MOLECULAR INTERACTIONS OF COAGULATION FACTOR X

凝血因子 X 的分子相互作用

• 批准号: 2734938
• 负责人: PETER J LARSON
• 金额: $ 8.79万
• 依托单位: CHILDREN'S HOSP OF PHILADELPHIA
• 依托单位国家: 美国
• 财政年份: 1995
• 资助国家: 美国
• 起止时间: 1995-07-01 至 2000-06-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/2734938
• 关键词: activation product; blood coagulation; carboxylation; chemical binding; chemical kinetics; coagulation factor V; coagulation factor X; cofactor; enzyme complex; enzyme substrate; immunoaffinity chromatography; intermolecular interaction; ion exchange chromatography; lipid bilayer membrane; molecular site; phospholipids; protein purification; prothrombin; recombinant proteins; site directed mutagenesis; tissue /cell culture

This proposal outlines a series of experiments designed to better delineate sites of specific intermolecular interactions between human coagulation factor X (FX) and its functional cofactors, phospholipid membranes, human factor Va (FVa), and a specific inhibitor, tissue factor pathway inhibitor (TFPI). The experiments proposed are designed to better delineate amino acid residues of human coagulation factor X which are involved in molecular interactions with its cofactor in the prothrombinase complex (FVa); phospholipid membrane (which is required for the normal activation and activity of FX); and with its specific inhibitor, TFPI. Using site- directed mutagenesis, individual amino acid residues of FX in regions thought to be involved with these intermolecular interactions will be modified and rFX expressed in mammalian cells. Purified recombinant variant proteins will be characterized in clotting assays; a functional prothrombinase assay using purified components (to determine interaction with the cofactor, FVa); and in kinetic and binding assays for the TFPI interaction with FXa and the TFPI-FXa inhibition of FVIIa/TF.

该提案概述了一系列旨在更好地 描绘人类之间特定分子间相互作用的位点 凝血因子 X (FX) 及其功能性辅助因子、磷脂 膜、人因子 Va (FVa) 和特定抑制剂组织因子 途径抑制剂（TFPI）。 所提出的实验旨在更好地描述氨基酸 人凝血因子 X 的残基参与分子 与其在凝血酶原酶复合物 (FVa) 中的辅因子相互作用； 磷脂膜（正常激活和 外汇活动）；及其特异性抑制剂 TFPI。使用网站- 定向诱变，FX区域中的单个氨基酸残基 被认为参与这些分子间相互作用的将是 修饰并在哺乳动物细胞中表达 rFX。纯化重组体 变异蛋白将在凝血试验中进行表征；功能性的 使用纯化成分进行凝血酶原酶测定（以确定相互作用 与辅因子 FVa)；以及 TFPI 的动力学和结合测定 与 FXa 的相互作用以及 TFPI-FXa 对 FVIIa/TF 的抑制。

项目成果

Structure/function analyses of recombinant variants of human factor Xa: factor Xa incorporation into prothrombinase on the thrombin-activated platelet surface is not mimicked by synthetic phospholipid vesicles.

人因子 Xa 重组变体的结构/功能分析：合成磷脂囊泡无法模拟因子 Xa 掺入凝血酶激活的血小板表面上的凝血酶原酶。

• DOI: 10.1021/bi972428p
• 发表时间: 1998
• 期刊: Biochemistry.
• 作者: Larson,PJ;Camire,RM;Wong,D;Fasano,NC;Monroe,DM;Tracy,PB;High,KA
• 通讯作者: High,KA