Regulation of von Willebrand Factor Binding to the Platelet Glycoprotein Ib-IX by a Membrane Skeleton-dependent Inside-out Signal

Graham D Englund,Richard J Bodnar,Zhenyu Li,Zaverio M Ruggeri,Xiaoping Du

doi:10.1074/jbc.m008048200

Abstract

The platelet receptor for von Willebrand factor (vWF), glycoprotein Ib-IX (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 A1 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 A1 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 E1 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.

Highlights

Platelet adhesion plays a critical role in thrombosis and hemostasis
glycoprotein Ib-IX complex (GPIb-IX) Mutants with Deletions in the Cytoplasmic Domain of GPIb␣—To examine the roles of the GPIb␣ cytoplasmic domain in regulating the receptor function of GPIb-IX, wild type GPIb␣ or cytoplasmic domain deletion mutants of GPIb␣ were coexpressed in Chinese hamster ovary (CHO) cells with GPIb␤ and GPIX as previously described [11, 31]
Consistent with a previous report that prostaglandin E1 (PGE1) inhibited ristocetin-induced platelet aggregation and this inhibition was reversed by cytochalasin D [41], we found that PGE1 reduced botrocetin-induced von Willebrand factor (vWF) binding to resting platelets, and this inhibitory effect was reversed by cytochalasin D (Fig. 8E)

Summary

Introduction

Platelet adhesion plays a critical role in thrombosis and hemostasis. Under the influence of shear forces created by blood flow, initial platelet adhesion is dependent on the interaction between a platelet receptor for von Willebrand factor (vWF),1 the glycoprotein Ib-IX complex (GPIb-IX), and matrixbound vWF [1,2,3,4,5]. Deletion of Filamin Binding Sites of GPIb␣ Enhances GPIbIX-mediated Static Cell Adhesion to vWF—The above results show that ristocetin- or botrocetin-induced binding of soluble vWF to GPIb-IX is regulated by the interaction of the membrane skeleton with the cytoplasmic domain of GPIb␣.

Results

Conclusion