Inhibition of ASK-1 partly attenuates HNE-induced TF activation. (A-B) THP-1 cells were treated with 20 µM HNE (A) or 25 µM curcumin (B) for varying time periods. Phosphorylation of ASK-1 analyzed by western blot analysis using antibodies against phosphorylated ASK-1 (Thr 845). ASK-1 phosphorylation levels in HNE-treated cells were quantified by densitometry of phosphorylated ASK-1 protein band on western blots (A, right). (C) THP-1 cells were pretreated with the ASK-1 inhibitors NQD1 (10 µM) or TC-ASK 10 (12.5 µM) overnight and stimulated with HNE (20 µM) for 4 hours, and cell surface TF activity was measured. (D) ASK-1 levels in untransfected THP-1 cells or THP-1 cells stably transfected with a scrambled (sc) siRNA or ASK-1 siRNA lentivirus. Western blot analysis (left) and quantification of ASK-1 band intensity by densitometry (right). (E) THP-1 cells transfected with a control or ASK-1 siRNA lentivirus were treated with HNE (20 µM) for 4 hours, and cell surface TF activity was measured in an FX activation assay. (F) THP-1 cells (wild-type or transfected with ASK-1 siRNA lentivirus) were treated with HNE (20 µM) for 4 hours, and cell surface prothrombinase activity was measured using a prothrombin activation assay. *P < .05; **P < .01; ***P < .001; ns, not significant (compared with the values obtained in their respective controls or as indicated in the figures). k/d, knocked-down.