This research proposal focuses on factor XIII, a blood clotting factor present in plasma and in blood cells (platelets, monocytes, megakaryocytes). Factor XIII is an important enzyme in the coagulation and fibrinolytic mechanisms. The function of factor XIIIa is to catalyze the formation of covalent bonds in fibrin and other substrates. These covalent bonds make a fibrin clot more stable and less susceptible to fibrinolysis. Deficiency of factor XIII leads to a bleeding diathesis. Too much activated factor XIII could led to thrombosis. The two specific goals of this research proposal are 1) to ascertain specific structure-function relationships and 2) to define the mechanism and regulation of factor XIII biosynthesis. The long-term objective is to have a better understanding of factor XIII's mechanism and in vivo regulation.
Specific Aim 1 is to localize several important functional properties to specific protein domains. The experimental design for this aim is to fragment purified intracellular factor XIII (A protein) by enzymatic (thrombin, trypsin) and chemical (CNBr) methods, to purify these fragments by HPLC and to identify them within the linear sequence of the molecule (by electrophoresis, blotting with monoclonal antibodies, sequence analysis) and to assign binding functions to the fragments. The functional properties that will be determined include thrombin binding, fibronectin binding, non-covalent binding of the two proteins of factor XIII (A and B), and fibrin binding. The mechanism of thrombin binding (an activation enzyme) and an alternative activation mechanism by prothrombinase complex will also be studied in detail. The inhibition of platelet aggregation by the B protein of factor XIII will be studied.
Specific Aim 2 is to understand the biosynthesis of factor XIII, its subunit assembly and its regulation. Experiments will be conducted with U937 cells (monocyte line) for intracellular factor XIII and with hepatoma cell lines for plasma factor XIII. Cells will be grown in 35 S-methionine and immunoprecipitated with specific antibodies. ELISA assays, specific for A protein, B protein and the complex, will be used for protein analysis. mRNA will be analyzed with a specific cDNA probe for A. It is important that both protein and mRNA be analyzed under the same conditions for these experiments. These experimental approaches will lead to a better understanding of factor XIII function and regulation.
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