STRUCTURAL STUDIES ON PROTEINS INVOLVED IN HEMOSTASIS

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Thrombin is the central regulatory protein in hemostasis, defined as the containment of circulating blood. Improper activation of thrombin is a leading cause of premature death in that it is the immediate cause of most heart attacks and strokes. A long term goal of this project is to rationalize the many diverse functions of thrombin by determining its three-dimensional structure with and without substrate analogs, inhibitors, and effectors. The specific goals are: (1) To complete the construction and refinement at 2.4 A resolution of the model of bovine alpha-thrombin. (2) To compare the structure of beta150-thrombin, which is a derivative of alpha-thrombin cut at position 150, with and without a covalent inhibitor in the active site. (3) To determine by molecular replacement the structures of alpha-thrombin or beta150-thrombin, complexed with important inhibitors. (4.) To crystallize proteins related to thrombin, such as recombinant prothrombin, and solve their structures by molecular replacement or heavy atom derivatives. Platelet factor 4 (PF4), which is released by activated platelets during the clotting process has procoagulation, antiheparin, chemotactic, and immunoregulatory activities. It is also an architypical member of a new superfamily of homologous proteins involved in hemostasis, inflammation, and cell growth. The specific aims of this subproject are: (1) To complete the refinement and analysis of the structure of native PF4. (2) To develop a structure for the PF4 / heparin complex from modeling and crystallographic studies. (3) To determine the structure of recombinant human PF4 and its variants by molecular replacement using the crystals now in hand. (4) To crystallize and determine the structures of homologs of PF4 which function as growth factors, mitogens, and wound healing agents. (5) To crystallize and determine the structures of selected heparin-binding proteins not homologous to PF4.