Factor VIII (fVIII) and von Willebrand factor (vWf) are plasma proteins that are necessary for normal hemostasis and which form a tightly-bound, non-covalent complex. After proteolytic activation by thrombin or factor Xa, activated fVIII (fVIIa) is a cofactor for the activation of factor X by factor IXa that occurs on lipid or cellular surfaces in the presence of calcium. vWf is necessary for normal adhesion of platelets to the vessel wall and may be involved in platelet-platelet interactions. vWf also prolongs the life of fVIII in vivo and probably promotes the release of fVIII from its site of synthesis. The study of the fVIII-vWf complex is important because hemophilia A (congenital fVIII deficiency) and von Willebrand's disease (congenital vWf deficiency) are relatively common bleeding disorders. Previous published findings made during initial funding of this project include 1) all subunits of multimeric vWf can bind fVIII, 2) intact but not thrombin-cleaved fVIII light chains bind vWf indicating that light chain cleavage is the key step in promoting dissociation of the fVIII-vWf complex, 3) vWf is a cofactor for fVIII light chain cleavage by thrombin, thus promoting dissociation of the complex, 4) cleavage of fVIII heavy chains but not light chains is required for expression of the factor IXa-cofactor (procoagulant) function of FVIIIa, 5) the product of thrombin activation of the fVIII- vWF complex is a 160-kDa A1/A2/A3-C1-C2 heterotrimer.
The aim of this project will be to continue a detailed investigation of the mechanism which governs the association of fVIII with vWf. Specifically, we propose to examine the mechanism of activation of the fVIII-vWf complex by factor Xa and the role of factor IXa in this process, 2) to examine the recognition of fVIII by thrombin by using inhibitors of fVIII activation, including heparin and peptides corresponding to N-terminal region of the fVIIi light chain and to the C-terminal domain of hirudin, 3) to measure the equilibrium binding of fVIII to vWf by using quantitative affinity chromatography, 4) to measure the kinetics of association and dissociation of the fVIII-vWf complex by stopped-flow right-angle light scattering, and 5) to examine the structure of the fVIII-vWf complex by scanning transmission electron microscopy. Taken together, these studies will provide the basis for a quantitative model of the fVIII-vWf complex.
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