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

Figure 4

Thrombin activation of PAR1 stimulates the calcium-dependent activation of calpain. ARPE-19 cells were serum-deprived for 24 hours prior to stimulation for 1 min with 10 nM thrombin or 2.5 μM PAR1-AP. PPACK, BAPTA-AM, and calpain inhibitors were included 30 minutes prior to stimulation. Calpain activation was measured as a function of luciferase activity as described in Methods. (a) Thrombin stimulates calpain activity by ~250% in a specific manner, prevented by PPACK. 10% FBS was included as positive control. PAR1-AP stimulates calpain activity by ~170% compared to negative control (- Control). (b) Calpain activation was prevented by the inclusion of BAPTA-AM (10 μM), calpastatin (1 μM), and PD150606 (100 μM). Results in Relative Luminescence Units (RLU) are the mean ± SEM of three independent experiments. Values for nonstimulated cultures maintained in serum-free DMEM/F12 (- Control) were set as 100%. Multiple comparison ANOVA and Tukey’s test: α = 0.001 () or α = 0.01 () referred to negative control; α = 0.001 (@@@@) referred to thrombin stimulation.

(a)

(b)