STRUCTURE FUNCTION RELATIONSHIPS OF FACTOR IX

本项目的目的是了解维生素K依赖的凝血因子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作用，可以促进对止血的了解。更好地了解因子IX活性的分子基础可能会在治疗血栓形成方面有临床应用。首先，我们将试图通过检测突变的因子IX无法结合IV型胶原的小鼠的表型来确定因子IX与IV型胶原结合的生理学相关性。其次，为了确定IV型胶原是在细胞内还是在动脉和静脉腔内的内皮细胞表面被发现，在那里它(在依赖维生素K的凝血因子中是唯一的)与IX因子共同定位，我们将使用单抗与胶体金和电子显微镜相结合。第三，为了确定因子IX结合的IV型胶原残基，我们将利用化学交联技术。第四，由于凝血因子IX GLA结构域的一个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该受体。第五，我们将继续我们对羧基酶底物的研究，通过检查几个新的构建体在体外羧化酶试验中的活性，并确定体内维生素K依赖蛋白的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期间发现的条件血友病B患者的缺陷，对于理解这些前肽突变是如何在华法林存在的情况下导致血友病B非常重要的。此外，了解羧化的机制和羧化过程中每个前肽残基的重要性，可能会开发出特定的维生素K依赖凝血因子的抗凝血剂。

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.