The intent of this proposal is to continue structure-function studies of the human factor VIII/von Willebrand (FVIII/vWF) glycoprotein complex. The following will be done: 1) The FVIII procoagulant protein will be purified and identified by inhibition of its activity by a human antibody, by acceleration of the activation of factor X by factor IXa, by the effect of thrombin and protein C on its activity, and by electrophoretic-immunoblotting with a human antibody. 2) The FVIII protein will be characterized biochemically, i.e., molecular weight by sedimentation equilibrium, subunit structure, amino acid composition, amino terminal analyses, and cyanogen bromide and proteolytic enzyme cleavage patterns. Our preliminary data show that activated FVIII has a molecular weight of 160,000 and is composed of several proteolytically degraded subunits above 50,000 daltons and one major, constant subunit of 52,000 daltons. 3) Proteolytic cleavage of FVIII by thrombin or protein C will be examined with time by high performance liquid chromatography (HPLC) as well as two-dimensional slab-gel electrophoresis and correlated with FVIII procoagulant activity. 4) Purified FVIII protein will be used for production of monoclonal antibodies for use in purifying FVIII from whole plasma and FVIII/vWF concentrates. The following will be done with the vWF protein: 1) By isoelectric focusing or various ion exchange and absorptive chromatographic techniques, including HPLC, we will determine if the 200,000 dalton dissulfide-linked subunits in vWF multimers are indeed identical. 2) The effect of removing sialic acid and then galactose on the electron microscopic appearance of vWF will be examined. 3) The vWF multimers will be chemically and proteolytically cleaved and separated to obtain fragments for electron microscopic, biochemical and functional analyses. 4) Cytoplasmic vWF will be quantitated in endothelial cells, its ability to support platelet interactions with or without ristocetin determined, and its size and subunit structure defined. 5) The effect of blocking N-linked oligosaccharide attachment to vWF by tunicamycin on cytoplasmic levels of vWF, its secretion, and its interaction with platelets will be examined. These studies should help define the role of these two closely associated, yet functionally different, clotting activities in normal blood coagulation, abnormal states of hemostasis, and thrombotic disorders.
