Effect of MMP-2 on the spreading and FAK phosphorylation of α_IIbβ₃-expressing CHO cells and platelets. (A) CHO-α_IIbβ₃ cells were pretreated with active MMP-2 (0.75 nM) (blue columns) or vehicle (red columns) for 5 minutes and then layered on a fibrinogen-coated surface for 30, 60, or 90 minutes at 37°C. Spreading was assessed as the percentage of surface covered and FAK phosphorylation by western blotting, and bands were measured using QUANTISCAN software. Data represent means ± SEM (n = 3); *P < .05 vs vehicle. (B) CHO-α_IIbβ₃ cells were pretreated with active MMP-2 (0.75 nM) or pro-MMP-2, heat denatured (Den) MMP-2, MMP-2 treated with TIMP-2 (47.5 nM), TIMP-2 alone, or vehicle for 5 minutes and then layered on a fibrinogen-coated surface for 60 minutes at 37°C. Spreading was assessed as the percentage of surface covered. Data represent means ± SEM (n = 4); *P < .05 vs vehicle. (C) CHO-α_IIbβ₃ cells were pretreated with active MMP-2 (0.75 nM) or the PEX domain or the catalytic (CAT) domain of MMP-2 (0.75 nM) for 5 minutes and then layered on a fibrinogen-coated surface for 60 minutes at 37°C. Spreading was assessed as the percentage of surface covered. Data represent means ± SEM (n = 3); *P < .05 vs vehicle. (D) Gel-filtered human platelets were pretreated with active MMP-2 (0.75 nM) (blue columns) or vehicle (red columns) for 5 minutes and then layered on a fibrinogen-coated surface for 15, 30, or 45 minutes at room temperature. Spreading was assessed as the percentage of surface covered and FAK phosphorylation by western blotting, and bands were measured using QUANTISCAN software. Data represent means ± SEM (n = 3); *P < .05 vs vehicle. (E) Gel-filtered human platelets were pretreated with active MMP-2 (0.75 nM) for 5 minutes, in the absence or presence of RWJ-56610 (1uM), and then layered on a fibrinogen-coated surface for 30 minutes at room temperature. Results are reported as percent increase in spreading compared with MMP-2-untreated platelets. Data represent means ± SEM (n = 4); *P < .05 vs vehicle.