MOLECULAR INTERACTIONS OF COAGULATION FACTOR X

这项提案概述了一系列旨在更好地 描述人与人之间特定分子间相互作用的位置 凝血因子X(FX)及其功能性辅因子磷脂 膜，人类因子Va(FVA)和一种特定的抑制物，组织因子 途径抑制物(TFPI)。 提出的实验旨在更好地描述氨基酸 参与分子生物学研究的人凝血因子X残基 凝血酶原复合体(FVA)中与辅因子的相互作用； 磷脂膜(这是正常激活和 Fx的活性)；以及其特异性抑制剂TFPI。使用Site- FX的定向突变，各区域的单个氨基酸残基 被认为与这些分子间相互作用有关的将是 修饰并在哺乳动物细胞中表达rFX。纯化重组 变异蛋白将在凝血试验中进行表征；功能 使用纯化组分的凝血酶原酶检测(以确定相互作用 辅因子FVA)；以及TFPI的动力学和结合分析 与FXA相互作用及TFP-FXA对FVIIa/Tf的抑制作用

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.