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

Figure 6

Thrombin stimulation decreases PAR1 membrane expression through calpain activation. Cells were serum-deprived for 24 hours prior to stimulation with thrombin or PAR1-AP. PAR1 membrane expression was assessed by epifluorescence microscopy. (a) PAR1 (CY3; red) and cell nuclei (Hoechst; blue) merged images. (b) Quantitative analysis of epifluorescence images shown in (a). Thrombin (10 nM) decreases PAR1 membrane expression through a specific process prevented by PPACK (25 μM). (c) Stimulation with PAR1-AP (25 μM) for 60 min decreases PAR1 membrane expression to the same extent as thrombin stimulation for 5 min. (d) Thrombin-induced decrease in PAR1 membrane expression requires calcium-dependent activation of calpain. Thrombin-induced decrease in PAR1 membrane expression is prevented by the joint addition of BAPTA-AM (10 μM) and EGTA (0.25 mM), by calpastatin (1 μM) and by PD150606 (100 μM). Results are expressed as the Corrected Total Cell Fluorescence (CTCF) compared to nonstimulated cells (- Control). Data are the mean ± SEM of three independent experiments. Multiple comparison ANOVA and Tukey’s test: α = 0.001 () referred to negative control. Or α = 0.001 (@@@@) referred to thrombin stimulation.

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