MOLECULAR BIOLOGY OF HUMAN COAGULATION FACTOR V

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Defects in regulation of the prothrombinase complex play a central role in the pathogenesis of thrombosis. Factor V regulates the activity of the prothrombinase complex which is responsible for the generation of thrombin during blood coagulation. Genetic defects in factor V are the cause of protein C resistance which is the most common inherited risk factor for thrombosis. Generation of factor Va and assembly of the prothrombinase complex is therefore a critical event in thrombosis. The long-term goal of this project is to understand the structure, function and regulation of factor V and the prothrombinase complex. During the first funding period we characterized the factor V gene and used factor V expression systems to define structure-function relationships in the protein. We determined that the Factor V C2 domain contains binding sites for phosphatidylserine and acquired factor V antibodies. We also found that alternative glycosylation of the C2 domain is the structural basis for the two forms of the factor V light chain that differ in their affinity for anionic phospholipid. We found that a large deletion within the B-domain results in a single chain protein that expresses partial cofactor activity. Finally, we have characterized molecular mechanisms of APC resistance. During the second funding period we propose to extend our studies on the structure and function of recombinant factor V. The goals of these studies will be to further define domains and specific amino acid residues that comprise the binding sites required for assembly of the prothrombinase complex. We will use alanine scanning mutagenesis to identify the amino acids within the light chain required for factor V binding to phospholipid and platelet membranes. We will use the same approach to identify amino acids within the A1 and A3 domains as required for the factor Xa binding site. We will localize the sites of tyrosine sulfation and glycosylation that are important for expression of cofactor activation and function. Finally we will investigate the functions of the factor V B-domain. We will determine whether novel B-domain cleavages by cellular proteases contribute to factor V action. We will also localize the sites of factor XIII mediated crosslinking within the B-domain. The proposed studies will provide new insights into the precise molecular interactions involved in assembly of the prothrombinase complex. This information will be critical for understanding the regulation of the prothrombinase complex under physiological and pathological conditions.