Molecular Biology Of Human Coagulation Factor V

人类凝血因子 V 的分子生物学

• 批准号: 7382501
• 负责人: WILLIAM H KANE
• 金额: $ 37.88万
• 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 2011-03-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7382501
• 关键词: Affinity; Alanine; Amino Acids; Antibodies; Bacteriophages; Basic Amino Acids; Binding; Binding Sites; Biological; Biological Assay; Blood Platelets; Blood Vessels; C2 Domain; Cell Line; Cell surface; Cells; Cellular Membrane; Charge; Cities; Clinical; Complement component C1s; Complex; DAG/PE-Binding Domain; Deep Vein Thrombosis; Disclosure; Disease; Electrostatics; Endothelial Cells; Enzymes; Epitopes; Face; Factor V; Factor V Deficiency; Factor Va; Factor Xa; Fluorescence; Goals; Head; Human; Human Resources; Immersion Investigative Technique; Indoles; Instruction; Last Name; Lead; Leucine; Libraries; Light; Localized; Location; Membrane; Modeling; Molecular; Molecular Biology; Mutation; Myocardial Infarction; Names; North Carolina; Numbers; Ontario; Pathogenesis; Patients; Phosphatidyl glycerol; Phosphatidylglycerols; Phosphatidylserines; Phospholipids; Play; Principal Investigator; Printing; Property; Prothrombin; Pulmonary Embolism; Recombinants; Regulation; Research Personnel; Research Project Grants; Role; Serine; Side; Site; Solvents; Specificity; Sphingomyelins; Stroke; Structure; Surface; Thrombin; Thromboplastin; Time; Tryptophan; Tyrosine; Universities; Vesicle; Work; analog; cofactor; human embryonic stem cell; indole; inorganic phosphate; mutant; novel; performance site; preference; programs; prothrombinase complex; research study

Thrombin generated by the prothrombinase complex contributes significantly to the pathogenesis of common clinical disorders such as myocardial infarction, deep vein thrombosis, pulmonary embolism, and stroke. The prothrombinase complex consists of the enzyme factor Xa, the cofactor factor Va and a phospholipid membrane surface. The interaction of factor Va with platelet membranes requires expression of phosphatidylserine (PS) on the surface of activated platelets or endothelial cells. 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. A membrane binding site was localized to the factor V C2 domain and the structures of two crystal forms of this domain have been elucidated. These structures have suggested a working model for the interaction of factor Va with phospholipid membranes that includes:(1) immersion of exposed hydrophobic residues in the apolar membrane core; (2) stereospecific interactions with PS head groups and (3) favorable electrostatic contacts of basic side chains with negatively charged membrane phosphate groups. Several aspects of this model have been validated. The indole side chains of two tryptophans located on a mobile solvent exposed loop within the C2 domain insert into the membrane bilayer and are required for high affinity membrane binding. Tyrosine and leucine residues located in an analogous location within the homologous factor V C1 domain play a role in binding to membranes containing low concentrations of PS. The goals of the present study are to validate and refine our working model for the association of factor Va with biological membranes. The specific aims of the present proposal are to define the PS specificity pocket(s) and membrane binding sites within the factor V C1 and C2 domains and to determine their contribution to prothrombinase assembly on both phospholipid vesicles and cellular membranes. Binding sites will be defined using recombinant factor Va mutants, recombinant light chain domains, scFv antibodies and soluble phospholipid analogues. Binding interactions will be characterized using quantitative fluorescence binding assays and functional prothrombinase assays. We hypothesize that complex interactions between factor Va and PS containing membranes allow for the fine regulation of the prothrombinase complex. The proposed experiments could lead to novel targets for anti-thrombotic therapy.

凝血酶原酶复合物产生的凝血酶对常见临床疾病（例如心肌梗塞、深静脉血栓形成、肺栓塞和中风）的发病机制有重要影响。凝血酶原酶复合物由酶因子 Xa、辅因子 Va 和磷脂膜表面组成。 Va 因子与血小板膜的相互作用需要活化的血小板或内皮细胞表面表达磷脂酰丝氨酸 (PS)。该项目的长期目标是利用综合分子、结构和生物物理方法来了解因子 Va 与生物膜的相互作用。膜结合位点位于因子 V C2 结构域，并且该结构域的两种晶型的结构已被阐明。这些结构提出了因子Va与磷脂膜相互作用的工作模型，包括：（1）将暴露的疏水残基浸入非极性膜核心中； (2) 与 PS 头部基团的立体特异性相互作用，以及 (3) 碱性侧链与带负电的膜磷酸基团的有利静电接触。该模型的几个方面已经得到验证。位于 C2 结构域内暴露于移动溶剂的环上的两个色氨酸的吲哚侧链插入膜双层，并且是高亲和力膜结合所必需的。酪氨酸和亮氨酸残基位于同源因子 V C1 结构域内的类似位置，在与含有低浓度 PS 的膜结合中发挥作用。本研究的目标是验证和完善我们的因子 Va 与生物膜关联的工作模型。本提案的具体目标是定义 V 因子 C1 和 C2 结构域内的 PS 特异性口袋和膜结合位点，并确定它们对磷脂囊泡和细胞膜上凝血酶原酶组装的贡献。将使用重组因子 Va 突变体、重组轻链结构域、scFv 抗体和可溶性磷脂类似物来定义结合位点。将使用定量荧光结合测定和功能性凝血酶原酶测定来表征结合相互作用。我们假设因子 Va 和含 PS 的膜之间复杂的相互作用可以对凝血酶原酶复合物进行精细调节。拟议的实验可能会产生抗血栓治疗的新靶标。