This proposal is focused on two long-term goals: 1) characterization of regulation of synthesis of blood coagulation Factor XIII, and 2) characterization of platelet membrane proteins and pathogenesis of immune platelet disorders. The specific objective of each goal are: 1. Factor XIII: to complete cloning of the cDNA for the a- subunit. This will be done by rescreening the cDNA lambda GT11 placental library with the 5'-most portion of a 32p-labelled cDNA which has already been purified and sequenced. In addition, a full-length cDNA clone will be obtained. ln vivo and in vitro synthesis of the a-subunit will also be characterized. Regulation of synthesis of the a-subunit will be studied in vivo by infusion of purified components of the coagulation and fibrinolytic systems into rats and analysis of mRNA from hepatic tissue by Northern hybridization with oligonucleotide probes. Similar studies will be done in vitro with cultured rat hepatocyte and human U937 cells. Structure-function relationships will be studied by site-directed mutagenesis of the a-subunit cDNA and expression in cultured cells and bacteria. The a-subunit gene structure in normal individuals and patients with congenital Factor XIII deficiency will be analyzed by Southern blot hybridization. The genetic defects will be characterIzed by cloning the genomic DNA for the a-subunit in patients and normals and studying expression following transfection Into cell lines. After completion of these experiments, a full- length cDNA for the Factor XIII b-subunit will be cloned using a human hepatocyte lambda GT11 library screened with polyclonal anti- b-subunit antibodies and oligonucleotide probes based on amino acid sequence of peptides of the protein. Similar studies to those performed on the a-subunit regarding sites of synthesis, structure, and regulation of synthesis will be done. 2. The specific objectives of the platelet membrane portion of the proposal are to: a) further characterize the role of a 210 kDa glycoprotein as an F(c) receptor and in the Ristocetininduced interaction of von Willebrand factor with platelets. Monoclonal antibodies against GP 210 will be made and their effect on von Willebrand factor binding to platelets determined. ln addition, crossed-immunoelectrophoresis and immunoprecipitation with these antibodies will be used to determine whether GP 210 is complexed with other platelet membrane glycoproteins. b) the role of glycoprotein V in the pathogenesis of quinidine purpura. Binding of (3H)-quinidine to purified GP V will be characterized by equilibrium dialysis. In addition, the mechanism by which antibody production is stimulated will be studied by culturing patients' mononuclear cells with quinidine with or without GP V. c) to identify and characterize other antigens involved in immune disorders of platelets.
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