Factor Va regulation of prothrombinase activity

凝血酶原酶活性的因子 Va 调节

• 批准号: 7758367
• 负责人: Jamila Hirbawi
• 金额: $ 3.28万
• 依托单位: CLEVELAND STATE UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2007
• 资助国家: 美国
• 起止时间: 2007-02-01 至 2011-01-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7758367
• 关键词: Acidic Amino Acids; Amino Acids; Applications Grants; Binding; Biological Assay; Blood Coagulation Factor; Blood coagulation; Cells; Coagulation Process; Data; Deep Vein Thrombosis; Development; Enzymes; Factor V; Factor Va; Factor Xa; Generations; Goals; Hemostatic function; Human; Human body; Individual; Ions; Kinetics; Life; Light; Measures; Membrane; Methods; Pathway interactions; Peptide Hydrolases; Plasmids; Process; Proteins; Prothrombin; Reaction; Recombinant Proteins; Regulation; Research; Role; Staging; Structure; Surface; Thrombin; Thromboplastin; Thrombosis; Western Blotting; base; cofactor; divalent metal; meizothrombin; mutant; prothrombinase complex

DESCRIPTION (provided by applicant): The LONG-TERM goal of our research is to study and analyze the structure and function of the factor V molecule in order to understand key mechanisms involved in maintaining hemostasis in the human body. In order to understand these functions, it is necessary to implement the SHORT-TERM goal of identifying the amino acid residue(s) of factor V that interact with the blood coagulation factor prothrombin (flla), during prothrombinase complex assembly and function. The specific aims of the project are stated as follows: 1) To identify the specific amino acid residue(s) of the carboxyl-terminus of the factor Va heavy chain that interact with prothrombin during prothrombinase complex formation. 2) To identify the individual amino acid(s) of factor Va that interact with prothrombin and factor Xa required to drive meizothrombin formation. 3) To identify specific acidic amino acid(s) of the factor Va heavy chain that are involved in prothrombinase complex formation. To obtain our data, factor V mutants will be constructed by PCR-based methods and purified plasmids will be transiently transfected into COS-7L cells in order to obtain recombinant proteins that will be anayzed by SDS-PAGE analysis and Western Blotting. Proteins will be screened for clotting activity in one-and two-stage clotting assays. Kinetic analyses will be done by measuring thrombin formation.

描述（由申请人提供）：我们研究的长期目标是研究和分析因子V分子的结构和功能，以了解维持人体止血的关键机制。为了理解这些功能，有必要在凝血酶复合物组装和功能期间实施与血液凝结因子凝血酶原（FLLA）相互作用的因子V的氨基酸残基的短期目标。该项目的具体目的如下：1）确定在凝血酶激酶复合物形成过程中与凝血酶蛋白相互作用的因子VA重链的羧基末端的特定氨基酸残基。 2）确定与凝血酶原相互作用的因子VA的单个氨基酸，以及驱动梅氏蛋白酶形成所需的因子XA。 3）确定与凝血酶酶络合物形成有关的VA因子重链的特异性酸性氨基酸。为了获得我们的数据，将通过基于PCR的方法构建因子V突变体，并将纯化的质粒瞬时转染到COS-7L细胞中，以获得通过SDS-PAGE分析和Western blotting将其anayaze蛋白的重组蛋白。蛋白质将在一个和两个阶段的凝血测定中进行筛选以进行凝血活性。动力学分析将通过测量凝血酶形成来完成。