MOLECULAR BIOLOGY OF HUMAN COAGULATION FACTOR V

人类凝血因子 V 的分子生物学

• 批准号: 2220856
• 负责人: WILLIAM H KANE
• 金额: $ 17.45万
• 依托单位: DUKE UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 1991
• 资助国家: 美国
• 起止时间: 1991-01-01 至 1996-06-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/2220856
• 关键词: binding proteins; chimeric proteins; coagulation factor V; coagulation factor VIII; coagulation factor X; enzyme activity; enzyme complex; enzyme induction /repression; enzyme mechanism; enzyme structure; gene deletion mutation; gene expression; genetic regulation; hemostasis; human tissue; molecular biology; phospholipids; platelets; polymerase chain reaction; protein structure function; prothrombin; receptor; receptor binding; site directed mutagenesis; thrombomodulin; thromboplastin

DESCRIPTION: (Adapted from the investigator's abstract). Factor V is a protein cofactor that is essential for the prothrombinase complex. This complex consists of factor Va, factor Xa, calcium, and a phospholipid surface. The generation of thrombin is a critical reaction during hemostasis. Thrombin catalyzes a number of important reactions during hemostasis including the activation of platelets, the activation of factors V, VIII and XIII, and the conversion of fibrinogen to fibrin. In addition, thrombin bound to its endothelial receptor thrombomodulin catalyzes activation of protein C which then inactivates factors V and VIII. In order to understand the mechanisms by which the activation of prothrombin is regulated we have undertaken an investigation of the structure and function of the factor V molecule. Initially, we developed methods to isolate human factor V from plasma and studied its proteolytic activation. Subsequently we investigated the role of factor Va as the factor Xa receptor on human platelets. More recently, we have determined the complete primary sequence of the molecule. We have demonstrated that factor V is a multidomain protein with homology to ceruloplasmin and factor VIII. We are currently using site directed mutagenesis to investigate structure function relationships in recombinant factor V expressed in mammalian cells. The goals of the present study are to identify regions of the molecule that constitute the binding sites for phospholipid, cell surface receptors, factor Xa and prothrombin. We will also define the critical proteolytic events required for the activation and inactivation of this protein. We will use both genetic and reverse genetic approaches to define the function of this important protein. Using the factor V cDNA and mammalian cell expression systems we will express recombinant factor V with specific mutations. We will also use PCR to amplify platelet mRNA isolated from patients with factor V deficiency and determine natural mutations that affect factor V function. The results of these experiments will ultimately define the exact molecular interactions involved in the assembly and regulation of the prothrombinase complex. Expression and characterization of chimeric factor V/factor VIII molecules will further enhance understanding of the structure function relationships in this family of related proteins. This knowledge may provide insight into the pathogenesis of thrombotic disorders.

描述：（改编自研究者的摘要）。 因子 V 是 凝血酶原酶复合物所必需的蛋白质辅因子。 这 复合物由因子 Va、因子 Xa、钙和磷脂组成 表面。 凝血酶的产生是凝血过程中的关键反应 止血。 凝血酶在过程中催化许多重要反应 止血包括血小板的活化、因子的活化 V、VIII和XIII，以及纤维蛋白原向纤维蛋白的转化。 此外， 凝血酶与其内皮受体结合，血栓调节蛋白催化 激活蛋白 C，然后使因子 V 和 VIII 失活。 为了了解激活的机制 凝血酶原受到监管，我们已对此进行了调查 V因子分子的结构和功能。 最初，我们开发了 从血浆中分离人因子V并研究其蛋白水解的方法 激活。 随后我们研究了因子 Va 的作用 人血小板上的 Xa 因子受体。 最近，我们确定 分子的完整一级序列。 我们已经证明了 V因子是一种多结构域蛋白，与铜蓝蛋白和因子具有同源性 八． 我们目前正在使用定点诱变来研究 重组因子 V 的结构功能关系 哺乳动物细胞。 本研究的目标是确定分子的区域 构成磷脂、细胞表面受体的结合位点， Xa 因子和凝血酶原。 我们还将定义关键蛋白水解 该蛋白质的激活和失活所需的事件。 我们 将使用遗传和反向遗传方法来定义功能 这种重要的蛋白质。 使用 V 因子 cDNA 和哺乳动物细胞 表达系统我们将用特定的表达重组因子V 突变。 我们还将使用 PCR 来扩增从分离的血小板 mRNA 患有 V 因子缺乏症的患者并确定自然突变 影响因子V功能。 这些实验的结果将最终定义确切的分子 参与凝血酶原酶组装和调节的相互作用 复杂的。 嵌合因子 V/因子 VIII 的表达和表征 分子将进一步增强对结构功能的理解 该相关蛋白质家族中的关系。 这些知识可能 深入了解血栓性疾病的发病机制。