FACTOR V GENE DEFECTS IN THROMBOPHILIA

血栓形成倾向中的 V 因子基因缺陷

• 批准号: 6330084
• 负责人: WILLIAM H KANE
• 金额: $ 26.16万
• 依托单位: DUKE UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 1996
• 资助国家: 美国
• 起止时间: 1996-12-01 至 2003-11-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6330084
• 关键词: activation product; active sites; binding proteins; chemical kinetics; coagulation factor V; coagulation factor X; cofactor; enzyme activity; enzyme complex; enzyme linked immunosorbent assay; gene expression; gene mutation; hemostasis; human subject; intermolecular interaction; laboratory mouse; laboratory rabbit; monoclonal antibody; phosphatidylethanolamines; phosphatidylserines; protein C; protein S; protein structure function; thrombin; thrombosis

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.

凝血酶原酶复合物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 通过灭活因子 V 来抑制凝血酶原复合物 必需蛋白质辅因子。对活化蛋白 C (APC) 的抗性 血栓形成最常见的遗传危险因素。 APC抗性 由 V 因子突变引起，该突变阻断了三种 APC 之一 活化辅因子中的切割位点。分子机制 有助于正常和异常调节 APC 的凝血酶原酶复合物尚未完全阐明。 我们采取了系统的方法来了解 三个 APC 切割位点在因子中的功能重要性 Va 和因子 Va 的 APC 失活机制。我们有 表达和分离的 V 因子突变体，其中一二处裂解 或者所有三个 APC 切割位点均被封闭。我们的初步研究 表明这些突变体将成为解剖 APC 耐药的分子机制。 在这个修订后的申请中，我们建议使用我们的重组因子 V表达系统进一步明确了调控 蛋白质 C 途径的凝血酶原酶复合物。首先，我们将定义 每个单独的 APC 切割位点在失活中的作用 纯化系统和血浆系统中的 V 因子。其次，我们将 表征磷脂酰丝氨酸和 磷脂酰乙醇胺促进辅因子失活 合成膜和天然膜。第三，我们会用生化， 免疫学和分子方法来定义结合位点 蛋白质 C 的因子 Va。最后，我们将使用因子 V 突变体，它们是 对凝血酶和 APC 均具有抗性，以确定其性质和 由原辅因子表达的 APC 辅因子活性的重要性， 因素V。 这些研究的结果将定义精确的分子 调节凝血酶原酶复合物失活的机制 装甲运兵车。这些信息将为我们提供重要的见解 APC 抵抗和血栓形成的病理生理学，最终可能 导致抗血栓治疗的新策略。