Substitution of _IIb amino acid residue D224 results in a loss of ligand binding function. FACS histograms depicting the binding of the _IIbβ₃complex-specific MoAb D57, the activation-independent ligand mimetic MoAb OPG2, or the ligand mimetic MoAb PAC1 to CHO cells transfected with wild-type _IIbβ₃ or _IIbD224Vβ₃. (A) MoAb D57 staining of cells transfected with _IIbβ₃ (shaded histogram) or _IIbD224Vβ₃ (open histogram). Untransfected CHO cells are depicted by the dotted histogram. (B) MoAb OPG2 staining of the _IIbβ₃ (shaded histogram) or _IIbD224Vβ₃ (open histogram) transfected cells. Untransfected CHO cells are depicted by the dotted histogram. Cells expressing wild-type _IIbβ₃ (C) or _IIbD224Vβ₃ (D) were activated by incubation with 4 μmol/L anti-LIBS6 for 30 minutes followed by the addition of MoAb PAC1 (IgM). Cells were washed, stained with fluorescein-conjugated goat antimouse IgM for 30 minutes, and analyzed by FACS. The binding of PAC1was analyzed in the presence (open histogram) and absence (shaded histogram) of the competitve inhibitor Ro43-5054.