Regulation of von Willebrand factor finding to the platelet glycoprotein Ib-IX by a membrane skeleton-dependent inside-out signal

The platelet receptor for von Willebrand factor (vWF), glycoprotein Ib-M (GPIb-IX), mediates initial platelet adhesion and activation. We show here that the receptor function of GPIb-IX is regulated intracellularly via its link to the filamin-associated membrane skeleton. Deletion of the filamin binding site in GPIb alpha markedly enhances ristocetin- (or botrocetin)-induced vWF binding and allows GPIb-IX-expressing cells to adhere to immobilized vWF under both static and flow conditions. Cytochalasin D (CD) that depolymerizes actin also enhances vWF binding to wild type GPIb-IX, Thus, VWF binding to GPIb-IX is negatively regulated by the filamin-associated membrane skeleton. In contrast to native vWF, binding of the isolated recombinant vWF Al domain to wild type and filamin binding-deficient mutants of GPIb-IX is comparable, suggesting that the membrane skeleton-associated GPIb-IX is in a state that prevents access to the Al domain in macromolecular vWF, In platelets, there is a balance of membrane skeleton-associated and free forms of GPIb-IX. Treatment of platelets with CD increases the free form and enhances vWF binding. CD also reverses the inhibitory effects of prostaglandin El on vWF binding to GPIb-IX. Thus, GPIb-IX-dependent platelet adhesion is doubly controlled by vWF conformation and a membrane skeleton-dependent inside-out signal.