FACTOR V GENE DEFECTS IN THROMBOPHILIA

血栓形成倾向中的 V 因子基因缺陷

• 批准号: 2029531
• 负责人: WILLIAM H KANE
• 金额: $ 23.25万
• 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 2001-11-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/2029531
• 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.

凝血酶激酶复合物调节的缺陷起着核心作用 在血栓形成的发病机理中。活化的蛋白C（APC）调节 通过灭活因子V作为凝凝结凝结物复合物 必需的蛋白质辅因子。对活化蛋白C（APC）的抗性为 血栓形成最常见的遗传危险因素。 APC电阻 是由一个因子V突变引起的，该因子V突变阻断了三个APC之一 激活的辅因子中的切割位点。分子机制 有助于正常和异常调节 APC的凝血酶酶配合物尚未完全阐明。 我们采取了一种系统的方法来理解 在因子中，三个APC切割位点中的每个位点的功能重要性 VA和APC灭活因子VA的机制。我们有 表达和孤立的因子V突变体，其中裂解在一个两个 或所有三个APC裂解位点都被阻塞。我们的初步研究 表明这些突变体将是剖析的宝贵工具 APC电阻的分子机制。 在此修订的应用中，我们建议使用我们的重组因素 V表达系统进一步定义了 蛋白C途径的凝血酶酶复合物。首先，我们将定义 每个单独的APC切割位点的作用在失活中 纯化和基于等离子体的系统中的因子V。第二，我们会的 表征磷脂酰丝氨酸和 磷脂酰乙醇胺促进辅因子在上灭活 合成和自然膜。第三，我们将使用生化， 免疫学和分子方法定义结合位点 蛋白质C的因子Va。最后，我们将使用因子V突变体 对凝血酶和APC都有抗性，以确定性质和 ProcoFactor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 通过调节黑凝结酶复合物失活的机制 APC。这些信息将为您提供重要的见解 APC耐药性和血栓形成的病理生理学，最终可能 导致抗血栓疗法的新策略。