Thr153in the LBS is critical for PAR4 activation. (A) Comparable expression levels of PAR4-WT, PAR4-T153A, and PAR4-T153S on the surface of the HEK293 Flp-In T-REx stable cell lines confirmed by flow cytometry using a V5-fluorescein isothiocyanate (FITC)–conjugated antibody. (B) HEK293 cell expresses PAR1 endogenously, which is inhibited by 100 nM vorapaxar. There is no intracellular calcium mobilization triggered by stimulating HEK293 Flp-In T-REx cells that express PAR4-WT, PAR4-T153A, or PAR4-T153S with 50 μM PAR1-AP (TFLLRN). Stimulating PAR4-WT with 250 μM of PAR4-AP initiated calcium flux, but PAR4-T153A and PAR4-T153S had no response to this dose of PAR4-AP. (C) PAR4-WT, PAR4-T153A, and PAR4-T153S were challenged with 5 different doses of PAR4-AP, and calcium mobilization responses to PAR4-AP stimulation were measured (n = 5). Data are presented as the area under curve (AUC) after stimulation of the Fura2-loaded HEK293 Flp-In T-REx stable cell lines. (D) PAR4-WT, PAR4-T153A, and PAR4-T153S were challenged with 6 different doses of α-thrombin, and calcium mobilization responses to α-thrombin stimulation were measured (n = 5). Data are presented as the AUC after stimulation of the Fura2-loaded HEK293 Flp-In T-REx stable cell lines. Data are mean ± standard deviation (SD) from 5 independent experiments at each concentration. N.S., not significant, unpaired Student t test. *P < .05; **P < .01; ***P < .001. The yellow asterisks indicate the comparison of PAR4-T153A to PAR4-WT and the blue asterisks indicate the comparison of PAR4-T153S to PAR4-WT.