MEKK3 regulates platelet activation through integrin αIIbβ3–mediated inside-out signaling. Aggregation traces of washed MEKK3^f/f and MEKK3^−/− platelets treated with thrombin (A), ADP (B), collagen (C), or U46619 (D) were measured by aggregometry. (E) ATP secretion by MEKK3^f/f and MEKK3^−/− platelets induced by 0.06 U/mL thrombin, 10 µmol/L ADP, 2 µmol/L U46619, or 1.5 µg/mL collagen (n = 3, mean ± SD). (F) Expression of P-selectin in MEKK3^f/f and MEKK3^−/− platelets stimulated with 0.05 U/mL thrombin, 10 µmol/L ADP, 5 µmol/L U46619 or 2 µg/mL collagen was analyzed by flow cytometry. P-selectin expression was presented as mean fluorescence density (n = 3, mean ± SD). (G) Binding of Alexa Fluor 647–conjugated Fg to washed MEKK3^f/f and MEKK3^−/− platelets stimulated with 0.05 U/mL thrombin, 10 µmol/L ADP, 5 µmol/L U46619, or 2 µg/mL collagen. The results are expressed as mean fluorescence intensity (n = 3, mean ± SD). (H) Representative phalloidin staining and quantification of washed MEKK3^f/f and MEKK3^−/− platelets spreading on immobilized Fg for 90 minutes. (I) Quantification of the areas of spreading platelets in (H) (n = 4, mean ± SD; P > .05). (J) Representative pictures of the clot retraction of washed MEKK3^f/f and MEKK3^−/− platelets stimulated by 1 U/mL thrombin. (K) The 2-dimensional area of clots in panel J. Data are expressed as retraction ratios (n = 3, mean ± SD; P > .05). *P < .05, **P < .01, ***P < .001.