STRUCTURE FUNCTION RELATIONSHIPS OF FACTOR IX

本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，即与IV型胶原和血小板结合的因子IX和羧基化的位置，可以提高对止血的认识。更好地了解因子IX活性的分子基础可以在血栓的治疗中有临床应用。首先，我们将尝试通过检查无法结合胶原IV的突变因子IX小鼠的表型来确定因子IX与胶原IV结合的生理相关性。其次，确定胶原IV是细胞内的，还是实际上是在动脉和静脉腔内的内皮细胞表面上发现的，在那里它与因子IX共定位（在维生素k依赖性凝血因子中是独一无二的）。我们将利用单克隆抗体、胶体金和电子显微镜。第三，为了鉴定与因子IX结合的胶原IV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-苯胺来鉴定和纯化受体。第五，我们将继续对羧化酶底物的研究，通过在体外羧化酶实验中检测几种新的结构体的活性，并确定维生素K依赖性蛋白的体内羧化是否需要特定的序列。一个合理的机制来解释在我们目前的项目任期内发现的条件B型血友病患者的缺陷，对于理解这些前肽突变如何在华法林存在下导致B型血友病非常重要。此外，了解羧基化的机制以及羧基化中前肽每个残基的重要性，可能有助于开发针对特定维生素K依赖性凝血因子的抗凝剂。

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.