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)

Excessive coagulation and impaired fibrinolysis lead to many hemostatic disorders, which enhance the risk of development of life-threatening cardiovascular diseases such as myocardial infarction, stroke, deep venous thrombosis and pulmonary embolism, belonging to the most important factors influencing morbidity and mortality in civilized societies. The adverse events induced by currently used drugs, the need for regular monitoring of coagulation parameters, inconvenient, in some cases, route of administration stimulate further search for novel, effective and safe methods of therapies of these disorders. In this paper, we describe those new agents which are now under experimental and clinical study, such us prostanoids, nitroaspirin, GP IIb/IIIa receptor antagonists, thienopyridine derivatives, collagen-GPVI and von Willebrand factor-GPIb-IX contact blockers, direct thrombin inhibitors, inhibitors of thrombin-platelet interactions, factor VII inhibitors and tissue factor-factor VII contact blockers. Based on the available literature, we discuss the possible role of these agents in the future prevention and treatment of thromboembolic diseases.
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PMID:Progress in pharmacotherapy of thrombosis. 1458 10

Platelet interaction with subendothelial collagen is crucial for hemostasis and thrombosis. Under conditions of elevated shear, platelet adhesion and activation on collagen requires the coordinated action of glycoprotein (GP) Ib and GPVI, which may be physically and functionally linked in the platelet membrane. While the surface expression of GPIb can be down-regulated by internalization and/or proteolytic cleavage of a 130 kDa fragment of GPIb (glycocalicin, GC), very little is known about the cellular regulation of GPVI. We have recently shown that GPIb on platelets is cleaved by metalloproteinase-dependent mechanisms in response to mitochondrial injury. In the current study, we examined a possible role of platelet metalloproteinases in the regulation of GPVI. Mitochondrial injury induced by incubation of mouse platelets with carbonyl cyanide m-chlorophenyl-hydrazone (CCCP) severely affected the cells' responses to collagen or the GPVI-specific agonist, collagen related peptide (CRP), but not to thrombin or the stable thromboxane A(2) analog U46619. This defect was due to a rapid proteolytic cleavage of GPVI, as shown by the release of the 55 kDa extracellular domain into the supernatant. Both the proteolysis of GPVI and the loss of its activity were inhibited in the presence of the broad range metalloproteinase inhibitor, GM6001. Platelet stimulation with thrombin or CRP, however, resulted in marked metalloproteinase-dependent shedding of GPIbalpha, but not GPVI suggesting that different metalloproteinases are involved in the regulation of the two receptors or, alternatively, an additional signal is required to render GPVI susceptible to cleavage.
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PMID:GPVI down-regulation in murine platelets through metalloproteinase-dependent shedding. 1511 56

Lipoxygenases (LOX) contribute to vascular disease and inflammation through generation of bioactive lipids, including 12-hydro(pero)xyeicosatetraenoic acid (12-H(P)ETE). The physiological mechanisms that acutely control LOX product generation in mammalian cells are uncharacterized. Human platelets that contain a 12-LOX isoform (p12-LOX) were used to define pathways that activate H(P)ETE synthesis in the vasculature. Collagen and collagen-related peptide (CRP) (1 to 10 microg/mL) acutely induced platelet 12-H(P)ETE synthesis. This implicated the collagen receptor glycoprotein VI (GPVI), which signals via the immunoreceptor-based activatory motif (ITAM)-containing FcRgamma chain. Conversely, thrombin only activated at high concentrations (> 0.2 U/mL), whereas U46619 and ADP alone were ineffective. Collagen or CRP-stimulated 12-H(P)ETE generation was inhibited by staurosporine, PP2, wortmannin, BAPTA/AM, EGTA, and L-655238, implicating src-tyrosine kinases, PI3-kinase, Ca2+ mobilization, and p12-LOX translocation. In contrast, protein kinase C (PKC) inhibition potentiated 12-H(P)ETE generation. Finally, activation of the immunoreceptor tyrosine-based inhibitory motif (ITIM)-containing platelet endothelial cell adhesion molecule (PECAM-1) inhibited p12-LOX product generation. This study characterizes a receptor-dependent pathway for 12-H(P)ETE synthesis via the collagen receptor GPVI, which is negatively regulated by PECAM-1 and PKC, and demonstrates a novel link between immune receptor signaling and lipid mediator generation in the vasculature.
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PMID:Platelet 12-lipoxygenase activation via glycoprotein VI: involvement of multiple signaling pathways in agonist control of H(P)ETE synthesis. 1521 16

Vessel wall damage exposes collagen fibres, to which platelets adhere directly via the collagen receptors glycoprotein (GP) VI and integrin alpha(2)beta(1) and indirectly by collagen-bound von Willebrand factor (vWF) via the GPIb-V-IX and integrin alphaIIbbeta3 receptor complexes. Platelet-collagen interaction under shear stimulates thrombus formation in two ways, by integrin-dependent formation of platelet aggregates and by surface exposure of procoagulant phosphatidylserine (PS). GPVI is involved in both processes, complemented by alpha2beta1. In mouse blood flowing over collagen, we investigated the additional role of platelet-vWF binding via GPIb and alphaIIbbeta3. Inhibition of GPIb as well as blocking of vWF binding to collagen reduced stable platelet adhesion at high shear rate. This was accompanied by delayed platelet Ca(2+) responses and reduced PS exposure, while microaggregates were still formed. Inhibition of integrin alphaIIbbeta3 with JON/A antibody, which blocks alphaIIbbeta3 binding to both vWF and fibrinogen, reduced PS exposure and aggregate formation. The JON/A effects were not enhanced by combined blocking of GPIb-vWF binding, suggesting a function for alphaIIbbeta3 downstream of GPIb. Typically, with blood from FcR gamma-chain +/- mutant mice, expressing 50% of normal platelet GPVI levels, GPIb blockage almost completely abolished platelet adhesion and PS exposure. Together, these data indicate that, under physiological conditions of flow, both adhesive receptors GPIb and alphaIIbbeta3 facilitate GPVI-mediated PS exposure by stabilizing platelet binding to collagen. Hence, these glycoproteins have an assistant procoagulant role in collagen-dependent thrombus formation, which is most prominent at reduced GPVI activity and is independent of the presence of thrombin.
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PMID:Facilitating roles of murine platelet glycoprotein Ib and alphaIIbbeta3 in phosphatidylserine exposure during vWF-collagen-induced thrombus formation. 1515 90

ATP binding cassette transporter A1 (ABCA1) is involved in regulation of intracellular lipid trafficking and export of cholesterol from cells to high density lipoproteins. ABCA1 defects cause Tangier disease, a disorder characterized by absence of high density lipoprotein and thrombocytopenia. In the present study we have demonstrated that ABCA1 is expressed in human platelets and that fibrinogen binding and CD62 surface expression in response to collagen and low concentrations of thrombin, but not to ADP, are defective in platelets from Tangier patients and ABCA1-deficient animals. The expression of platelet membrane receptors such as GPVI, alpha2beta1 integrin, and GPIIb/IIIa, the collagen-induced changes in phosphatidylserine and cholesterol distribution, and the collagen-induced signal transduction examined by phosphorylation of LAT and p72syk and by intracellular Ca2+ mobilization were unaltered in Tangier platelets. The electron microscopy of Tangier platelets revealed reduced numbers of dense bodies and the presence of giant granules typically encountered in platelets from Chediak-Higashi syndrome. Further studies demonstrated impaired release of dense body content in platelets from Tangier patients and ABCA1-deficient animals. In addition, Tangier platelets were characterized by defective surface exposure of dense body and lysosomal markers (CD63, LAMP-1, LAMP-2, CD68) during collagen- and thrombin-induced stimulation and by abnormally high lysosomal pH. We conclude that intact ABCA1 function is necessary for proper maturation of dense bodies in platelets. The impaired release of the content of dense bodies may explain the defective activation of Tangier platelets by collagen and low concentrations of thrombin, but not by ADP.
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PMID:Impaired platelet activation in familial high density lipoprotein deficiency (Tangier disease). 1516 65

Homophilic engagement of platelet endothelial cell adhesion molecule-1 (PECAM-1/CD31) induces 'outside-in' signal transduction that results in phosphorylation events and recruitment and activation of signalling molecules. The formation of signalling scaffolds with PECAM-1 are important signalling events that modulate platelet secretion, aggregation and platelet thrombus formation. In this study, we describe a novel interaction between PECAM-1 and cytosolic calmodulin (CaM) in platelets. Reciprocal co-immunoprecipitation studies revealed that cytosolic CaM is constitutively associated with PECAM-1 in resting, thrombin activated and aggregated human platelets. Our studies demonstrate that CaM directly interacts with a PECAM-1 peptide (594-604) C595A containing the sequences (594)KAFYLRKAKAK(604). This CaM:PECAM-1 interaction has a threefold higher affinity than CaM:GPVI interaction. It is potentiated by the addition of calcium ions, and dissociated by the CaM inhibitor, trifluoperazine. Treatment of platelets with CaM inhibitors triggers cleavage of PECAM-1 in a time- and dose-dependent manner. Furthermore, this membrane proximal portion of PECAM-1 is conserved across mammalian species and the helical representation of basic/hydrophobic residues reveals a charge distribution analogous to other CaM-binding motifs in other proteins. Taken together, these results suggest that this highly charged cluster of amino acids in the PECAM-1 cytoplasmic domain directly interacts with CaM and this novel interaction appears to regulate cleavage of PECAM-1.
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PMID:Proteolytic cleavage of platelet endothelial cell adhesion molecule-1 (PECAM-1/CD31) is regulated by a calmodulin-binding motif. 1519 23

Collagen- and thrombin-activated (COAT) platelets were first described in 2000 and have attracted considerable interest, changing the interpretation of the way in which platelets contribute to thrombin generation and how their procoagulant activity is organized. Platelets activated by two agonists coming from glycoprotein VI or Fc gamma-receptor IIA agonists on the one hand and thrombin on the other produce a population of approximately 50% highly procoagulant active platelets. This subgroup is formed by tissue transglutaminase and factor XIIIa linking of serotonin to the procoagulant proteins from granules or plasma, and these serotonylated proteins bind to fibrinogen or thrombospondin on the platelet surface. Serotonylation in the platelet cytoplasm has recently been shown to be an important regulating mechanism governing the activation of small GTPases and their function in granule release. Recent studies with Tph-/- mice in which the peripheral serotonin, including that in platelets, is very strongly reduced, have shown a prolonged bleeding time, suggesting it has an important hemostatic role in the release of platelet von Willebrand factor. More knowledge about how COAT platelets are formed will be important for a better understanding of the physiology and pathology of hemostasis.
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PMID:All platelets are not equal: COAT platelets. 1534

A high throughput 384-well plate assay of blood function in 60 microl reactions with the fluorogenic thrombin substrate, boc-VPR-MCA, allowed for real-time monitoring of coagulation under a diverse set of reaction conditions. Using recalcified, citrated whole blood diluted 3-fold with corn trypsin inhibitor (to block Factor XIIa), addition of 0 to 13.8 pM of tissue factor (TF) reduced the time of maximal rate of thrombin production T(max) from 45 min to 11 min. Over this range of TF,T(max) was reduced from 35 min to 6 min by co-addition of 10 nM convulxin to activate platelets via GPVI. The maximal rate of thrombin production at T(max) was not a function of exogenously-added TF,Va, or reVIIa, but increased 30% with added convulxin. Addition of 0.07 to 0.7 pM TF along with convulxin produced small, but detectable reductions in T(max). Addition of up to 0.67 nM reVIIa reduced T(max) by up to 53% in the range of 0.7 to 7 pM TF. Interestingly, platelet factor 4 (2.7 microM) caused a prolongation of T(max) from 45 min to 78 min at 0 TF, while protamine (1.8 microM) reduced T(max) to 30 min at 0 TF. Finally, combinatorial reaction studies with exogenously-added ADP, histamine, fMLP, indomethacin, anti-CD18, and fibrinogen revealed no unusual synergies amongst the agents, but demonstrated a striking procoagulant activity of added fibrinogen, due to protease contaminants in the "purified" fibrinogen. This high throughput approach allowed automated profiling of blood (50 reactions/ml of blood) to generate large data sets for testing cellular-proteomic kinetic models, screening drug interactions, and potentially monitoring subtle changes in the functional phenotype of a patient blood sample.
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PMID:Blood coagulation kinetics: high throughput method for real-time reaction monitoring. 1546 20

Systemic inflammation has been shown to be a contributing factor to the instability of atherosclerotic plaques in patients with acute coronary syndromes (ACS). VX-702, a novel p38 mitogen-activated protein kinase (MAPK) inhibitor, is currently under investigation in ACS patients with unstable angina to evaluate its safety and efficacy during percutaneous coronary intervention (PCI). The role of p38 MAPK in platelet aggregation of normal individuals was examined using the selective second generation p38 MAPK inhibitor VX-702. Treatment of platelets with thrombin (activates PAR1 and PAR4 thrombin receptors), SFLLRN (PAR1), AYPGKF (PAR4), collagen (alpha2beta1 and GPVI/FCgammaIIR receptors) and U46619 (TXA(2)) resulted in strong activation of p38 MAPK. Activation of the GPIb von Willebrand factor receptor with ristocetin did not stimulate p38 MAPK. Pre-treatment of platelets with 1 microM VX-702 completely inhibited activation of p38 MAPK by thrombin, SFLLRN, AYPGKF, U46619, and collagen. There was no effect of VX-702 on platelet aggregation induced by any of the agonists in the presence or absence of aspirin, heparin or apyrase. It has been postulated that a potential role of p38 MAPK is to activate phospholipase A(2) (cPLA(2)) which catalyses formation of arachidonic acid leading to production of thromboxane. Interestingly, we show contrasting effects of p38 MAPK inhibition as compared to aspirin inhibition on platelet aggregation in response to collagen. Blockade of TXA(2) production by aspirin results in significant inhibition of collagen activation. However,VX-702 has no effect on collagen-mediated platelet aggregation, suggesting that blocking p38 MAPK does not effect thromboxane production in human platelets. Therefore, unlike aspirin blockade of thromboxane production in platelets, p38 MAPK inhibitors such as VX-702 do not significantly affect platelet function and would not be expected to contribute to an elevated risk of bleeding side-effects in treated patients.
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PMID:Effect of selective inhibition of the p38 MAP kinase pathway on platelet aggregation. 1558 48

The activation of the small GTPase Rap2B in resting and agonist-stimulated human platelets was investigated. Both thrombin, that stimulates heterotrimeric G-protein-coupled receptors, and the GPVI ligand convulxin, that activates a tyrosine-kinase based signaling pathway, were able to induced the rapid and sustained binding of GTP to Rap2B. Similarly, a number of other agonists tested, previously known to activate the highly related protein Rap1B, were also able to stimulate Rap2B. In contrast, platelet antagonists that increase the intracellular concentration of cAMP did not signal to Rap2B. Thrombin- and convulxin-induced activation of Rap2B was not dependent on thromboxane A2, did not require the interaction of the protein with the cytoskeleton, and was not regulated by integrin alphaIIbbeta3-dependent outside-in signaling. When secreted ADP was neutralized, activation of Rap2B induced by thrombin, but not by convulxin, was significantly reduced. ADP itself was found to induce the rapid and sustained binding of GTP to Rap2B, and this effect was predominantly mediated by stimulation of the Gi-coupled P2Y12 receptor. Activation of Rap2B promoted by both thrombin and convulxin was regulated by intracellular Ca2+, while protein kinase C was found to be involved in convulxin- but not in thrombin-induced activation of Rap2B. Moreover, Rap2B activation induced by thrombin, but not by convulxin, was totally dependent on phosphatidylinositol 3-kinase activity. These results demonstrate that the small GTPase Rap2B is involved in platelet activation, and outline some important differences between the regulation of highly related GTPases Rap2B and Rap1B in human platelets.
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PMID:Activation of the small GTPase Rap2B in agonist-stimulated human platelets. 1561 30


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