This proposal will determine the molecular mechanisms which result in the regulation of human factor VIII coagulant activity. The primary aim will focus on the interaction of factor VIII with thrombin. This interaction results in a potentiation of factor VIII activity which is followed by an inactivation of factor VIII to levels below that of the pre-activated level. Since this overall effect may result from more than one mechanism, a multifaceted analysis will be performed. Kinetic analyses will offer insights into the rates and extents of thrombin activation and inactivation of factor VIII; and the inclusion of thrombin-specific inhibitors will aid in the dissection of these mechanisms. Specific events will be correlated to altered polypeptide composition and amino acid sequencing analysis will be applied to selective proteolytic events. The formation of transient yet functionally active molecular complexes will be stabilized using chemical cross-linking reagents, thus allowing for detection. Monoclonal antibodies will be used to probe functional domains on the factor VIII which may reflect binding or catalytic sites. These same techniques will be used to evaluate the activation of factor VIII by factors IXa and Xa; the generation of factor Xa using a purified system; and the inactivation of factor VIII by activated Protein C in the absence and presence of Protein S. The elucidation of these mechanisms will offer insights into the regulation of a plasma coagulation protein that is central to the hemostatic process and which will have implications for the understanding and management of the most common of the congenital coagulation disorders.
