Signaling triggered by MMP-2 and DPR-TRAP in human platelets. (A) Platelets were incubated with MMP-2 (0.75 nM), TRAP (10 µM), DPR-TRAP (500 µM), or vehicle (veh) for 5 minutes at 37°C, without (red columns) or with (blue columns) RWJ-56610 (1 µM). Phospho-p38 MAPK was assessed by western blotting, and bands were analyzed using QUANTISCAN software. Data represent means ± SEM (n = 3); *P < .005 vs without RWJ-56610. (B) Platelets were incubated with MMP-2 (0.75 nM), TRAP (10 µM), DPR-TRAP (500 µM), or vehicle (veh) for 5 minutes at 37°C, without (red columns) or with (blue columns) RWJ-56610 (1 µM). Phospho-Akt was assessed by western blotting, and bands were analyzed using QUANTISCAN software. Data represent means ± SEM (n = 4); * P < .005 vs without RWJ-56610. (C) Changes in cytosolic free Ca²⁺ in 5 mM fluo-3–labeled platelets activated with 0.75 nM MMP-2 in the absence or presence of 1 µM RWJ-56610. The agonists were added, and changes in green fluorescence over time were measured in 5 mM fluo-3–loaded GFPs. Data represent means ± SEM (n = 4). (D) Changes in cytosolic free Ca²⁺ in 5 mM fluo-3–labeled platelets activated with DPR-TRAP (10 and 500 µM) in the absence or presence of RWJ-56610 (1 µM). Agonists were added and changes in green fluorescence in function of time were measured. Data represent means ± SEM (n = 4). (E) Gel-filtered platelets were preincubated with active MMP-2 (0.75 nM) (red columns) or DPR-TRAP (500 µM) (blue columns) for 5 minutes and then activated with a subthreshold dose of convulxin (1.8 ng/mL) in the presence or absence of U73122 (10 µM). Results are reported as percent increase in CD62P expression vs convulxin. Data represent means ± SEM (n = 5); *P < .05 vs vehicle. (F) Gel-filtered platelets were preincubated with active MMP-2 (0.75 nM) (red columns) or DPR-TRAP (500 µM) (blue columns) for 5 minutes and then activated with a subthreshold dose of convulxin (1.8 ng/mL) in the presence or absence of GSK429286 (100 µM). Results are reported as percent increase in CD62P expression vs convulxin. Data represent means ± SEM (n = 4); *P < .05 vs vehicle. (G) Gel-filtered platelets were preincubated with iloprost (100 ng/mL) for 2 minutes before addition of vehicle (veh), MMP-2 (0.75 nM), TRAP-6 (10 µM), DPR-TRAP (10 and 500 µM), and TRAP-6 (10 µM) plus DPR-TRAP (500 µM). The reaction was stopped after 5 minutes by centrifugation at 12 000g for 2 minutes, pellets were lysed by 0.1 N HCl, and the supernatant was analyzed for cAMP using a competition-based assay. Data represent means ± SEM (n = 4); *P < .05 vs vehicle. (H) Gel-filtered platelets were pretreated with active MMP-2 (0.75 nM) (red columns) or DPR-TRAP (500 µM) (blue columns) for 5 minutes and then activated with a subthreshold dose of convulxin (1.8 ng/mL) without or with 1 µg/mL PTX. Results are reported as percent increase in CD62P expression vs convulxin. Data represent means ± SEM (n = 3); *P < .05 vs vehicle. (I) Gel-filtered platelets were pretreated with active MMP-2 (0.75 nM) without (red columns) or with (blue columns) PTX (1 µg/mL) and then induced to adhere on a fibrinogen-coated surface for 30 minutes. Results are reported as percent increase of platelet spreading. Data represent means ± SEM (n = 3).