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

• Published in: Biochemistry (Easton) 1988-01, Vol.27 (2), p.633-640
• Main Authors: Berndt, Michael C, Du, Xiaoping, Booth, William J
• Format: Article
• Language: English
• Subjects: Animals; Antibodies, Monoclonal; Antigen-Antibody Complex; Biological and medical sciences; Blood coagulation. Blood cells; Cattle; Epitopes - analysis; Fundamental and applied biological sciences. Psychology; Humans; Iodine Radioisotopes; Kinetics; man; membrane glycoproteins; Molecular and cellular biology; Platelet; Platelet Membrane Glycoproteins - immunology; Platelet Membrane Glycoproteins - metabolism; platelets; Protein Binding; Ristocetin - pharmacology; von Willebrand factor; von Willebrand Factor - metabolism
• ISSN: 0006-2960; 1520-4995
• DOI: 10.1021/bi00402a021

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 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 at ristocetin concentrations greater than or equal to 1 mg/mL. Ristocetin-dependent specific binding of 125I-labeled vWF was 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 Ib-IX complex. Similar correspondence between platelets and purified GP Ib-IX complex with respect to the ristocetin-dependent binding of vWF was obtained with anti-vWF monoclonal antibodies.