing
amounts of PLG, 0–3 μg. (▲) Competition assay with 50 mM εACA included during PLG incubation. (●)
Wells coated with BSA used as negative control. Each point is the mean ± SD from three independent
experiments. The asterisk (*) designates significant (p<0.05) differences.

3.3. rDiACT and rDiFBAL enhance the activation of PLG by tPA

In order to determine the ability of rDiACT and rDiFBAL to activate PLG and
generate plasmin on their own, the amidolytic activity of plasmin generated in the

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presence or absence of tPA was measured. Negative controls replacing each recombinant
protein for BSA or tPA were also used. Fig 5 shows the capacity of rDiGAPDH and
rDiGAL to stimulate plasmin generation by tPA obtaining ODs significantly higher than
the negative controls (p<0.05). Both proteins obtained similar results and PLG activation
did not occur in the absence of tPA. In addition, this effect is inhibited by 50 mM εACA,
indicating the involvement of lysine residues in the process (Fig 5).

Fig. 5. PLG activation assay of rDiACT and rDiFBAL. PLG activation and plasmin generation by
rDiACT (A) or rDiFBAL (B). (□) 15 ng of tPA was added to mixtures which contained 2 μg of human
PLG, 3 μg of D-Val-Leu-Lys 4-nitroanilide dihydrochloride (Sigma) and 1 μg of each recombinant protein
(or BSA as negative control) in the presence or absence of 50 mM of εACA in a test volume of 100 μl. (■)
Reaction mixtures in absence of tPA. Each point is the mean ± SD from three independent experiments.
The asterisk (*) designates significant (p<0.05) differences.

3.4. Effect of rDiACT and rDiFBAL on the fibrinolytic system activators (tPA and
uPA) expression in CnAOEC and CnAOSMC
To complete the study of the effect of rDiACT and rDiFBAL on the fibrinolytic
system activation, proteins from rDiACT or rDiFBAL-treated or untreated CnAOEC and
CnAOSMC extracts were separated by SDS-PAGE and analyzed by Western blotting
using anti-tPA and anti-uPA antibodies. rDiACT and rDiFBAL induced a marked
increase in the expression of the main fibrinolytic activators tPA and uPA in cultured
endothelial cells (p<0.05) (Fig 6A and 6C). This increase was greater in the case of the
uPA expression and significantly higher in cells stimulated with rDiFBAL (Fig 6C).
Significant differences in the expression of tPA and uPA in CnAOSMC between
rDiACT/rDiFBAL-treated or untreated cultures were not found (Fig 6B and 6D).

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Fig. 6. Effect of rDiACT and rDiFBAL on the fibrinolytic system activators expression. Effect of
rDiACT and rDiFBAL on the expression of tPA (A and B) and uPA (C and D) in canine vascular endothelial
(A and C) and smooth muscle cells (B and D). Protein extracts from lysed rDiACT or rDiFBAL treated or
untreated confluent cell cultures were analyzed by Western blot for tPA and uPA. α-tubulin served as a
protein control. Data are shown as representative images or means ± SD from three independent
experiments. The asterisk (*) designates significant (p<0.05) differences from control cells. (■) Stimulated
cells with 1µg/ml of rDiACT. (■) Stimulated cells with 1µg/ml of rDiFBAL. (■) Non-treated control cells.

3.5. Immunolocalization of DiACT and DiFBAL in sections from D. immitis adult

worms
Immunolocalization of proteins was carried out by the use of a commercially
available high-affinity ligand (in the case of ACT) and a rabbit polyclonal antisera (in the
case of FBAL). The reactivity and specificity of this antiserum was tested in ELISA and

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Western blot prior to their use in the immunolocalization studies. The antibody titers of
this antiserum were higher than 1/500, with an OD of 1.15 while the negative serum
showed an OD of 0.13 at the same dilution. The anti-rDiFBAL antiserum reacted strongly
and specifically against the recombinant protein, while the negative serum showed no
reactivity in the Western blot analysis (not shown).

The anatomical localization of DiACT and DiFBAL was carried out in

histological sections of D. immitis adult worms by immunofluorescence. As shown in Fig
7, all sections showed green fluorescence throughout the soma of the parasite, as a result
of the binding of phalloidin-Alexa Fluor 488, ACT high-affinity ligand which serves also
as a positive control of the technique. Sections incubated with the anti-rDiFBAL
antiserum showed, in addition, specific reactivity (in red) against the parasitic FBAL due
to the binding of the anti-rabbit IgG antibody conjugated to Alexa Fluor 594. Both
proteins are located scattered throughout all the soma, being especially abundant in the
cuticle (reflected by an orange color in the overlay of Phalloidin-Alexa Fluor 488 + Alexa
Fluor 594 images). Sections incubated with a rabbit negative serum showed no specific
red fluorescence from recombinant proteins.

3.6. rDiACT, but not rDiFBAL, produces proliferation of CnAOEC and CnAOSMC
via PLG/plasmin system
The effect of rDiACT or rDiFBAL and PLG on the proliferation of endothelial
and smooth muscle cells was quantified using the crystal violet technique in a period of
10 days (Fig 8). Both cultures showed typical curves of cell growth in all experimental
groups with a progressive growth between days 0 and 6 or 8 post-treatment in the
CnAOEC or CnAOSMC cultures, experiencing cell death and an evident decrease of
viable cells from there until the end of the experiment (day 10 post-treatment). Crystal
violet staining showed an increase significantly greater in the number of viable cells in
cultures stimulated with rDiACT + PLG than that showed by other experimental groups
on days 4 and 6 post-treatment (CnAOEC) or day 8 post-treatment (CnAOSMC)
(p<0.05), indicating that this treatment stimulates the proliferation of CnAOEC and
CnAOSMC in culture. Significant differences in cell proliferation between cells
stimulated with rDiFBAL + PLG and other experimental groups were not found in both
types of cultures.

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Fig. 7. Immunolocalization of DiACT and DiFBAL in sections from D. immitis adult worms.
Representative images from three independent experiments of parasite sections incubated with phalloidin-
Alexa Fluor 488 (in green, specific binding to ACT) plus the negative or the anti-rDiFBAL rabbit sera and
an anti-rabbit IgG-Alexa Fluor 594 (in red). Corresponding transmitted light images are also addressed.
Magnification 4X.

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Fig. 8. Cell proliferation assay performed by the crystal violet technique measuring cell viability over
a 10 days period. The experiment was carried out in canine endothelial (A and C) and smooth muscle cells
(B and D) untreated ( ) or treated with 1 µg/ml of rDiACT or rDiFBAL + 10 µg/ml of PLG ( ), 1 µg/ml
of rDiACT or rDiFBAL ( ), 10 µg/ml of PLG ( ), or with 1 µg/ml of rDiACT or rDiFBAL + 10 µg/ml of
PLG + 50 mM of the εACA ( ). Results were expressed as number of viable cells (x 10,000). Each point
is the mean ± SD from three independent experiments. The asterisk (*) designates significant (p<0.05)
differences between rDiACT + PLG treatment and control groups.

3.7. rDiACT and rDiFBAL produce migration of CnAOEC and CnAOSMC via
PLG/plasmin system
A Wound Healing assay was performed to assess migration of endothelial (Fig 9)
and smooth muscle cells (Fig 10). The quantification was carried out by measuring the
distance of migration in comparison with negative control (untreated cells) to 8 hours
post-treatment. In both CnAOEC and CnAOSMC cultures a significant increase of cell
migration after stimulation with rDiACT or rDiFBAL + PLG with respect to the other
experimental groups (p<0.05) occurred, being this increase most pronounced in cultured
smooth muscle cells. After comparing the effect of both parasitic proteins, rDiACT
showed higher values of migration ability in both types of cell cultures with respect to
those obtained by rDiFBAL.

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Fig. 9. CnAOEC migration by Wound-Healing assay. Confluent cell cultures were wounded post-
treatment and migration distances were measured at 8 hours. The experiment was carried out in canine
endothelial cells untreated ( ) or treated with 10 µg/ml of PLG ( ), 1 µg/ml of rDiACT/rDiFBAL ( / ), 1
µg/ml of rDiACT/rDiFBAL + 10 µg/ml of PLG ( / ) or with 1 µg/ml of rDiACT/rDiFBAL + 10 µg/ml of
PLG + 50 mM of the εACA ( / ). The results were expressed as percentage of the migration ability of the
negative control cells (100%). Data are shown as representative images or means ± SD from three
independent experiments. The asterisk (*) designates significant (p<0.05) differences between rDiACT or
rDiFBAL + PLG treatment and control groups.

3.8. rDiACT and rDiFBAL produce ECM degradation of CnAOEC and CnAOSMC
via PLG/plasmin system
To examine ECM degradation, Type I Collagen in the culture supernatant of
treated and untreated CnAOEC and CnAOSMC were measured by ELISA (Fig 11). A
lower concentration of Type I Collagen and therefore a further degradation of the secreted
collagen by the cells was observed in the culture media of CnAOEC and CnAOSMC
stimulated with rDiACT/rDiFBAL + PLG than that obtained by the control cells (p<0.05).
In addition, the same culture media from treated and untreated cells were analyzed with
gelatin zymography for MMP-2 and MMP-9 levels (Fig 12). Density of the bands was
measured by the Quantity One Software (Bio-Rad). Treatment with rDiACT or rDiFBAL
+ PLG shows a significantly higher MMP-2 level in the CnAOEC and CnAOSMC culture
media and MMP-9 level in the CnAOEC culture media than that obtained by the other
treatments (p<0.05). In addition, treatment with rDiFBAL + PLG shows an activation of

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the latent form of the MMP-9 in the CnAOSMC culture media (show by a clear band of
82 kDa), which does not appear with other treatments. No significant differences in the
MMP-9 levels in the culture media of CnAOSMC were observed (Fig 12).

Fig. 10. CnAOSMC migration by Wound-Healing assay. Confluent cell cultures were wounded post-
treatment and migration distances were measured at 8 hours. The experiment was carried out in canine
smooth muscle cells untreated ( ) or treated with 10 µg/ml of PLG ( ), 1 µg/ml of rDiACT/rDiFBAL
( / ), 1 µg/ml of rDiACT/rDiFBAL + 10 µg/ml of PLG ( / ) or with 1 µg/ml of rDiACT/rDiFBAL + 10
µg/ml of PLG + 50 mM of the εACA ( / ). The results were expressed as percentage of the migration
ability of the negative control cells (100%). Data are shown as representative images or means ± SD from
three independent experiments. The asterisk (*) designates significant (p<0.05) differences between
rDiACT or rDiFBAL + PLG treatment and control groups.

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Fig. 11. Type I Collagen degradation assay. Collagen degradation measured in culture supernatants from
canine endothelial (A) and smooth muscle cells (B) untreated ( ) or treated with 10 µg/ml of PLG ( ), 1
µg/ml of rDiACT/rDiFBAL ( / ), 1 µg/ml of rDiACT/rDiFBAL + 10 µg/ml of PLG ( / ) or with 1 µg/ml
of rDiACT/rDiFBAL + 10 µg/ml of PLG + 50 mM of the εACA ( / ). The results were expressed as
percentage of the Type I Collagen concentration in the culture supernatant from negative control cells
(100%). Each point is the mean ± SD from three independent experiments. The asterisk (*) designates
significant (p<0.05) differences between rDiACT or rDiFBAL + PLG treatment and control groups.

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Fig. 12. MMP-2 and 9 levels assay. Representative zymography of MMP-2 (solid bars) and MMP-9
(hatched bars) levels in the culture supernatants from canine endothelial (A) and smooth muscle cells (B)
untreated ( ) or treated with 10 µg/ml of PLG ( ), 1 µg/ml of rDiACT/rDiFBAL ( / ), 1 µg/ml of
rDiACT/rDiFBAL + 10 µg/ml of PLG ( / ) or with 1 µg/ml of rDiACT/rDiFBAL + 10 µg/ml of PLG +
50 mM of the εACA ( / ). Note the gelatinolytic bands associated with MMP-2 (72 KDa) and MMP-9 (92
KDa) levels, as well as with the MMP-9 activated form (marked with a white asterisk at 82KDa. The results
were expressed as percentage of the MMPs levels in the culture supernatant from negative control cells
(100%). Data are shown as representative images or means ± SD from three independent experiments. The
asterisk (*) designates significant (p<0.05) differences between rDiACT or rDiFBAL treatment and control
groups.

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4. Discussion
The main finding of this study lies in relating how the activation of the fibrinolytic
system by two proteins of the blood-borne parasite D. immitis (a priori beneficial for both
the parasite and host), may cause long-term pathological effects based on the participation
of generated plasmin in the emergence of a process of proliferative endarteritis. This study
was conducted with the ACT and FBAL of D. immitis, highly conserved proteins that
were selected among parasite antigens that had been identified as PLG-binding proteins
in previous works [17,18]. Both proteins have previously been linked to pro-fibrinolytic
activities. The interaction between actin and PLG is well known, as well as the fact that
specific binding occurs through lysine residues, which stimulate the tPA-dependent
plasmin generation [29]. In addition, its function as PLG receptor has been demonstrated
on the surface of endothelial cells [30] and in the tegument of S. bovis [31]. Meanwhile,
FBAL has been identified as PLG-binding protein in the bacterium Mycobacterium
tuberculosis [32], the fungal pathogens Candida albicans [33] and Cryptococcus
neoformans [34] and in the helminth parasite S. bovis [31].

In this paper, two peptide sequences of 376 and 363 amino acids were respectively
obtained by cloning and sequencing of the D. immitis ACT and FBAL cDNAs. The
subsequent bioinformatic analyses based on the multiple sequence alignments carried out
with homologous proteins from other helminth parasites and the homology modelling of
their 3D structures have highlighted their high degree of conservation. None of the
proteins showed structural motifs for their transport or expression on the cell surface
(signal peptide, transmembrane motifs or GPI anchors), despite the fact that both proteins
have been previously identified in the secretome and on the surface of D. immitis [18,35].
This may be related to unconventional mechanisms of protein transport, as for example
with the association of these proteins with exosome-like secretion vesicles. This fact has
been recently postulated as an extracellular transport mechanism for glycolytic enzymes
from several groups of parasites [36].

In order to assess the interaction of these proteins with the host fibrinolytic system
we study their abilities to bind PLG, enhance plasmin generation, stimulate the production
of the fibrinolytic activators tPA and uPA, as well as their locations in the adult parasite.
Both proteins rDiACT and rDiFBAL showed ability to bind PLG and stimulate plasmin
generation by tPA, which are capabilities mediated by the participation of lysine residues,

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as it has been demonstrated by competition assays carried out with εACA. Interaction
with PLG has been historically associated with the presence of carboxyl-terminal lysine
residues in their receptors [37]. However, conserved internal lysine residues have been
more recently described as PLG-binding domains as in the case of enolase of
Streptococcus pneumoniae [38] or human beta-actin, in which a PLG-binding domain
within amino acids 55 to 69 (GDEAQSKRGILTLKY) has been identified indicating that
Lys61 and Lys68 are essential for this action [39]. This domain has been conserved in the
ACT of D. immitis (see Fig 1). In addition, after viewing the spatial location of the
conserved lysine residues of the DiACT and DiFBAL in their 3D models, these residues
seem to be located externally in these molecules, which would facilitate the accessibility
of PLG.

Despite the fact that the generation of plasmin by rDiACT and rDiFBAL is
dependent on tPA availability, we demonstrate that both proteins produce a significant
stimulation of the basal production not only of this fibrinolytic activator, but also of uPA
in canine endothelial cells in culture. This result reinforces the pro-fibrinolytic condition
of these proteins, since the participation of tPA and uPA in fibrinolysis is essential in the
effective activation of PLG [40]. On the other hand, high levels in the expression of both
tPA and uPA have been related to several physiological and pathological processes like
tissue remodeling and chronic inflammatory diseases, such as atherosclerosis and arthritis
[41,42]. Finally, immunolocalization studies showed that DiACT and DiFBAL, as well
as having an intracellular location, they are particularly abundant in the cuticle of D.
immitis. This fact is essential, so that the interaction of these proteins with the fibrinolytic
system may have relevance in vivo, it is necessary that DiACT and DiFBAL are expressed
in tissues in direct contact with the blood of the host [43].

Secondly, in order to study the effect of plasmin resulting from the fibrinolytic
activation by DiACT and DiFBAL on the proliferative endarteritis in the canine arterial
wall, we have developed an “in vitro” model of canine endothelial and smooth muscle
cells. Our data demonstrate that stimulation with rDiACT + PLG causes the proliferation
of CnAOEC and CnAOSMC, and treatments with rDiACT or rDiFBAL + PLG enhance
migration of both types of cells. This would be consistent with the formation of
intravascular microvilli occurring during dirofilariosis, which is result of the
multiplication and migration of the arterial wall cells [44]. In addition, the binding of both

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proteins to PLG causes a significant increase in the degradation of collagen type I and in
the levels of MMP-2 in the culture media of CnAOEC and CnAOSMC, as well as in the
levels of MMP-9 in the culture media of CnAOEC. Moreover, the binding of rDiFBAL
and PLG seems to induce an activation of the latent form of the MMP-9 in the culture
media of CnAOSMC. These facts highlight the participation of the rDiACT/rDiFBAL +
PLG interaction in the degradation of the ECM needed for the formation of intravascular
microvilli. Type I Collagen represents the main component of the ECM of elastic arteries.
Its alteration has been associated with vascular disease and its degradation products with
the proliferation and migration of smooth muscle cells in remodeling arteries [45]. These
results are consistent with those observed in vivo by Wang et al. (2005) who reported a
significantly lower amount of collagen in heartworm-infected dogs than in clinically
normal dogs [15]. On the other hand, MMPs function in the extracellular environment of
cells and degrade ECM molecules from the tissue. Among them, Gelatinases (MMP-2
and MMP-9) can digest a large number of the ECM molecules including type IV, V and
XI collagens, laminin, aggrecan core protein, etc. MMP-2, but not MMP-9, also digests
collagens I, II and III [46]. In addition, the pathophysiological study of the action of
gelatinases shows that an increase in its activity can be responsible for tissue remodeling,
hypertrophy, angiogenesis and chronic inflammation [47]. Finally, inhibition of all
positive results by including the 50 mM εACA in the stimulations demonstrates the final
participation of plasmin generated by binding between DiACT or DiFBAL and PLG on
the proliferation and migration of CnAOEC and CnAOSMC, as well as the degradation
of the ECM.

These results seem to indicate that D. immitis could use DiACT and DiFBAL to
shift the fibrinolytic balance towards the generation of plasmin, which might constitute a
survival mechanism to avoid the clot formation in its intravascular habitat. On the other
hand, in long-term infections as cardiopulmonary dirofilariasis, this overproduction of
plasmin could be related to pathological phenomena described in the emergence of
proliferative endarteritis. These findings contribute to understand a very complex part of
the host-pathogen relationships of dirofilariasis, showing how a process related to the
survival of the parasite and the host can lead to a pathogenic mechanism of great
importance. Since the ability to bind PLG and enhance plasmin generation of proteins of
many pathogens has been shown, and that ACT and FBAL are highly conserved
pathogenic antigens, similar events could occur in other infections caused by vascular
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pathogens developing chronic processes. The knowledge of these mechanisms could be

critical for the treatment and prevention of diseases caused by infectious agents.

Author Contributions
Conceived and designed the experiments: JGM, FS. Performed the experiments:
JGM, RM, MSL. Analyzed the data: JGM, FS. Contributed reagents/materials/analysis
tools: JGM, RM, MSL. Wrote the paper: JGM, FS.

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CONCLUSIONES
 CONCLUSIONES

1. Dirofilaria immitis activa el sistema fibrinolítico de su hospedador mediante la

acción conjunta de sus antígenos excretores/secretores y de superficie. Esto podría
ser utilizado por el parásito para desplazar el equilibrio fibrinolítico hacia la
generación de plasmina, lo que supondría un mecanismo de supervivencia para el
parásito al controlar la formación de coágulos en su hábitat intravascular inmediato.

2. Se identifican 10 y 11 proteínas fijadoras de plasminógeno, respectivamente, en los

compartimentos antigénicos excretor/secretor y de superficie del parásito. De ellas,
se han producido en su forma recombinante la actina, fructosa-bifosfato aldolasa,
gliceraldehído-3-fosfato deshidrogenasa y galectina de D. immitis, demostrando
individualmente sus propiedades profibrinolíticas. Todas ellas, en mayor o menor
medida, son capaces de fijar plasminógeno y potenciar la generación de plasmina a
través de la implicación de sus residuos de lisina. Además, son capaces de estimular
la expresión de activadores fibrinolíticos en cultivos de células endoteliales caninas
y se localizan en la interfase D. immitis/hospedador.

3. La plasmina, producto de la activación fibrinolítica causada por los antígenos de D.

immitis, participa en la generación de los procesos patológicos descritos en la
aparición de la endarteritis proliferativa en la dirofilariosis cardiopulmonar. Esto
incluye la proliferación y migración de las células de la pared arterial, así como la
degradación de la matriz extracelular, demostrándose estos hechos mediante la
utilización tanto de los antígenos excretores/secretores del parásito, como de las dos
proteínas con mayor capacidad profibrinolítica entre las estudiadas (actina y fructosa-
bifosfato aldolasa) en un modelo in vitro.

4. Los resultados obtenidos en la presente Tesis Doctoral contribuyen al conocimiento

de una parte muy compleja de las relaciones parásito/hospedador a nivel molecular
en la dirofilariosis cardiopulmonar, mostrando por primera vez cómo un proceso
relacionado con la supervivencia del parásito puede desencadenar mecanismos
patogénicos de gran importancia. Puesto que la capacidad para unir plasminógeno y
potenciar la generación de plasmina ha sido demostrada en proteínas de muchos
patógenos y debido al alto grado de conservación evolutiva de algunos de los
antígenos estudiados, mecanismos similares podrían ocurrir en otras infecciones
provocadas por patógenos sanguíneos que desarrollen procesos crónicos.

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ABSTRACT
 UNIVERSITY OF SALAMANCA
FACULTY OF BIOLOGY

DEPARTMENT OF ANIMAL BIOLOGY, PARASITOLOGY,

ECOLOGY, EDAFOLOGY AND AGRICULTURAL CHEMISTRY

DOCTORAL THESIS

The role played by plasmin in the survival of

Dirofilaria inmitis and in the vascular pathology of
the host during cardiopulmonary dirofilariosis

Javier González Miguel

Salamanca, 2015
 ABSTRACT

INTRODUCTION, MAIN THESIS AND KEY GOALS

Dirofilaria immitis is the causing filaroid nematode of canine and feline

cardiopulmonary dirofilariosis, a vector-borne transmitted parasitosis with cosmopolitan
distribution. D. immitis is, furthermore, responsible of human pulmonary dirofilariosis,
a clinical entity characterized by the formation of benign pulmonary nodules, which
could be wrongly taken for carcinoma during radiology sessions (Simón et al., 2012).
The dog acts as definitive host and reservoir of the parasite. In it, adult worms can
survive inside the pulmonary arteries and in the right ventricle of the heart for 7 years or
more, causing a chronic inflammatory pathology on a vascular level (Venco et al.,
2011). One of the key facts about this pathology is the appearance of proliferative
endarteritis, which has as a consequence the formation of intravascular microvilli. It has
been reported that this process goes hand in hand with the proliferation and migration of
cells belonging to the arterial wall towards the interior of the blood vessels, alongside
the destruction of the extracellular matrix (Adcook, 1961; Atwell et al., 1986; Hidaka et
al., 2004; Wang et al., 2005; Kawabata et al., 2008).

The aforementioned alterations cause the lack of organization of the

endothelium and the reduction of vascular lumen in the pulmonary vessels, with the
subsequent apparition of hypertension and edema. As a consequence of the damages
reported during the late stages of the disease the cardiac function can be affected,
leading to the apparition of hypertrophy and cardiac-congestive failure. Besides this
chronic development, acute processes involving an immediate life-risk for the infected
animals may appear. They emerge when the sudden and simultaneous death of many
adult worms occurs, either in a natural way or as a consequence of a filaricide treatment.
The massive liberation of antigenic products of parasites and their symbiotic bacteria
Wollbachia to the circulatory system is responsible of the exacerbation of inflammatory
reactions in the vascular endothelium and the formation of thromboembolisms of
different entity (Venco, 2007). However, D. immitis possesses mechanisms enabling the
regulation of these pathological processes, contributing to its survival in the
intravascular habitat during several years.

With the aim of maintaining and spreading to the blood vessels, many pathogens
not only require adaptations in order to avoid the activity of the host’s immune system,

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 ABSTRACT

but they must also hinder the coagulation of blood through the interaction with the
fibrinolytic system (Mebius et al., 2013). Fibrinolysis is one of the most important
anticoagulant mechanisms of the haemostatic system. One of its key elements is
plasminogen, a single-chain glycoprotein with a molecular mass of 92 KDa.
Plasminogen is produced in the liver, and is present in blood and other extravascular
fluids. Plasminogen is a pro-enzyme, which is transformed into plasmin (the serine
protease responsible for the degradation of the fibrin present in clots) after its activation.
The transformation of plasminogen into plasmin is regulated by its binding to the
receptors through its five “kringle” domains with affinity for lysine residues and
plasminogen activators [tissue plasminogen activator (tPA) and urokinase-type
plasminogen activator (uPA)] (Cesarman-Maus and Hajjar, 2005). The plasminogen
receptors are present in the fibrin net and also in diverse types of cells, such as
monocytes, macrophages, endothelial and neuronal cells, fibroblasts, platelets and
tumor cells (Hawley et al., 2000). These have also been identified on the surface of
diverse bacteria, fungi, protozoa and helminths (Bhattacharya et al., 2012; Figuera et al.,
2013)

Given that the mechanisms of these alterations are not completely known in
cardiopulmonary dirofilariosis, their study is of paramount importance, as its
understanding could facilitate the management of these situations by clinical vets,
contributing to a considerable improvement in the life quality of the affected animals.
Due to the survival capacity of D. immitis in its hosts, and also because
thromboembolisms appear when the worms die, our first hypothesis was that the
parasite controls and modifies the sanguine habitat in order to facilitate its survival
through molecules present in its antigenic products, generating a clear anti-thrombotic
effect through the use of pro-fibrinolytic products.

On the other hand, the activation of the fibrinolytic system through the
participation of the antigenic extracts of D. immitis as plasminogen receptors and the
subsequent maintenance of the haemostasis, which a priori has beneficial results for
both the parasite and the host, could have pathologic consequences. An over-production
of the plasminogen/plasmin pathway has been related to cell invasion and to the intra-
organic migration of diverse pathogens (Jong et al., 2003; Bernal et al., 2004).
Furthermore, cardiovascular research conducted on humans has linked the over-

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 ABSTRACT

production of plasmin with the proliferation and migration of vascular cells and the
degradation of the extracellular matrix (Nicholl et al., 2005; Yang et al., 2005; Roth et
al., 2006; Hayashi et al., 2009). These mechanisms are similar to those observed in the
formation of microvilli in cardiopulmonary dirofilariosis, but its molecular aspects have
not been hitherto conveniently studied in this parasitic disease. As a result of this, our
second hypothesis was to consider the over-activation of the fibrinolytic route by D.
immitis as directly related to the apparition of such pathologic processes in the vessel
wall of the infected animals. In order to demonstrate both hypotheses, we proposed the
following objectives of the present doctoral dissertation:

1. Analyze the interaction of the antigens of D. immitis with the fibrinolytic system
of its host in relation to the survival mechanisms on a vascular level.

2. Study if the activation of the fibrinolytic system by the parasite has an influence
on the pathological processes described on the development of proliferative
endarteritis in cardiopulmonary dirofilariosis.

RESULTS

1. Collection of excretory/secretory and surface associated extracts of proteins

from D. immitis adult worms

As a first step, in order to perform the proposed objectives, we start by

developing excretory/secretory (DiES) and surface associated (DiSAA) extracts of
proteins from D. immitis adult worms. DiES and DiSAA extracts were respectively
prepared following the methodology described by Morchón et al. (2010) and
Wedrychowicz et al. (1994) with minor modifications. Proteins were extracted in saline
solution mixed with a cocktail of protease inhibitors and their concentration measured
by DC protein assay commercial kit (Bio-Rad).

2. Proteins of DiES and DiSAA extracts bind plasminogen and enhance its
activation by tPA

To assess the capability of the antigenic extracts of D. immitis to interact with

the host fibrinolytic system, their ability to bind plasminogen and stimulate plasmin
generation was analyzed. The binding capacity of plasminogen to DiES and DiSAA

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 ABSTRACT

extracts was measured by ELISA. Multiwell microplates (Costar) were coated with 1
µg/well of DiES or DiSAA extracts. The wells were blocked with 1% BSA in PBS and
incubated successively with increasing amounts (from 0 μg to 3 μg) of human
plasminogen (Acris Antibodies), with a sheep anti-human plasminogen IgG and then
with a peroxidase-conjugated donkey anti-sheep IgG. Competition assays were
performed by including 50 mM of the lysine analogue ε-aminocaproic acid (εACA)
during plasminogen incubation. Some wells coated with BSA only were used as
negative controls.

The test showed that DiES and DiSAA bind plasminogen obtaining optical
densities statistically higher (p < 0.05) than those of the control wells (coated only with
BSA) (Figure 1). This binding is also directly proportional to the amount of
plasminogen. The competition assay showed that the inclusion of 50 mM εACA inhibits
the plasminogen-binding (Figure 1), demonstrating that this union is dependent on
lysine residues.

Figure 1. Plasminogen binding to 1 μg of DiES (A) or DiSAA (B) extracts of D. immitis measured over a
range of plasminogen amounts using a microtiter plate method. (■) Incubation with increasing amounts of
plasminogen, 0-3 μg. (●) Competition assay with 50 mM εACA included during plasminogen incubation.
(▲) Negative control consisted of wells coated only with BSA. Each point is the mean of three replicates
± SD. The asterisk (*) designates significant (p < 0.05) differences.

The ability to activate plasminogen by DiES or DiSAA extracts and to generate

plasmin was assessed by measuring the amidolytic activity of plasmin generated in the
presence of the antigenic extracts and plasminogen. This effect was measured in the

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 ABSTRACT

presence or absence of a physiological activator of the process, tPA, to observe the

ability of the DiES and DiSAA extracts proteins of activating plasminogen on their
own. Negative controls replacing DiES or DiSAA by BSA or t-PA were also used. As
shown in Figure 2, the generation of plasmin by tPA is enhanced by DiES and DiSSA
reaching optical density values significative higher (p < 0.05) than the negative controls
in the presence of tPA. However, DiES and DiSAA extracts are unable to generate
plasmin without tPA resulting in optical density values identical to the negative
controls.

Figure 2. Plasminogen activation and plasmin generation by DiES (A) and DiSAA (B) extracts of D.
immitis. (□) 15 ng of t-PA was added to mixtures containing 2 μg of human plasminogen, 3 μg of S-2251
(Sigma) and 1 μg of DiES or DiSAA extracts (or BSA as negative control) in the presence or absence of
50mM of εACA in a test volume of 100 μl. (■) No t-PA was added to reaction mixtures. Each point is the
mean of three replicates ± SD. The asterisk (*) designates significant (p < 0.05) differences.

3. Effect of DiES on the fibrinolytic system components (tPA, uPA, Annexin A2

and PAI-1) expression in CnAOEC and CnAOSMC

The study on the interaction between the antigens from D. immitis and the host
fibrinolytic system was completed by analyzing the effect of DiES on the expression of
the fibrinolytic activators tPA and uPA, the inhibitor PAI-1 and the plasminogen
receptor Annexin A2 in vascular cell cultures. For this purpose, we have developed an
“in vitro” model of canine endothelial (CnAOEC) and smooth muscle cells
(CnAOSMC).

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 ABSTRACT

Confluent cultures of CnAOEC and CnAOSMC were previously treated with 1

μg/ml of DiES for 24 h and lysed in ice-cold lysis buffer. Non-stimulated cells were
used as controls under the same conditions. Extracted proteins from DiES-treated or
untreated CnAOEC and CnAOSMC extracts were separated by SDS–PAGE and
analyzed by Western blotting using anti-tPA, anti-uPA, anti-Annexin A2 and anti-PAI-1
antibodies. DiES induced a marked increase in the expression of the main fibrinolytic
activators tPA and uPA in cultured endothelial cells (p˂0.05) (Figure 3A and 3B), as
well as a slight decrease in the expression of the main fibrinolytic inhibitor PAI-1 in
both types of cultures (p˂0.05) (Figure 3D). Significant differences in the expression of
tPA and uPA in CnAOSMC (Figure 3A and 3B) and in the expression of Annexin A2 in
both cell types between DiES-treated or untreated cultures were not found (Figure 3C).

Figure 3. Effect of DiES on the expression of tPA (A), uPA (B), annexin A2 (C) and PAI-1 (D) in canine
vascular endothelial (EC) and smooth muscle cells (SMC). Protein extracts from lysed DiES untreated or
treated confluent cell cultures were analyzed by Western blot for tPA, uPA annexin A2 and PAI-1. α-
tubulin served as a protein control. Results were expressed as the mean ± SD of at least 3 independent
experiments. The asterisk (*) designates significant (p < 0.05) differences from control cells. (■) Non-
treated control cells. (■) Stimulated endothelial or smooth muscle cells with 1µg/ml of DiES.

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 ABSTRACT

4. Two-dimensional analysis of DiES and DiSSA extracts and identification of

plasminogen-binding proteins by mass spectrometry

Two-dimensional electrophoresis of DiES and DiSAA extracts were performed

in order to obtain an overall view of all the proteins of both extracts. In order to improve
spot resolution and detection, once the spot MW and pI ranges were determined, both
extracts were electrofocused in 5–8 and 7–10 IPG strips. With these conditions, silver
staining respectively revealed a total of 636 and 347 spots in the DiES and DiSSA
extracts proteomes (Figures 4A, 4B, 5A and 5B).

Figure 4. Representative 2-DE of 60 µg of the DiES extract from adult D. immitis worms. The gels were
in the 5-8 and 7-10 pH ranges, 12% polyacrylamide and silver-stained (A and B). Plasminogen-binding
spots revealed on ligand blots from gels A and B (C and D). Reference molecular masses are indicated on
the left. The plasminogen-binding spots analyzed by MS are circled and numbered.

211
 ABSTRACT

Next, to determine which proteins of DiES and DiSAA extracts bind

plasminogen an immunoblot was performed. The 2-D gels were transferred to
nitrocellulose membranes which were blocked and then incubated with plasminogen.
After incubating the membranes with the corresponding antibodies, proteins were
revealed with 4-chloro naphthol and spots analyzed using the PDQuest Software v.8.0.1
(Bio-Rad).

MW Sequence
Spot pI Mascot
Accesion code Protein definition Species (kDa) coverage
number theor/exp score
theor/exp (%)

23 ACY25666 Chaperonin-like Brugia malayi 61.4/67.1 5.7/5.6 11 130

protein HSP60
27 AF121264_1 Chaperonin protein Onchocerca 64.5/67.0 5.7/5.8 17 145
HSP60 volvulus
28 AF121264_1 Chaperonin protein Onchocerca 64.5/65.2 5.7/5.8 16 130
HSP60 volvulus
31 ACT1_CAEEL Actin-1/3 Caenorhabditis 42.1/65.2 5.3/5.9 11 41
elegans
32 XP_001894819 Actin Brugia malayi 42.1/43.3 5.3/5.8 4 64

33 NP_508842 ACTin family Caenorhabditis 37.5/39.4 5.4/6.0 17 142

member (act-4) elegans
37 AAC24752 Transglutaminase Dirofilaria 57.6/61.0 5.7/6.3 19 91
precursor immitis
66 XP_001899850 Glyceraldehyde 3- Brugia malayi 32.1/40.8 8.5/7.5 20 207
phosphate
dehydrogenase
67 XP_001899850 Glyceraldehyde 3- Brugia malayi 32.1/40.7 8.5/7.8 25 292
phosphate
dehydrogenase
69 AAD00843 Ov87 Onchocerca 36.7/36.4 8.9/8.2 24 161
volvulus
71 AAD00843 Ov87 Onchocerca 36.7/36.5 8.9/9.0 16 157
volvulus
72 XP_003150284 Hypothetical Loa loa 13.3/33.7 6.7/6.3 11 94
protein
LOAG_14743
73 AAF37720 Galectin Dirofilaria 32.2/30.1 6.0/6.6 11 118
immitis
78 AAD11968 P22U Dirofilaria 24.4/22.0 8.9/9.2 66 499
immitis
79 AAD11968 P22U Dirofilaria 24.4/22.0 8.9/9.4 62 458
immitis
80 AAD11968 P22U Dirofilaria 24.4/22.0 8.9/9.6 62 489
immitis
81 AAD11968 P22U Dirofilaria 24.4/22.0 8.9/9.8 54 201
immitis

Table 1. Plasminogen-binding protein spots of DiES extract identified by MALDI-TOF MS. Exp,
experimental; theo, theoretical.

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 ABSTRACT

As shown in Figures 4C, 4D, 5C and 5D, 81 and 61 plasminogen-binding spots

were revealed in the DiES and DiSAA membranes. In the control blots, in which
plasminogen incubation was omitted, the anti-plasminogen antibody did not reveal any
spots (not shown). The matching of spots revealed by ligand-blotting with their
homologous in the silver-stained 2-D gels allowed us to select a total of 53
plasminogen-binding spots of D. immitis, which were manually excised from 2-D gels
and submitted to analysis by mass spectrometry. Seventeen spots corresponded to 10
different proteins and 16 spots corresponded to 11 different proteins were respectively
identified in the DiES and DiSAA 2-D gels. Tables 1 and 2 show the identity of these
proteins and their MWs and pIs (theoretical and experimental), the number of access to
similar information available in the NCBI database, the sequence coverage and the
Mascot score.

Figure 5. Representative 2-DE of 60 µg of the DiSAA extract from adult D. immitis worms. The gels
were in the 5-8 and 7-10 pH ranges, 12% polyacrylamide and silver-stained (A and B). Plasminogen-
binding spots revealed on ligand blots from gels A and B (C and D). Reference molecular masses are
indicated on the left. The plasminogen-binding spots analyzed by MS are circled and numbered.

213
 ABSTRACT

MW
Spot pI Queries Mascot
Accesion code Protein definition Species (kDa)
number theor/exp matched score
theor/exp

17 EFV54220 Actin-5C Trichinella 41.8/54.1 5.3/5.7 4 154

spiralis
18 P02578 Actin-1 Acanthamoeba 41.7/52.8 5.4/5.8 6 190
castellanii
20 XP_001896281 Enolase Brugia malayi 47.5/59.6 6.0/6.3 6 248
22 Q7YZX3 Enolase Onchocerca 47.1/55.5 6.0/6.6 8 64
volvulus
26 AAB52600 Fructose- Onchocerca 39.2/40.5 7.7/7.7 8 247
bisphosphate volvulus
aldolase
42 AAB52600 Fructose- Onchocerca 39.2/39.0 7.7/7.9 2 59
bisphosphate volvulus
aldolase
28 P48812 GAPDH Brugia malayi 36.1/40.0 7.7/7.2 13 128
30 P48812 GAPDH Brugia malayi 36.1/39.8 7.7/7.4 17 259
32 P48812 GAPDH Brugia malayi 36.1/37.4 7.7/7.8 11 84
46 P48812 GAPDH Brugia malayi 36.1/36.0 7.7/8.0 9 71
35 XP_001900868 MSP domain protein Brugia malayi 18.1/40.1 5.5/6.0 2 64
with Glu-rich
domain
60 P13263 Major sperm protein Onchocerca 14.3/15.7 7.8/8.8 18 265
2 volvulus
37 AAA20541 Beta-galactosidase- Onchocerca 32.0/33.6 6.0/6.6 20 143
binding-lectin volvulus
38 XP_001900812 Galectin Brugia malayi 31.8/30.3 6.4/7.8 3 68
39 XP_003139445 Immunoglobulin I- Loa loa 22.5/24.2 6.6/7.6 8 307
set domain-
containing protein
58 AAC47233 Cyclophilin Ovcyp- Onchocerca 18.5/20.9 8.3/9.4 1 71
2 volvulus

Table 2. Plasminogen-binding protein spots of DiSAA extract identified by MALDI-TOF MS. GAPDH,
Glyceraldehyde 3-phosphate dehydrogenase; Exp, experimental; theo, theoretical.

5. Amplification, cloning, sequencing, and expression of D. immitis actin, fructose-

bisphosphate aldolase, glyceraldehyde 3-phosphate dehydrogenase and galectin

Among the plasminogen-binding proteins identified by mass spectrometry in the

D. immitis antigenic extracts, four proteins were selected for the following experiments
based on the availability of homologous sequences from other filarial parasites, their
evolutionary conservation and if they had been previously related with plasminogen-
binding activities. We select actin (ACT), fructose-bisphosphate aldolase (FBAL),
glyceraldehyde 3-phosphate dehydrogenase (GAPDH) and galectin (GAL).

After designing the primers of the proteins, the genetic material was amplified
and isolated. DNA fragments were inserted in a PSC-A cloning vector, and the obtained

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 ABSTRACT

sequences were developed in their recombinant form in the TOPO/pDEST expression

system.

Amplification of D. immitis ACT, FBAL, GAPDH and GAL cDNAs by RT-

PCR respectively resulted in 4 PCR products of around 1100, 1000, 1000 and 850 bp.
After their cloning into the pSC-A vector, fragments were fully sequenced and their
identities demonstrated as actin, fructose-bisphosphate aldolase, glyceraldehyde 3-
phosphate dehydrogenase and galectin by BLAST analysis. The ACT, FBAL and
GAPDH new sequences were respectively deposited in the Gen-Bank under accession
numbers JQ780093.1, JQ780094.1 and JQ780095.1. The full D. immitis ACT, FBAL,
GAPDH and GAL cDNAs respectively contained 1131, 1092, 1020 and 846
nucleotides, encoded proteins of 376, 363, 339 and 280 amino acids, with theoretical
molecular weights of 41820, 39423, 36179 and 32085 Da, and pIs of 5.29, 7.65, 7.11
and 6.08.

The bioinformatics analyses of the deduced amino acid sequences did not reveal
a signal peptide, transmembrane helices or glycosyl-phosphatidyl inositol anchors. The
percentage identity between recombinant proteins and homologous sequences from
other organisms was analyzed using multiple sequence alignment with the ClustalW
program. The analysis revealed that DiACT, DiFBAL, DiGAPDH and DiGAL are
highly conserved proteins. Additionally, a plasminogen-binding domain to actin within
amino acids 56 to 70 (GDEAQSKRGILTLKY) and 19, 12, 7 and 16 conserved lysine
residues in the DiACT, DiFBAL, DiGAPDH and DiGAL alignments were respectively
found as possible plasminogen-binding sites.

Prediction of the secondary structures and three-dimensional modelling were

done with the Swiss-Model server (http://swissmodel.expasy.org/). In silico three-
dimensional modelling of the molecules predicted the 3D structures showing in the case
of DiACT a monomer with 20 α-helices and 19 β-sheets (Figure 6A). Molecular
modelling of DiFBAL showing a homo-tetramer with the presence of 14 α-helices and
13 β-sheets (Figure 6B). DiGAPDH appeared as a homo-tetramer with 15 α-helices and
4 β-sheets (Figure 6C), whereas modellling of DiGAL showed a monomer with the
presence of 2 α-helices and 26 β-sheets (Figure 6D). The plasminogen-binding domain

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 ABSTRACT

(GDEAQSKRGILTLKY) and the conserved lysine residues were highlighted and were
visualized on the outside of the proteins.

Proteins were finally purified under denaturing conditions using nickel affinity
chromatography. The purified rDiACT, rDiFBAL, rDiGAPDH and rDiGAL
respectively had molecular weights of 43.6 kDa, 41.6 kDa, 38.6 kDa and 34.6 kDa in
polyacrylamide gel.

Figure 6. Molecular modelling of D. immitis ACT, FBAL, GAPDH and GAL. The secondary structure of
the proteins was predicted with the Swiss-Model web server (http://swissmodel.expasy.org/) by analogy
with the X-ray crystallography available models. The three-dimensional models of DiACT (A), DiFBAL
(B), DiGAPDH (C) and DiGAL (D) were visualized with the RasMol application v. 2.7.5.2. Conserved
lysine residues of proteins were highlighted as red balls. The PLG-binding domain
(GDEAQSKRGILTLKY) is highlighted in yellow.

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6. rDiACT, rDiFBAL, rDiGAPDH and rDiGAL bind plasminogen and enhance its
activation by tPA

To analyze the ability of recombinant proteins as plasminogen-binding proteins,

experiments performed on antigenic extracts of D. immitis described in section 2 of this
abstract were repeated. After performing the corresponding ELISAs, these showed that
rDiACT, rDiFBAL, rDiGAPDH and rDiGAL bind plasminogen and that this binding is
directly proportional to the amount of plasminogen.

Figure 7. Plasminogen binding to 0.5 µg of rDiACT (A), rDiFBAL (B), rDiGAPDH (C) or rDiGAL (D)
analyzed over a range of plasminogen amounts using a microtiter plate method. (■) Incubation with
increasing amounts of plasminogen, 0–3 µg. (▲) Competition assay with 50 mM εACA included during
plasminogen incubation. (●) Wells coated with BSA used as negative control. Each point is the mean ±
SD from three independent experiments. The asterisk (*) designates significant (p < 0.05) differences.

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 ABSTRACT

Comparing the results obtained, rDiACT and rDiFBAL showed higher

plasminogen-binding capacity than rDiGAPDH, being rDiGAL the protein with less
binding capacity (Figure 7). The negative control consisting of wells coated only with
BSA showed some non-specific binding activity, but always with values significantly
lowers than those obtained by recombinant proteins (p < 0.05). To determine whether or
not lysine residues are involved in binding, a competition experiment including 50 mM
εACA was carried out. In this case, the binding was inhibited about 90% in the case of
rDiFBAL and rDiGAPDH and approximately 70% in the case of rDiACT and rDiGAL,
resulting in slightly higher optical densities than the negative control (Figure 7).

Figure 8. Plasminogen activation and plasmin generation by rDiACT (A), rDiFBAL (B), rDiGAPDH (C)
or rDiGAL (D). (□) 15 ng of tPA was added to mixtures which contained 2 µg of human plasminogen, 3
µg of D-Val-Leu-Lys 4-nitroanilide dihydrochloride (Sigma) and 1 µg of each recombinant protein (or
BSA as negative control) in the presence or absence of 50 mM of εACA in a test volume of 100 µl. (■)
Reaction mixtures in absence of tPA. Each point is the mean ± SD from three independent experiments.
The asterisk (*) designates significant (p < 0.05) differences.

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 ABSTRACT

In order to assess the ability of rDiACT, rDiFBAL, rDiGAPDH and rDiGAL to

activate plasminogen and generate plasmin on their own, the amidolytic activity of
plasmin generated in the presence or absence of tPA was measured. Negative controls
replacing each recombinant protein for BSA or tPA were also used. Figure 8 shows the
capacity of rDiACT, rDiFBAL, rDiGAPDH and rDiGAL to stimulate plasmin
generation by tPA obtaining optical densities significantly higher than the negative
controls (p < 0.05). rDiACT and rDiFBAL obtained higher optical densities than those
obtained by rDiGAPDH and rDiGAL and plasminogen-activation did not occur in the
absence of tPA. Furthermore this effect is inhibited by 50 mM εACA, indicating the
involvement of lysine residues in the process.

7. Effect of rDiACT, rDiFBAL, rDiGAPDH and rDiGAL on the fibrinolytic system

activators (tPA and uPA) expression in CnAOEC

To study the possible effect of rDiACT, rDiFBAL, rDiGAPDH and rDiGAL on

the expression of the main activators of fibrinolysis (tPA and uPA), the parasitic
proteins were employed to stimulate CnAOEC in culture. Following a similar procedure
to that described in section 3 of this abstract. Proteins from
rDiACT/rDiFBAL/rDiGAPDH/rDiGAL-treated or untreated vascular endothelial cell
extracts were separated by SDS-PAGE and analyzed by Western blotting using anti-tPA
or anti-uPA antibodies. rDiACT and rDiFBAL induced a significant increase in the
expression of the main fibrinolytic activators tPA in cultured endothelial cells (p ˂ 0.05)
(Figure 9A), being this increase slightly higher in the case of stimulation with
rDiFBAL. Significant differences in the expression of tPA in CnAOEC between
rDiGAPDH/rDiGAL-treated or untreated cultures were not found (Figure 9B). On the
other hand, all the proteins induced a marked increase in the expression of uPA in
CnAOEC cultures (p ˂ 0.05) (Figures 9C and 9D). This increase was greater in the case
of the rDiFBAL stimulation, rDiACT and rDiGAPDH showed similar optical density
values, whereas rDiGAL showed the lowest differences.

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 ABSTRACT

Figure 9. Effect of rDiACT, rDiFBAL, rDiGAPDH and rDiGAL on the expression of the fibrinolytic
system activators tPA (A and B) and uPA (C and D) in canine vascular endothelial cells. Protein extracts
from lysed rDiACT, rDiFBAL, rDiGAPDH or rDiGAL treated or untreated confluent cell cultures were
analyzed by Western blot for tPA and uPA. α-tubulin served as a protein control. Data are shown as
representative images or means ± SD from three independent experiments. The asterisk (*) designates
significant (p < 0.05) differences from control cells. (■) Stimulated cells with 1µg/ml of rDiACT. (■)
Stimulated cells with 1µg/ml of rDiFBAL. (■) Stimulated cells with 1µg/ml of rDiGAPDH. (■)
Stimulated cells with 1µg/ml of rDiGAL. (■) Non-treated control cells.

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8. Immunolocalization of DiACT, DiFBAL, DiGAPDH and DiGAL

The immunolocalization of proteins was conducted in order to know if these

were localized in tissues in contact with the blood of the host. This fact is essential, so
that the interaction of these proteins with the fibrinolytic system may have relevance “in
vivo”.

Figure 10. Immunolocalization of DiACT, DiFBAL, DiGAPDH and DiGAL in sections from D. immitis
adult worms. Representative images from three independent experiments of parasite sections incubated
with phalloidin-Alexa Fluor 488 (in green, specific binding to ACT) plus the negative or the anti-
rDiFBAL, anti-rDiGAPDH or anti-rDiGAL rabbit serum and an anti-rabbit IgG-Alexa Fluor 594 (in red).
Corresponding transmitted light images are also addressed. Magnification 4X.

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Thus, the anatomical localization of selected proteins was carried out in

histological sections of D. immitis adult worms by immunofluorescence using a
commercially available high-affinity ligand (in the case of DiACT) and rabbit
polyclonal antiserum (in the case of DiFBAL, DiGAPDH and DiGAL). As shown in
Figure 10, all sections showed green fluorescence throughout the soma of the parasite,
as a result of the binding of phalloidin-Alexa Fluor 488, actin high-affinity ligand which
serves also as a positive control of the technique. Sections incubated with the anti-
rDiFBAL, anti-rDiGAPDH or anti-rDiGAL antiserum respectively showed, in addition,
specific reactivity (in red) against the parasitic FBAL, GAPDH or GAL due to the
binding of the anti-rabbit IgG antibody conjugated to Alexa Fluor 594. All proteins are
located scattered throughout all the soma, being especially abundant in the cuticle
(reflected by an orange color in the overlay of Phalloidin-Alexa Fluor 488 + Alexa
Fluor 594 images). Sections incubated with a rabbit negative serum showed no specific
red fluorescence from recombinant proteins.

9. Reagents and stimulation of CnAOEC and CnAOSMC

To analyze the effects of plasmin generated by the interaction of plasminogen-

binding proteins of D. immitis with the fibrinolytic system of the host on the
proliferation and cell migration, as well as the destruction of extracellular matrix
(ECM), we use the previously optimized CnAOEC and CnAOSMC cultures. In
addition, the excretory/secretory extract of proteins from D. immitis (DiES) and the two
recombinant proteins, whose optical density levels in experiments of binding of
plasminogen, enhancement of plasmin generation and stimulation on the expression of
the fibrinolytic activators had been higher, were employed.

CnAOEC and CnAOSMC were grown for 4 days to obtain confluent cultures
and were treated with 1 µg/ml of DiES, rDiACT or rDiFBAL, 10 µg/ml of plasminogen
(PLG) (Acris Antibodies) or with a mixture of both treatments (DiES + PLG, rDiACT +
PLG or rDiFBAL + PLG). Untreated cells and cells treated with
DiES/rDiACT/rDiFBAL + PLG + 50 mM of εACA as an inhibitor of plasminogen
activation were used as control cells under the same conditions.

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10. DiES and rDiACT, but not rDiFBAL, produces proliferation of CnAOEC and
CnAOSMC via PLG/plasmin system

Cell proliferation was analyzed by crystal violet nuclei staining over 10 days
determining the number of viable cells. Cells were plated on 24-well plates to a density
of 104 CnAOEC/well or 1.5 x 104 CnAOSMC/well, allowed to attach overnight, rinsed,
fixed and stained. After staining, the absorbance of the samples was measured at 595
nm and transformed to “number of viable cells” using a curve that correlated
absorbance and number of endothelial or smooth muscle cells previously determined.

Both cultures showed typical curves of cell growth in all experimental groups
with a progressive growth between days 0 and 6 or 8 post-treatment, experiencing cell
death and an evident decrease of viable cells from there until the end of the experiment
(day 10 post-treatment) (Figure 11). Crystal violet staining showed an increase
significantly greater in the number of viable cells in cultures stimulated with DiES +
PLG (on day 8 post-treatment, p < 0.05) and with rDiACT + PLG [on days 4 and 6
post-treatment (CnAOEC) or day 8 post-treatment (CnAOSMC), p < 0.05] than that
showed by other experimental groups, indicating that these treatments stimulates the
proliferation of CnAOEC and CnAOSMC in culture. Significant differences in cell
proliferation between cells stimulated with rDiFBAL + PLG and other experimental
groups were not found in both types of cultures (Figure 11).

11. DiES, rDiACT and rDiFBAL produce migration of CnAOEC and CnAOSMC
via PLG/plasmin system

A Wound Healing assay was performed to assess migration of endothelial

(Figure 12) and smooth muscle cells (Figure 13). The quantification was carried out by
measuring the distance of migration in comparison with negative control (untreated
cells) to 8 hours post-treatment. In both CnAOEC and CnAOSMC cultures a significant
increase of cell migration after stimulation with DiES + PLG, rDiACT + PLG or
rDiFBAL + PLG with respect to the other experimental groups (p < 0.05) occurred.
This increase was higher in cultured endothelial cells in the case of DiES + PLG
treatment and in cultured smooth muscle cells in the case of rDiACT/rDiFBAL + PLG
treatment. After comparing the effect of both parasitic proteins, rDiACT showed higher

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values of migration ability in both types of cell cultures with respect to those obtained
by rDiFBAL.

Figure 11. Cell proliferation assay performed by the crystal violet technique measuring cell viability over
a 10 days period. The experiment was carried out in canine endothelial (A, C and E) and smooth muscle
cells (B, D and F) untreated ( ) or treated with 1 µg/ml of DiES, rDiACT or rDiFBAL + 10 µg/ml of
PLG ( ), 1 µg/ml of DiES, rDiACT or rDiFBAL ( ), 10 µg/ml of PLG ( ), or with 1 µg/ml of DiES,
rDiACT or rDiFBAL + 10 µg/ml of PLG + 50 mM of the εACA ( ). Results were expressed as number
of viable cells (x 10,000). Each point is the mean ± SD from three independent experiments. The asterisk
(*) designates significant (p < 0.05) differences between DiES/rDiACT + PLG treatment and control
groups.

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Figure 12. CnAOEC migration by Wound-Healing assay. Confluent cell cultures were wounded post-
treatment and migration distances were measured at 8 hours. The experiment was carried out in canine
endothelial cells untreated or treated with 10 µg/ml of PLG, 1 µg/ml of DiES/rDiACT/rDiFBAL (A/B/C),
1 µg/ml of DiES/rDiACT/rDiFBAL + 10 µg/ml of PLG (A/B/C) or with 1 µg/ml of
DiES/rDiACT/rDiFBAL + 10 µg/ml of PLG + 50 mM of the εACA (A/B/C). The results were expressed
as percentage of the migration ability of the negative control cells (100%). Data are shown as
representative images or means ± SD from three independent experiments. The asterisk (*) designates
significant (p < 0.05) differences between DiES/rDiACT/rDiFBAL + PLG treatment and control groups.

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 ABSTRACT

Figure 13. CnAOSMC migration by Wound-Healing assay. Confluent cell cultures were wounded post-
treatment and migration distances were measured at 8 hours. The experiment was carried out in canine
endothelial cells untreated or treated with 10 µg/ml of PLG, 1 µg/ml of DiES/rDiACT/rDiFBAL (A/B/C),
1 µg/ml of DiES/rDiACT/rDiFBAL + 10 µg/ml of PLG (A/B/C) or with 1 µg/ml of
DiES/rDiACT/rDiFBAL + 10 µg/ml of PLG + 50 mM of the εACA (A/B/C). The results were expressed
as percentage of the migration ability of the negative control cells (100%). Data are shown as
representative images or means ± SD from three independent experiments. The asterisk (*) designates
significant (p < 0.05) differences between DiES/rDiACT/rDiFBAL + PLG treatment and control groups.

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12. DiES, rDiACT and rDiFBAL produce ECM degradation of CnAOEC and
CnAOSMC via PLG/plasmin system

To examine ECM degradation, Type I Collagen in the culture supernatant of

treated and untreated CnAOEC and CnAOSMC were measured by ELISA (Figure 14).
A lower concentration of Type I Collagen and therefore a further degradation of the
secreted collagen by the cells was observed in the CnAOEC and CnAOSMC stimulated
with DiES + PLG, rDiACT + PLG or rDiFBAL + PLG than that obtained by the control
cells (p < 0.05). There were no large differences between these treatments
(DiES/rDiACT/rDiFBAL + PLG) but always a further degradation of Type I Collagen
in cultured smooth muscle cells (Figures 14B and 14D) than in endothelial cells
(Figures 14A and 14C) was found.

Figure 14. Type I Collagen degradation measured in culture supernatants from canine endothelial (A and
C) and smooth muscle cells (B and D) untreated or treated with 10 µg/ml of PLG, 1 µg/ml of
DiES/rDiACT/rDiFBAL, 1 µg/ml of DiES/rDiACT/rDiFBAL + 10 µg/ml of PLG or with 1 µg/ml of
DiES/rDiACT/rDiFBAL + 10 µg/ml of PLG + 50 mM of the εACA. The results were expressed as
percentage of the Type I Collagen concentration in the culture supernatant from negative control cells
(100%). Each point is the mean ± SD from three independent experiments. The asterisk (*) designates
significant (p < 0.05) differences between DiES/rDiACT/rDiFBAL + PLG treatment and control groups.

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 ABSTRACT

In addition, the same culture media from treated and untreated cells was analyzed
with gelatin zymography for metalloproteinase-2 (MMP-2) and metalloproteinase-9
(MMP-9) levels (Figure 15). Media samples employed in the collagen degradation assays
were electrophoresed on polyacrylamide gel copolymerized with gelatin. The gels were
washed in 2.5% Triton X-100, incubated at 37 °C in agitation in an enzymatic activation
buffer and stained with Coomassie blue.

Figure 15. Representative zymography of MMP-2 (solid bars) and MMP-9 (hatched bars) levels in the
culture supernatants from canine endothelial (A and C) and smooth muscle cells (B and D) untreated or
treated with 10 µg/ml of PLG, 1 µg/ml of DiES/rDiACT/rDiFBAL, 1 µg/ml of DiES/rDiACT/rDiFBAL
+ 10 µg/ml of PLG or with 1 µg/ml of DiES/rDiACT/rDiFBAL + 10 µg/ml of PLG + 50 mM of the
εACA. Note the gelatinolytic bands associated with MMP-2 (72 KDa) and MMP-9 (92 KDa) levels, as
well as with the MMP-9 activated form (marked with a white asterisk at 82KDa. The results were
expressed as percentage of the MMPs levels in the culture supernatant from negative control cells
(100%). Data are shown as representative images or means ± SD from three independent experiments.
The asterisk (*) designates significant (p < 0.05) differences between DiES/rDiACT/rDiFBAL + PLG
treatment and control groups.

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The positivity was assessed as appearance of clear bands on a dark background

with molecular weights of 72 kDa (MMP-2) and 92 kDa (MMP-9). The radius of the
MMPs levels was calculated after measuring the density of the existing bands, which is
directly proportional to the amount of gelatin degraded into the gel. Treatment with DiES,
rDiACT or rDiFBAL + PLG shows a significantly higher MMP-2 level in the CnAOEC
and CnAOSMC culture media (p < 0.05) (Figure 15). The results also show a significantly
higher level of MMP-9 in the CnAOEC treated with rDiACT or rDiFBAL + PLG than that
obtained in the control cells (p < 0.05) (Figure 15C). In addition, treatment with rDiFBAL
+ PLG shows an activation of the latent form of the MMP-9 in the CnAOEC culture media
(show by a clear band of 82 kDa), which does not appear with other treatments (Figure
15C). No significant differences in the MMP-9 levels in the culture media of both types of
cultures in the case of DiES experiment (Figures 15A and 15B) or in smooth muscle cells
cultures in the case of recombinant proteins experiments (Figure 15D) were observed.

CONCLUSIONS

D. immitis produces chronic infections characterized by the persistence of its

adult worms in the vascular system of its host. There, the parasites are exposed to a
wide range of defense mechanisms which are highly aggressive for their integrity. One
of these mechanisms is the generation of thromboembolisms, a process physiologically
regulated by the fibrinolytic system, whose final product, plasmin, is capable of
degrading fibrin clots.

In the present doctoral dissertation, we demonstrate how D. immitis is capable of

modifying this route towards the generation of plasmin through the use of different
antigens of the host/parasite interface. This fact would imply a benefit for both the host
and the parasite, as it would enable them to maintain an antithrombotic state in the
immediate vascular medium of D. immitis. Due to this fact and given that plasmin has
been related with the lysis of extra-cytoplasmic matrices, a fact which has been
interpreted as a mechanism related to cell invasion and intra-organic migration, the
activation of fibrinolysis has been historically considered as a beneficial mechanism for
the survival of blood-borne pathogens and also for its invasive capacity.

Nevertheless, the great number of substrata on which plasmin can carry out its
proteolytic function has shown, in other contexts, the implication of the activation of the

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fibrinolytic system in different pathological processes on a vascular level. We

demonstrate a direct relationship between the plasmin derived from the pro-fibrinolytic
capacity of the antigens of D. immitis with the proliferation and migration of the host’s
cells located in the arterial wall; as well as the degradation of the extracellular matrix in
an “in vitro” model. These mechanisms are directly related with proliferative
endarteritis, a key pathological process in the development of the subsequent pulmonary
and cardiac pathology in cardiopulmonary dirofilariosis, resulting in the formation of
intravascular microvilli.

From the findings obtained in the present doctoral dissertation, we derive the
following conclusions:

1. D. immitis activates the fibrinolytic system of its host through the joint action of
its excretory/secretory and surface antigens. This could be used by the parasite
to displace the fibrinolytic balance towards the generation of plasmin, which
would imply a change of the survival mechanism of the parasite in order to
control the formation of clots in its immediate intravascular habitat.

2. Ten and 11 plasminogen-binding proteins are respectively identified in the

excretory/secretory and surface antigenic compartments of the parasite. Of these,
actin, fructose-bisphosphate aldolase, glyceraldehyde 3-phosphate
dehydrogenase and galectin of D. immitis have been produced in their
recombinant form, individually demonstrating their pro-fibrynolitic properties.
All of them, in a greater or lesser extent, are capable of binding plasminogen and
enhancing the generation of plasmin through the implication of its lysine
residues. Furthermore, they are capable of stimulating the expression of
fibrinolytic activators in cultures of canine endothelial cells, and are located in
the host/D. immitis interface.

3. Plasmin, a product of the fibrinolytic activation caused by the antigens of D.

immitis, participates in the generation of the pathologic processes described in
the apparition of proliferative endarteritis in cardiopulmonary dirofilariosis. This
includes the proliferation and migration of the arterial wall cells, as well as the
degradation of the extracellular matrix, all these facts being demonstrated

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through the use of both the parasite’s excretory/secretory antigens and the two
studied proteins with a higher pro-fibrinolytic capacity in an “in vitro” model.

4. The results obtained in the present doctoral dissertation contribute to the

understanding of a very complex part of the relationships between parasite and
host on a molecular level in cardiopulmonary dirofilariosis, demonstrating for
the first time how a process related to the survival of the parasite can unleash
pathogenic mechanisms of great importance. Given that the capacity of binding
plasminogen and enhancing the generation of plasmin has been demonstrated in
the proteins of many pathogens, and taking into account the high degree of
evolutionary conservation of some of the studied antigens, similar mechanisms
could occur in other infections caused by blood-borne pathogens capable of
developing chronic processes.

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