PAR4p-induced activation of αIIbβ3 in CalDAG-GEFI–deficient platelets requires signaling by PKC and Gαi-coupled receptors. (A) WT platelets pretreated with 5 μg/mL Ro31-8220 or CalDAG-GEFI–deficient (CalDAG-GEFI^−/−) platelets were activated with 1.25 mM of PAR4p in the presence of (1) the P2Y1 inhibitor MRS2179 (100 μM), (2) the P2Y12 inhibitor 2-MesAMP (50 μM), or (3) the ADP degrading enzyme apyrase (8 U/mL). Aggregation of platelets was recorded for 10 minutes. (B) WT () or CalDAG-GEFI–deficient () platelets were stimulated with 1.25 mM of PAR4p in the presence or absence of 2-MesAMP or MRS2179. Binding of JON/A-PE was measured to determine the level of αIIbβ3 activation by flow cytometry; n = 6 (***P < .001). (C) CalDAG-GEFI–deficient platelets pretreated with apyrase (8 U/mL) were activated with 1.25 mM of PAR4p, followed by 10 μM of epinephrine or 10 μM of serotonin. Platelet aggregation was recorded for 10 minutes. (D) CalDAG-GEFI–deficient platelets were preincubated with 2-MesAMP, followed by stimulation with 1.25 mM of PAR4p in combination with epinephrine or serotonin. Binding of JON/A-PE was measured to determine the level of αIIbβ3 activation by flow cytometry; n = 6 (***P < .001; ns indicates not significant).