CalDAG-GEFI and PKC synergize in αIIbβ3 activation in platelets activated through the PAR4 receptor. (A) Dose response for PAR4p-induced aggregation of wild-type (WT) and CalDAG-GEFI–deficient (KO) platelets. The data shown represent the percentage aggregation measured 5 minutes after addition of the agonist; n = 6 (P values given in “Results”). (B) WT or CalDAG-GEFI–deficient platelets were stimulated with 1.25 mM PAR4p in the presence or absence of the broad-range PKC inhibitor Ro31-8220 (5 μg/mL). (Top panel) Representative aggregation traces. (Bottom panel) Representative histograms for the activation of integrin αIIbβ3 as measured by flow cytometry. Gray curve represents untreated; black curve, treated with PAR4p. The results are representative of 5 individual experiments. (C) WT () or CalDAG-GEFI–deficient () platelets were stimulated with 1.25 mM of PAR4p in the presence or absence of the broad-range PKC inhibitor Ro31–8220. Binding of JON/A-PE was measured to determine the level of αIIbβ3 activation by flow cytometry; n = 6 (***P < .001). (D) Calcium flux was measured over time in Fluo-3–loaded WT or CalDAG-GEFI–deficient platelets stimulated with the indicated concentrations of PAR4p. The bar graph shows the maximum fluorescence intensity (Fluo-3) measured in WT () and CalDAG-GEFI–deficient () platelets within 1 minute after addition of PAR4p; n = 6. No significant difference in Fluo-3 fluorescence signals between WT and KO platelets was observed.