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.
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