FACTOR V GENE DEFECTS IN THROMBOPHILIA

凝血酶原酶复合物调节的缺陷起着核心作用 在血栓形成的发病机制中。活化蛋白C（APC）调节 凝血酶原复合物通过灭活因子V， 必需蛋白辅因子对活化蛋白C（APC）的抗性是 血栓形成最常见的遗传风险因素。APC抗性 是由因子V突变引起的，该突变阻断了三种APC中的一种， 活化辅因子中的裂解位点。的分子机制 有助于正常和异常的调节， 尚未完全阐明APC的凝血酶原酶复合物。 我们采取了一种系统的方法来了解 因子中三个APC切割位点中每一个的功能重要性 Va和因子Va的APC失活的机制。我们有 表达和分离的因子V突变体，其中在一个两个切割 或者所有三个APC切割位点都被阻断。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。 在这个修订的申请中，我们建议使用我们的重组因子 V表达系统，以进一步确定调控 凝血酶原酶复合物通过蛋白C途径。首先，我们将定义 每个单独的APC切割位点在失活中的作用 在纯化的和基于血浆的系统中的因子V。二是 表征磷脂酰丝氨酸和 磷脂酰乙醇胺促进辅因子的失活， 合成和天然膜。第三，我们将使用生物化学， 免疫学和分子学方法来确定 因子Va代表蛋白C。最后，我们将使用因子V突变体， 对凝血酶和APC都有抗性，以确定其性质和 原辅因子表达的APC辅因子活性的重要性， 第五因子 这些研究的结果将确定精确的分子 调节凝血酶原酶复合物失活的机制， 装甲运兵车这些信息将提供重要的见解， APC抵抗和血栓形成的病理生理学，并可能最终 从而导致抗血栓治疗新策略。

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.