The transcripts were made personally by me from photocopies of documents in the archives of the C... more The transcripts were made personally by me from photocopies of documents in the archives of the Claimant. Every attempt has been made to ensure that the transcripts are faithful reproductions of these photocopies. This means that typographical errors in the original documents have been retained and transcribed. Any errors of transcription are my responsibility and will be corrected on notification. A full unredacted transcript and copies of original documents will be supplied on request. Personal addresses will remain redacted.
SummaryThe major activation-inducing collagen receptor glycoprotein VI (GPVI) has been cloned wit... more SummaryThe major activation-inducing collagen receptor glycoprotein VI (GPVI) has been cloned within the last two years. It is a member of the Ig superfamily of proteins and is constitutively associated with the ITAM-bearing Fc receptor γ-chain (FcR γ-chain). GPVI signals through a pathway that involves several of the proteins used by Fc, B- and T-lymphocyte receptors and which takes place in glycolipid-enriched membrane domains in the plasma membrane known as GEMs. Responses to GPVI are regulated by PECAM-1 (CD31) and possibly other ITIM-bearing receptors. Despite a pivotal role for GPVI, there are important differences between signalling events to collagen and GPVI-specific ligands. This may reflect a role for co-receptors in the response to collagen.
Glycoprotein VI (GPVI) mediates collagen-induced platelet activation after vascular damage and is... more Glycoprotein VI (GPVI) mediates collagen-induced platelet activation after vascular damage and is an important contributor to the onset of thrombosis, heart attack, and stroke. Animal models of thrombosis have identified GPVI as a promising target for antithrombotic therapy. Although for many years the crystal structure of GPVI has been known, the essential details of its interaction with collagen have remained elusive. Here, we present crystal structures of the GPVI ectodomain bound to triple-helical collagen peptides, which reveal a collagen-binding site across the β-sheet of the D1 domain. Mutagenesis and binding studies confirm the observed binding site and identify Trp76, Arg38, and Glu40 as essential residues for binding to fibrillar collagens and collagen-related peptides (CRPs). GPVI binds a site on collagen comprising two collagen chains with the core formed by the sequence motif OGPOGP. Potent GPVI-binding peptides from Toolkit-III all contain OGPOGP; weaker binding peptid...
Background: Platelet glycoprotein (GP) Ib-IX-V supports platelet adhesion on damaged vascular wal... more Background: Platelet glycoprotein (GP) Ib-IX-V supports platelet adhesion on damaged vascular walls by binding to von Willebrand factor (VWF). For several decades it has been recognized that the a-subunit of GP (GPIba) also binds thrombin but the physiological relevance, if any, of this interaction was unknown. Previous studies have shown that a sulfated tyrosine 276 (Tyr276) is essential for thrombin binding to GPIba. Objectives: This study investigated the in vivo relevance of GPIba residue Tyr276 in hemostasis and thrombosis. Methods: Transgenic mouse colonies expressing the normal human GPIba subunit or a mutant human GPIba containing a Phe substitution for Tyr276 (hTg Y276F) were generated. Both colonies were bred to mice devoid of murine GPIba. Results: Surface-expressed GPIba levels and platelet counts were similar in both colonies. hTg Y276F platelets were significantly impaired in binding a-thrombin but displayed normal binding to type I fibrillar collagen and human VWF in the presence of ristocetin. In vivo thrombus formation as a result of chemical damage (FeCl 3) demonstrated that hTg Y276F mice have a delayed time to occlusion followed by unstable blood flow indicative of embolization. In models of laser-induced injury, thrombi developing in hTg Y276F animals were also less stable. Conclusions: The results demonstrate that GPIba residue Tyr276 is physiologically important, supporting stable thrombus formation in vivo.
Citalopram, a selective serotonin reuptake inhibitor (SSRI), inhibits platelet function in vitro.... more Citalopram, a selective serotonin reuptake inhibitor (SSRI), inhibits platelet function in vitro. We have previously shown that this action is independent of citalopram's ability to block serotonin uptake by the serotonin transporter and must therefore be mediated via distinct pharmacological mechanisms. We now report evidence for two novel and putative mechanisms of citalopram-induced platelet inhibition. Firstly, in platelets, citalopram blocked U46619-induced Rap1 activation and subsequent platelet aggregation, but failed to inhibit U46619-induced increases in cytosolic Ca 2+. Similarly, in neutrophils, citalopram inhibited Rap1 activation and downstream functions but failed to block PAF-induced Ca 2+ mobilisation. In a cell-free system, citalopram also reduced CalDAG-GEFI-mediated nucleotide exchange on Rap1B. Secondly, the binding of anti-GPVI antibodies to resting platelets was inhibited by citalopram. Furthermore, citalopram-induced inhibition of GPVI-mediated platelet aggregation was instantaneous, reversible and displayed competitive characteristics, suggesting that these effects were not caused by a reduction in GPVI surface expression, but by simple competitive binding. In conclusion, we propose two novel, putative and distinct inhibitory mechanisms of action for citalopram: (1) inhibition of CalDAG-GEFI/Rap1 signalling, and (2) competitive antagonism of GPVI in platelets. These findings may aid in the development of novel inhibitors of CalDAG-GEFI/Rap1-dependent nucleotide exchange and novel GPVI antagonists. Citalopram, a selective serotonin reuptake inhibitor (SSRI), is widely used as an antidepressant 1. Its primary pharmacological target is the serotonin transporter (SERT) 2,3 inhibition of which prevents cellular uptake of serotonin (5-hydroxytryptamine, 5-HT) 4. SSRIs are widely believed to exert psychiatric benefit by inhibiting SERT and modifying serotonergic neurotransmission in the central nervous system 5. SERT is also found on non-neuronal cells, including platelets, which store 5-HT in dense granules that resemble neurotransmitter vesicles 6,7. Citalopram not only inhibits platelet SERT, but also platelet aggregation, adhesion, thromboxane A 2 (TxA 2) synthesis and dense granule release 8-12. However, this functional inhibition is not caused by blockade of 5-HT uptake and must therefore be mediated by distinct pharmacological mechanisms of action 10. Citalopram inhibits platelet aggregation induced by both collagen and the TxA 2 mimetic, U46619, countering the claim that it is a specific inhibitor of collagen-induced platelet activation 9. Nevertheless, citalopram is a more potent inhibitor of collagen 10 , which activates platelets predominantly via glycoprotein VI (GPVI), than U46619, a thromboxane prostanoid (TP) receptor agonist, suggesting differential mechanisms of action. Reduced phosphorylation of signalling proteins in the GPVI pathway 10 points to GPVI as a possible site of action for citalopram, which could act as a classical competitive antagonist or allosteric inhibitor. For example, citalopram could disrupt GPVI dimers that mediate collagen binding and platelet activation 13. However, GPVI inhibition would not account for citalopram's effect on U46619-induced responses. Both GPVI and TP receptor activation raise cytosolic calcium concentrations ([Ca 2+ ] cyt), a shared signalling pathway for collagen, TxA 2 , and many other platelet agonists. Although there are numerous reports of citalopram inhibiting platelet aggregation in vitro 8-12 few have directly measured its effect on Ca 2+ signalling. Tseng et al. 14 reported that citalopram did not inhibit ADP-induced Ca 2+ signalling, suggesting some specificity in the action of citalopram, perhaps downstream of increased [Ca 2+ ] cyt. Elevated [Ca 2+ ] cyt upregulates GTP binding
Integrins are unusual signalling proteins that function to signal both from the extracellular env... more Integrins are unusual signalling proteins that function to signal both from the extracellular environment into the cell, but also from the cytoplasm to the external of the cell. The intracellular signalling cascades associated with integrin activation focus on protein kinase activities, such as focal adhesion kinase and Src. Based on this association between extracellular signals and intracellular protein kinase activity, we have chosen to include integrins in the 'Catalytic receptors' section of the database until more stringent criteria from NC-IUPHAR allows precise definition of their classification.Integrins are heterodimeric entities, composed of α and β subunits, each 1TM proteins, which bind components of the extracellular matrix or counter-receptors expressed on other cells. One class of integrin contains an inserted domain (I) in its α subunit, and if present (in α1, α2, α10, α11, αD, αE, αL, αM and αX), this I domain contains the ligand binding site. All β subunits...
... P2Y[sub 12]--the hare cathes up with the tortoise! Autores: Gavin Jarvis; Localización: Trend... more ... P2Y[sub 12]--the hare cathes up with the tortoise! Autores: Gavin Jarvis; Localización: Trends in pharmacological sciences, ISSN 0165-6147, Vol. 22, Nº 3, 2001 , pág. 111. Fundación Dialnet. Acceso de usuarios registrados. Acceso ...
The drug discovery paradigm is changing. The sequencing of the human genome has ushered in a brav... more The drug discovery paradigm is changing. The sequencing of the human genome has ushered in a brave new world of genomics, proteomics, cloning, chips and arrays, which the global pharmaceutical companies are falling over themselves in an effort to exploit. Even Time magazine has noticed and sees a change from a ‘halting, inefficient approach’ to ‘something much more rational and systematic’. The promised prize is impressive, but the enabling technology perhaps even more so. Little wonder that Time magazine does not run features on the guinea-pig ileum.And in the vanguard of this revolution are the molecular biologists whose technological wizardry occasionally leave old-fashioned pharmacologists like me gasping, trying to keep up. The latest trophy in the molecular adventure is the cloning, after many years waiting, of the Gi-linked platelet ADP receptor reported in Nature 1xIdentification of the platelet ADP receptor targeted by antithrombotic drugs. Hollopeter, G et al. Nature. 2001; 409: 202–207Crossref | PubMed | Scopus (1059)See all References1. The fact that the platelet contains no DNA has made the molecular identification of this receptor particularly challenging – several groups have tried and failed. It is fitting, therefore, that this receptor finally surrendered its identity in the face of an impressive scientific effort, comprising electrophysiological, pharmacological and molecular methods, which was not only technically faultless, but intellectually imaginative and creative. Hollopeter et al. 1xIdentification of the platelet ADP receptor targeted by antithrombotic drugs. Hollopeter, G et al. Nature. 2001; 409: 202–207Crossref | PubMed | Scopus (1059)See all References1 have successfully proven the distinct identity and distribution of the receptor, confirmed it as the target for the anti-thrombotic agent clopidogrel, and demonstrated that a rare bleeding disorder is a result of a defect in this receptor's gene.The cloning of the P2Y12 (also known as P2T, P2cyc, P2YADP, P2TAC, P2YAC, among others) receptor marks a milestone in platelet purine pharmacology. Indeed, a perfunctory glance at the scientific literature might suggest that it is the molecular biologist who has finally laid to rest the doubts over whether this receptor is P2Y1 or P2Y2 or even exists at all! Yet it remains the case that, since 1994, this receptor has been perhaps the best characterized of all purine receptors. Pharmacological data then and subsequently has shown that it is a molecule with a limited tissue distribution and an identity distinct from all other purine receptors. Furthermore, the lack of the clone did not prevent the recognition that this receptor represented a highly attractive therapeutic target, and one successful product has already reached the market, with several in its developmental wake. It can only be hoped, even expected, that the identification and sequencing of P2Y12 will accelerate the discovery and development of much-needed anti-thrombotic drugs.There is little doubt that molecular biology has transformed medical and pharmaceutical research and that in the future, it will lead the way into unimagined opportunities. However, the combined use of old and new techniques, as exemplified in the study by Hollopeter et al., will be required to exploit these opportunities to their fullest extent. In the meantime, for those pharmacologists who have worked on ADP and platelets for many years now, we are merely content that the hi-tech hare of molecular medicine has finally caught up with the technological tortoise of pharmacology.
The Journal Club section of Trends in Endocrinology & Metabolism provides summaries of recently p... more The Journal Club section of Trends in Endocrinology & Metabolism provides summaries of recently published papers across the broad spectrum of endocrinology and metabolism. A typical Journal Club contribution has a short introduction that sets the scene, a description of the approach taken, the key results and conclusions of the paper, and implications for further research.
The β isoform of phosphoinositide 3-kinase may be an effective therapeutic target in inflammatory... more The β isoform of phosphoinositide 3-kinase may be an effective therapeutic target in inflammatory diseases.
Recently, the ability of polymeric collagen-like peptides to regulate cell behavior has generated... more Recently, the ability of polymeric collagen-like peptides to regulate cell behavior has generated great interest. A triple-helical peptide known as collagen-related peptide (CRP) contains the sequence (Gly-Pro-Hyp) 10. With Gly-Pro-Cys triplets appended to both of its termini, designated CRP cys , chemical cross-linking using heterobifunctional reagents generates CRP cys-XL, a potent, widely used, polymeric agonist for platelet Glycoprotein VI, whereas non-cross-linked, monomeric CRP cys antagonizes Glycoprotein VI. Here, we describe how cysteine in these triplets may also undergo random air-induced oxidation, especially upon prolonged storage or repeated freeze-thawing, to form disulphide bonds, resulting in a lesser degree of polymerization than with chemical cross-linking. We investigated the monomeric and polymeric states of these and other cysteine-containing collagen-derived peptides, using gel filtration and dynamic light scattering, allowing the size of a CRP-XL aggregate to be estimated. The effect of cysteine thiols upon peptide adsorption to surfaces and subsequent platelet responses was investigated. This demonstrated that cysteine is required for strong binding to glass coverslips and to plastic plates used in ELISA assays.
Comprehensive mapping of protein-binding sites within human collagen III has allowed the recognit... more Comprehensive mapping of protein-binding sites within human collagen III has allowed the recognition motifs for integrin a 2 b 1 and VWF A3 domain to be identified. Glycoprotein VI-binding sites are understood, although less well defined. This information, together with recent developments in understanding collagen fiber architecture, and crystal structures of the receptor collagen-binding domains, allows a coherent model for the interaction of collagen with the platelet surface to be developed. This complements our understanding of the orchestration of receptor presentation by membrane microdomains, such that the polyvalent collagen surface may stabilize signaling complexes within the heterogeneous receptor composition of the lipid raft. The ensuing interactions lead to the convergence of signals from each of the adhesive receptors, mediated by FcR c-chain and/or FccRIIa, leading to concerted and cooperative platelet activation. Each receptor has a sheardependent role, VWF/GpIb essential at high shear, and a 2 b 1 at low and intermediate shear, whilst GpVI provides core signals that contribute to enhanced integrin affinity, tighter binding to collagen and consequent platelet activation.
A set of 57 synthetic peptides encompassing the entire triplehelical domain of human collagen III... more A set of 57 synthetic peptides encompassing the entire triplehelical domain of human collagen III was used to locate binding sites for the collagen-binding integrin ␣ 2  1. The capacity of the peptides to support Mg 2؉-dependent binding of several integrin preparations was examined. Wild-type integrins (recombinant ␣ 2 I-domain, ␣ 2  1 purified from platelet membranes, and recombinant soluble ␣ 2  1 expressed as an ␣ 2-Fos/ 1-Jun heterodimer) bound well to only three peptides, two containing GXXGER motifs (GROGER and GMOGER, where O is hydroxyproline) and one containing two adjacent GXXGEN motifs (GLKGEN and GLOGEN). Two mutant ␣ 2 I-domains were tested: the inactive T221A mutant, which recognized no peptides, and the constitutively active E318W mutant, which bound a larger subset of peptides. Adhesion of activated human platelets to GER-containing peptides was greater than that of resting platelets, and HT1080 cells bound well to more of the peptides compared with platelets. Binding of cells and recombinant proteins was abolished by anti-␣ 2 monoclonal antibody 6F1 and by chelation of Mg 2؉. We describe two novel high affinity integrinbinding motifs in human collagen III (GROGER and GLOGEN) and a third motif (GLKGEN) that displays intermediate activity. Each motif was verified using shorter synthetic peptides.
After 3 years of devoted work for Laboratory Animals as deputy editor, Arieh Bomzon retired at th... more After 3 years of devoted work for Laboratory Animals as deputy editor, Arieh Bomzon retired at the end of September 2018. With the upcoming 50-year reunion of veterinary graduates in Sydney, Australia, he felt that it is time to step aside in order to let the younger generation of laboratory animal veterinarians and animal welfare scientists take the lead in determining the journal’s future. In his letter of resignation, he thanked the journal’s editorial team, the editorial board members, the reviewers, the publisher and the executive committee and council of management of LAL Ltd for their support. It is now our turn to thank him for all his work, his constructive input, and his contribution to the oversight of the review process. It is also thanks to Arieh that case reports were valued and again found acceptance. His reflections on the future direction of the journal will be taken into account. In August, Gavin Jarvis was appointed as Deputy Editor and will succeed Arieh Bomzon. We would like to take this opportunity to welcome him and to introduce him to the readership of Laboratory Animals.
The transcripts were made personally by me from photocopies of documents in the archives of the C... more The transcripts were made personally by me from photocopies of documents in the archives of the Claimant. Every attempt has been made to ensure that the transcripts are faithful reproductions of these photocopies. This means that typographical errors in the original documents have been retained and transcribed. Any errors of transcription are my responsibility and will be corrected on notification. A full unredacted transcript and copies of original documents will be supplied on request. Personal addresses will remain redacted.
SummaryThe major activation-inducing collagen receptor glycoprotein VI (GPVI) has been cloned wit... more SummaryThe major activation-inducing collagen receptor glycoprotein VI (GPVI) has been cloned within the last two years. It is a member of the Ig superfamily of proteins and is constitutively associated with the ITAM-bearing Fc receptor γ-chain (FcR γ-chain). GPVI signals through a pathway that involves several of the proteins used by Fc, B- and T-lymphocyte receptors and which takes place in glycolipid-enriched membrane domains in the plasma membrane known as GEMs. Responses to GPVI are regulated by PECAM-1 (CD31) and possibly other ITIM-bearing receptors. Despite a pivotal role for GPVI, there are important differences between signalling events to collagen and GPVI-specific ligands. This may reflect a role for co-receptors in the response to collagen.
Glycoprotein VI (GPVI) mediates collagen-induced platelet activation after vascular damage and is... more Glycoprotein VI (GPVI) mediates collagen-induced platelet activation after vascular damage and is an important contributor to the onset of thrombosis, heart attack, and stroke. Animal models of thrombosis have identified GPVI as a promising target for antithrombotic therapy. Although for many years the crystal structure of GPVI has been known, the essential details of its interaction with collagen have remained elusive. Here, we present crystal structures of the GPVI ectodomain bound to triple-helical collagen peptides, which reveal a collagen-binding site across the β-sheet of the D1 domain. Mutagenesis and binding studies confirm the observed binding site and identify Trp76, Arg38, and Glu40 as essential residues for binding to fibrillar collagens and collagen-related peptides (CRPs). GPVI binds a site on collagen comprising two collagen chains with the core formed by the sequence motif OGPOGP. Potent GPVI-binding peptides from Toolkit-III all contain OGPOGP; weaker binding peptid...
Background: Platelet glycoprotein (GP) Ib-IX-V supports platelet adhesion on damaged vascular wal... more Background: Platelet glycoprotein (GP) Ib-IX-V supports platelet adhesion on damaged vascular walls by binding to von Willebrand factor (VWF). For several decades it has been recognized that the a-subunit of GP (GPIba) also binds thrombin but the physiological relevance, if any, of this interaction was unknown. Previous studies have shown that a sulfated tyrosine 276 (Tyr276) is essential for thrombin binding to GPIba. Objectives: This study investigated the in vivo relevance of GPIba residue Tyr276 in hemostasis and thrombosis. Methods: Transgenic mouse colonies expressing the normal human GPIba subunit or a mutant human GPIba containing a Phe substitution for Tyr276 (hTg Y276F) were generated. Both colonies were bred to mice devoid of murine GPIba. Results: Surface-expressed GPIba levels and platelet counts were similar in both colonies. hTg Y276F platelets were significantly impaired in binding a-thrombin but displayed normal binding to type I fibrillar collagen and human VWF in the presence of ristocetin. In vivo thrombus formation as a result of chemical damage (FeCl 3) demonstrated that hTg Y276F mice have a delayed time to occlusion followed by unstable blood flow indicative of embolization. In models of laser-induced injury, thrombi developing in hTg Y276F animals were also less stable. Conclusions: The results demonstrate that GPIba residue Tyr276 is physiologically important, supporting stable thrombus formation in vivo.
Citalopram, a selective serotonin reuptake inhibitor (SSRI), inhibits platelet function in vitro.... more Citalopram, a selective serotonin reuptake inhibitor (SSRI), inhibits platelet function in vitro. We have previously shown that this action is independent of citalopram's ability to block serotonin uptake by the serotonin transporter and must therefore be mediated via distinct pharmacological mechanisms. We now report evidence for two novel and putative mechanisms of citalopram-induced platelet inhibition. Firstly, in platelets, citalopram blocked U46619-induced Rap1 activation and subsequent platelet aggregation, but failed to inhibit U46619-induced increases in cytosolic Ca 2+. Similarly, in neutrophils, citalopram inhibited Rap1 activation and downstream functions but failed to block PAF-induced Ca 2+ mobilisation. In a cell-free system, citalopram also reduced CalDAG-GEFI-mediated nucleotide exchange on Rap1B. Secondly, the binding of anti-GPVI antibodies to resting platelets was inhibited by citalopram. Furthermore, citalopram-induced inhibition of GPVI-mediated platelet aggregation was instantaneous, reversible and displayed competitive characteristics, suggesting that these effects were not caused by a reduction in GPVI surface expression, but by simple competitive binding. In conclusion, we propose two novel, putative and distinct inhibitory mechanisms of action for citalopram: (1) inhibition of CalDAG-GEFI/Rap1 signalling, and (2) competitive antagonism of GPVI in platelets. These findings may aid in the development of novel inhibitors of CalDAG-GEFI/Rap1-dependent nucleotide exchange and novel GPVI antagonists. Citalopram, a selective serotonin reuptake inhibitor (SSRI), is widely used as an antidepressant 1. Its primary pharmacological target is the serotonin transporter (SERT) 2,3 inhibition of which prevents cellular uptake of serotonin (5-hydroxytryptamine, 5-HT) 4. SSRIs are widely believed to exert psychiatric benefit by inhibiting SERT and modifying serotonergic neurotransmission in the central nervous system 5. SERT is also found on non-neuronal cells, including platelets, which store 5-HT in dense granules that resemble neurotransmitter vesicles 6,7. Citalopram not only inhibits platelet SERT, but also platelet aggregation, adhesion, thromboxane A 2 (TxA 2) synthesis and dense granule release 8-12. However, this functional inhibition is not caused by blockade of 5-HT uptake and must therefore be mediated by distinct pharmacological mechanisms of action 10. Citalopram inhibits platelet aggregation induced by both collagen and the TxA 2 mimetic, U46619, countering the claim that it is a specific inhibitor of collagen-induced platelet activation 9. Nevertheless, citalopram is a more potent inhibitor of collagen 10 , which activates platelets predominantly via glycoprotein VI (GPVI), than U46619, a thromboxane prostanoid (TP) receptor agonist, suggesting differential mechanisms of action. Reduced phosphorylation of signalling proteins in the GPVI pathway 10 points to GPVI as a possible site of action for citalopram, which could act as a classical competitive antagonist or allosteric inhibitor. For example, citalopram could disrupt GPVI dimers that mediate collagen binding and platelet activation 13. However, GPVI inhibition would not account for citalopram's effect on U46619-induced responses. Both GPVI and TP receptor activation raise cytosolic calcium concentrations ([Ca 2+ ] cyt), a shared signalling pathway for collagen, TxA 2 , and many other platelet agonists. Although there are numerous reports of citalopram inhibiting platelet aggregation in vitro 8-12 few have directly measured its effect on Ca 2+ signalling. Tseng et al. 14 reported that citalopram did not inhibit ADP-induced Ca 2+ signalling, suggesting some specificity in the action of citalopram, perhaps downstream of increased [Ca 2+ ] cyt. Elevated [Ca 2+ ] cyt upregulates GTP binding
Integrins are unusual signalling proteins that function to signal both from the extracellular env... more Integrins are unusual signalling proteins that function to signal both from the extracellular environment into the cell, but also from the cytoplasm to the external of the cell. The intracellular signalling cascades associated with integrin activation focus on protein kinase activities, such as focal adhesion kinase and Src. Based on this association between extracellular signals and intracellular protein kinase activity, we have chosen to include integrins in the 'Catalytic receptors' section of the database until more stringent criteria from NC-IUPHAR allows precise definition of their classification.Integrins are heterodimeric entities, composed of α and β subunits, each 1TM proteins, which bind components of the extracellular matrix or counter-receptors expressed on other cells. One class of integrin contains an inserted domain (I) in its α subunit, and if present (in α1, α2, α10, α11, αD, αE, αL, αM and αX), this I domain contains the ligand binding site. All β subunits...
... P2Y[sub 12]--the hare cathes up with the tortoise! Autores: Gavin Jarvis; Localización: Trend... more ... P2Y[sub 12]--the hare cathes up with the tortoise! Autores: Gavin Jarvis; Localización: Trends in pharmacological sciences, ISSN 0165-6147, Vol. 22, Nº 3, 2001 , pág. 111. Fundación Dialnet. Acceso de usuarios registrados. Acceso ...
The drug discovery paradigm is changing. The sequencing of the human genome has ushered in a brav... more The drug discovery paradigm is changing. The sequencing of the human genome has ushered in a brave new world of genomics, proteomics, cloning, chips and arrays, which the global pharmaceutical companies are falling over themselves in an effort to exploit. Even Time magazine has noticed and sees a change from a ‘halting, inefficient approach’ to ‘something much more rational and systematic’. The promised prize is impressive, but the enabling technology perhaps even more so. Little wonder that Time magazine does not run features on the guinea-pig ileum.And in the vanguard of this revolution are the molecular biologists whose technological wizardry occasionally leave old-fashioned pharmacologists like me gasping, trying to keep up. The latest trophy in the molecular adventure is the cloning, after many years waiting, of the Gi-linked platelet ADP receptor reported in Nature 1xIdentification of the platelet ADP receptor targeted by antithrombotic drugs. Hollopeter, G et al. Nature. 2001; 409: 202–207Crossref | PubMed | Scopus (1059)See all References1. The fact that the platelet contains no DNA has made the molecular identification of this receptor particularly challenging – several groups have tried and failed. It is fitting, therefore, that this receptor finally surrendered its identity in the face of an impressive scientific effort, comprising electrophysiological, pharmacological and molecular methods, which was not only technically faultless, but intellectually imaginative and creative. Hollopeter et al. 1xIdentification of the platelet ADP receptor targeted by antithrombotic drugs. Hollopeter, G et al. Nature. 2001; 409: 202–207Crossref | PubMed | Scopus (1059)See all References1 have successfully proven the distinct identity and distribution of the receptor, confirmed it as the target for the anti-thrombotic agent clopidogrel, and demonstrated that a rare bleeding disorder is a result of a defect in this receptor's gene.The cloning of the P2Y12 (also known as P2T, P2cyc, P2YADP, P2TAC, P2YAC, among others) receptor marks a milestone in platelet purine pharmacology. Indeed, a perfunctory glance at the scientific literature might suggest that it is the molecular biologist who has finally laid to rest the doubts over whether this receptor is P2Y1 or P2Y2 or even exists at all! Yet it remains the case that, since 1994, this receptor has been perhaps the best characterized of all purine receptors. Pharmacological data then and subsequently has shown that it is a molecule with a limited tissue distribution and an identity distinct from all other purine receptors. Furthermore, the lack of the clone did not prevent the recognition that this receptor represented a highly attractive therapeutic target, and one successful product has already reached the market, with several in its developmental wake. It can only be hoped, even expected, that the identification and sequencing of P2Y12 will accelerate the discovery and development of much-needed anti-thrombotic drugs.There is little doubt that molecular biology has transformed medical and pharmaceutical research and that in the future, it will lead the way into unimagined opportunities. However, the combined use of old and new techniques, as exemplified in the study by Hollopeter et al., will be required to exploit these opportunities to their fullest extent. In the meantime, for those pharmacologists who have worked on ADP and platelets for many years now, we are merely content that the hi-tech hare of molecular medicine has finally caught up with the technological tortoise of pharmacology.
The Journal Club section of Trends in Endocrinology & Metabolism provides summaries of recently p... more The Journal Club section of Trends in Endocrinology & Metabolism provides summaries of recently published papers across the broad spectrum of endocrinology and metabolism. A typical Journal Club contribution has a short introduction that sets the scene, a description of the approach taken, the key results and conclusions of the paper, and implications for further research.
The β isoform of phosphoinositide 3-kinase may be an effective therapeutic target in inflammatory... more The β isoform of phosphoinositide 3-kinase may be an effective therapeutic target in inflammatory diseases.
Recently, the ability of polymeric collagen-like peptides to regulate cell behavior has generated... more Recently, the ability of polymeric collagen-like peptides to regulate cell behavior has generated great interest. A triple-helical peptide known as collagen-related peptide (CRP) contains the sequence (Gly-Pro-Hyp) 10. With Gly-Pro-Cys triplets appended to both of its termini, designated CRP cys , chemical cross-linking using heterobifunctional reagents generates CRP cys-XL, a potent, widely used, polymeric agonist for platelet Glycoprotein VI, whereas non-cross-linked, monomeric CRP cys antagonizes Glycoprotein VI. Here, we describe how cysteine in these triplets may also undergo random air-induced oxidation, especially upon prolonged storage or repeated freeze-thawing, to form disulphide bonds, resulting in a lesser degree of polymerization than with chemical cross-linking. We investigated the monomeric and polymeric states of these and other cysteine-containing collagen-derived peptides, using gel filtration and dynamic light scattering, allowing the size of a CRP-XL aggregate to be estimated. The effect of cysteine thiols upon peptide adsorption to surfaces and subsequent platelet responses was investigated. This demonstrated that cysteine is required for strong binding to glass coverslips and to plastic plates used in ELISA assays.
Comprehensive mapping of protein-binding sites within human collagen III has allowed the recognit... more Comprehensive mapping of protein-binding sites within human collagen III has allowed the recognition motifs for integrin a 2 b 1 and VWF A3 domain to be identified. Glycoprotein VI-binding sites are understood, although less well defined. This information, together with recent developments in understanding collagen fiber architecture, and crystal structures of the receptor collagen-binding domains, allows a coherent model for the interaction of collagen with the platelet surface to be developed. This complements our understanding of the orchestration of receptor presentation by membrane microdomains, such that the polyvalent collagen surface may stabilize signaling complexes within the heterogeneous receptor composition of the lipid raft. The ensuing interactions lead to the convergence of signals from each of the adhesive receptors, mediated by FcR c-chain and/or FccRIIa, leading to concerted and cooperative platelet activation. Each receptor has a sheardependent role, VWF/GpIb essential at high shear, and a 2 b 1 at low and intermediate shear, whilst GpVI provides core signals that contribute to enhanced integrin affinity, tighter binding to collagen and consequent platelet activation.
A set of 57 synthetic peptides encompassing the entire triplehelical domain of human collagen III... more A set of 57 synthetic peptides encompassing the entire triplehelical domain of human collagen III was used to locate binding sites for the collagen-binding integrin ␣ 2  1. The capacity of the peptides to support Mg 2؉-dependent binding of several integrin preparations was examined. Wild-type integrins (recombinant ␣ 2 I-domain, ␣ 2  1 purified from platelet membranes, and recombinant soluble ␣ 2  1 expressed as an ␣ 2-Fos/ 1-Jun heterodimer) bound well to only three peptides, two containing GXXGER motifs (GROGER and GMOGER, where O is hydroxyproline) and one containing two adjacent GXXGEN motifs (GLKGEN and GLOGEN). Two mutant ␣ 2 I-domains were tested: the inactive T221A mutant, which recognized no peptides, and the constitutively active E318W mutant, which bound a larger subset of peptides. Adhesion of activated human platelets to GER-containing peptides was greater than that of resting platelets, and HT1080 cells bound well to more of the peptides compared with platelets. Binding of cells and recombinant proteins was abolished by anti-␣ 2 monoclonal antibody 6F1 and by chelation of Mg 2؉. We describe two novel high affinity integrinbinding motifs in human collagen III (GROGER and GLOGEN) and a third motif (GLKGEN) that displays intermediate activity. Each motif was verified using shorter synthetic peptides.
After 3 years of devoted work for Laboratory Animals as deputy editor, Arieh Bomzon retired at th... more After 3 years of devoted work for Laboratory Animals as deputy editor, Arieh Bomzon retired at the end of September 2018. With the upcoming 50-year reunion of veterinary graduates in Sydney, Australia, he felt that it is time to step aside in order to let the younger generation of laboratory animal veterinarians and animal welfare scientists take the lead in determining the journal’s future. In his letter of resignation, he thanked the journal’s editorial team, the editorial board members, the reviewers, the publisher and the executive committee and council of management of LAL Ltd for their support. It is now our turn to thank him for all his work, his constructive input, and his contribution to the oversight of the review process. It is also thanks to Arieh that case reports were valued and again found acceptance. His reflections on the future direction of the journal will be taken into account. In August, Gavin Jarvis was appointed as Deputy Editor and will succeed Arieh Bomzon. We would like to take this opportunity to welcome him and to introduce him to the readership of Laboratory Animals.
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Papers by Gavin E Jarvis