Parmodulins bind outside the ligand-binding pocket and act at the intracellular face of PAR1. (A) Equilibrium radioligand-binding studies were performed as described in “Methods.” The effect of vehicle (closed triangles), parmodulin 1 (open diamonds), parmodulin 2 (closed squares), and SCH79797 (open circles) on bound (B) and free (F) [³H]TRAP after incubation with platelet membranes was evaluated. The effect of vehicle (N/A), 10 μM parmodulin 1 (PM1), 3 μM parmodulin 2 (PM2), and 1 μM SCH79797 (S) on agonist-induced [Ca²⁺]_i flux was evaluated in COS-7 transfected with (B) wild-type (WT) PAR1, (C) WT PAR4, (D) the ΔH8 PAR1 mutant, (E) a chimeric PAR1 in which il2 was replaced with il2 of PAR4, and (F) a chimeric PAR1 in which il3 was replaced with il3 of PAR4. [Ca²⁺]_i flux was evaluated after stimulation with either 5 μM SFLLRN (B,D-F) or 300 μM AYPGKF (C). Data are presented as means ± SEM (n = 3-6). Results were analyzed using a Bonferroni multiple comparison test as described in supplemental Table 1 (*P < .05).