MEKK3 regulates platelet activation through ERK1/2 and JNK2. (A) Expression levels of β3, GPIbα, GPVI, α2b, and α2 proteins in MEKK3^f/f and MEKK3^−/− platelets were detected using flow cytometry. The results are expressed as mean fluorescence intensity (n = 3, mean ± SD; P > .05). (B) Lysates of washed MEKK3^f/f and MEKK3^−/− platelets were probed for the expression of p38, ERK1/2, JNK, and ERK5 proteins. Washed MEKK3^f/f and MEKK3^−/− platelets were stimulated with 0.06 U/mL thrombin or 10 µmol/L ADP (C) or with 2 µmol/L U46619 or 1.5 μg/mL collagen (D) for 3 minutes, and the lysates were probed with phospho-specific antibodies for p38, ERK1/2, JNK, or ERK5 to quantify MAPK activation. GAPDH was used to verify equal loading. Aggregation of washed MEKK3^f/f and MEKK3^−/− mouse platelets in response to 0.05 U/mL thrombin (E), 10 µM ADP (F), 1.5 µg/mL collagen (G), and 2 µM U46619 (H) in the presence of 5 µM JNK inhibitor SP600125 or 5 µM MEK1/2 inhibitor U0126. At least 3 independent experiments were performed.