REGULATION OF HUMAN PLATELET PROTHROMBINASE ACTIVITY

努力集中在两个截然不同但又相互关联的领域。一个目标 是为了定义人类血小板凝血酶的生成是如何 表面是受影响和调节的。我们的第二个目标是开始 确定凝血酶一旦形成，如何与血小板蛋白相互作用 调节其活性和纤溶反应。因为凝血酶是 通过凝血酶原酶的正确组装和功能产生 在血小板表面，我们将测试几个相关的假说 功能复合体组装的发生和血小板如何活跃 调节这些过程。的坐标绑定的量化 凝血因子Va和Xa对血小板的影响 在各种条件下的功能活动将使我们能够 检验以下假设：1)影响蛋白水解酶受体1 (EPR-1)是凝血酶原酶受体(S)的一部分。 活化的血小板表面；2)受体的表达和功能 凝血酶原酶组装可由血小板黏附到 细胞外基质蛋白和3)激动剂诱导的释放和 结合血小板因子V9a)在脑出血中起着重要作用 凝血酶原酶的调节，因为血小板因子Va在功能上(和 可能在结构上)在以下方面不同于血浆因子Va APC、纤溶酶、弹力酶和组织蛋白酶等酶的失活 G. 最后，我们假设被激活的血小板继续促进 通过抑制纤溶而产生的促凝血反应。为了测试这一点 假设我们将确定纤溶反应是否延长 通过从激活的血小板中释放因子Va以及 血栓调节蛋白的表达，因为这两个分子都会增强， 尽管通过不同的机制，凝血酶催化的激活 对于凝血酶激活的纤溶抑制物(TAFI)， 原羧基肽酶B类分子。 我们的成就包括证明：1) 被称为因子V魁北克的出血性疾病是由于 储存在血小板α颗粒内的辅因子池；2)活化蛋白 C(APC)通过调节TAFI的激活促进纤溶；3) 与促凝血因子突变相关的血栓前效应， 抵抗APC灭活的因子V Leiden应在 部分原因是凝血酶的持续生成和TAFI的激活 导致持续和长时间的血栓形成。 我们目前的计划是：1)鉴定和鉴定凝血酶 血小板膜表面高亲和力结合部位；2) 继续定义血小板膜的功能意义 蛋白糖蛋白Ib与血小板高亲和力的关系 结合部位；3)确定激活蛋白C， 纤溶酶和弹性酶催化激活和/或失活 血小板因子V和Va；4)鉴定和表征 血小板膜表面血栓调节蛋白样分子，5) 建立均质血小板因子的分离方法 V/Va用于生化表征并与血浆进行比较- 衍生蛋白质。

Efforts are focused in two distinct, yet interrelated areas. One goal is to define how the generation of thrombin at the human platelet surface is effected and regulated. Our second goal is to begin to define how thrombin, once formed, interacts with platelet proteins to modulate its activity and the fibrinolytic response. Since thrombin is generated through the proper assembly and function of Prothrombinase at the platelet surface we will test several hypotheses relevant to how functional complex assembly occurs and how the platelet actively regulates these processes. Quantitation of the coordinate binding of factors Va and Xa to platelets followed by assessment of their functional activity under a variety of conditions will allow us to the test the following hypotheses: 1) that Effector Protease Receptor 1 (EPR-1) functions as part of the Prothrombinase receptor(s) at the activated platelet surface; 2) that receptor expression and functional Prothrombinase assembly can be modulated by platelet adherence to extracellular matrix proteins and 3) agonist-induced release and binding of platelet factor V9a) plays a preeminent role in Prothrombinase regulation since platelet factor Va is functionally (and perhaps structurally) different than plasma factor Va with respect to inactivation by proteases such as APC, plasmin, elastase and cathepsin G. Finally, we hypothesize that the activated platelet continues to promote a procoagulant response by inhibiting fibrinolysis. To test this hypothesis we will determine if the fibrinolytic response is prolonged through the release of factor Va from the activated platelet as well as the expression of thrombomodulin since both molecules will enhance, albeit through different mechanisms, the thrombin-catalyzed activation of the thrombin activatable fibrinolysis inhibitor (TAFI), a procarboxypeptidase B-like molecule. Our accomplishments included demonstrating that: 1) the defect in the bleeding disorder termed Factor V Quebec is due to proteolysis of the cofactor pool stored within the platelet a-granule; 2) activated protein C (APC) promotes fibrinolysis by regulating the activation of TAFI; 3) the prothrombotic effect associated with procoagulation factor mutant, Factor V Leiden, which is resistant to inactivation by APC, is due in part to its sustained generation of thrombin and activation of TAFI leading to sustained and prolonged thrombus formation. Our current plans are: 1) to identify and characterize the thrombin high affinity binding site on the platelet membrane surface; 2) to continue to define the functional significance of the platelet membrane protein glycoprotein Ib as it relates to the platelet high affinity binding site; 3) to define the mechanisms by which activated protein C, plasmin and elastase catalyze the activation and/or inactivation of platelet factors V and Va; 4) to identify and characterize the thrombomodulin-like molecule on the platelet membrane surface, and 5) to develop protocols for the isolation of homogeneous platelet factor V/Va for biochemical characterization and comparison to the plasma- derived protein.