The Physical Exchange of Factor VIII (FVIII) between von Willebrand Factor and Activated Platelets and the Effect of the FVIII B-Domain on Platelet Binding

Abstract

Normal hemostasis proceeds through the assembly of coagulant complexes on a lipid surface derived from activated platelets. The activation complex assembly is governed by multiple factors including the binding constants (K_d) of the coagulant factors for the lipid surface. The formation of the tenase complex requires delivery of factor VIII (FVIII) to the activated lipid surface by von Willebrand factor (vWF). Using electrophoretic quasi-elastic light scattering (ELS), we have examined the interaction of FVIII in the presence and absence of vWF with both resting and activated gel-filtered human platelets. Resting platelets do not bind FVIII. Platelets activated by thrombin, epinephrine, or SFLLRN, but not ADP or collagen, bind unactivated FVIII if vWF is not present. In the absence of vWF, unactivated FVIII binds to activated platelets with a K_d of 10.4 nM. B-domain deleted FVIII binds to activated platelets with a K_d of 5.1 nM. Thrombin -activated FVIII (FVIII_a) binds to activated platelets with a K_d of 1.7 nM. The activation of FVIII while bound to the platelet surface can be monitored as a function of time. In the presence of vWF, binding of unactivated FVIII to activated platelets was inhibited, but not the binding of FVIII_a. Displacement of bound unactivated FVIII from the platelet surface occurs when vWF is added to the FVIII−platelet complex. The binding of FVIII to activated platelets is affected by the B-domain, the state of FVIII activation, and the presence of soluble vWF and proceeds as a multistep process. FVIII binding by activated platelets is not affected by platelet gpIIb/IIIa or by platelet vWF.