c7E3 binding before and after washout procedure and fibrinogen (fg) binding to unstimulated platelets after incubation with various GP IIb/IIIa inhibitors and following washout. (A) Initially bound biotinylated-c7E3 (solid line) and the remaining c7E3 binding after washout procedure (dashed line) as detected by R-phycoerythrin–conjugated streptavidin. Biotinylated unspecific Fab was used as negative control (pointed line). (B) Typical flow cytometric histogram of fg binding without c7E3 and after incubation with c7E3 and following washout. The marker that is used to determine the percentage of cells binding fg is shown. Platelets were incubated with increasing concentrations of GRGDSP (C ), c7E3 (D), 7E3 IgG (E), P2 (F), or PAC-1 (G) and then fixed and washed as described in Materials and Methods. After incubation with fibrinogen solution (3 g/L), fibrinogen binding was measured with an FITC-labeled polyclonal chicken antifibrinogen antibody in flow cytometry. The results are expressed as the percentage of platelets binding fibrinogen as the mean ± standard deviation of five determinations.