CalDAG-GEFI is critical for collagen-dependent TxA₂ generation. (A) Time course of TxB₂ release from WT (black line, circle) or CalDAG-GEFI^−/− (knockout [KO], gray line, triangle) platelets. Cells were stimulated with 25 μg/mL fibrillar type I collagen under stirring conditions in the presence (open symbols) or absence (filled symbols) of 75 μM 2-MesAMP. Data shown are mean ± SEM (n = 4). Significantly higher levels of TxB₂ were detected at indicated time points in the supernatant of activated WT platelets compared with KO or KO/MesAMP platelets. (B) Aggregation traces representative of 3 independent experiments. (C-D) Time course of ERK (top panel) and Rap1 (middle panel) activation in platelets activated with collagen in the presence (D) or absence (C) of 75 μM 2-MesAMP. Representative of 3 independent experiments. (E) TxB₂ levels in the supernatant of WT platelets stimulated for 10 minutes with 500 ng/mL Cvx (high dose [HD] Cvx), or 25 μg/mL fibrillar collagen (HD collagen). Cells were activated in the presence (□) or absence (■) of 50 μM U0126 (MEK inhibitor). U0126 totally abolished ERK phosphorylation in response to Cvx or collagen (data not shown). Data are shown as mean ± SEM. The amount of TxB₂ released from collagen-activated platelets was defined as 100%. n = 3. *P < .05, **P < .01, ***P < .001.