Gene/Protein Disease Symptom Drug Enzyme Compound
Pivot Concepts:   Target Concepts:
Query: EC:3.4.21.5 (thrombin)
 33,306 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

Bernard-Soulier syndrome (BSS) is a rare hereditary bleeding disorder and macrothrombocytopenia which is caused by a defect in the platelet glycoprotein Ib/IX/V (GP Ib/IX/V) complex, the receptor for von Willebrand factor and thrombin. Here we report the molecular basis of the classical form of BSS in two unrelated Finnish patients, both with a life-long history of severe bleeding. Flow cytometry and immunoblotting showed no expression of GP Ib/IX, GP Ib alpha, GP Ib beta or GP IX (less than 10%) in the patients' platelets. No expression of GP V (< 10%) was observed in propositus 1, but a residual amount was found in propositus 2 (24%). DNA sequencing analysis revealed that propositus 1 was compound heterozygous for a two-base-pair deletion at Tyr505(TAT) and a point mutation Leu129(CTC)Pro(CCC) in the GP Ib alpha gene. Propositus 2 was homozygous for the Tyr505(TAT) deletion. The nine relatives who were heterozygous for either of the mutations also had low levels of GP Ib alpha (74-90%). Hence, Bernard-Soulier patients homozygous or compound heterozygous for Tyr505(TAT) are severely affected. Interestingly, both mutations have independently been found in three other families in previous reports, suggesting their ancient age or mutational 'hot spot'.
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PMID:Molecular characterization of two mutations in platelet glycoprotein (GP) Ib alpha in two Finnish Bernard-Soulier syndrome families. 1008 93

Thrombin, the most potent platelet agonist, plays a central role in haemostasis and in the occurrence of thrombotic events. This agonist activates platelets by cleaving the PAR G-protein coupled receptors and by binding to glycoprotein (GP) Ib and also cleaves GPV at the platelet surface to liberate the soluble 69 kDa fragment GPVf1. Monoclonal antibodies (MoAbs) to GPV were developed as tools to study the mechanism of platelet GPV cleavage and measure release of GPV in pathological situations. Specificity of the MoAbs for GPV was confirmed by flow cytometry and immunoprecipitation of proteins from human platelets and Dami megakaryocytic cells. A sensitive immunocapture sandwich ELISA for soluble GPV was developed using two MoAbs recognizing different epitopes of GPV and purified platelet or recombinant GPV as reference protein. This ELISA was employed to determine the mean plasma concentration of GPV in 100 normal individuals (17.3 ng/ml), to demonstrate the dose-dependent release of GPVf1 from washed platelets stimulated with thrombin and to follow the progressive release of GPVf1 during storage of therapeutic platelet concentrates. The present report describes a sensitive GPV ELISA of direct application to survey the processing and storage of platelet concentrates for transfusion and of potential value to monitor platelet activation in thrombotic states.
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PMID:Measurement of GPV released by activated platelets using a sensitive immunocapture ELISA--its use to follow platelet storage in transfusion. 1034 4

A role for glycoprotein (GP)V in platelet function has been proposed on the basis of observations that GP V is the major thrombin substrate on intact platelets cleaved during thrombin-induced platelet aggregation, and that GP V promotes GP Ib-IX surface expression in heterologous cells. We tested the hypotheses that GP V is involved in thrombin-induced platelet activation, in GP Ib-IX expression, and in other platelet responses by generating GP V null mice. Contrary to expectations, GP V -/- platelets were normal in size and expressed normal amounts of GP Ib-IX that was functional in von Willebrand factor binding, explaining why defects in GP V have not been observed in Bernard-Soulier syndrome, a bleeding disorder caused by a lack of functional GP Ib-IX-V. Moreover, in vitro analysis demonstrated that GP V -/- platelets were hyperresponsive to thrombin, resulting in increased fibrinogen binding and an increased aggregation response. Consistent with these findings, GP V -/- mice had a shorter bleeding time. These data support a role for GP V as a negative modulator of platelet activation. Furthermore, they suggest a new mechanism by which thrombin enhances platelet responsiveness independent of activation of the classical G-protein-coupled thrombin receptors.
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PMID:Increased thrombin responsiveness in platelets from mice lacking glycoprotein V. 1055 21

Glycoprotein (GP) Ib (alpha and beta) in platelets forms a noncovalent hetero-oligomeric complex with GPIX and GPV and serves as a receptor for von Willebrand factor (vWF), which mediates the initial adhesion of platelets to the subendothelium after vascular damage and also plays a role in thrombin-induced platelet activation. We investigated the interaction between GPIbalpha and FcgammaIIA receptor using a yeast two-hybrid system and mutagenesis, and we identified residues R542G543R544 in GPIbalpha and D298D299D300 in FcgammaIIA receptor as the primary interaction sites. These results further confirmed the interaction between GPIbalpha and FcgammaIIA receptor and support the hypothesis that the signal transduction of GPIb-IX-V that leads to platelet activation may be partially mediated through FcgammaIIA receptor.
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PMID:Interaction between GPIbalpha and FcgammaIIA receptor in human platelets. 1058 Nov 59

Adhesion of platelets to extracellular matrix via von Willebrand factor (vWF) and activation of platelets by thrombin are critical steps in hemostasis. Glycoprotein (GP) V is a component of the GPIb-V-IX complex, the platelet receptor for vWF. GPV is also cleaved by thrombin. Deficiency of GPIb or GPIX results in Bernard-Soulier syndrome (BSS), a bleeding disorder in which platelets are giant and have multiple functional defects. Whether GPV-deficiency might also cause BSS is unknown as are the roles of GPV in platelet-vWF interaction and thrombin signaling. We report that GPV-deficient mice developed normally, had no evidence of spontaneous bleeding, and had tail bleeding times that were not prolonged compared with wild-type mice. GPV-deficient platelets were normal in size and structure as assessed by flow cytometry and electron microscopy. GPV-deficient and wild-type platelets were indistinguishable in botrocetin-mediated platelet agglutination and in their ability to adhere to mouse vWF A1 domain. Platelet aggregation and ATP secretion in response to low and high concentrations of thrombin were not decreased in GPV-deficient platelets compared with wild-type. Our results show that (1) GPV is not necessary for GPIb expression and function in platelets and that GPV deficiency is not likely to be a cause of human BSS and (2) GPV is not necessary for robust thrombin signaling. Whether redundancy accounts for the lack of phenotype of GPV-deficiency or whether GPV serves subtle or as yet unprobed functions in platelets or other cells remains to be determined.
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PMID:Glycoprotein V-deficient platelets have undiminished thrombin responsiveness and Do not exhibit a Bernard-Soulier phenotype. 1059 56

Thrombin plays a central role in the genesis of thrombotic events and is the most potent known platelet agonist. This enzyme activates platelets by cleaving G-protein coupled protease activated receptors (PARs) and by binding to glycoprotein (GP) Ib. Thrombin also cleaves platelet GPV to liberate a soluble 69 kDa fragment (GPVf1), leaving a 20 kDa fragment (GPVf2) attached to the membrane. The aim of this study was to assess the value of GPV as an in vivo marker of the activation of platelets by thrombin. Newly developed monoclonal and polyclonal antibodies recognizing rat GPVf1 and GPVf2 respectively were used to detect soluble GPV by ELISA and the new NH2-terminus exposed by thrombin using flow cytometry. These assays were employed in a rat thrombosis model designed to trigger thrombin formation in vivo. When thromboplastin (4.8 ml/kg/h) was infused for 30 min, thrombin generation was reflected by a rapid increase in thrombin-antithrombin (TAT) complexes in plasma and by the appearance of GPVf2 at the surface of circulating platelets. Simultaneously, GPVf1 disappeared from the surface of platelets and accumulated as a soluble fragment in plasma, where it was detected by GPV ELISA. These effects were inhibited by pretreatment of the rats with hirudin. Levels of plasma PF4 also increased in this model, but unlike GPV levels which returned slowly (> 2 hours) to baseline, PF4 had a very short half-life. In conclusion, GPV is cleaved by thrombin in vivo, circulates and is a reliable in vivo marker of the activation of platelets by thrombin. Monitoring of GPV levels in rats should be useful to evaluate the effects of antithrombotic and antiplatelet drugs, while further studies will be required to confirm the potential interest of GPV as a marker of thrombotic states in humans.
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PMID:GPV is a marker of in vivo platelet activation--study in a rat thrombosis model. 1073 94

Lesions in the genes for GPIb alpha, GPIb beta or GPIX result in a bleeding diathesis, the Bernard-Soulier syndrome (BSS), which associates a platelet adhesion defect with thrombocytopenia, giant platelets and abnormal megakaryocytes (MK). The role of GPV, also absent in BSS, was recently addressed by gene targeting in mice. While a negative modulator function for GPV on thrombin-induced platelet responses was found in one model, the absence of GP V had no effect on GPIb-IX expression or platelet adhesion. Our study extends previous results and reports that electron microscopy of bone marrow from the GPV knockout mice revealed a normal MK ultrastructure and development of the demarcation membrane system (DMS). There was a usual presence of MK fragments in the bone marrow vascular sinus. Immunogold labelling of MK from the knockout mice showed a normal distribution of GPIb-IX in the DMS and on the cell surface. The distribution of fibrinogen, vWF and P-selectin was unchanged with, interestingly, P-selectin also localised within the DMS in both situations. Thus GPV is not crucial to MK development and platelet production, consistent with the fact that no mutation in the GPV gene has as yet been described in BSS.
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PMID:Ultrastructural analysis of megakaryocytes in GPV knockout mice. 1095 6

The only known function of the 41 amino acid cleaved peptide (TR1-41) of the seven transmembrane domain thrombin receptor (PARI) is to activate platelets (as determined by aggregation, surface P-selectin, and fibrinogen binding to activated GPIIb-IIIa). We now demonstrate that TR1-41 results in a concentration-dependent decrease in the platelet surface expression of each component of the GPIb-IX-V complex, as determined by flow cytometry with a panel of monoclonal antibodies (including 6D1, directed against the von Willebrand factor binding site on GPIbalpha, and TM60, directed against the thrombin binding site on GPIbalpha). TR1-41 also decreased ristocetin-induced platelet agglutination. Immunoblotting after incubation of platelets with TR1-41 revealed neither a loss of platelet GPIb nor increase in supernatant GPIb fragments. As demonstrated by immunoelectron microscopy, TR1-41 resulted in a redistribution of GPIb, GPIX, and GPV from the platelet surface to the surface-connected canalicular system (SCCS). In summary, the cleaved peptide (TR1-41) of PAR1 results in a redistribution of the platelet surface GPIb-IX-V complex to the SCCS, thereby negatively regulating the GPIbalpha binding sites for von Willebrand factor and thrombin.
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PMID:The cleaved peptide of PAR1 results in a redistribution of the platelet surface GPIb-IX-V complex to the surface-connected canalicular system. 1112 74

The mouse- and rat-platelet-specific hamster monoclonal antibody (MAb) 1C2, previously found to react with a thrombin-sensitive 74-kD glycoprotein, was now shown to recognize platelet glycoprotein V (GPV, CD42d). 1C2 reacted with NIH-3T3 cells in which recombinant mouse or rat GPV was expressed. Both 1C2 and 4A5, another mouse-platelet-specific rat MAb, immunoprecipitated GVP, although they recognized different epitopes. Side-by-side comparison confirmed that 1C2 as well as RPM.9, a MAb against rat GPV, recognized the same rat platelet molecule. In a mouse bone marrow culture, 1C2+ megakaryocytes emerged from CD41 (GPIIb)+1C2- megakaryocytes. Because 1C2+ megakaryocytes exhibited higher DNA ploidy distribution than CD41+ cells, GPV likely appears in the late stage of megakaryocyte maturation. This study established 1C2 as a MAb against mouse and rat GPV, namely CD42d, and as useful tool to study rodent megakaryopoiesis.
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PMID:Characterization of monoclonal antibodies against mouse and rat platelet glycoprotein V (CD42d). 1115 97

Glycoprotein (GP) V is a major substrate cleaved by the protease thrombin during thrombin-induced platelet activation. Previous analysis of platelets from GP V-null mice suggested a role for GP V as a negative modulator of platelet activation by thrombin. We now report the mechanism by which thrombin activates GP V -/- platelets. We show that proteolytically inactive forms of thrombin induce robust stimulatory responses in GP V null mouse platelets, via the platelet GP Ib--IX--V complex. Because proteolytically inactive thrombin can activate wild-type mouse and human platelets after treatment with thrombin to cleave GP V, this mechanism is involved in thrombin-induced platelet aggregation. Platelet activation through GP Ib-IX depends on ADP secretion, and specific inhibitors demonstrate that the recently cloned P2Y(12) ADP receptor (G(i)-coupled ADP receptor) is involved in this pathway, and that the P2Y(1) receptor (G(q)-coupled ADP receptor) may play a less significant role. Thrombosis was generated in GP V null mice only in response to catalytically inactive thrombin, whereas thrombosis occurred in both genotypes (wild type and GP V null) in response to active thrombin. These data support a thrombin receptor function for the platelet membrane GP Ib--IX--V complex, and describe a novel thrombin signaling mechanism involving an initiating proteolytic event followed by stimulation of the GP Ib--IX via thrombin acting as a ligand, resulting in platelet activation.
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PMID:A thrombin receptor function for platelet glycoprotein Ib-IX unmasked by cleavage of glycoprotein V. 1117 48


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