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

Figure 3

Thrombin promotes []i increase mainly by the release from intracellular pools. Serum-deprived cells were stimulated with thrombin or PAR1-AP. [Ca²⁺]i was determined by fluorescence microscopy as described in Methods. (a) Thrombin-induced [Ca⁺²]i increase is partially prevented by EGTA (0.25 mM) and abolished by thapsigargin (Tg; 2 μM) + EGTA. (b) Thrombin-induced calcium release is mimicked by PAR1-AP. (c) Blockage of plasma membrane calcium channels by Lanthanum (La³⁺; 100 μM) decreases thrombin-induced and (d) PAR1-AP-induced calcium increase to a similar extent as EGTA. Results are expressed as the area under the curve (AUC) compared to nonstimulated (NS) cells (negative control). Data are the mean ± SEM of three independent experiments. Multiple comparison ANOVA and Tukey’s test: α = 0.001 () or α = 0.01 () referred to negative control. And α = 0.001 (@@@@) or α = 0.01 (@@@) referred to thrombin stimulation (10 nM) or PAR1 peptide agonist (2.5 μM).

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