Antibodies to GPIIbα (300-312) Inhibit Fg Binding,Clot Retraction,and Platelet Adhesion to Multiple Ligands

Abstract

We previously reported that a peptide with the sequence Gly-Ala-Pro-Leu (GAPL) found as residues 309—312 in glycoprotein IIbα (GPIIb) comprises at least part of a Fg binding site on GPIIb (1). Subsequent studies demonstrated that a peptide corresponding to residues 300-312 of GPIIbα can bind to Fg and Vn, and is a potent inhibitor of platelet aggregation and the adhesion of activated platelets to at least four adhesive ligands: Fg, Fn, Vn, and vWf (2). Here, the production and initial characterization of polyclonal antibodies against this peptide are described. ELISAs and dot-blot assays reveal the specificity of the antibodies for the peptide immunogen. In immunoblots, the antibodies recognize GPIIb under reducing conditions. The binding of Fg to immobilized GPIIb/IIIa, the rate of clot retraction and the adhesion of stimulated platelets to Fg, fibronectin (Fn), vitronectin (Vn), and von Willebrand factor (vWf) were inhibited by these antibodies, but not by a control IgG. These results together with our earlier published data indicate that GPIIbα (300-312) comprises at least part of a common ligand binding site within the integrin α_IIIb subunit.