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)

Thrombin activates human platelets through three different membrane receptors, the protease-activated receptors PAR-1 and PAR-4 and the glycoprotein Ib (GPIb)-IX-V complex. We investigated the contribution of these three receptors to thrombin-induced activation of the small GTPase Rap1B. We found that, similarly to thrombin, selective stimulation of either PAR-1 or PAR-4 by specific activating peptides caused accumulation of GTP-bound Rap1B in a dose-dependent manner. By contrast, in PAR-1- and PAR-4-desensitized platelets, thrombin failed to activate Rap1B. Thrombin, PAR-1-, or PAR-4-activating peptides also induced the increase of intracellular Ca(2+) concentration and the release of serotonin in a dose-dependent manner. We found that activation of Rap1B by selected doses of agonists able to elicit comparable intracellular Ca(2+) increase and serotonin release was differently dependent on secreted ADP. In the presence of the ADP scavengers apyrase or phosphocreatine-phosphocreatine kinase, activation of Rap1B induced by stimulation of either PAR-1 or PAR-4 was totally inhibited. By contrast, thrombin-induced activation of Rap1B was only minimally affected by neutralization of secreted ADP. Concomitant stimulation of both PAR-1 and PAR-4 in the presence of ADP scavengers still resulted in a strongly reduced activation of Rap1B. A similar effect was also observed upon blockade of the P2Y12 receptor for ADP, as well as in P2Y12 receptor-deficient human platelets, but not after blockade of the P2Y1 receptor. Activation of Rap1B induced by thrombin was not affected by preincubation of platelets with the anti-GPIbalpha monoclonal antibody AK2 in the absence of ADP scavengers or a P2Y12 antagonist but was totally abolished when secreted ADP was neutralized or after blockade of the P2Y12 receptor. Similarly, cleavage of the extracellular portion of GPIbalpha by the cobra venom mocarhagin totally prevented Rap1B activation induced by thrombin in the presence of apyrase and in P2Y12 receptor-deficient platelets. By contrast, inhibition of MAP kinases or p160ROCK, which have been shown to be activated upon thrombin binding to GPIb-IX-V, did not affect agonist-induced activation of Rap1B in the presence of ADP scavengers. These results indicate that although both PAR-1 and PAR-4 signal Rap1B activation, the ability of thrombin to activate this GTPase independently of secreted ADP involves costimulation of both receptors as well as binding to GPIb-IX-V.
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PMID:Contribution of protease-activated receptors 1 and 4 and glycoprotein Ib-IX-V in the G(i)-independent activation of platelet Rap1B by thrombin. 1507 82

Stimulating human platelets with thrombin induces the activation of the extracellular signal-regulated kinase 2 (ERK2). We demonstrate that this effect is highly dependent on ADP secretion and P2Y12 receptor signalling. AR-C69931MX (10 microM), a specific antagonist of the Gi-coupled P2Y12 ADP receptor, inhibits ERK2 activation induced by thrombin. Antagonists of the Gq-coupled P2Y1 ADP receptor, A3P5P (500 microM) and MRS2179 (100 microM), have no effect. ADP and its more potent analogue 2-methylthio-ADP alone (both up to 100 microM) do not induce ERK2 activation. Furthermore, we show that the inhibitory effect of AR-C69931MX on ERK2 activation induced by 0.1 U/ml thrombin as well as on platelet aggregation can be bypassed by epinephrine (1 and 10 microM), whereas epinephrine alone has no effect. Epinephrine acts on platelets mainly via alpha(2A)-adrenergic receptors, which, like P2Y12 receptors, couple to inhibitory G proteins. In addition, 2-methylthio-ADP as well as epinephrine provoke ERK2 activation at a thrombin concentration that alone has no detectable effect (0.05 U/ml). Thromboxane A2 (TXA2), which, like ADP, is released by activated platelets, acts as a positive feedback mediator. Stimulating the Gq-coupled TXA2 -receptor with U46619 (10 microM), which leads to ADP secretion and P2Y12 receptor-dependent platelet aggregation, also induces P2Y12-related ERK2 activation. The inhibition of U46619-induced ERK2 activation and platelet aggregation by AR-C69931MX are also rescued by epinephrine. Pretreatment with aspirin inhibits ERK2 activation induced by 0.1 U/ml thrombin, but has no effect at high concentrations of thrombin. The combination of U46619 and thrombin, at concentrations which alone have no effect, provokes ERK2 activation, suggesting that thrombin and released TXA2 act synergistically. Our data indicate that both primary signalling through Gq, which evokes ADP secretion, as well as subsequent coupling via Gi by the P2Y12 receptor are required for ERK2 activation.
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PMID:ADP secretion and subsequent P2Y12 receptor signalling play a crucial role in thrombin-induced ERK2 activation in human platelets. 1521 52

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

Photochemically induced thrombosis (a thrombin-dependent process) was measured in rats treated with moderate doses of anticoagulants, but which appeared to be unchanged. We considered the possibility that platelet-inhibiting agents, which also indirectly inhibit coagulation, would act as more potent antithrombotic agents. Inhibitors used as such were prostaglandin E1 (PGE1), which elevates cyclic AMP levels, and the P2Y12 ADP-receptor antagonist, AR-C69931MX. Effects of these agents were investigated in an ex vivo model system, in which whole blood under coagulant conditions was perfused over fibrinogen at defined wall shear rate. Perfusion of blood (rat or human) in the presence of tissue factor resulted in deposition of activated platelets and subsequent aggregate formation, along with exposure of procoagulant phosphatidylserine (PS) on the platelet surface and formation of fibrin fibers. In the presence of PGE1 aggregation was completely inhibited, but platelet adhesion and PS exposure were only party reduced, while fibrin formation was hardly affected. Treatment with AR-C69931MX caused similar, but less complete effects. These results indicate that in tissue factor-triggered blood under conditions of flow: (i) the platelet procoagulant response is independent of aggregate formation; (ii) the platelet-inhibiting effect of PGE1 and AR-C69931MX is sufficient to suppress aggregation, but not platelet adhesion and coagulation. These platelet inhibitors thus maintain their aggregation-inhibiting effect at sites of thrombin formation.
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PMID:Regulation of tissue factor-induced coagulation and platelet aggregation in flowing whole blood. 1563 Apr 98

In thrombin-stimulated human platelets several proteins undergo rapid and transient changes in tyrosine phosphorylation. We demonstrate that a set of proteins of 27, 29, 31, 34, and 39 kDa is affected by released ADP and P2Y12 receptor signaling during platelet activation. AR-C69931MX, an antagonist of the Gi(2)-coupled P2Y12 ADP receptor, inhibits initial tyrosine phosphorylation of p27 and p31 and prevents subsequent dephosphorylation of p29, p34, and p39. Antagonists of the Gq-coupled P2Y1 ADP receptor have no effect. Precluding integrin alpha(IIb)beta(3) outside-in signaling with RGDS or S1197 does not affect the increase in tyrosine phosphorylation of the set of proteins but inhibits their subsequent dephosphorylation. Besides the ADP analogue 2-MeS-ADP, other platelet agonists such as collagen and the TXA(2)-mimetic U46619 also induce p27 and p31 tyrosine phosphorylation in a P2Y12 receptor-dependent manner. Tyrosine phosphorylation of p27 and p31 in response to collagen, but not thrombin, is prevented by aspirin and the TXA(2) receptor antagonist SQ29548, indicating that the effect of collagen strongly relies on TXA(2) signaling. Furthermore, epinephrine, acting via inhibitory Gz-coupled alpha(2A)-adrenoceptors, bypasses the inhibitory effect of AR-C69931MX on thrombin-induced p27 and p31 tyrosine phosphorylation. Finally, we demonstrate that tyrosine phosphorylation of p27 and p31 downstream of P2Y12 receptors is due to the inhibition of adenylyl cyclase but not phosphoinositide 3-kinase (PI 3-K) activation. Elevating cAMP levels with PGI(2) or forskolin precludes thrombin-induced p27 and p31 tyrosine phosphorylation. Moreover, direct inhibition of adenylyl cyclase by SQ22536 reverses the effect of AR-C69931MX. Our data indicate that the observed changes in tyrosine phosphorylation are the result of both primary Gq signaling, initiating the release of ADP, as well as subsequent P2Y12 receptor-mediated Gi coupling.
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PMID:P2Y12 ADP receptor-dependent tyrosine phosphorylation of proteins of 27 and 31 kDa in thrombin-stimulated human platelets. 1588 4

Platelet activation by ADP and ATP plays a crucial role in haemostasis and thrombosis, and their so-called P2 receptors are potential targets for antithrombotic drugs. The ATP-gated channel P2X1 and the 2 G protein-coupled P2Y1 and P2Y12 ADP receptors selectively contribute to platelet aggregation. The P2Y1 receptor is responsible for ADP-induced shape change and weak and transient aggregation, while the P2Y12 receptor is responsible for the completion and amplification of the response to ADP and to all platelet agonists, including thromboxane A2 (TXA2), thrombin, and collagen. The P2X1 receptor is involved in platelet shape change and in activation by collagen under shear conditions. Due to its central role in the formation and stabilization of a thrombus, the P2Y12 receptor is a well-established target of antithrombotic drugs like ticlopidine or clopidogrel, which have proved efficacy in many clinical trials and experimental models of thrombosis. Competitive P2Y12 antagonists have also been shown to be effective in experimental thrombosis as well as in several clinical trials. Studies in P2Y1 and P2X1 knockout mice and experimental thrombosis models using selective P2Y1 and P2X1 antagonists have shown that, depending on the conditions, these receptors could also be potential targets for new antithrombotic drugs.
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PMID:The platelet P2 receptors as molecular targets for old and new antiplatelet drugs. 1595 65

There have been major advances in our understanding of thrombosis and antithrombotic drugs. This review focuses on the molecular aspects of thrombus formation and antithrombotic therapy. Molecules involved in arterial thrombosis are derived from inflammatory cells in the atherosclerotic plaque and blood platelets. These molecules work in concert to promote plaque instability and thrombogenicity. Thrombus formation on the ruptured plaque is mediated by platelet and coagulation activation. By contrast, molecules involved in venous thrombosis are derived from the activated coagulation cascade. Platelets appear to play a secondary role. The antithrombotic drugs are classified according to their targeted constituents: antiplatelet agents and anticoagulants; the latter are further divided into non-specific anticoagulants, such as vitamin K antagonists and heparin, and direct thrombin inhibitors, including hirudin and argatroban. Currently available antiplatelet agents target glycoprotein IIbIIIa (abciximab, tirofiban, eptifibatide), cyclooxygenase-1 (aspirin) or adenosine diphosphate receptor, P2Y12 (clopidogrel).
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PMID:Molecular aspects of thrombosis and antithrombotic drugs. 1604 40

Regulation of platelet activation plays a central role in hemostasis and pathophysiological processes such as coronary artery disease. Thrombin is the most potent activator of platelets. Human platelets express two thrombin receptors, PAR1 and PAR4, both of which signal platelet activation. Evidence is lacking on the mechanism by which PAR1 and PAR4 may differentially signal platelet aggregation. Here we show that at the relatively high concentration of agonist most likely found at the site of a local thrombus, dual inhibition of the P2Y12 receptor and calcium mobilization result in a complete inhibition of PAR4-induced aggregation, while having no effect on either thrombin or PAR1-mediated platelet aggregation. Both PAR1- and PAR4mediated aggregation are independent of calcium mobilization. Furthermore, we show that P2Y12 receptor activation is not required for protease-activated receptor-mediated aggregation at higher agonist concentrations and is only partially required for Rap1 as well as GPIIbIIIa activation. P2Y12 receptor inhibitors clinically in use such as clopidogrel are postulated to decrease platelet aggregation through partial inhibition of PAR1 signaling. Our data, however, indicate that at high local concentrations of thrombin, it is the signaling through PAR4 rather than PAR1 that may be regulated through purinergic feedback. Thus, our data identify an intra-platelet mechanism that may function as a future site for therapeutic intervention.
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PMID:PAR4, but not PAR1, signals human platelet aggregation via Ca2+ mobilization and synergistic P2Y12 receptor activation. 1683 56

The effect of the direct platelet P2Y12 receptor inhibitor, AR-C69931MX, on activation of blood induced by stents with and without heparin coating was investigated using a whole blood Chandler loop model in vitro. Stents were deployed in Chandler loops. Fresh human blood with heparin and AR-C69931MX was rotated for 1 h at 37 degrees C and used for measurements of platelets, microparticles, thrombin-antithrombin complex (TAT), fibrinogen binding to platelets, P-selectin expression by platelets, CD11b, Prothrombin Fragment F1+2, FXIa-AT, FXIIa-AT, C3a, sC5b-9 and stent score. In the first experiment there were four study groups with unmodified stents: 1a, no AR-C69931MX; 1b, 250 nmol/L; 1c, 750 nmol/L; 1d, 2250 nmol/L of AR-C69931MX. In the second experiment the concentration of AR-C69931MX was 500 nmol/L: 2a; tubings without stent; 2b; tubings with heparin-coated stent; 2c; tubings with unmodified stents. Heparin-coated stents were used in the third experiment: 3a; no AR-C69931MX; 3b; 500 nmol/L of AR-C69931MX. In the first experiment there were significant differences in all parameters analysed except for C3a, and stent score when the group with no AR-C69931MX was compared to all the groups with AR-C69931MX. In the second experiment there were significant differences in platelet count, TAT, FXIa-AT, FXIIa-AT and stent score when unmodified stents were compared to loops with no stents and partly to loops with heparin-coated stents. In the third experiment there was a significant reduction in generation of TAT, stent score and better preservation of platelet number by combining the platelet inhibitor and heparin-coated stents as compared to heparin-coated stents alone. The conclusion is that the direct P2Y12 receptor inhibitor AR-C69931MX reduced the different aspects of activation of blood induced by both unmodified and heparin-coated stents.
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PMID:Effects on blood compatibility in vitro by combining a direct P2Y12 receptor inhibitor and heparin coating of stents. 1692 4

p38 MAP kinase in human platelets is activated by platelet agonists including thrombin, thromboxane A2 (TxA2), ADP, and others. However, both upstream mechanisms of p38 MAP kinase activation, and their downstream sequelae, are presently controversial and essentially unclear. Certain studies report sequential activation of cGMP-dependent protein kinase (PKG) and p38/ERK pathways by platelet agonists, leading to integrin activation and secretion, whereas others establish an essential role of Src/ERK-mediated TxA2 generation for fibrinogen receptor activation in human platelets. Here, we show that ADP secreted from platelet-dense granules, and subsequent activation of P2Y12 receptors, as well as TxA2 release are important upstream mediators of p38 MAP kinase activation by thrombin. However, p38 MAP kinase activation did not significantly contribute to calcium mobilization, P-selectin expression, alphaIIbbeta3 integrin activation, and aggregation of human platelets in response to thrombin. Finally, PKG activation did not stimulate, but rather inhibited, p38 MAP kinase in human platelets.
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PMID:Thrombin stimulation of p38 MAP kinase in human platelets is mediated by ADP and thromboxane A2 and inhibited by cGMP/cGMP-dependent protein kinase. 1699 May 90


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