DEFINITION OF FUNCTIONAL SITES ON COAGULATION FACTOR X

凝血因子 X 功能位点的定义

• 批准号: 3471960
• 负责人: William R Church
• 金额: $ 9.62万
• 依托单位: UNIVERSITY OF VERMONT & ST AGRIC COLLEGE
• 依托单位国家: 美国
• 财政年份: 1989
• 资助国家: 美国
• 起止时间: 1989-07-01 至 1994-06-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/3471960
• 关键词: antibody formation; antigens; chemical binding; coagulation factor V; coagulation factor X; laboratory mouse; laboratory rabbit; monoclonal antibody; phospholipids; protein sequence; protein structure function; prothrombin; synthetic peptide; thromboplastin

Both the intrinsic and extrinsic coagulation pathways terminate with the activation of the blood protein Factor X. Activated Factor X in turn forms a supramolecular complex, termed prothrombinase, consisting of an enzyme (Factor X alpha), a protein cofactor (Factor V alpha), calcium ion, and a phospholipid surface. This assembled complex catalyzes the proteolytic activation of prothrombin to the protease thrombin. The expedient formation of thrombin at wound sites thus requires the precise recognition and binding of the enzyme to a cofactor, a surface, and a substrate. Each of these interactions is mediated by distinct regions on the surface of Factor X. The approach outlined in this proposal will define those peptide sites on Factor X involved in recognition and binding to prothrombin, Factor V alpha, and phospholipid. The approach is to use isolated peptide fragments of Factor X, synthetic peptides from the known sequence of Factor X, and antibodies to specific topographic sites on Factor X in well- defined functional assays for Factor X. Peptides that will be synthesized include the variable region sequences of Factor X and sequences from the Gla domain. Several available monoclonal antibodies will be examined for their ability to inhibit Factor X activity and the antigenic determinant recognized by the inhibitory antibody alpha Beta F chi-2 beta will be localized. Antibodies produced to the synthetic peptides will also be examined for their ability to bind Factor X and to inhibit prothrombin activation, prothrombinase complex assembly or Factor X binding to phospholipid vesicles. This combined approach on the structure/function of Factor X using peptide fragments, synthetic peptides, and site- specific antibodies will provide more information about the relevant surface features of the Factor X molecule important for its function than would each approach used separately. Such topographic information will be extremely valuable in understanding the assembly, function, and regulation of prothrombinase, and at the same time, assist the design and modification of genetically engineered blood coagulation proteins.

内源性和外源性凝血途径均终止 血液蛋白X因子的激活 激活 因子X又形成一种超分子复合物，称为 凝血酶原酶，由酶（因子X α）、蛋白质 辅因子（因子V α）、钙离子和磷脂表面。 这种组装的复合物催化蛋白水解活化， 凝血酶原转化为凝血酶蛋白酶。 的权宜形成 因此，伤口部位的凝血酶需要精确识别， 酶与辅因子、表面和底物的结合。 这些相互作用中的每一种都是由细胞膜上的不同区域介导的。 因子X的表面。 本提案中概述的方法将 定义因子X上参与识别的那些肽位点， 与凝血酶原、因子V α和磷脂结合。 的 方法是使用分离的因子X的肽片段， 来自因子X的已知序列的合成肽，和 抗体对特定地形的网站上因子X在井- 定义了因子X的功能测定。 这些肽 合成的包含因子X的可变区序列和 来自Gla结构域的序列。 几种可用的单克隆 将检查抗体抑制因子X的能力 活性和由抑制剂识别的抗原决定簇 抗体α β F chi-2 β将被定位。 抗体 还将检查合成肽的 能够结合X因子并抑制凝血酶原激活， 凝血酶原酶复合物组装或因子X与磷脂结合 囊泡 这种关于结构/功能的综合方法 使用肽片段、合成肽和位点- 特异性抗体将提供更多关于 因子X分子的相关表面特征对于 其功能比单独使用每种方法要好。 等 地形信息对于理解 凝血酶原酶的组装、功能和调节，以及 同时，协助设计和修改基因 工程化凝血蛋白。