REGULATION OF HUMAN PLATELET PROTHROMBINASE ACTIVITY

人血小板表面凝血酶的产生是通过 凝血酶原酶复合体的组装和功能，包括 辅因子Va和辅因子Va的1：1化学计量比的钙/2+络合物 丝氨酸蛋白酶Xa因子。在血小板激活后，释放 血小板因子V(A)和/或血浆衍生因子Va与血小板结合 膜表面，并在这样做时形成至少一部分受体 因子Xa。因此，几种蛋白质/蛋白质和蛋白质/膜 相互作用决定了功能复杂的组装。这项研究的目标之一 项目是定义血小板如何积极参与和调节 复杂的装配。因为血小板已经被证明可以表达这种因子 Xa膜受体效应蛋白受体-1(EPR-1) 激动剂诱导的激活，将投入大量的努力来确定 它在功能性凝血酶原酶复合体中的作用和调节。这个 血小板调节酶复合体组装的关键事件的能力 也将评估激动剂诱导的激活之后， 特别是与1)功能性EPR-1表达和2) 抗活化蛋白的血小板因子Va分子的表达 C失活，也许还有其他的蛋白酶。因为凝血酶 一旦形成，正向调节凝血酶原酶复合体的组装，并 的功能，第二个目标是了解如何 凝血酶与血小板膜蛋白的相互作用影响其功能。 将致力于定义和分离凝血酶的高亲和力 似乎使凝血酶分子瞬间呈现的结合部位 失活并阐明糖蛋白Ib/IX与此的关系 地点。最后，观察到一种血栓调节蛋白样分子 血小板膜成分表达和血小板源 凝血因子Va通过凝血酶依赖机制抑制纤溶 被探索，特别是与最近发现的凝血酶有关 可激活纤溶抑制物(TAFI)。这样做的最终目的是 研究项目是确定血小板活跃的机制 调节其膜上凝血酶的生成和功能 浮出水面。定义调节细胞和分子的事件 凝血酶的产生是我们全面了解 止血和血栓形成过程。

Thrombin generation at the human platelet surface is effected through the assembly and function of the enzymatic complex, Prothrombinase, consisting of a 1:1 stoichiometric, Ca/2+ complex of the cofactor factor Va and the serine protease factor Xa. Subsequent to platelet activation, released platelet factor V(a) 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 dictate functional complex assembly. A goal of this research project is to define how platelets actively participate in and regulate complex assembly. Because platelets have been shown to express the factor Xa membrane receptor Effector Protease Receptor-1 (EPR-1) following agonist-induced activation, substantial effort will be placed on defining its role in, and regulation of, a functional Prothrombinase complex. The platelet's ability to regulate events critical for enzyme complex assembly subsequent to agonist-induced activation will be assessed as well, specifically as related to 1) functional EPR-1 expression and 2) expression of a platelet factor Va molecule resistant to activated protein C inactivation, and perhaps other proteases as well. Because thrombin once formed positively regulates Prothrombinase complex assembly and function through platelet activation, a second goal is to understand how thrombin interactions with platelet membrane proteins affect its function. Effort will be placed on defining and isolating the thrombin high affinity binding site which appears to render the thrombin molecule transiently inactive and delineating the relationship of glycoprotein Ib/IX to this site. Finally, the observation that both a thrombomodulin-like molecule constitutively expressed on the platelet membrane and platelet-derived factor Va inhibit fibrinolysis through a thrombin-dependent mechanism will be explored, especially as related to the recently identified Thrombin Activatable Fibrinolysis Inhibitor (TAFI). The ultimate goal of this research project is to define the mechanisms by which platelets actively regulate both the generation and function of thrombin at their membrane surface. Defining the cellular and molecular events which regulate thrombin production is fundamental to our overall understanding of the hemostatic and thrombotic processes.