STRUCTURE FUNCTION RELATIONSHIPS OF FACTOR IX

本研究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，即因子IX与IV型胶原和血小板结合的位置以及羧化的位置，可以推进止血知识。更好地了解因子IX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，我们将试图确定因子IX的结合到IV型胶原蛋白的生理相关性，通过检查小鼠的表型与突变的因子IX不能结合到IV型胶原蛋白。第二，为了确定IV型胶原蛋白是在细胞内还是实际上在动脉和静脉的管腔内的内皮细胞表面上发现的，在那里它（在维生素K依赖性凝血因子中是唯一的）与因子IX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，为了确定因子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-苯丙氨酸来鉴定和纯化受体。第五，我们将继续研究羧化酶的底物，通过在体外羧化酶测定中检查几种新构建体的活性，并确定体内维生素K依赖性蛋白羧化是否需要特定序列。一个合理的机制来解释在我们目前的项目任期内确定的条件性血友病B患者的缺陷将是非常重要的了解这些前肽突变是如何导致血友病B在华法林的存在。此外，羧化机制和羧化中前肽的每个残基的重要性的知识可以允许开发对特定维生素K依赖性凝血因子特异性的抗凝剂。

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.