A MMP-2-generated tethered ligand (DPR-TRAP) potentiates human platelet activation. (A) CHO-α_IIbβ₃ cells transiently transfected with T7-tagged wild type (WT), T37D, L38S, D39S, P40N, R41A, S42D, and F43R PAR1 were incubated for 20 minutes at 37°C with thrombin (20 nM) (red columns) or active MMP-2 (1.5 nM) (blue columns). Loss of T7 epitope was assessed by flow cytometry. Data represent means ± SEM (n = 5); *P < .05; #P < .005 vs WT. (B) Gel-filtered platelets were preincubated with active MMP-2 (0.75 nM), DPR-TRAP (10-500 µM), or DPRSFLLR (500 µM) for 5 minutes and then activated with a subthreshold dose of adenosine 5′-diphosphate (ADP). Preincubation with 1 µM RWJ-56610 together with active MMP-2 (0.75 nM) or DPR-TRAP (500 µM) was also tested. Results are reported as percent increase in CD62P expression vs ADP. Data represent means ± SEM (n = 4); *P < .05 vs MMP-2; #P < .05 vs DPR-TRAP (500 µM). (C) Gel-filtered platelets were preincubated with active MMP-2 (0.75 nM) or DPR-TRAP (10-500 µM) with or without 1 µM RWJ-56610 and then activated with a subthreshold dose of collagen. Aggregation tracings are representative of 3 experiments. (D) Gel-filtered platelets were incubated with MMP-2 (0.75 nM), thrombin (3 nM), TRAP-6 (1-5 µM), or DPR-TRAP (10-5000 µM) for 1 hour at 37°C. PAR1 internalization was assessed by flow cytometry using the phycoerythrin-WEDE15 antibody. Data represent means ± SEM (n = 3); *P < .05 vs resting.