MOLECULAR BIOLOGY OF HUMAN COAGULATION FACTOR V

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DESCRIPTION: (Adapted from the investigator's abstract). Factor V is a protein cofactor that is essential for the prothrombinase complex. This complex consists of factor Va, factor Xa, calcium, and a phospholipid surface. The generation of thrombin is a critical reaction during hemostasis. Thrombin catalyzes a number of important reactions during hemostasis including the activation of platelets, the activation of factors V, VIII and XIII, and the conversion of fibrinogen to fibrin. In addition, thrombin bound to its endothelial receptor thrombomodulin catalyzes activation of protein C which then inactivates factors V and VIII. In order to understand the mechanisms by which the activation of prothrombin is regulated we have undertaken an investigation of the structure and function of the factor V molecule. Initially, we developed methods to isolate human factor V from plasma and studied its proteolytic activation. Subsequently we investigated the role of factor Va as the factor Xa receptor on human platelets. More recently, we have determined the complete primary sequence of the molecule. We have demonstrated that factor V is a multidomain protein with homology to ceruloplasmin and factor VIII. We are currently using site directed mutagenesis to investigate structure function relationships in recombinant factor V expressed in mammalian cells. The goals of the present study are to identify regions of the molecule that constitute the binding sites for phospholipid, cell surface receptors, factor Xa and prothrombin. We will also define the critical proteolytic events required for the activation and inactivation of this protein. We will use both genetic and reverse genetic approaches to define the function of this important protein. Using the factor V cDNA and mammalian cell expression systems we will express recombinant factor V with specific mutations. We will also use PCR to amplify platelet mRNA isolated from patients with factor V deficiency and determine natural mutations that affect factor V function. The results of these experiments will ultimately define the exact molecular interactions involved in the assembly and regulation of the prothrombinase complex. Expression and characterization of chimeric factor V/factor VIII molecules will further enhance understanding of the structure function relationships in this family of related proteins. This knowledge may provide insight into the pathogenesis of thrombotic disorders.