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-羧基谷氨酸 (Gla) 结构域的功能。了解 Gla 结构域（IX 因子与 IV 型胶原蛋白和血小板结合的位置以及羧化的位置）可以增进有关止血的知识。更好地了解 IX 因子活性的分子基础可能在血栓形成的治疗中具有临床应用。首先，我们将尝试通过检查具有无法结合 IV 型胶原蛋白的突变因子 IX 的小鼠的表型来尝试确定 IX 因子与 IV 型胶原蛋白结合的生理相关性。其次，为了确定 IV 型胶原蛋白是在细胞内还是实际上存在于动脉和静脉管腔内衬的内皮细胞表面（在维生素 K 依赖性凝血因子中独一无二）与 IX 因子共定位，我们将利用单克隆抗体以及胶体金和电子显微镜。第三，为了鉴定与因子 IX 结合的 IV 型胶原蛋白的残基，我们将利用化学交联技术。第四，由于因子 IX Gla 结构域的特定区域与血小板结合，我们建议使用光化学交联剂苯甲酰基苯丙氨酸来识别和纯化受体。第五，我们将继续对羧化酶底物进行研究，通过在体外羧化酶测定中检查几种新构建体的活性，并确定维生素 K 依赖性蛋白质体内羧化是否需要特定序列。解释我们当前项目期间发现的条件性血友病 B 患者缺陷的合理机制对于理解这些前肽突变如何在华法林存在下导致血友病 B 非常重要。此外，了解羧化机制和羧化中前肽的每个残基的重要性可以允许开发针对特定维生素K依赖性凝血因子的特异性抗凝血剂。