This program project establishes a coordinated multi-laboratory investigation on the synthesis of membrane binding structures on blood clotting proteins, the mechanism by which blood clotting proteins assemble on phospholipid vesicles and cell surface membranes, and the function of granule membrane and plasma membrane proteins in cells essential for normal hemostasis. The program involves collaborations among seven laboratories, with many of these activities focused at the Center for Hemostasis and Thrombosis Research at New England Medical Center. In Project #1, Vitamin K-dependent carboxylase: mechanist studies, Dr. Christopher Walsh will study the mechanism of action of the bovine liver vitamin K-dependent carboxylase. In Project #2, Structure of the vitamin K-dependent carboxylase, Dr. Barbara Furie will purify the vitamin k-dependent carboxylase using an affinity purification strategy based upon the propeptide of prothrombin which contains the gamma-carboxylation recognition site. The carboxylase will be characterized and cloned. The three dimensional structure of the prothrombin propeptide will be determined by two dimensional NMR techniques. In Project #3, Assembly of Factor IX and Factor VIII on membranes, Dr. bruce Furie will examine the assembly of Factor IXa and Factor VIII on membrane surfaces. The role of beta- hydroxyaspartic acid will be determined, and the structure-function relationship of Factor IX membrane binding ascertained by the examination of naturally occurring mutants from hemophilia B patients. In Project #4, Endothelial cell receptors and membrane proteins, Dr. Denisa Wagner is concerned with the identification of cell receptors and membrane proteins on endothelial cells that may play an essential role in hemostasis, specifically those that are part of the Weibel-Palade bodies in endothelial cells after stimulation. In Project #5, Alpha granule membrane proteins translocated to plasma membranes, Dr. Bruce Furie and Dr. Robert Rosenberg will identify and functionally characterize alpha granule membrane proteins similar to and including PADGEM. In addition, PADGEM will be used as a target for antibody-mediated clot- specific thrombolysis in baboons. In Project #6, Adhesive protein receptors on vascular cell, Dr. John Lawler will examine the interactions of various adhesive proteins, including von Willebrand factor, thrombospondin, fibrinogen, and vitronectin, with the vitronectin receptor on endothelial and smooth muscle cells. Core support facilities include and Administration Core (Core A), and Analytical and Preparative Core (Core B), and a Tissue Culture core (Core C).
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