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.

调节凝血酶酶复合物的缺陷在 血栓形成的发病机理。 因子V调节 凝血酶激酶复合物，负责凝血酶的产生 在血液凝血过程中。 因子V中的遗传缺陷是 蛋白质抗性是最常见的遗传风险因素 血栓形成。 VA因子的产生和凝血酶酶的组装 因此，复合物是血栓形成的关键事件。 长期目标 该项目的结构，功能和调节 因子V和凝血酶酶复合物。 在第一个资金期间 我们将因子V基因和使用因子V表达系统表征为 定义蛋白质中的结构功能关系。 我们确定 因子V C2结构域包含磷脂酰丝氨酸的结合位点 并获得因子V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 因子V轻链的亲和力与阴离子的亲和力不同 磷脂。 我们发现B域中的大删除结果很大 在表达部分辅因子活性的单链蛋白中。 最后，我们表征了APC抗性的分子机制。 在第二个资金期间，我们建议扩展有关 重组因子V的结构和功能。这些目标的目标 研究将是进一步定义域和特定的氨基酸残基 这包括组装所需的绑定位点 凝血酶酶复合物。 我们将使用丙氨酸扫描诱变 识别因子V所需的轻链中的氨基酸 与磷脂和血小板膜结合。 我们将使用相同的 根据需要识别A1和A3域内氨基酸的方法 对于因子XA结合位点。 我们将定位酪氨酸的地点 硫酸和糖基化对于表达辅因子很重要 激活和功能。 最后，我们将研究 因子V B域。 我们将确定新颖的B域是否通过 细胞蛋白酶有助于因子V作用。 我们还将本地化 XIII因子的位点介导了B域内的交联。这 拟议的研究将为精确的分子提供新的见解 凝血酶酶复合物组装涉及的相互作用。 这 信息对于理解调节至关重要 在生理和病理条件下，凝血酶酶复合物。