Exogenous TxA₂ restores aggregation, PKC activation, and dense granule secretion in Cvx-stimulated CalDAG-GEFI^−/− platelets. (A) Left: Aggregation of WT platelets stimulated with low dose Cvx (100 ng/mL). Right: Aggregation traces for CalDAG-GEFI^−/− platelets activated with 100 ng/mL Cvx, 500 nM U46619, or the combination of both agonists. Platelet aggregation was studied in the presence of 75 μM 2-MesAMP or 5 μg/mL Ro31-8220 to block signaling by P2Y12 and PKC, respectively. Results are representative of 5 individual experiments. (B) CalDAG-GEFI–deficient platelets were stimulated with low dose (LD) Cvx (100 ng/mL) and/or U46619 (3 μM) in the presence or absence of 2-MesAMP. Binding of JON/A-PE was measured to determine the level of α_IIbβ₃ activation by flow cytometry. n = 6. **P < .001, *P < .05. (C) Detection of phosphorylated PKC substrates in platelet lysates (Western blotting). WT (left lanes) and CalDAG-GEFI^−/− (knockout [KO], central lanes) platelets were stimulated for 10 minutes with 100 ng/mL Cvx, 500 nM U46619, or the combination of both agonists. Phosphorylation of PKC substrates, including PLEK (←), in WT platelets pretreated with Ro31-8220 (10 μg/mL) or activated with phorbol myristate acetate (100 nM) were determined as controls (right panel). Results are representative of 3 independent experiments. (D) ³H-serotonin release in WT (black line, ●) or CalDAG-GEFI^−/− (gray line, gray triangle) platelets activated with 100 ng/mL Cvx. After 3 minutes, 500 nM U46619 was added to CalDAG-GEFI^−/− platelets. Experiments were performed in the presence (open symbols) or absence (filled symbols) of the PKC inhibitor Ro31-8220 (n = 3).