The interaction of Factor VIII-von Willebrand Factor with phospholipid vesicles has been studied by using sucrose-density-gradient ultracentrifugation. When purified Factor VIII-von Willebrand Factor was run alone. Factor VIII activity and Factor VIIIR-Ag sedimented together to the lower half of the tube. Addition of phosphatidylserine/phosphatidylethanolamine vesicles at concentrations above 250 microgram/ml resulted in complete separation of Factor VIII activity and Factor VIIIR-Ag, the former appearing with the phospholipid on the top of the tube and the latter sedimenting as before. This separation was obtained even in the presence of proteinase inhibitors. Activation of Factor VIII-von Willebrand Factor by thrombin resulted in formation of a slow sedimenting component containing essentially all the Factor VIII activity, whereas the Factor VIIIR-Ag sedimented towards the bottom of the tube as before. The thrombin-induced Factor VIII activity was strongly bound to phospholipid vesicles as determined by density-gradient centrifugations at various Factor VIII concentrations and low concentrations of phospholipid. Based on certain assumptions a dissociation constant of 2.5 nM was calculated, a mechanism for the formation in vivo of the Factor X-activator complex is suggested.

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