MOLECULAR BIOLOGY OF HUMAN COAGULATION FACTOR V

人类凝血因子 V 的分子生物学

• 批准号: 2445189
• 负责人: WILLIAM H KANE
• 金额: $ 23.91万
• 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 2000-06-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/2445189
• 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

Defects in regulation of the prothrombinase complex play a central role in the pathogenesis of thrombosis. Factor V regulates the activity of the prothrombinase complex which is responsible for the generation of thrombin during blood coagulation. Genetic defects in factor V are the cause of protein C resistance which is the most common inherited risk factor for thrombosis. Generation of factor Va and assembly of the prothrombinase complex is therefore a critical event in thrombosis. The long-term goal of this project is to understand the structure, function and regulation of factor V and the prothrombinase complex. During the first funding period we characterized the factor V gene and used factor V expression systems to define structure-function relationships in the protein. We determined that the Factor V C2 domain contains binding sites for phosphatidylserine and acquired factor V antibodies. We also found that alternative glycosylation of the C2 domain is the structural basis for the two forms of the factor V light chain that differ in their affinity for anionic phospholipid. We found that a large deletion within the B-domain results in a single chain protein that expresses partial cofactor activity. Finally, we have characterized molecular mechanisms of APC resistance. During the second funding period we propose to extend our studies on the structure and function of recombinant factor V. The goals of these studies will be to further define domains and specific amino acid residues that comprise the binding sites required for assembly of the prothrombinase complex. We will use alanine scanning mutagenesis to identify the amino acids within the light chain required for factor V binding to phospholipid and platelet membranes. We will use the same approach to identify amino acids within the A1 and A3 domains as required for the factor Xa binding site. We will localize the sites of tyrosine sulfation and glycosylation that are important for expression of cofactor activation and function. Finally we will investigate the functions of the factor V B-domain. We will determine whether novel B-domain cleavages by cellular proteases contribute to factor V action. We will also localize the sites of factor XIII mediated crosslinking within the B-domain. The proposed studies will provide new insights into the precise molecular interactions involved in assembly of the prothrombinase complex. This information will be critical for understanding the regulation of the prothrombinase complex under physiological and pathological conditions.

凝血酶原酶复合物的调节缺陷在 血栓形成的发病机制。 因子 V 调节 凝血酶原酶复合物负责产生凝血酶 血液凝固过程中。 V 因子的遗传缺陷是导致 蛋白 C 抵抗是最常见的遗传风险因素 血栓形成。 因子 Va 的产生和凝血酶原酶的组装 因此，复合物是血栓形成的关键事件。 长期目标 该项目的目的是了解结构、功能和调节 V 因子和凝血酶原复合物。 在第一个资助期内 我们表征了 V 因子基因并使用 V 因子表达系统 定义蛋白质的结构-功能关系。 我们确定 因子 V C2 结构域包含磷脂酰丝氨酸的结合位点 并获得了 V 因子抗体。 我们还发现了替代方案 C2 结构域的糖基化是两种形式的结构基础 V因子轻链对阴离子的亲和力不同 磷脂。 我们发现 B 域内的大量删除导致 在表达部分辅因子活性的单链蛋白质中。 最后，我们描述了 APC 抗性的分子机制。 在第二个资助期间，我们建议扩展我们的研究 重组因子 V 的结构和功能。这些目标 研究将进一步定义结构域和特定氨基酸残基 包括组装所需的结合位点 凝血酶原复合物。 我们将使用丙氨酸扫描诱变 鉴定因子 V 所需的轻链内的氨基酸 与磷脂和血小板膜结合。 我们将使用相同的 根据需要鉴定 A1 和 A3 结构域内氨基酸的方法 为因子 Xa 结合位点。 我们将定位酪氨酸的位点 对辅因子表达很重要的硫酸化和糖基化 激活和功能。 最后我们将研究一下其功能 因子 V B 结构域。 我们将确定是否存在新的 B 结构域裂解 细胞蛋白酶有助于因子 V 的作用。 我们也会本地化 B 结构域内因子 XIII 介导的交联位点。这 拟议的研究将为精确的分子生物学提供新的见解 涉及凝血酶原复合物组装的相互作用。 这 信息对于理解监管规定至关重要 生理和病理条件下的凝血酶原酶复合物。