The intact rigidity of ECL3 is essential for the interaction between the tethered ligand and the binding site. (A) Comparable expression levels of PAR4-WT, PAR4-P310L, PAR4-S311A, and PAR4-P312L on the surface of the HEK293 Flp-In T-REx stable cell lines were confirmed by flow cytometry using a V5-FITC–conjugated antibody. (B) Endogenous PAR1 in HEK293 cells is inhibited by 100 nM vorapaxar. No intracellular calcium mobilization was triggered by stimulating HEK293 Flp-In T-REx cells that express PAR4-WT, PAR4-P310L, PAR4-S311A, and PAR4-P312L with 50 μM PAR1-AP. Stimulating PAR4-WT with 250 μM of PAR4-AP initiated calcium flux, and PAR4-P310L had a small but unmistakable response to the same dose of PAR4-AP. At this dose of PAR4-AP, PAR4-S311A cells showed a receptor reactivity similar to that of PAR4-WT, and PAR4-P312L had a hyporesponse to PAR4-AP stimulation. (C) PAR4-WT, PAR4-P310L, PAR4-S311A, and PAR4-P312L 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 AUC after stimulation of the Fura2-loaded HEK293 Flp-In T-REx stable cell lines. (D) PAR4-WT, PAR4-P310L, PAR4-S311A, and PAR4-P312L were challenged with 6 different doses of α-thrombin, and calcium mobilization responses to 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 ± SD from 5 independent experiments at each concentration. Unpaired Student t test; *P < .05; **P < .01; ***P < .001.