A NOVEL REGULATORY EPITOPE DEFINED BY A MURINE MONOCLONAL-ANTIBODY TOTHE PLATELET GPIIB-IIIA COMPLEX (ALPHA-IIB-BETA-3 INTEGRIN)

We characterized a murine monoclonal antibody, PT25-2 (IgG(1)), raised against washed human platelets. The antibody and its Fab fragments we re both capable of inducing platelet aggregation in a fibrinogen-depen dent manner and induced I-125-fibrinogen binding to unstimulated plate lets (120,000 molecules/platelet at a 100 nM IgG concentration). The a ntibody immunoprecipitated the alpha IIb beta 3 complex from lysates o f iodinated platelets but did not react with the respective subunits w hen complex formation was disrupted by treatment with 5 mM EDTA at 37 degrees C for 30 min. However, simply removing the extracellular dival ent cation with EDTA had no effect on antibody binding indicating that the antibody's epitope depends upon a conformational structure mainta ined by alpha beta subunit association. Antibody binding to unstimulat ed, washed platelets yielded binding parameters (K-d=40 nM, B-max=100, 000 molecules/platelet), which were found to be virtually unchanged wh en binding was performed using thrombin or RGDS-peptide-stimulated pla telets. Thus, the PT25-2 antibody defines a novel regulatory epitope e xpressed by the alpha IIb beta 3 integrin on unstimulated, quiescent p latelets.