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。其中一个目标是 为了定义人类血小板表面凝血酶的生成是如何 受到影响并受到规范。我们的第二个目标是开始定义如何 凝血酶一旦形成，就会与血小板蛋白相互作用，调节其 活性和纤溶反应。因为会产生凝血酶 通过凝血酶原酶在血小板上的正确组装和功能 表面上，我们将测试几个与函数复杂程度相关的假设 组装的发生以及血小板如何主动调节这些过程。 因子Va和Xa与血小板配位结合的定量研究 然后评估它们在各种情况下的功能活动 条件允许我们检验以下假设：1) 效应蛋白受体1(EPR-1)的功能是 活化的血小板表面凝血酶原酶受体(S)；2) 受体表达和功能性凝血酶原酶组装可以 受血小板与细胞外基质蛋白的黏附调节和3) 激动剂诱导的血小板因子V(A)释放和结合 由于血小板因子Va在凝血酶原酶调节中的重要作用 在功能上(可能在结构上)不同于血浆因子Va 关于APC、纤溶酶、弹性酶等酶的失活 和组织蛋白酶G凝血酶与血小板的多重相互作用 膜蛋白及其对凝血酶诱导的血小板的调节 激活是我们研究的中心，因为凝血酶激活 血小板对血管的组装和功能有很大影响 凝血酶原酶。假设血小板膜蛋白 对凝血酶活性有正向和负向调节作用 测试：1)鉴定和表征血小板的高亲和力 凝血酶的结合部位；2)定义凝血酶如何以及是否相互作用 与血小板GPIB或GPIB/IX络合物；以及3)定义存在和 血栓调节蛋白或血栓调节蛋白样分子在血栓形成过程中的作用 血小板表面。最后，我们假设被激活的血小板 继续通过抑制纤溶来促进促凝血反应。 为了验证这一假设，我们将确定纤溶反应是否为 通过从活化的血小板释放Va因子而延长 以及血栓调节蛋白的表达，因为这两个分子 尽管通过不同的机制，但增强凝血酶催化的 激活凝血酶激活的纤溶抑制物(TAFI)，a 原羧基肽酶B类分子。 这些合并的数据表明，血小板是 同时影响凝血酶的产生和调节凝血酶的活性 功能。血小板似乎在一定程度上通过 受调控的“受体”介导的蛋白分解活性在其 血小板活化后的膜表面。或者， 蛋白质可以被释放或结构性表达，这有助于 积极或积极地调节血小板的反应和功能 消极的。显然，有几个潜在的重要机制 这里讨论的是凝血酶的产生和调节 需要额外的研究。由于凝血酶是一种积极的参与者 止血、血栓和纤溶过程的描述 调控过程是理解动态平衡的核心 维护好了。 我们的研究成果包括：1)效应细胞蛋白酶 受体-1(EPR-1)，一种依赖于血小板活化的膜蛋白 通过促进凝血酶原酶催化的凝血酶生成 凝血因子Xa与血小板表面的结合；2)抗凝剂 体外对血小板活化的不同影响；3) 溶栓治疗可导致快速和完全失活 因子V/Va；和4)激活的血小板保护因子Va 被激活的蛋白C灭活我们目前的计划是：1) 鉴定和鉴定凝血酶高亲和力结合部位 血小板膜表面；2)继续定义功能 血小板膜蛋白糖蛋白Ib的意义 与血小板高亲和力结合部位有关；3)定义 活化的蛋白C、纤溶酶和弹性蛋白酶催化血管生成的机制 激活和/或失活血小板因子V和Va；以及4) 均相血小板因子V/Va分离方法的建立 用于生化表征并与血浆来源的 蛋白。

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 V(a) 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. The multiple interactions of thrombin with platelet membrane proteins and how they modulate thrombin-induced platelet activation is central to our studies since thrombin activation of platelets has a dramatic effect on the assembly and function of Prothrombinase. Hypotheses suggesting that platelet membrane proteins serve to modulate thrombin activity both positively and negatively will be tested by: 1) identifying and characterizing the platelet's high affinity binding site for thrombin; 2) defining how, and if, thrombin interacts with platelet GPIb or GPIb/IX complexes; and 3) defining the presence and function of thrombomodulin or a thrombomodulin-like molecule at the platelet surface. 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. These combined data indicate that platelets are active participants in both effecting thrombin production and modulating thrombin activity and function. Platelets appear to perform these functions in part through the regulated "receptor"-mediated assembly of proteolytic activities at their membrane surface subsequent to platelet activation. Alternatively, proteins may be released or constitutively expressed which serve to regulate platelet responsiveness and function either positively or negatively. Clearly, several potentially important mechanisms have been discussed here with respect to thrombin production and regulation which require additional study. Since thrombin is an active participant in the hemostatic, thrombotic, and fibrinolytic processes, delineation of these regulatory processes is central to understanding how homeostasis is maintained. Our accomplishments included demonstrating that: 1) effector cell protease receptor-1 (EPR-1), a platelet activation-dependent membrane protein regulates prothrombinase-catalyzed thrombin generation by facilitating the binding of factor Xa to the platelet surface; 2) anticoagulants have differential effects on the activation of platelets ex vivo; 3) thrombolytic therapy can result in the rapid and complete inactivation of factor V/Va; and 4) activated platelets protect factor Va from inactivation by activated protein C. 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; and 4) to develop protocols for the isolation of homogeneous platelet factor V/Va for biochemical characterization and comparison to the plasma-derived protein.