In acute coronary syndrome (ACS) cardiac cell damage is preceded by thrombosis. Therefore, plasma... more In acute coronary syndrome (ACS) cardiac cell damage is preceded by thrombosis. Therefore, plasma coagulation markers may have additional diagnostic relevance in ACS. By using novel coagulation assays this study aims to gain more insight into the relationship between the coagulation system and ACS. We measured plasma thrombin generation, factor XIa and D-dimer levels in plasma from ACS (n = 104) and non-ACS patients (n = 42). Follow-up measurements (n = 73) were performed at 1 and 6 months. Associations between coagulation markers and recurrent cardiovascular events were calculated by logistic regression analysis. Thrombin generation was significantly enhanced in ACS compared to non-ACS patients: peak height 148±53 vs. 122±42 nM. There was a significantly diminished ETP reduction (32 vs. 41%) and increased intrinsic coagulation activation (25 vs. 7%) in ACS compared to non-ACS patients. Furthermore, compared to non-ACS patients factor XIa and D-dimer levels were significantly elevat...
The use of mouse models for the study of thrombotic disorders has gained increasing importance. M... more The use of mouse models for the study of thrombotic disorders has gained increasing importance. Methods for measurement of coagulation activation in mice are, however, scarce. The primary aim of this study was to develop a specific mouse thrombin-antithrombin (TAT) ELISA for measurement of coagulation activation and to compare it with two commercially available assays for human TAT complexes. In addition, we aimed to improve methods for mouse plasma anticoagulation and preparation.
In a clinical setting, fresh frozen plasma (FFP) is transfused to diluted patients with complicat... more In a clinical setting, fresh frozen plasma (FFP) is transfused to diluted patients with complicated surgery or trauma, as guided by prolonged conventional coagulation times or low fibrinogen levels. However, the limited sensitivity of these coagulation tests may restrict their use in measuring the effect of transfusion and hence predicting the risk of perioperative bleeding. We used the more sensitive, calibrated automated thrombogram (CAT) method to evaluate the result of therapeutic FFP transfusion to 51 patients with dilutional coagulopathy. Thrombin generation was measured in pre- and post-transfusion plasma samples in the presence of either platelets or phospholipids. For all patients, the transfusion led to higher plasma coagulation factor levels, a shortened activated partial thromboplastin time, and a significant increase in thrombin generation (peak height and endogenous thrombin potential). Interestingly, thrombin generation parameters and fibrinogen levels were higher in post-transfusion plasmas from patients who stopped bleeding (n = 32) than for patients with ongoing bleeding (n = 19). Plasmas from 15 of the 19 patients with ongoing bleeding were markedly low in either thrombin generation or fibrinogen level. We conclude that the thrombin generation method detects improved haemostatic activity after plasma transfusion. Furthermore, the data suggest that thrombin generation and fibrinogen are independent determinants of the risk of perioperative bleeding in this patient group.
Background: Exposure to ambient particulate matter (PM) has been associated with adverse cardiova... more Background: Exposure to ambient particulate matter (PM) has been associated with adverse cardiovascular effects in epidemiological studies. Current knowledge of independent effects of individual PM characteristics remains limited.
Background: Thrombin is a key protease in coagulation also implicated in complex pathology includ... more Background: Thrombin is a key protease in coagulation also implicated in complex pathology including atherosclerosis. To address the role of thrombin in relation to myocardial infarction (MI) we explored thrombin generation analysis in plasma from patients and controls that had participated in the Glasgow MI Study (GLAMIS).
Resistance to activated protein C (APC) is often associated with a mutation in factor V (factor V... more Resistance to activated protein C (APC) is often associated with a mutation in factor V (factor V Leiden ). Individuals without factor V Leiden who exhibit a response in functional APC-resistance tests similar to that of carriers of factor V Leiden are considered to be acquired APC resistant. This phenomenon is particularly observed in women using oral contraceptives (OC).
Biochimica et Biophysica Acta (BBA) - Molecular Cell Research, 2001
Store-regulated Ca 2 entry (SOCE) is an important mechanism of elevating cytosolic [Ca 2 ] i in p... more Store-regulated Ca 2 entry (SOCE) is an important mechanism of elevating cytosolic [Ca 2 ] i in platelets, though the Ca 2 influx channels involved are still unclear. We screened human platelets and their precursor cells (human stem cells and megakaryocytes) for the presence of candidate influx channels, i.e., isoforms of the Trp family of proteins. Primary stem cells were cultured with thrombopoietin to allow differentiation into megakaryocytes. The undifferentiated stem cells (CD34 ) showed mRNA expression of only a spliced variant Trp1A. Immature (CD61 /CD42b low ) and mature (CD61 /CD42b high ) megakaryocytes as well as platelets expressed in addition unspliced Trp1 as well as Trp4 (less abundant) and Trp6 isoforms. This unspliced isoform appeared to be specific for cells of the megakaryocyte/platelet lineage, since immature (CD14 / CD61 3 /CD42b 3 ) and mature monocytes expressed only the Trp1A isoform. This conclusion was confirmed by the presence of Trp1A, 3, 4 and 6 transcripts in the immature megakaryocytic Dami cell line, and of Trp1, 1A, 4 and 6 transcripts in the more mature CHRF-288 cell line. The up-regulation of Trp1, 4 and 6 in the lineage from primary stem cells to mature megakaryocytes and platelets was accompanied by increased influx of extracellular Ca 2 after pretreatment of the cells with thapsigargin or thrombin. Expression of new Trp isoforms in the differentiated cells is thus accompanied by increased SOCE. ß
Coronary artery bypass grafting (CABG) with cardiopulmonary bypass (CPB) is frequently associated... more Coronary artery bypass grafting (CABG) with cardiopulmonary bypass (CPB) is frequently associated with low platelet count (PC) and disturbed platelet function (PF). While PC is easy to measure, PF is more difficult to assess. Moreover, the time-related platelet dysfunction and recovery after CPB is not fully elucidated. Platelet dysfunction could lead to bleeding but also to coronary graft failure. Laboratory tests could provide more insights into PF after CABG. The aim of the current study was to investigate the time-related PF induced by CPB. Blood samples of 20 patients with a preoperative PC of more than 250 × 10(9)/L were collected before incision, after weaning from CPB, and 24 h postoperative. Platelet contribution to coagulation was quantified by PLTEM (calculated by means of EXTEM and FIBTEM results). PF was assessed by multiple electrode impedance aggregometry (MEIA) in whole blood and by light transmission aggregometry (LTA) in platelet-rich plasma after stimulation with arachidonic acid (AA), adenosine diphosphate, collagen, and thrombin-receptor-activating peptide. LTA and MEIA analysis demonstrated significant platelet dysfunction after CPB, with partial recovery within 24 h after surgery. AA-induced platelet aggregation increased to higher levels within 24 h after surgery compared to baseline values as measured by LTA. PLTEM maximum clot firmness remained unchanged throughout the study. Correlation analyses revealed that MEIA and rotational thromboelastometry (ROTEM), but not LTA, were dependent on PC and hematocrit. No correlations were found between LTA, MEIA, ROTEM, PC, and clinical outcome parameters. Our results demonstrate a reversible platelet dysfunction recovering within 24 h after CPB. Interestingly, AA-induced platelet aggregation increases to higher levels during the first 24 h postoperatively, which might be important for early initiation of antiplatelet therapy after CABG. MEIA as POC test is able to detect platelet dysfunction during cardiac surgery with a PC of ≥150 × 10(9)/L.
5th ICTHIC Abstracts: Poster Sessions / Thrombosis Research 125 (2010) S166-S191 thrombogram, CAT... more 5th ICTHIC Abstracts: Poster Sessions / Thrombosis Research 125 (2010) S166-S191 thrombogram, CAT) and the PCA (by the P-PPL/1 assay), of plasma MP in 15 ET (5M/10F) and 15 matched control (C) (5M/10F) subjects. Materials and Methods: Platelet Free Plasma (PFP) was obtained by two serial centrifugations (4,000 rpm for 15 min, 11,000 rpm for 10 min) and MP-free plasma (MP-FP) by re-centrifuging PFP at 14,000 rpm for 30 min. MP were isolated from the pellet. For CAT assay, 80 ml of P-FP or MP-FP and 20 ml of buffer were mixed, and TG started adding CaCl2. The results were expressed as LagTime, Peak, area under the curve (ETP), and Time-to-Peak (ttPeak). For P-PPL/1 assay, 100 ml of P-FP or MP-FP were mixed with 50 ml of phospholipid-depleted plasma. Clotting was started by adding FXa and CaCl2 and results expressed in seconds. Results: PFP from ET patients produced significantly (p < 0.05) higher TG (LagTime: 14.8±3.5 min) and PCA (76.7±8.1 sec) then controls (LagTime: 20.5±7.9 min; PCA: 89.2±7.6 sec). This increase was due to the presence of MP, as no TG and PCA was observed in MP-FP from both patients and controls. The addition of isolated MP to autologous MP-FP restored the TG and PCA of the samples, which was identical to the original values of PFP for both assays. There was a significant correlation between PCA by the P-PPL/1 assay and the different parameters of TG assay [lagtime (R2 = 0.749), Peak (R2 = −0.809), ETP (R2 = −0.745) and TTPeak (R2 = −0.773)]. Conclusions: The hypercoagulable state of ET patients is characterized by an increased MP-associated PCA. Prospective studies are needed to evaluate whether MP-associated TG and PCA can predict for thrombosis in these patients.
Inhibition of specific coagulation pathways such as the factor VIIa-tissue factor complex has bee... more Inhibition of specific coagulation pathways such as the factor VIIa-tissue factor complex has been shown to attenuate ischemia/reperfusion (I/R) injury, but the cellular mechanisms have not been explored. To determine the cellular mechanisms involved in the working mechanism of active site inhibited factor VIIa (ASIS) in the protection against myocardial I/R injury. We investigated the effects of a specific mouse recombinant in a mouse model of myocardial I/R injury. One hour of ischemia was followed by 2, 6 or 24 h of reperfusion. Mouse ASIS or placebo was administered before and after induction of reperfusion. ASIS administration reduced myocardial I/R injury by more than 40% at three reperfusion times. Multiplex ligation dependent probe amplification (MLPA) analysis showed reduced mRNA expression in the ischemic myocardium of CD14, TLR-4, interleukin-1 (IL-1) receptor-associated kinase (IRAK) and IkappaBalpha upon ASIS administration, indicative of inhibition of toll-like receptor-4 (TLR-4) and subsequent nuclear factor-kappaB (NF-kappaB) mediated cell signaling. Levels of nuclear activated NF-kappaB and proteins influenced by the NF-kappaB pathway including tissue factor (TF) and IL-6 that were increased after I/R, were attenuated upon ASIS administration. After 6 and 24 h of reperfusion, neutrophil infiltration into the area of infarction was decreased upon ASIS administration. There was, however, no evidence of an effect of ASIS on apoptosis (Tunel staining and MLPA analysis). We conclude that the diminished amount of myocardial I/R injury after ASIS administration is primarily due to attenuated inflammation-related lethal I/R injury, probably mediated through the NF-kappaB mechanism.
To cite this article: Dielis AWJH, Castoldi E, Spronk HMH, van Oerle R, Hamulyá k K, ten Cate H, ... more To cite this article: Dielis AWJH, Castoldi E, Spronk HMH, van Oerle R, Hamulyá k K, ten Cate H, Rosing J. Coagulation factors and the protein C system as determinants of thrombin generation in a normal population. J Thromb Haemost 2008; 6: 125-31.
Thrombin generation monitoring has the potential to be used as a clinical diagnostic tool in the ... more Thrombin generation monitoring has the potential to be used as a clinical diagnostic tool in the near future. However, robust preanalytical conditions may be required, and one factor that has been reported is in-vitro contact activation that might influence in-vitro measurements of thrombin generation and thereby act as an unpredictable pre-analytical variable. The aim of the current study was to investigate the influence of contact activation and the necessity of corn trypsin inhibitor (CTI) to abolish contact activation in thrombin generation measurements at low tissue factor (TF) concentrations. Thrombin generation was performed using the calibrated automated thrombinoscopy (CAT), thereby determining the endogenous thrombin potential (ETP), peak height, and the lag time, in plasma obtained from healthy volunteers. Addition of CTI after plasma preparation had no significant influence on thrombin generation triggered with 0.5 pM TF or higher, as demonstrated by unaltered ETP and lag time values between analyses with and without CTI. Addition of CTI before blood collection reduced thrombin generation triggered with 0.5 pM TF: both the ETP and peak height were significantly reduced compared to no CTI addition. In contrast, thrombin generation remained unaltered at a 1 pM TF trigger or above. This study demonstrates that addition of CTI after plasma separation is not necessary when triggering with TF concentrations of 0.5 pM and higher. Furthermore, it was demonstrated that it is not needed to pre-fill blood collecting tubes with CTI when measuring thrombin generation at TF concentrations of &amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;gt;/=1 pM.
Background: Familial combined hyperlipidemia (FCHL) is characterized by a varied expression of hy... more Background: Familial combined hyperlipidemia (FCHL) is characterized by a varied expression of hypertriglyceridemia and hypercholesterolemia within a family, and a high risk of premature coronary artery disease. The present study evaluated a number of potential prothrombotic markers in familial combined hyperlipidemia, and studied their relationship to the hypercholesterolemic (Fredrickson type IIa) and hypertriglyceridemic (IIb and IV) phenotypes. Methods and results: Selected prothrombotic markers were studied in 68 subjects: 34 hyperlipidemic subjects with familial combined hyperlipidemia and 34 controls. FCHL patients exhibited significantly higher Thrombin-Antithrombin complex (TAT), activated coagulation factor XII (F XIIa), von Willebrand Factor (vWF), Plasminogen Activator Inhibitor-1 (PAI-1) and tissue derived Plasminogen Activator (t-PA) values in comparison to controls. Within the subgroup of familial combined hyperlipidemia subjects, elevated PAI-1 activity and soluble Thrombomodulin levels were particularly associated with features of the metabolic syndrome, including hyperinsulinemia, hypertriglyceridemia and predominance of small dense low density lipoprotein (LDL). Conclusions: A general pattern of activated blood coagulation and endothelial activation is present in all hyperlipidemic subjects studied, independent of metabolic phenotype. In those familial combined hyperlipidemia subjects with features of the metabolic syndrome, impaired fibrinolysis can provide an additional cardiovascular risk factor.
Thus far, validated whole blood assays used in in vitro fibrinolysis experiments using thromboela... more Thus far, validated whole blood assays used in in vitro fibrinolysis experiments using thromboelastometry (ROTEM) are lacking or have yet to be tested in humans. The objective was first, to establish a standardized modified ROTEM approach to detect both hypo- and hyperfibrinolysis. And second, to perform a technical and clinical validation of the assay. Blood was used of healthy volunteers, patients with sepsis, patients after cardiothoracic surgery, pregnant women, and cirrhotic liver disease patients. A whole blood tissue factor (TF) activated ROTEM assay with and without the addition of recombinant tissue plasminogen activator (rTPA) was developed. Plasma fibrinolysis determinants were measured in all volunteers and patients. Thirty five pM TF and additions of 125 and 175 ng/ml rTPA resulted in full lysis within 60 min in healthy volunteers. Coefficients of variation were below 10 % without and below 20 % with rTPA addition. In sepsis the hypofibrinolytic ROTEM profiles with 175 ...
In acute coronary syndrome (ACS) cardiac cell damage is preceded by thrombosis. Therefore, plasma... more In acute coronary syndrome (ACS) cardiac cell damage is preceded by thrombosis. Therefore, plasma coagulation markers may have additional diagnostic relevance in ACS. By using novel coagulation assays this study aims to gain more insight into the relationship between the coagulation system and ACS. We measured plasma thrombin generation, factor XIa and D-dimer levels in plasma from ACS (n = 104) and non-ACS patients (n = 42). Follow-up measurements (n = 73) were performed at 1 and 6 months. Associations between coagulation markers and recurrent cardiovascular events were calculated by logistic regression analysis. Thrombin generation was significantly enhanced in ACS compared to non-ACS patients: peak height 148±53 vs. 122±42 nM. There was a significantly diminished ETP reduction (32 vs. 41%) and increased intrinsic coagulation activation (25 vs. 7%) in ACS compared to non-ACS patients. Furthermore, compared to non-ACS patients factor XIa and D-dimer levels were significantly elevat...
The use of mouse models for the study of thrombotic disorders has gained increasing importance. M... more The use of mouse models for the study of thrombotic disorders has gained increasing importance. Methods for measurement of coagulation activation in mice are, however, scarce. The primary aim of this study was to develop a specific mouse thrombin-antithrombin (TAT) ELISA for measurement of coagulation activation and to compare it with two commercially available assays for human TAT complexes. In addition, we aimed to improve methods for mouse plasma anticoagulation and preparation.
In a clinical setting, fresh frozen plasma (FFP) is transfused to diluted patients with complicat... more In a clinical setting, fresh frozen plasma (FFP) is transfused to diluted patients with complicated surgery or trauma, as guided by prolonged conventional coagulation times or low fibrinogen levels. However, the limited sensitivity of these coagulation tests may restrict their use in measuring the effect of transfusion and hence predicting the risk of perioperative bleeding. We used the more sensitive, calibrated automated thrombogram (CAT) method to evaluate the result of therapeutic FFP transfusion to 51 patients with dilutional coagulopathy. Thrombin generation was measured in pre- and post-transfusion plasma samples in the presence of either platelets or phospholipids. For all patients, the transfusion led to higher plasma coagulation factor levels, a shortened activated partial thromboplastin time, and a significant increase in thrombin generation (peak height and endogenous thrombin potential). Interestingly, thrombin generation parameters and fibrinogen levels were higher in post-transfusion plasmas from patients who stopped bleeding (n = 32) than for patients with ongoing bleeding (n = 19). Plasmas from 15 of the 19 patients with ongoing bleeding were markedly low in either thrombin generation or fibrinogen level. We conclude that the thrombin generation method detects improved haemostatic activity after plasma transfusion. Furthermore, the data suggest that thrombin generation and fibrinogen are independent determinants of the risk of perioperative bleeding in this patient group.
Background: Exposure to ambient particulate matter (PM) has been associated with adverse cardiova... more Background: Exposure to ambient particulate matter (PM) has been associated with adverse cardiovascular effects in epidemiological studies. Current knowledge of independent effects of individual PM characteristics remains limited.
Background: Thrombin is a key protease in coagulation also implicated in complex pathology includ... more Background: Thrombin is a key protease in coagulation also implicated in complex pathology including atherosclerosis. To address the role of thrombin in relation to myocardial infarction (MI) we explored thrombin generation analysis in plasma from patients and controls that had participated in the Glasgow MI Study (GLAMIS).
Resistance to activated protein C (APC) is often associated with a mutation in factor V (factor V... more Resistance to activated protein C (APC) is often associated with a mutation in factor V (factor V Leiden ). Individuals without factor V Leiden who exhibit a response in functional APC-resistance tests similar to that of carriers of factor V Leiden are considered to be acquired APC resistant. This phenomenon is particularly observed in women using oral contraceptives (OC).
Biochimica et Biophysica Acta (BBA) - Molecular Cell Research, 2001
Store-regulated Ca 2 entry (SOCE) is an important mechanism of elevating cytosolic [Ca 2 ] i in p... more Store-regulated Ca 2 entry (SOCE) is an important mechanism of elevating cytosolic [Ca 2 ] i in platelets, though the Ca 2 influx channels involved are still unclear. We screened human platelets and their precursor cells (human stem cells and megakaryocytes) for the presence of candidate influx channels, i.e., isoforms of the Trp family of proteins. Primary stem cells were cultured with thrombopoietin to allow differentiation into megakaryocytes. The undifferentiated stem cells (CD34 ) showed mRNA expression of only a spliced variant Trp1A. Immature (CD61 /CD42b low ) and mature (CD61 /CD42b high ) megakaryocytes as well as platelets expressed in addition unspliced Trp1 as well as Trp4 (less abundant) and Trp6 isoforms. This unspliced isoform appeared to be specific for cells of the megakaryocyte/platelet lineage, since immature (CD14 / CD61 3 /CD42b 3 ) and mature monocytes expressed only the Trp1A isoform. This conclusion was confirmed by the presence of Trp1A, 3, 4 and 6 transcripts in the immature megakaryocytic Dami cell line, and of Trp1, 1A, 4 and 6 transcripts in the more mature CHRF-288 cell line. The up-regulation of Trp1, 4 and 6 in the lineage from primary stem cells to mature megakaryocytes and platelets was accompanied by increased influx of extracellular Ca 2 after pretreatment of the cells with thapsigargin or thrombin. Expression of new Trp isoforms in the differentiated cells is thus accompanied by increased SOCE. ß
Coronary artery bypass grafting (CABG) with cardiopulmonary bypass (CPB) is frequently associated... more Coronary artery bypass grafting (CABG) with cardiopulmonary bypass (CPB) is frequently associated with low platelet count (PC) and disturbed platelet function (PF). While PC is easy to measure, PF is more difficult to assess. Moreover, the time-related platelet dysfunction and recovery after CPB is not fully elucidated. Platelet dysfunction could lead to bleeding but also to coronary graft failure. Laboratory tests could provide more insights into PF after CABG. The aim of the current study was to investigate the time-related PF induced by CPB. Blood samples of 20 patients with a preoperative PC of more than 250 × 10(9)/L were collected before incision, after weaning from CPB, and 24 h postoperative. Platelet contribution to coagulation was quantified by PLTEM (calculated by means of EXTEM and FIBTEM results). PF was assessed by multiple electrode impedance aggregometry (MEIA) in whole blood and by light transmission aggregometry (LTA) in platelet-rich plasma after stimulation with arachidonic acid (AA), adenosine diphosphate, collagen, and thrombin-receptor-activating peptide. LTA and MEIA analysis demonstrated significant platelet dysfunction after CPB, with partial recovery within 24 h after surgery. AA-induced platelet aggregation increased to higher levels within 24 h after surgery compared to baseline values as measured by LTA. PLTEM maximum clot firmness remained unchanged throughout the study. Correlation analyses revealed that MEIA and rotational thromboelastometry (ROTEM), but not LTA, were dependent on PC and hematocrit. No correlations were found between LTA, MEIA, ROTEM, PC, and clinical outcome parameters. Our results demonstrate a reversible platelet dysfunction recovering within 24 h after CPB. Interestingly, AA-induced platelet aggregation increases to higher levels during the first 24 h postoperatively, which might be important for early initiation of antiplatelet therapy after CABG. MEIA as POC test is able to detect platelet dysfunction during cardiac surgery with a PC of ≥150 × 10(9)/L.
5th ICTHIC Abstracts: Poster Sessions / Thrombosis Research 125 (2010) S166-S191 thrombogram, CAT... more 5th ICTHIC Abstracts: Poster Sessions / Thrombosis Research 125 (2010) S166-S191 thrombogram, CAT) and the PCA (by the P-PPL/1 assay), of plasma MP in 15 ET (5M/10F) and 15 matched control (C) (5M/10F) subjects. Materials and Methods: Platelet Free Plasma (PFP) was obtained by two serial centrifugations (4,000 rpm for 15 min, 11,000 rpm for 10 min) and MP-free plasma (MP-FP) by re-centrifuging PFP at 14,000 rpm for 30 min. MP were isolated from the pellet. For CAT assay, 80 ml of P-FP or MP-FP and 20 ml of buffer were mixed, and TG started adding CaCl2. The results were expressed as LagTime, Peak, area under the curve (ETP), and Time-to-Peak (ttPeak). For P-PPL/1 assay, 100 ml of P-FP or MP-FP were mixed with 50 ml of phospholipid-depleted plasma. Clotting was started by adding FXa and CaCl2 and results expressed in seconds. Results: PFP from ET patients produced significantly (p < 0.05) higher TG (LagTime: 14.8±3.5 min) and PCA (76.7±8.1 sec) then controls (LagTime: 20.5±7.9 min; PCA: 89.2±7.6 sec). This increase was due to the presence of MP, as no TG and PCA was observed in MP-FP from both patients and controls. The addition of isolated MP to autologous MP-FP restored the TG and PCA of the samples, which was identical to the original values of PFP for both assays. There was a significant correlation between PCA by the P-PPL/1 assay and the different parameters of TG assay [lagtime (R2 = 0.749), Peak (R2 = −0.809), ETP (R2 = −0.745) and TTPeak (R2 = −0.773)]. Conclusions: The hypercoagulable state of ET patients is characterized by an increased MP-associated PCA. Prospective studies are needed to evaluate whether MP-associated TG and PCA can predict for thrombosis in these patients.
Inhibition of specific coagulation pathways such as the factor VIIa-tissue factor complex has bee... more Inhibition of specific coagulation pathways such as the factor VIIa-tissue factor complex has been shown to attenuate ischemia/reperfusion (I/R) injury, but the cellular mechanisms have not been explored. To determine the cellular mechanisms involved in the working mechanism of active site inhibited factor VIIa (ASIS) in the protection against myocardial I/R injury. We investigated the effects of a specific mouse recombinant in a mouse model of myocardial I/R injury. One hour of ischemia was followed by 2, 6 or 24 h of reperfusion. Mouse ASIS or placebo was administered before and after induction of reperfusion. ASIS administration reduced myocardial I/R injury by more than 40% at three reperfusion times. Multiplex ligation dependent probe amplification (MLPA) analysis showed reduced mRNA expression in the ischemic myocardium of CD14, TLR-4, interleukin-1 (IL-1) receptor-associated kinase (IRAK) and IkappaBalpha upon ASIS administration, indicative of inhibition of toll-like receptor-4 (TLR-4) and subsequent nuclear factor-kappaB (NF-kappaB) mediated cell signaling. Levels of nuclear activated NF-kappaB and proteins influenced by the NF-kappaB pathway including tissue factor (TF) and IL-6 that were increased after I/R, were attenuated upon ASIS administration. After 6 and 24 h of reperfusion, neutrophil infiltration into the area of infarction was decreased upon ASIS administration. There was, however, no evidence of an effect of ASIS on apoptosis (Tunel staining and MLPA analysis). We conclude that the diminished amount of myocardial I/R injury after ASIS administration is primarily due to attenuated inflammation-related lethal I/R injury, probably mediated through the NF-kappaB mechanism.
To cite this article: Dielis AWJH, Castoldi E, Spronk HMH, van Oerle R, Hamulyá k K, ten Cate H, ... more To cite this article: Dielis AWJH, Castoldi E, Spronk HMH, van Oerle R, Hamulyá k K, ten Cate H, Rosing J. Coagulation factors and the protein C system as determinants of thrombin generation in a normal population. J Thromb Haemost 2008; 6: 125-31.
Thrombin generation monitoring has the potential to be used as a clinical diagnostic tool in the ... more Thrombin generation monitoring has the potential to be used as a clinical diagnostic tool in the near future. However, robust preanalytical conditions may be required, and one factor that has been reported is in-vitro contact activation that might influence in-vitro measurements of thrombin generation and thereby act as an unpredictable pre-analytical variable. The aim of the current study was to investigate the influence of contact activation and the necessity of corn trypsin inhibitor (CTI) to abolish contact activation in thrombin generation measurements at low tissue factor (TF) concentrations. Thrombin generation was performed using the calibrated automated thrombinoscopy (CAT), thereby determining the endogenous thrombin potential (ETP), peak height, and the lag time, in plasma obtained from healthy volunteers. Addition of CTI after plasma preparation had no significant influence on thrombin generation triggered with 0.5 pM TF or higher, as demonstrated by unaltered ETP and lag time values between analyses with and without CTI. Addition of CTI before blood collection reduced thrombin generation triggered with 0.5 pM TF: both the ETP and peak height were significantly reduced compared to no CTI addition. In contrast, thrombin generation remained unaltered at a 1 pM TF trigger or above. This study demonstrates that addition of CTI after plasma separation is not necessary when triggering with TF concentrations of 0.5 pM and higher. Furthermore, it was demonstrated that it is not needed to pre-fill blood collecting tubes with CTI when measuring thrombin generation at TF concentrations of &amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;gt;/=1 pM.
Background: Familial combined hyperlipidemia (FCHL) is characterized by a varied expression of hy... more Background: Familial combined hyperlipidemia (FCHL) is characterized by a varied expression of hypertriglyceridemia and hypercholesterolemia within a family, and a high risk of premature coronary artery disease. The present study evaluated a number of potential prothrombotic markers in familial combined hyperlipidemia, and studied their relationship to the hypercholesterolemic (Fredrickson type IIa) and hypertriglyceridemic (IIb and IV) phenotypes. Methods and results: Selected prothrombotic markers were studied in 68 subjects: 34 hyperlipidemic subjects with familial combined hyperlipidemia and 34 controls. FCHL patients exhibited significantly higher Thrombin-Antithrombin complex (TAT), activated coagulation factor XII (F XIIa), von Willebrand Factor (vWF), Plasminogen Activator Inhibitor-1 (PAI-1) and tissue derived Plasminogen Activator (t-PA) values in comparison to controls. Within the subgroup of familial combined hyperlipidemia subjects, elevated PAI-1 activity and soluble Thrombomodulin levels were particularly associated with features of the metabolic syndrome, including hyperinsulinemia, hypertriglyceridemia and predominance of small dense low density lipoprotein (LDL). Conclusions: A general pattern of activated blood coagulation and endothelial activation is present in all hyperlipidemic subjects studied, independent of metabolic phenotype. In those familial combined hyperlipidemia subjects with features of the metabolic syndrome, impaired fibrinolysis can provide an additional cardiovascular risk factor.
Thus far, validated whole blood assays used in in vitro fibrinolysis experiments using thromboela... more Thus far, validated whole blood assays used in in vitro fibrinolysis experiments using thromboelastometry (ROTEM) are lacking or have yet to be tested in humans. The objective was first, to establish a standardized modified ROTEM approach to detect both hypo- and hyperfibrinolysis. And second, to perform a technical and clinical validation of the assay. Blood was used of healthy volunteers, patients with sepsis, patients after cardiothoracic surgery, pregnant women, and cirrhotic liver disease patients. A whole blood tissue factor (TF) activated ROTEM assay with and without the addition of recombinant tissue plasminogen activator (rTPA) was developed. Plasma fibrinolysis determinants were measured in all volunteers and patients. Thirty five pM TF and additions of 125 and 175 ng/ml rTPA resulted in full lysis within 60 min in healthy volunteers. Coefficients of variation were below 10 % without and below 20 % with rTPA addition. In sepsis the hypofibrinolytic ROTEM profiles with 175 ...
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