Gene/Protein Disease Symptom Drug Enzyme Compound
Pivot Concepts: Gene/Protein Disease Symptom Drug Enzyme Compound   Target Concepts: Gene/Protein Disease Symptom Drug Enzyme Compound

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Query: EC:3.4.21.5 (thrombin)
33,306 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

The glycoprotein (GP) Ib-V-IX complex plays a critical role in initiating platelet adhesion to von Willebrand factor (vWF) at the site of vascular injury. The complex also forms a high-affinity binding site for thrombin. Using an intravital microscopy mouse model, it was previously established that vWF plays a critical role in mediating platelet adhesion and thrombus formation following mesenteric arteriolar injury induced by ferric chloride. Further characterization of this model showed that these thrombotic events were also thrombin dependent. Using this vWF- and thrombin-dependent model, this study shows that GP V gene deficiency significantly accelerates both platelet adhesion and thrombus formation in mice following arteriolar injury. The time required for vessel occlusion in GP V-deficient (GP V(-/-)) mice was significantly shorter than that in wild-type mice. Interestingly, large emboli were also produced in GP V(-/-) mice, but not in wild-type mice, causing frequent downstream occlusion. However, when the 2 genotypes were compared in the in vitro perfusion chamber where thrombin was inhibited by heparin, no significant differences were found in either initial single-platelet adhesion or thrombus volume. These results demonstrate that GP V(-/-) mice have accelerated thrombus growth in response to vascular injury and suggest that this is caused by enhanced thrombin-induced platelet activation rather than enhanced binding of GPIb-V-IX to vWF. Absence of GP V also compromises thrombus stability.
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PMID:Increased thrombogenesis and embolus formation in mice lacking glycoprotein V. 1143 5

Thrombocytopenia is a frequent comorbid condition in many in hospital patients. In some patients, drugs are the cause of low platelet counts. While cytotoxic effects of anti-tumor therapy are the most frequent cause, immune mechanisms should also be considered. This review addresses thrombocytopenias in four groups. Heparin-dependent thrombocytopenia (HIT), by far the most frequent drug-induced immune-mediated type of thrombocytopenia, has a unique pathogenesis and clinical consequences. HIT is a clinicopathological syndrome in which antibodies mostly directed against a multimolecular complex of platelet factor 4 and heparin cause paradoxical thromboembolic complications. The mechanisms through which heparin can enhance thrombin generation are discussed and treatment alternatives for affected patients are presented in detail. It is of primary importance to recognize these patients as early as possible and to substitute heparin with a compatible anticoagulatory drug, such as hirudin, danaparoid or argatroban. Patients seem to benefit from therapeutic doses of alternative treatment rather than from low-dose prophylactic doses. With the increasing use of glycoprotein (GP) IIb/IIIa inhibitors in patients with acute coronary syndromes, thrombocytopenias are increasingly recognized as an adverse effect of these drugs. Up to 4% of treated patients are affected. Most important, pseudothrombocytopenia, a laboratory artefact, is as frequent as real drug-induced thrombocytopenia and must be excluded before changes in treatment are considered. The pathogenesis of these thrombocytopenias is still debated; an immune mechanism involving preformed antibodies is likely. However, since these antibodies are also detectable in a high percentage of normal controls and of patients not developing thrombocytopenia, their impact is still unclear. Patients with real thrombocytopenia are at an increased risk of bleeding; treatment consists of cessation of the GP IIb/IIIa inhibitor and platelet transfusions in cases of severe hemorrhage. Classic immune thrombocytopenia can be induced by some drugs, e.g. gold, which trigger anti-platelet antibodies indistinguishable from platelet autoantibodies found in autoimmune thrombocytopenia. Drug-induced and drug-dependent immune thrombocytopenia is induced by antibodies recognizing an epitope on platelet GP formed after binding of a drug to a platelet glycoprotein. Still unresolved is whether antibody binding is the consequence of a conformational change of the antigen, the antibody, or both. These antibodies typically react with monomorphic epitopes on platelet GP, but only in the presence of the drug or a metabolite. Although several platelet GP have been identified as antibody target (GPIb/IX, GPV, GP IIb/IIIa), antibodies in an individual patient are highly specific for a single GP. Clinically, these patients present with very low platelet counts and acute, sometimes severe, hemorrhage. Treatment is restricted to withdrawal of the drug and symptomatic treatment of bleeding.
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PMID:Drug-induced and drug-dependent immune thrombocytopenias. 1170 14

GPIbbeta is disulfide-linked to GPIbalpha to form GPIb, a platelet receptor for von Willebrand factor (vWF). GPIb is in turn non covalently linked to GPIX and GPV to form the GPIb/V/IX complex. Apart from its contribution to controlling surface expression of the complex, the exact function of GPIbbeta is not well established due to a lack of suitable ligands or antibodies. The present report describes a monoclonal antibody (RAM.1) that labeled the 26 kDa GPIbbeta subunit on western blots and coprecipitated the three subunits of the GPIb/IX complex from lysates of platelets and transfected CHO and K562 cells. RAM.1 bound to GPIbbeta deleted of its intracellular domain whereas Gi27, directed against intracellular GPIbbeta, did not. Using synthetic peptides, the RAM.1 epitope was mapped to a putative cysteine loop within the COOH-terminal leucine-rich flanking region. In functional assays, RAM.1 had no effect on platelet aggregation induced by ADP, collagen or thrombin, but inhibited ristocetin induced platelet agglutination and botrocetin induced vWF binding. RAM.1 inhibited adhesion of GPIb/V/IX transfected K562 cells to a vWF matrix under flow, increased their rolling velocity and decreased the resistance of cells to detachment at high shear. This study suggests a role of GPIbbeta in modulating the adhesive properties of GPIb/V/IX and describes a useful tool to analyze the exact functions of GPIbbeta.
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PMID:A novel monoclonal antibody against the extracellular domain of GPIbbeta modulates vWF mediated platelet adhesion. 1181 13

We evaluated assay systems for detection of in vivo thrombin activity in patients with acute myocardial infarction. The study included 31 consecutive patients with acute myocardial infarction treated either with fibrinolytic therapy (FLT), or acute PTCA. Blood samples were drawn at admission, after 30 min, 1 h, 3 h, 6 h, 12 h, 24, and 48 h. Assays related to the enzymatic action of thrombin included fibrinopeptide A ELISA, a novel latex-enhanced photometric immunoassay for soluble fibrin complexes, fibrin monomer antigen, D-dimer antigen, and soluble platelet glycoprotein V, which is released from the platelet surface by thrombin cleavage. The soluble fibrin assay displayed a high degree of correlation with fibrinopeptide A, and no correlation with D-dimer antigen, indicating that this parameter allows monitoring of in vivo thrombin activity in patients with acute myocardial infarction. Fibrin monomer antigen, and D-dimer antigen, were influenced by FLT, indicating cross-reactivity with proteolytic fragments of fibrin. No significant changes in soluble glycoprotein V were observed. The results show that the novel soluble fibrin assay appears to be a promising method for measurement of in vivo fibrin formation in patients with acute myocardial infarction, irrespective of the primary treatment decision for FLT or acute PTCA.
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PMID:Course of indicators of thrombin activity in the early phase of acute myocardial infarction: effect of fibrinolytic therapy and acute percutaneous coronary angioplasty. 1287 38

The platelet receptor GPIb/IX/V mediates a crucial role in hemostasis, yet the signaling mechanisms involved are incompletely understood. The complex consists of four polypeptides GPIb alpha, GPIb beta, GPIX and GPV. We identified an amino acid sequence in the cytoplasmic tail of the GPIb beta subunit between residues R151 and A161 that is highly conserved across species and hypothesized that it has functional importance. To target this motif, we synthesized a corresponding cell-permeable palmitylated peptide (Pal-RRLRARARARA) and investigated its effect on platelet function. Pal-RRLRARARARA completely inhibited low dose thrombin- and ristocetin-induced aggregation in washed platelets but only partially inhibited collagen- and U46619-induced aggregation. Thromboxane production in platelets stimulated with thrombin was significantly reduced by Pal-RRLRARARARA compared with collagen. Activation of the integrin alpha IIb beta 3 in response to thrombin was significantly reduced when platelets were preincubated with Pal-RRLRARARARA. The adhesion of washed platelets to von Willebrand factor (VWF) under static conditions was significantly reduced by Pal-RRLRARARARA. Under conditions of high shear, the velocity of platelets rolling on VWF was significantly increased when platelets are preincubated with Pal-RRLRARARARA. This study defines a novel function for the RRLRARARARA motif of GPIb beta in platelet activation.
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PMID:A palmitylated peptide derived from the glycoprotein Ib beta cytoplasmic tail inhibits platelet activation. 1467 1

During myocardial infarction (MI), platelet activation and endothelial apoptosis are responsible for the release of procoagulant membrane-derived microparticles (MP) in the blood flow. MP prothrombotic and proinflammatory properties may be crucial for coronary prognosis. Elevated amounts of circulating procoagulant MP were described in diabetes mellitus (DM), and could be of particular significance in a MI context. We evaluated the prothrombotic status of DM and non-DM (NDM) patients at days 1 and 6 after MI, by measurement of circulating procoagulant MP and soluble GPV (sGPV), the platelet glycoprotein V major fragment released upon thrombin cleavage. Variations were compared to values measured in healthy volunteers (HV). Procoagulant MP were captured onto insolubilized annexin V and quantified by prothrombinase assay. Their cellular origin was assessed. With respect to HV, the levels of procoagulant MP detected at D1 and D6 were elevated in DM and NDM, MP being significantly higher in DM vs. NDM. The high amounts of platelet-derived MP and the correlation between procoagulant MP and sGPV, testify to the central role of thrombin-activated platelets during MI in both DM and NDM subsets. The release of platelet and endothelial cell-derived MP persisted at D6 and was more important in DM, the associated prothrombotic risk being also reflected by higher levels of sGPV. The endothelial damage revealed by endothelial-derived MP was twice that observed in NDM patients. In DM patients presenting cardio-vascular events at 6 month follow-up, MP levels were significantly higher at D1 after MI than in those without complication (24.9 +/- 4.8 vs. 12.3 +/- 2.7 nM PhtdSer, p = 0.02), suggesting a prognostic potential for MP.
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PMID:Sustained elevated amounts of circulating procoagulant membrane microparticles and soluble GPV after acute myocardial infarction in diabetes mellitus. 1496 Nov 63

Circulating procoagulant microparticles (MP) were measured as markers of vascular damage and prothrombotic risk in patients undergoing ST-segment myocardial infarction (STEMI) treated by primary percutaneous transluminal coronary angioplasty (PTCA) and additional GPIIb-IIIa antagonists. Cells possibly more responsive to GPIIb-IIIa (alpha(IIb)beta(3)) antagonists were evidenced through MP phenotypes by comparison with healthy volunteers (HV) and STEMI patients treated by PTCA without GPIIb-IIIa antagonist (CP). In 50 STEMI patients, blood samples were collected at day 1 and day 6. Circulating procoagulant MP were captured on annexin V and quantified by prothrombinase assay as nanomolar phosphatidylserine equivalents (nm PhtdSer). Platelet activation by thrombin was confirmed through independent measurement of soluble GPV (sGPV). With respect to HV, procoagulant MP levels were high in patients with STEMI or unstable angina, platelet-derived MP and elevated sGPV testifying to significant platelet activation. A substantial release of endothelial-derived MP was evidenced simultaneously. In abciximab-treated patients, procoagulant MP, mainly of platelet origin, decreased precociously at day 1 (4.2 +/- 0.6 vs. CP 15.5 +/- 2.1 nm PhtdSer; P = 0.001) together with sGPV (36 +/- 3 vs. CP 58 +/- 8 ng mL(-1); P = 0.02). Leukocyte-derived MP decreased at day 6 (0.12 +/- 0.04 vs. CP 0.56 +/- 0.12 nm PhtdSer; P = 0.01) suggesting a possible effect on underlying inflammatory status. In patients presenting cardiovascular events at 6-month follow-up, procoagulant MP levels at day 1 could be indicative of a worsened outcome. MP could constitute a relevant parameter for the follow-up of STEMI patients treated by GPIIb-IIIa antagonists.
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PMID:Circulating procoagulant microparticles and soluble GPV in myocardial infarction treated by primary percutaneous transluminal coronary angioplasty. A possible role for GPIIb-IIIa antagonists. 1521 95

Glycoprotein (GP) V is noncovalently linked to GPIbalpha, GPIbbeta and GPIX within the platelet GPIb-V-IX complex, a receptor for von Willebrand factor and thrombin. Two functions have been ascribed to GPV, namely, the modulation of thrombin- and collagen-dependent platelet responses. The biosynthesis of this molecule was investigated in pulse-chase metabolic labelling experiments performed in CHO cell lines transfected with GPV, alone or in the presence of GPIb-IX. GPV could not be detected at the surface of cells expressing the single subunit but was found instead as a soluble form in the culture medium. In pulse-chase studies, an immature 70 kDa protein was detected in cell lysates, whereas a fully processed 80-82 kDa form was only observed in the culture supernatants at later chase times. Immature GPV was N-glycosylated and retained before the medial Golgi while the secreted molecule contained complex sialylated sugars. The mature soluble form of GPV was produced by an enzymatic cleavage which was not affected by inhibitors of proteasome, calpain or metalloproteinases. When GPV was cotransfected with GPIb-IX, the former was no longer found in the culture supernatant but was retained in the cell membrane as shown by fluorescence-activated cell sorting and confocal microscopy analyses. Surface expressed GPV was processed from an immature 70 kDa form to produce a mature 80 kDa protein, processing similar to the intracellular trafficking of GPIbalpha. These results indicate that correct biosynthesis and surface expression of GPV in platelets requires the presence of the other subunits of the GPIb-V-IX complex.
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PMID:Biosynthesis of platelet glycoprotein V expressed as a single subunit or in association with GPIb-IX. 1535 44

The glycoprotein Ib-V-IX is one of the major adhesive receptors expressed on the surface of circulating platelets. It is composed of four different polypeptides-GPIbalpha, GPIbbeta, GPIX, and GPV-and represents a multifunctional receptor able to interact with a number of ligands, including the adhesive protein von Willebrand factor, the coagulation factors thrombin, factors XI and XII, and the membrane glycoproteins P-selectin and Mac-1. Interaction of GPIb-V-IX with the subendothelial von Willebrand factor is essential for primary haemostasis, as it initiates platelet adhesion to the subendothelial matrix at the sites of vascular injury even under high flow conditions. Upon interaction with von Willebrand factor, GPIb-V-IX initiates transmembrane signalling events for platelet activation, which eventually result in integrin alpha(IIb)beta(3) stimulation and platelet aggregation. The investigation of the biochemical mechanisms for platelet activation by GPIb-V-IX has attracted increasing attention during the last years. This review will describe and discuss recent findings that have provided new insights into the events underlying GPIb-V-IX transmembrane signalling. In particular, it will summarise basic concepts on the structure of this receptor, extracellular ligands, and intracellular interactors potentially involved in transmembrane signalling. The recently suggested role of membrane Fc receptors in GPIb-V-IX-initiated platelet activation will also be discussed, along with the involvement of lipid metabolising enzymes, tyrosine kinases, and the cytoskeleton in the crosstalk between GPIb-V-IX and integrin alpha(IIb)beta(3).
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PMID:Signalling through the platelet glycoprotein Ib-V-IX complex. 1538 Dec 49

Green pit viper (Trimeresurus albolabris) is the most common venomous snake responsible for bites in Bangkok. It causes local edema and systemic hypofibrinogenemia resulted from the thrombin-like, as well as the fibrinolytic effects of the venom. However, the amino acid sequences of these venom proteins have never been reported. In this study, we have cloned five novel serine proteases from the Thai T. albolabris venom gland cDNA library. They were all closely homologous to the corresponding serine proteases from Chinese green viper (Trimeresurus stejnegeri), suggesting the evolutionary proximity of the two species. In addition, their functional activities could be deduced. There were predicted to be two thrombin-like enzymes (GPV-TL1 and GPV-TL-2), two isoforms of a fibrinogenolytic enzyme (albofibrase) and a plasminogen activator (GPV-PA), suggesting that defibrination syndrome in patients is a combination of these enzymatic effects. By multiple sequence alignment, no conserved residue or motif responsible for distinct functions of snake venom serine proteases could be observed. Moreover, one Lys 49 and one Asn 49 phospholipase A2 (PLA2) genes were cloned. Lys 49 PLA2 was predicted to devoid of catalytic activity, but showed a carboxy terminal cytotoxic region. No Asp 49 PLA2 was found in 150 clones screened. This explains the marked limb edema but no hemolysis in patients. These novel serine proteases have potentials to be therapeutic anti-thrombotic and thrombolytic agents in the future.
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PMID:Molecular cloning of novel serine proteases and phospholipases A2 from green pit viper (Trimeresurus albolabris) venom gland cDNA library. 1637 75


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