This proposal seeks continuation of support for long-term studies of molecules responsible for regulation of thrombosis by the protein C pathway. Hereditary protein C (PC) and protein S deficiencies are associated with thrombotic disease and activated protein C (APC) exhibits potent antithrombotic activity in animal models of thrombosis. APC is a normal circulating component in human blood which probably regulates basal levels of thrombin generation in vivo. Clinical trials of PC concentrates for antithrombotic therapy have been initiated. Plasma from many thrombophilia patients gives a poor anticoagulant response to APC. This property of plasma, called """"""""APC resistance"""""""", is the most common identifiable defect among venous thrombophilia patients. This proposal will test the hypothesis, based on our preliminary results, that abnormal factor V is responsible for APC resistance. Another hypothesis to be tested is that molecular mechanisms for the regulation of the anticoagulant activity of PC and APC are determined by specific amino acids on the surface of PC and APC. Thus, the five specific aims include the following. (1) To characterize abnormalities of factor V hypothesized to be responsible for APC resistance, we will sequence patients' factor V cDNA and perform studies involving purification and characterization of normal and patients' factor V to identify abnormalities at the molecular level. (2) To extend analysis of three-dimensional homology models to the entire molecules of PC and APC and to APC:serpin complexes, we will continue development of computer modelling studies to provide models which will be used to explain clinical phenotypes for naturally occurring mutations and to design and interpret structure-function studies. (3) To identify functional roles of specific surface loops of residues of PC and APC, we will use synthetic oligopeptides and polypeptides and antipeptide antibodies to identify essential sites and residues on the surface of PC and APC. Studies will employ synthetic oligopeptides and polypeptides, including among other, synthetic individual N- and C-terminal EGF domains, chemically-ligated polypeptides containing both EGF domains, and synthetic peptides containing the putative Ca2+-binding loop of the protease domain (residues 225-235). (4) To identify functionally essential residues at specific surface sites of PC and APC, we will use site-directed mutagenesis. Studies will focus on amino acids hypothesized to be involved in Ca2+-ion binding by the protease domain and in intermolecular interactions of PC or APC with thrombin:thrombomodulin, factor Va, and protein S. The roles of positive residues in a novel exosite will be probed. New inhibitor-resistant APC mutants will be sought. Studies will include defining the mechanism of anticoagulant action of the inhibitor-resistant mutant, S360A-APC, and using this molecule to study interactions of APC with normal and abnormal factor Va and with protein S. (5) To prepare PC and APC crystals suitable for x-ray diffraction determination of their three-dimensional structures. The proposed studies will provide new knowledge about natural anticoagulant mechanisms and may lead to new approaches to control thrombosis.
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