MOLECULAR BIOLOGY OF HUMAN COAGULATION FACTOR V

人类凝血因子 V 的分子生物学

• 批准号: 6389104
• 负责人: WILLIAM H KANE
• 金额: $ 34.65万
• 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 2005-06-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6389104
• 关键词: binding proteins; binding sites; cell membrane; coagulation factor V; coagulation factor X; enzyme activity; enzyme structure; fluorescent dye /probe; gene expression; gene mutation; hemostasis; human tissue; laboratory mouse; laboratory rabbit; lipid bilayer membrane; molecular biology; monoclonal antibody; mutant; phosphatidylserines; phospholipids; platelets; protein structure function; site directed mutagenesis; thromboplastin; vascular endothelium

DESCRIPTION: (Investigator's abstract) In the United States, cardiovascular disease results in one death every 30 seconds. Clinical disorders such as myocardial infarction, deep vein thrombosis and pulmonary embolism, and stroke are usually precipitated by thrombotic events. Although basic research in thrombosis has lead to significant advances in the diagnosis and treatment of thrombotic disorders current approaches remain sub optimal. Generation of thrombin by the prothrombinase complex plays a particularly important role in the pathogenesis venous thrombosis. The prothrombinase complex consists of the enzyme factor Xa, the cofactor factor Va and a phospholipid membrane surface. The interaction of factor Xa with the factor Va requires cofactor activation for expression of factor Xa binding sites. The interaction of factor Va with platelet membranes requires expression of phosphatidylserine on the surface of activated platelets or endothelial cells. The binding sites for factor Xa and phospholipid membranes are discontinuous and are located in several different domains. The complexity of these binding sites may allow for the fine regulation of the prothrombinase complex. The molecular bases for these interactions remain poorly understood. The long-term goal of this project is to use integrated molecular, structural and biophysical approaches to understand the interaction of factor Va with biological membranes. During the previous funding period the factor C2 domain was expressed using insect cells and the structures of two crystal forms were elucidated. Expression of factor Va mutants in mammalian cells demonstrated that glycosylation of the C2 domain modulates membrane binding and that two tryptophans located in a mobile solvent exposed loop play a critical role in high affinity binding of factor V to phospholipid membranes containing low concentrations of phosphatidylserine. The specific aims of the present proposal are to further define the binding sites in the factor Va light chain for phospholipid membranes and cellular membranes. Binding sites will be localized using recombinant factor Va mutants, recombinant light chain domains, domain specific and monoclonal antibodies. Experiments will be designed using available crystal structures or molecular models for individual domains. Binding interactions will be characterized using surface plasmon resonance and fluorescence binding assays. This information will provide important new insights into regulation of the prothrombinase complex and may identify sites that could be exploited as novel targets for anti-thrombotic therapy.

描述：（研究者的摘要）在美国，心血管 疾病每30秒一次死亡。临床疾病，例如 心肌梗塞，深静脉血栓形成和肺栓塞和中风 通常是由血小板事件沉淀的。尽管基础研究 血栓形成在诊断和治疗方面取得了重大进展 血栓性疾病当前方法仍然是最佳的。产生 凝血酶激酶复合物的凝血酶在 发病机理静脉血栓形成。凝血酶酶复合物由 酶因子Xa，辅因子因子VA和磷脂膜表面。 因子Xa与因子VA的相互作用需要辅助因子激活 用于表达因子Xa结合位点。因子VA与 血小板膜需要在表面表达磷脂酰丝氨酸 活化的血小板或内皮细胞。因子Xa和 磷脂膜不连续，位于几种不同的 域。这些结合位点的复杂性可能允许罚款 凝血酶酶复合物的调节。这些分子碱基 相互作用仍然很少理解。该项目的长期目标是 使用集成的分子，结构和生物物理方法来理解 因子VA与生物膜的相互作用。在上一个 资金期因子C2结构域使用昆虫细胞和 阐明了两种晶体形式的结构。因子VA的表达 哺乳动物细胞中的突变体表明C2结构域的糖基化 调节膜结合，并在移动溶剂中的两个色氨酸人 暴露环在因子V对高亲和力结合中起关键作用 含有低浓度磷脂酰丝氨酸的磷脂膜。这 本提案的具体目的是进一步定义结合位点 在VA因子轻链中，用于磷脂膜和细胞膜。 结合位点将使用重组因子VA突变体进行定位， 重组轻链结构域，特异性和单克隆抗体。 实验将使用可用的晶体结构或分子设计 单个领域的模型。使用绑定的相互作用将使用 表面等离子体共振和荧光结合测定。此信息 将为调节凝血酶酶的调节提供重要的新见解 复杂的，可以识别可以被利用的站点作为新的目标 抗栓性疗法。