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

Figure 1

Thrombin induces a specific, dose-dependent increase in []i. Cells were serum-deprived for 24 hours prior to stimulation with increasing concentrations of thrombin. [Ca²⁺]i was monitored by fluorescence microscopy as described in the Methods. (a) Thrombin induces a transient, saturable [Ca²⁺]i increase, sustained for ~3 min. [Ca⁺²]i was plotted as a function of fluorescence intensity (Arbitrary Fluorescence Units (AFU)) over time (minutes). Specificity of thrombin effect was assessed by inclusion of the thrombin inhibitor PPACK (25 μM). 10% FBS was included as positive control. (b) The area under the curves (AUC) plotted in (a) was calculated from the stimulation point up to the 4th minute of stimulation. (c) Thrombin Ec₅₀ = 0.55 nM was calculated from the logarithmic transformation of data in (b). Results are expressed as the mean ± SEM of three independent experiments compared to nonstimulated cells (- Control). 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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(b)

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