Thrombin signaling in Hemostasis and thrombosis

描述(申请人提供)：我的实验室的中心目标是确定凝血酶如何调节参与止血、血栓形成、炎症和其他过程的细胞行为，并阐明凝血酶信号在体内的作用。这笔赠款的重点是止血和血栓形成。我们以前的工作表明，蛋白酶激活的受体(PARs)是凝血酶激活血小板所必需的，对小鼠模型中的血栓形成和止血也很重要。这些和其他研究支持探索PAR拮抗剂用于预防或治疗人类血栓形成。我们现在建议进行研究，以更详细地确定PARS的作用，并确定凝血酶信号如何与体内其他血小板激活和凝血机制相结合。我们会问：1)vWF、胶原和凝血酶是体内血小板活化的主要起始物吗？这些途径是如何相互作用的？使用复杂的小鼠模型，我们将检验这一假设，即GPIB和GP-VI信号是推动损伤部位的血小板黏附和血栓形成的必要条件和充分条件，而PAR信号是血栓从血管壁传播出去所必需的。将使用遗传和药理学方法。PAR与P2Y12和Tbxa2r(Tp)的相互作用也将被探讨。2)凝血酶诱导的血小板活化和纤维蛋白形成如何在止血和血栓形成中相互作用？当凝血酶生成或活性减少或被抑制时，它们的相对重要性是否会改变？我们将确定凝血酶激活的血小板是否对凝血酶的产生和纤维蛋白的形成在远离血管壁的传播中起重要作用。我们还将询问，在凝血酶生成较低的情况下，PAR信号的中断是否对血栓形成或止血有协同作用。3)血管内皮细胞和造血细胞中组织因子的表达在止血和血栓形成中的作用是什么？内毒素血症？这种组织因子的作用能通过敲除凝血传播的替代机制而被发现或放大吗？组织因子在血管内皮细胞和造血组织中的表达将被消融，以探索循环组织因子的来源和作用。这些研究将阐明止血和血栓形成的关键影响因素是如何相互作用的。

DESCRIPTION (provided by applicant): A central goal of my laboratory has been to determine how thrombin regulates cellular behaviors involved in hemostasis, thrombosis, inflammation and other processes, and to elucidate the roles of thrombin signaling in vivo. This grant has focused on hemostasis and thrombosis. Our previous work showed that protease-activated receptors (PARs) are necessary for platelet activation by thrombin and important for thrombosis and hemostasis in mouse models. These and other studies support exploration of PAR antagonism for the prevention or treatment of thrombosis in humans. We now propose studies to define the roles of PARs in more detail and to determine how thrombin signaling integrates with other platelet activation and coagulation mechanisms in vivo. We shall ask: 1) Are vWF, collagen and thrombin the major initiators of platelet activation in vivo? How do these pathways interact? Using sophisticated mouse models, we will test the hypothesis that GPIb and GP-VI signaling is necessary and sufficient to drive platelet adhesion and juxtamural thrombus formation at a site of injury but that PAR signaling is necessary for propagation of the thrombus away from the vessel wall. Genetic and pharmacological approaches will be used. PAR interactions with P2Y12 and Tbxa2r (TP) will also be probed. 2) How do thrombin-induced platelet activation and fibrin formation interact in hemostasis and thrombosis? Does their relative importance change when thrombin generation or activity is reduced or inhibited? We shall determine whether platelet activation by thrombin is important for propagation of thrombin generation and fibrin formation away from the vessel wall. We shall also ask whether disruption of PAR signaling in the setting of low thrombin generation has synergistic effects on thrombosis or hemostasis. 3) What is the role of tissue factor expression in endothelial and hematopoietic cells in hemostasis and thrombosis? In endotoxemia? Can roles for such tissue factor be uncovered or amplified by knockout of alternative mechanisms for propagation of coagulation? Tissue factor expression will be ablated in endothelial and hematopoietic tissues to probe the source and roles of "circulating tissue factor". These studies will illuminate how key effectors of hemostasis and thrombosis interact.