STRUCTURE FUNCTION RELATIONSHIPS OF FACTOR IX

因子 IX 的结构功能关系

• 批准号: 6109346
• 负责人: HAROLD Ross ROBERTS
• 金额: $ 29.23万
• 依托单位: UNIV OF NORTH CAROLINA CHAPEL HILL
• 依托单位国家: 美国
• 财政年份: 1999
• 资助国家: 美国
• 起止时间: 1999-01-05 至 1999-12-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6109346
• 关键词: SDS polyacrylamide gel electrophoresis; affinity chromatography; carboxylation; coagulation factor IX; collagen; crosslink; electron microscopy; gamma carboxyglutamate; gene mutation; glutamine; hemophilia B; hemostasis; laboratory mouse; molecular pathology; monoclonal antibody; phenotype; platelets; protein C; protein binding; protein protein interaction; protein purification; protein sequence; protein structure function; vascular endothelium; vitamin K

The purpose of this project is to understand the function of the gamma- carboxyglutamic (Gla) domain of vitamin K-dependent coagulation factor IX. Understanding the Gla domain, the location both of factor IX binding to collagen IV and platelets and also of carboxylation, can advance knowledge about hemostasis. A better understanding of the molecular basis of factor IX's activity could have clinical applications in the treatment of thrombosis. First we will attempt to determine the physiological relevance of factor IX's binding to collagen IV by examining the phenotype of a mouse with a mutant factor IX unable to bind collagen IV. Second, to determine if collagen IV is intracellular or is actually found on the surface of endothelial cells lining the lumen of both arteries and veins where it is (uniquely among vitamin K-dependent clotting factors) co-localized with factor IX, we will utilize monoclonal antibodies together with colloidal gold and electron microscopy. Third, to identify residues of collagen IV to which factor IX binds, we will utilize chemical cross-linking techniques. Fourth, since a specific region of the factor IX Gla domain binds to platelets, we propose to use the photochemical cross-linker benzoyl- phenylanine to identify and purify the receptor. Fifth, we will continue our studies on substrates for the carboxylase by examining several new constructs for their activity in an in-vitro carboxylase assay and determining if a specific sequence is required for the carboxylation of vitamin K- dependent protein in-vivo. A plausible mechanism to explain the defects in conditional hemophilia B patients identified during the tenure of our current project will be very important to understanding how these pro-peptide mutations are leading to hemophilia B in the presence of warfarin. Moreover, knowledge of the mechanism of carboxylation and the importance of each residue of the pro-peptide in carboxylation may allow the development of anticoagulents specific for a particular vitamin K- dependent coagulation factor.

该项目的目的是了解维生素K依赖性凝血因子IX的γ-羧谷氨酸（GLA）结构域的功能。了解GLA结构域，即IX因子与胶原蛋白IV和血小板的结合以及羧化的位置，都可以提高有关止血的知识。更好地理解因子IX活性的分子基础，可以在血栓形成治疗中具有临床应用。首先，我们将尝试通过检查突变因子IX无法结合胶原蛋白IV的小鼠的表型来确定因子IX与胶原蛋白IV的结合的生理相关性。其次，为了确定胶原蛋白IV是在细胞内还是在与因子IX共定位的动脉和静脉内衬里的内皮细胞表面（在维生素K依赖性的凝结因子中唯一的独特），我们将利用单克隆抗体和胶体微元素显微镜将其共定位。第三，为了确定IX因子结合的胶原蛋白IV的残基，我们将利用化学交联技术。第四，由于IX GLA域的特定区域与血小板结合，因此我们建议使用光化学交叉链苯甲酰苯胺来识别和净化受体。第五，我们将通过检查几种在体外羧化酶测定中的活性的新构建体，并确定是否需要特定的序列维生素K-依赖性蛋白在体内，我们将继续对羧化酶的底物进行研究。解释在我们当前项目任期期间确定的有条件血友病患者缺陷的合理机制对于理解这些促肽突变如何导致在华法林存在下导致血友病B非常重要。此外，对羧化机理的了解以及促肽在羧化中的每个残基的重要性可能允许开发针对特定维生素K-依赖性凝血因子的抗凝剂。