MOLECULAR INTERACTIONS OF COAGULATION FACTOR X

该提案概述了一系列旨在更好地 描绘出人类与人类之间特定分子间相互作用的位点， 凝血因子X（FX）及其功能辅因子、磷脂 膜，人因子Va（FVa）和特异性抑制剂，组织因子 途径抑制剂（TFPI）。 所提出的实验旨在更好地描绘氨基酸 人凝血因子X的残基，其参与分子 与凝血酶原酶复合物（FVa）中的辅因子的相互作用; 磷脂膜（其是正常激活和 FX的活性）;以及其特异性抑制剂TFPI。使用网站- 定向诱变，区域中FX的单个氨基酸残基 被认为与这些分子间相互作用有关的是 在哺乳动物细胞中修饰和表达rFX。纯化的重组 变体蛋白质将在凝血测定中表征;功能性蛋白质将在凝血测定中表征。 使用纯化组分的凝血酶原酶测定（以确定相互作用 与辅因子FVa）;以及在TFPI的动力学和结合测定中， 与FXa的相互作用和FVIIa/TF的TFPI-FXa抑制。

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.