Factor Va regulation of prothrombinase activity

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，有必要实现短期目标，即鉴定在凝血酶原酶复合物组装和功能期间与凝血因子凝血酶原（flla）相互作用的因子V的氨基酸残基。本课题的具体目的如下：1）鉴定凝血因子Va重链羧基端在凝血酶原酶复合物形成过程中与凝血酶原相互作用的特异性氨基酸残基。2)鉴定与凝血酶原和Xa因子相互作用的Va因子的单个氨基酸，这些氨基酸是驱动美佐凝血酶形成所需的。3)鉴定凝血酶原酶复合物形成中涉及的因子Va重链的特定酸性氨基酸。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COS-7 L细胞，以获得重组蛋白，将通过SDS-PAGE分析和Western Blotting分析。将在一阶段和两阶段凝血试验中筛选蛋白质的凝血活性。将通过测量凝血酶形成来进行动力学分析。

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.