A novel anti-platelet monoclonal antibody (3C7) specific for the complex of integrin αIIbβ3 inhibits platelet aggregation and adhesion

Abstract

Activation or ligand binding induces conformational changes in αIIbβ3, resulting in exposure of neoepitopes named ligand-induced binding sites. We reported here a novel monoclonal antibody developed by using Chinese hamster ovary (CHO) cells expressing an activated αIIbβ3 mutant (CHO αIIbβ 3Δ717) as the immunogen. This IgG2bκ named 3C7 was specific for the complex of αIIbβ3 as demonstrated by flow cytometry, immunoprecipitation, and EDTA chelating. The binding of 3C7 to platelets increased significantly when platelets were activated by ADP/thrombin or occupied by RGDS peptides, fibrinogen, or PAC-1, suggesting that 3C7 was an anti-ligand-induced binding site antibody. The antibody failed to bind to the CHO cells expressing another α IIbβ3 mutant (β3Y178A) suggesting that the Cys177-Cys184 loop of β3 was likely the epitope for 3C7. 3C7 inhibited platelet aggregation, which was initiated by ADP or thrombin in a dose-dependent manner (IC50s of 5.6 and 0.05 μg/ml, respectively). The antibody also inhibited platelet adhesion to immobilized fibrinogen but not to fibronectin or collagen. These findings suggested that 3C7 was a potent antagonist of integrin α IIbβ3 and a potential anti-thrombotic agent. © 2005 by The American Society for Biochemistry and Molecular Biology, Inc.