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

Figure 5

Thrombin inducesα-spectrin degradation through PAR1-mediated -dependent activation of calpain. Serum-deprived ARPE-19 cells were stimulated for 1 minute with 10 nM thrombin or 2.5 μM PAR1-AP. PPACK, calcium chelators, and calpain inhibitors were included 30 minutes ahead of stimulation. α-Spectrin proteolysis into 150 kDa and 120 kDa fragments was assessed by Western blot. (a) Thrombin stimulates α-spectrin proteolysis by ~250% in a specific manner prevented by PPACK (25 μM). 10% FBS was included as positive control. (b) Thrombin effect was mimicked by 2.5 μM PAR1-AP. (c) Chelation of intracellular Ca²⁺ (10 μM BAPTA-AM), calpastatin (1 μM), and PD150606 (100 μM) prevented calpain activation. Results are expressed as percentage of α-spectrin proteolysis compared to nonstimulated cells (- 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. Or α = 0.001 (@@@@) referred to thrombin stimulation.

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