FACTOR V GENE DEFECTS IN THROMBOPHILIA

凝血酶原酶复合体的调节缺陷起着核心作用 在血栓形成的发病机制中。活化蛋白C(APC)调节 凝血酶原复合体通过灭活因子V而起作用 必需蛋白质辅因子。对活化蛋白C(APC)的抵抗力是 血栓形成最常见的遗传危险因素。APC电阻 是由因子V突变引起的，它阻止了三种APC中的一种 激活的辅因子中的裂解位点。分子机制 促成了正常和不正常的监管 APC的凝血酶原酶复合体尚未完全阐明。 我们已经采取了系统的方法来理解 三个APC裂解位点在因子中的功能重要性 Va和APC失活因子Va的机制。我们有 表达和分离出在1~2处切割的因子V突变体 或者所有三个APC裂解位点都被阻断。我们的初步研究 表明这些突变体将是解剖 APC抗性的分子机制。 在这个修改后的申请中，我们建议使用我们的重组因子 V表达系统进一步定义了对 凝血酶原酶复合体通过蛋白C途径。首先，我们将定义 每个单独的APC裂解位点在失活APC中的作用 净化系统和基于血浆的系统中的因子V。第二，我们将 描述磷脂酰丝氨酸和磷脂酰丝氨酸的作用机制 磷脂酰乙醇胺促进辅因子失活 合成膜和天然膜。第三，我们将使用生化， 用免疫学和分子生物学方法确定结合部位 蛋白C的Va因子。最后，我们将使用V因子突变体 同时对凝血酶和APC耐药以确定性质和 由原辅因子表达的APC辅因子活性的重要性， 因子V 这些研究的结果将定义精确的分子 凝血酶原酶复合体失活的调控机制 APC。这些信息将为我们提供对 APC抵抗和血栓形成的病理生理学机制 导致抗血栓治疗的新策略。

Defects in regulation of the prothrombinase complex play a central role in the pathogenesis of thrombosis. Activated protein C (APC) regulates the prothrombinese complex by inactivating factor V which serves as an essential protein cofactor. Resistance to activated protein C (APC) is the most common inherited risk factor for thrombosis. APC resistance is caused by a factor V mutation which blocks one of the three APC cleavage sites in the activated cofactor. The molecular mechanisms contributing to the normal and abnormal regulation of the prothrombinase complex by APC have not been completely elucidated. We have undertaken a systematic approach to understanding the functional importance of each of the three APC cleavage sites in factor Va and the mechanism for APC inactivation of factor Va. We have expressed and isolated factor V mutants in which cleavage at one two or all three APC cleavage sites is blocked. Our preliminary studies indicate that these mutants will be invaluable tools for dissecting the molecular mechanisms for APC resistance. In this revised application we propose to use our recombinant factor V expression system to further define the regulation of the prothrombinase complex by the protein C pathway. First, we will define the role of each individual APC cleavage site in the inactivation of factor V in both purified and plasma based systems. Second, we will characterize the mechanisms by which phosphatidylserine and phosphatidylethanolamine promote inactivation of the cofactor on synthetic and natural membranes. Third, we will use biochemical, immunological and molecular approaches to define the binding sites on factor Va for protein C. Finally, we will use factor V mutants that are resistant to both thrombin and APC to determine the nature and importance of the APC cofactor activity expressed by the procofactor, factor V. The results of these studies will define the precise molecular mechanisms that regulate inactivation of the prothrombinase complex by APC. This information will provide important insights into the pathophysiology of APC resistance and thrombosis and may ultimately lead to novel strategies for antithrombotic therapy.