Ristocetin-dependent reconstitution of binding of von Willebrand factor to purified human platelet membrane glycoprotein Ib-IX complex

Abstract

Whether the human platelet membrane glycoprotein (GP) Ib-IX complex is the receptor for ristocetin-dependent binding of von Willebrand factor (vWF) has been examined by reconstitution with the purified components using a solid-phase bead assay. Purified GP Ib-IX complex was bound and orientated on the beads via a monoclonal antibody, FMC 25, directed monoclonal antibody, FMC 25, directed against the membrane-associated region of the complex. Specific binding of 125I-labeled vWF to the GP Ib-IX complex coated beads was strictly ristocetin dependent with maximal binding occurring to ristocetin concentrations .gtoreq. 1 mg/mL. Ristocetin-dependent specific binding of 125I-labeled vWF saturable. The observed binding was specific to the interaction between vWF and the GP Ib-IX complex since there was no ristocetin-dependent specific binding of vWF if the physicochemically related platelet membrane glycoprotein, GP IIb, was substituted for the GP Ib-IX complex in a corresponding bead assay. Further, neither bovine serum albumin nor other adhesive glycoproteins, such as fibrinogen or fibronectin, specifically bound to the GP Ib-IX complex in the presence of ristocetin. Ristocetin-dependent binding of vWF to platelets and to GP Ib-IX complex coated beads was inhibited by monoclonal antibodies against a 45,000 molecular weight N-terminal region of GP Ib but not by monoclonal antibodies directed against other regions of the GP-IX complex. Similar correspondence between platlets and purified GP Ib-IX complex with respect to the ristocetin-dependent binding of vWF was obtained with anti-vWF monoclonal antibodies. The combined data indicate that the GP Ib-IX complex is the receptor involved in the ristocetin-dependent binding of vWF and that the specificity of this interaction is identical with that for the ristocetin-dependent binding of vWF to platelets.