Thromboxane generation is defective in the absence of Ask1. (A) Quantitation of TxA₂ generation measured as thromboxane B₂ (TxB₂) in washed platelets isolated from WT and Ask1^−/− mice and stimulated with different agonists, as indicated. Data presented are from 3 independent experiments. (B) Quantitation of thrombin-induced ¹⁴C-serotonin secretion in WT and Ask1^−/− platelets in the presence or absence of aspirin. (C) Quantitation of TxA₂ generation induced by various agonists in WT and Ask1^−/− platelets pretreated with pyrrophenone (1 μM) for 30 minutes. (D-E) Western blot of lysates of resting or thrombin-stimulated washed platelets (D) or indicated agonist-stimulated washed platelets (E) isolated from WT and Ask1^−/− mice blotted using phosphospecific anti-cPLA₂. The same blot was reprobed with anti-cPLA₂ to ensure equal protein loading in all the lanes. A representative blot from 3 independent experiments is shown. (F) Quantitation of JON/A-binding in platelets from WT and Ask1^−/− mice resting or treated with AA (100 μg/mL). (G) Quantitation of P-selectin expression in WT and Ask1^−/− platelets resting or treated with U46619 (1 μM). (H) Quantitation of ATP secretion in platelets from WT and Ask1^−/− mice resting or treated with U46619 (100 nM). ASA, acetylsalicylic acid. *P < .05; **P < .01; ***P < .001.