Inhibition of Trx contributes to p38 MAPK activation– and PS-dependent TF decryption. (A) THP-1 cells were treated with 20 µM HNE for 2 hours at 37°C and then washed and lysed in assay buffer. Equal amounts of protein were used to measure Trx activity using a Trx activity assay kit. (B) THP-1 cells were treated with HNE as described above. Cells were lysed, and equal amounts of protein were pulled down using anti-Trx antibody. Immunoprecipitates were probed with either anti-HNE antibody or anti-Trx antibody. Total cell lysates were probed with anti-HNE antibodies (right). (C-D) THP-1 cells were treated with PX-12 (40 µM), a specific inhibitor of Trx, for varying time intervals, and cell surface TF activity (C) and prothrombinase activity (D) were determined. (E) THP-1 cells were treated with PX-12 (40 µM) for 3 hours, fixed, and stained with 4′-6-diamidino-2-phenylindole (DAPI) and AF488-annexin V; the fluorescence intensity of the staining was then quantified. Images were obtained at 63× magnification (oil immersion). (F) THP-1 cells were treated with PX-12 for 3 hours, followed by annexin V (400 nM) for 30 min. Cell surface TF activity was measured with an FX activation assay. (G) THP-1 cells were treated with PX-12 (40 µM) for varying time periods, and the phosphorylation of MKK 3/6 and p38 MAPK were analyzed by western blot analysis. (H) THP-1 cells were pretreated with SB203580 (20 µM) for 1 hour, followed by PX-12 (40 µM) for 3 hours. At the end of PX-12 treatment, cell surface TF activity was measured in an FX 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). IB, immunoblot; IP, immunoprecipitation.