Thrombin-Induced Calpain Activation Promotes Protease-Activated Receptor 1 Internalization

Figure 7

Stimulation of living cells by thrombin decreases PAR1 membrane expression through a - and calpain-dependent process. Cells were serum-deprived for 24 hours prior to stimulation with thrombin or PAR1-AP. The decrease in thrombin-induced PAR1 membrane expression measured in fixed preparations (Figure 6) was confirmed in living cells by flow cytometry. (a) Cell population selected for analysis. (b) Thrombin (10 nM) stimulation decreases PAR1 membrane expression by a specific process prevented by PPACK (25 μM). (c) PAR1-AP (25 μM) induces time-dependent decrease in PAR1 membrane expression. (d) Thrombin induces the decrease in PAR1 membrane expression through the calcium-dependent activation of calpain. This effect was prevented by the joint addition of BAPTA-AM (10 μM) and EGTA (0.25 mM), calpastatin (1 μM), or PD150606 (100 μM). Results are expressed as a percent of fluorescence in nonstimulated cells (- Control). Data are the mean ± SEM of three independent experiments. Multiple comparison ANOVA and Tukey’s test: α = 0.001 (), α = 0.01 (), or α = 0.1 () referred to negative control. And α = 0.001 (@@@@) or α = 0.01 (@@@) referred to thrombin stimulation.

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