MOLECULAR BIOLOGY OF HUMAN COAGULATION FACTOR V

描述：(研究人员摘要)在美国，心血管 每30秒就有一人死于疾病。临床疾病，如 心肌梗死、深静脉血栓形成和肺栓塞，以及中风 通常是由血栓事件引起的。虽然中国的基础研究 血栓形成在诊断和治疗方面取得了重大进展 血栓性疾病目前的治疗方法仍然不太理想。新一代 凝血酶由凝血酶原酶复合体组成，在 静脉血栓形成的发病机制。凝血酶原酶复合体由 酶因子Xa、辅因子Va和磷脂膜表面。 因子Xa与因子Va的相互作用需要辅因子激活 用于Xa因子结合位点的表达。凝血因子Va与蛋白质的相互作用 血小板膜表面需要表达磷脂酰丝氨酸 激活的血小板或内皮细胞。Xa因子和Xa因子的结合位点 磷脂膜是不连续的，位于几个不同的 域名。这些结合位点的复杂性可能会导致罚款 凝血酶原酶复合体的调节。这些物质的分子基础 相互作用仍然鲜为人知。这个项目的长期目标是 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。在上一次 资助期因子C2结构域是用昆虫细胞表达的 阐明了两种晶型的结构。凝血因子Va的表达 哺乳动物细胞中的突变体证明C2结构域的糖基化 调节膜结合和位于流动溶剂中的两种色氨酸 暴露环在因子V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。这个 本提案的具体目标是进一步界定结合位点 在因子Va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、结构域特异性抗体和单抗。 实验将使用可用的晶体结构或分子来设计 各个域的模型。绑定交互作用的特征将使用 表面等离子体共振和荧光结合分析。此信息 将为凝血酶原酶的调节提供重要的新见解 复杂，并可能确定可被用作新目标的地点 抗血栓治疗。

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.