MMP-2 cleaves the PAR1 N-terminal domain. (A) Control or PAR1-desensitized platelets were pretreated with active MMP-2 (0.75 nM) for 5 minutes and then activated with a subthreshold dose of the PAR4 agonist peptide, and aggregation was followed for 5 minutes. Results are expressed as percent increase of aggregation over agonist alone. Data represent means ± SEM (n = 3); *P < .05 vs control. (B) Control or PAR1-desensitized platelets were pretreated with active MMP-2 (0.75 nM) for 5 minutes and then activated with a subthreshold dose of PAR4 agonist peptide. Tyr phosphorylation was assessed by western blotting, and bands were measured using QUANTISCAN software. Data represent means ± SEM (n = 3); *P < .05 vs control. (C) Gel-filtered platelets were treated for 10 minutes with thrombin (0.075-3 nM) or MMP-2 (0.075-0.75 nM) in the absence or presence of hirudin (HIR; 3 U/mL) or TIMP-2 (1 µg/mL), respectively, at 37°C. PAR1 N-terminus (N-term) expression was assessed by western blotting, and bands were measured using QUANTISCAN software. Data represent means ± SEM (n = 3). (D) Platelet-rich plasma was treated for 10 minutes with thrombin (0.1 nM), in the presence of GPRP (2.5 mM) or MMP-2 (0.75-3 nM), in the absence or presence of a monoclonal antibody blocking MMP-2 (MoAb Block), at 37°C. PAR1 N terminus expression was assessed by western blotting, and bands were measured using QUANTISCAN software. Data represent means ± SEM (n = 3). (E) CHO cells stably expressing α_IIbβ₃ (CHO-α_IIbβ₃-PAR1), or not (CHO-PAR1), were transiently transfected with T7-tagged WT PAR1, incubated with increasing concentrations of active MMP-2 for 20 minutes at 37°C, and stained with saturating concentrations of mouse anti-T7 tag. In the inset, CHO cells expressing α_IIbβ₃ and PAR1 were incubated with MMP-2 (1.5 nM) for increasing periods of time. Loss of T7 epitope was assessed by flow cytometry. Data represent means ± SEM (n = 4). (F) CHO cells stably expressing α_IIbβ₃ (red columns) or not (blue columns) were transiently transfected with T7-tagged PAR1 and incubated for 20 minutes at 37°C with thrombin (20 nM) or active MMP-2 or pro-MMP-2 (1.5 nM). Loss of T7 epitope was assessed by flow cytometry. Data represent means ± SEM (n = 5); *P < .05 vs CHO-PAR1. (G) EC₅₀ of MMP-2-induced cleavage of PAR1 in CHO cells expressing only PAR1 (CHO PAR1) or both α_IIbβ₃ and PAR1 (CHO-α_IIbβ₃-PAR1). Data represent means ± SEM (n = 3); *P < .05 vs CHO- PAR1. (H) CHO-α_IIbβ₃ cells expressing T7-tagged PAR1 were incubated with thrombin (20 nM), active MMP-2 (1.5 nM), pro-MMP-2, heat-denatured MMP-2, MMP-2 inhibited with TIMP-2 (47.5 nM), or MMP-2 inhibited with a blocking anti–human MMP-2 monoclonal antibody (4 µg/mL) for 20 minutes at 37°C. Loss of T7 epitope was assessed by flow cytometry. Data represent means ± SEM (n = 4); *P < .05 vs MMP-2. (I) Gel-filtered platelets were preincubated with active MMP-2 (0.75 nM) for 5 minutes and then activated with a subthreshold dose of TRAP-6 (2 µM) in the presence of the monoclonal antibody SPAN12, an immunoglobulin G isotype antibody control (control), or the PAR1 antagonist RWJ-56610 (1 µM). Results are reported as percentage increase CD62P expression vs TRAP-6. Data represent means ± SEM (n = 5); *P < .05 vs control.