CalDAG-GEFI is central to Cvx-dependent TxA₂ generation. Washed platelets in the presence of 1 mM Ca²⁺ and 50 μg/mL fibrinogen were stimulated under stirring conditions at 37°C (standard aggregometry) with (A,C) low (LD, 100 ng/mL) or (B,D) high (HD, 500 ng/mL) dose Cvx. Samples were withdrawn at the indicated time points to measure the levels of TxB₂, the stable product of the nonenzymatic hydration of TxA₂. (A-B) TxB₂ levels in the supernatant of WT (black line, ●) or CalDAG-GEFI^−/− (knockout [KO], gray line, ▴) platelets activated in the presence (open symbols) or absence (filled symbols) of 75 μM 2-MesAMP (P2Y12 inhibitor). Data shown are mean ± SEM (n = 4). At a high dose of Cvx (B), significantly higher levels of TxB₂ were detected at all time points in the supernatant of activated WT platelets compared with WT/MesAMP, KO, or KO/MesAMP platelets. (C-D) Aggregation traces representative of 3 independent experiments. (E) WT (■) or CalDAG-GEFI–deficient (▩) platelets were stimulated with LD (100 ng/mL) or HD (750 ng/mL) Cvx 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. (F) TxB₂ release from WT and CalDAG-GEFI^−/− (KO) platelets stimulated for 10 minutes with HD Cvx in the presence (□) or absence (■) of 40 μg/mL α_IIbβ₃ blocking antibody Leo.H4. Data shown are mean ± SEM (n = 3), *P < .05, **P < .01, ***P < .001.