REGULATION OF HUMAN PLATELET PROTHROMBINASE ACTIVITY

血小板调节其膜上凝血酶生成的能力 表面是它们在止血、血栓形成和 动脉硬化。凝血酶的产生是通过组装来实现的 和酶复合体的功能，以及动脉粥样硬化。凝血酶 生成是通过酶的组装和功能实现的 凝血酶原酶复合体，由I.化学计量比钙离子组成。 辅因子Va与丝氨酸蛋白酶的依赖复合体 因子Xa。在血小板激活后，释放血小板因子Va 和/或血浆衍生因子Va与血小板膜表面结合 在这样做的过程中，至少形成了Xa因子的部分受体。因此， 几种蛋白质/蛋白质和蛋白质/膜相互作用参与 并调节复杂的组装。该项目的一个目标是定义如何 血小板积极参与和调节凝血酶原酶装配 和功能。已经提出并将提出以下假设 测试过。血小板通过1)表达凝血酶调节凝血酶的生成 两个离散的血小板亚群--都是结合因子Va，只有一个结合 Va和Xa这两个因子；3)通过激动剂诱导释放 抗通过激活而失活的血小板因子Va 活化的蛋白C和纤溶酶，在功能和物理上都是独一无二的 与血浆衍生因子Va相比。我们将在以下方面作出努力 分离和鉴定这些离散的血小板群体，分离 和/或血小板膜因子Va受体的表达克隆 和Xa，并表征了血小板衍生因子Va的功能和 生理上与其参与凝血酶原酶有关。因为 凝血酶一旦形成，就会积极地调节凝血酶原酶的组装和 通过激活和释放血小板因子Va、a发挥作用 第二个目标是确定凝血酶如何通过血小板相互作用 激活和释放血小板因子Va，第二个目标是定义如何 凝血酶与血小板膜蛋白相互作用调节其 活动。我们将努力验证这样一种假设，即 凝血酶的高亲和力结合部位是1)独特的血小板膜 一种类似水飞蓟素的蛋白质，提供凝血酶分子 暂时无效，2)不同于糖蛋白1b。这将是 部分是通过表达克隆这种独特的血小板来实现的 膜蛋白及抗糖蛋白1b抗体的证明 抑制凝血酶诱导的血小板激活是通过它们的 与PAR1的交叉反应。成功完成这些目标将 演示了血小板主动调节的机制 凝血酶在其膜表面的产生和功能。

The ability of platelets to regulate thrombin generation at their membrane surface is central to their role in hemostasis, thrombosis, and atherosclerosis. Thrombin generation is effected through the assembly and function of the enzymatic complex, and atherosclerosis. Thrombin generation is effected through the assembly and function of the enzymatic complex, Prothrombinase, consisting of a I.I stoichiometric Ca2+- dependent complex of the cofactor factor Va and the serine protease factor Xa. Subsequent to platelet activation, release platelet factor Va and/or plasma-derived factor Va bind to the platelet membrane surface and in so doing form at least part of the receptor for factor Xa. Thus, several protein/protein and protein/membrane interactions participate in and regulate complex assembly. One goal of this project is to define how platelets actively participate in and regulate Prothrombinase assembly and function. The following hypotheses have been formulated and will be tested. Platelets regulate thrombin generation through 1) the expression of two discrete platelet subpopulations-both bind factor Va, only one binds both factors Va and Xa; and 3) through the agonist-induced release of platelet factor Va which is resistant to inactivation by activation by activated protein C and plasmin, and functionally and physically unique when compared to plasma-derived factor Va. Effort will be placed on isolating and characterizing these discrete platelet populations, isolating and/or expression cloning the platelet membrane receptors for factor Va and Xa, and characterizing platelet-derived factor Va, functionally and physically as related to its participation in Prothrombinase. Because thrombin once formed positively regulates Prothrombinase assembly and function through platelet activation and release of platelet factor Va, a second goal is to define how thrombin interacts through platelet activation and release of platelet factor Va, a second goal is to define how thrombin interacts with platelet membrane proteins to modulate its activity. Effort will be place don testing the hypothesis that the platelet high affinity binding site for thrombin is 1) a unique platelet membrane protein that resembles hirudin and renders the thrombin molecule transiently inactive and, 2) is distinct from glycoprotein 1b. This will be accomplished in part through expression cloning of this unique platelet membrane protein and demonstration that anti-glycoprotein 1b antibodies shown to inhibit thrombin-induced platelet activation do so through their cross reactivity with PAR1. Successful completion of these goals will demonstrate the mechanisms by which platelets actively regulate both the generation and function of thrombin at their membrane surface.