Factor VIII (fVIII) and Willebrand factor (WF) are two plasma proteins that are necessary for normal hemostasis and which form a tightly bound noncovalent complex. After proteolytic activation by thrombin or factor Xa, activated factor VIII (fVIIIa) is a cofactor for the factor IXa-catalyzed activation of factor X which occurs on lipid or cellular surfaces in the presence of calcium. WF is necessary for normal platelet adhesion to the blood vessel wall and may be involved in normal platelet-platelet interactions. It also prolongs the life of fVIII in the circulation. Deficiencies in fVIII and WF result in hemophilia A and von Willebrand disease which are two important disease states in hematology. Additionally, interactions of WF and platelets with the abnormal vessel wall are implicated in the pathogenesis of atherosclerosis. The purpose of this investigation will be to study the detailed mechanism which governs the association of fVIII with WF and the events which lead to the dissociation of fVIII from WF during the development of the procoagulant activity of fVIII. Specific questions that will be addressed are: 1) What are the stoichiometry, dissociation constant(s), and values of the rate constants that govern the interaction of fVIII with WF? 2) Does activated fVIII bind WF and if not what are the kinetic and proteolytic events that control the dissociation process? 3) Must activated fVIII dissociate from WF to become a cofactor in the activation of factor X by factor IXa in the presence of phospholipid and calcium, and if so, what is rate-limiting: activation, dissociation, or assembly? 4) Which part of the fVIII molecule binds WF? Preliminary studies in our laboratory have led to several technical developments that have made it feasible to address these questions for the first time. First, a procedure for isolating milligram quantities of a homogeneous 240 kDa species of fVIII has been developed. Second, velocity sedimentation studies have shown that fVIII, WF, and the fully-saturated fVIII- WF complex can be clearly resolved. Electrophoretic studies using 125I-fVIII have shown that multimeric WF can be labeled and that fVIII binds to all multimers. This technique will allow determination of whether fVIIIa binds WF and by competitive binding studies, which subunits or fragments of fVIII bind WF. Finally, stopped-flow light scattering studies have shown the binding of fVIII and WF is accompanied by a large increase in light scattering so that the kinetics of the interaction can be measured.

National Institute of Health (NIH)
National Heart, Lung, and Blood Institute (NHLBI)
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Hematology Subcommittee 2 (HEM)
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Emory University
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