Human active MMP-2 and mouse PAR3 TIKSFNGGP peptide potentiate mouse platelet activation. (A) Alignment of human PAR1 and mouse PAR3 exodomains (h, human; m, mouse). (B) Washed mouse platelets were preincubated with active MMP-2 (1.5 nM) or TIKSFNGGP peptide (100-500 µM) for 5 minutes and then activated with a subthreshold dose of convulxin (10 ng/mL). Results are reported as percent increase CD62P expression vs convulxin. Data represent means ± SEM (n = 5). (C) Washed platelets from wild-type (WT) and PAR3^−/− mice were pretreated with active MMP-2 (1.5 nM) for 5 minutes and then activated with a subthreshold dose of convulxin (10 ng/mL), and aggregation was followed for 5 minutes. Results are reported as percent increase light transmission aggregometry vs convulxin. Data represent means ± SEM (n = 10); *P < .05 vs WT. (D) Washed platelets from wild-type (WT) (red columns) and PAR3^−/− (blue columns) mice were pretreated with active human MMP-2 (1.5 nM) for 5 minutes, without or with tcY-NH2 (1 mM), and then activated with subthreshold dose of convulxin (10 ng/mL). Results are reported as percent increase CD62P expression vs convulxin. Data represent means ± SEM (n = 6); *P < .05 vs WT without tcY-NH2. (E) Washed platelets from WT (red columns) and PAR3^−/− (blue columns) mice were pretreated with TIKSFNGGP (500 µM) for 5 minutes, without or with tcY-NH2 (1 mM), and then activated with a subthreshold dose of convulxin (10 ng/mL). Results are reported as percent increase in CD62P expression vs convulxin. Data represent means ± SEM (n = 4); *P < .05 vs WT without tcY-NH2; #P < .05 vs PAR3^−/− without tcY-NH2. (F) Washed platelets from wild-type (red columns) and PAR3^−/− (blue columns) mice were pretreated with human active MMP-2 (1.5 nM) for 5 minutes at 37°C, and platelets were lysed. Rho-GTP and total Rho were assessed by western blot analysis, and bands were measured using QUANTISCAN software. Data represent means ± SEM (n = 4); *P < .05 vs WT baseline; #P < .05 vs WT MMP-2.