The Arg-Gly-Asp (RGD) recognition site of platelet glycoprotein IIb-IIIa on nonactivated platelets is accessible to high-affinity macromolecules

We have characterized a murine IgG monoclonal antibody, OP-G2, specific for platelet glycoprotein (GP) IIb-IIIa (α_IIbβ₃). OP-G2 Fab fragments inhibit fibrinogen-mediated platelet aggregation and competitively inhibit adenosine diphosphate-induced binding of ¹²⁵I-fibrinogen to washed platelets. OP-G2 binding to GPIIb-IIIa is specifically inhibited by RGD-containing peptides but not the fibrinogen γ-chain carboxy-terminal peptide, and OP-G2 Fab fragments, like RGD-containing peptides, alter the conformation of GPIIb-IIIa resulting in the expression of a ligand-induced binding site (LIBS) recognized by PMI-1. OP-G2 fails to bind to the recombinant Cam variant of GPIIb-IIIa (α_IIbβ_3Cam) wherein an Asp¹¹⁹ to Tyr¹¹⁹ substitution in GPIIIa abrogates the ability to recognize RGD. These data indicate that OP-G2 recognizes an epitope at or in very close proximity to the RGD recognition site of GPIIb-IIIa and that, in every aspect tested, OP-G2 behaves like a macromolecular RGD ligand. Interestingly, two-color flow cytometry shows that OP-G2 IgG can bind to nonactivated platelets. Quantitative binding assays indicate that nonactivated platelets bind approximately 50,000 ¹²⁵I-OP-G2 molecules/ platelet. Furthermore, the affinity of OP-G2 for platelets activated with thrombin is roughly fivefold higher (nonactivated, kd = 24.8 nmol/L; activated, kd = 4.9 nmol/L). These results suggest that the RGD recognition site of GPIIb-IIIa is available to macromolecules that contain RGD even on nonactivated platelets, provided that the affinity of the ligand is adequate.