by Shabbir A. Ansari, Usha R. Pendurthi, and L. Vijaya Mohan Rao The lipid peroxidation product 4-hydroxy-2-nonenal induces tissue factor decryption via ROS generation and the thioredoxin system by Shabbir A. Ansari, Usha R. Pendurthi, and L. Vijaya Mohan Rao BloodAdv Volume 1(25):2399-2413 November 28, 2017 © 2017 by The American Society of Hematology

Shabbir A. Ansari et al. Blood Adv 2017;1:2399-2413 © 2017 by The American Society of Hematology

HNE-induced ROS generation in mitochondria and its role in TF decryption. HNE-induced ROS generation in mitochondria and its role in TF decryption. (A) THP-1 cells were labeled with the mitochondrial staining dye MitoTracker (200 nM) for 30 minutes and then loaded with 5 µM H2DCFDA for 5 minutes at 37°C. Cells were then washed and treated with 20 µM HNE for 20 min at 37°C. After HNE treatment, cells were washed and analyzed for their fluorescence by confocal microscopy. (B) THP-1 cells were treated with specific electron transport chain inhibitors for 1 hour and then loaded with 5 µM H2DCFDA for 5 minutes at 37°C. Cells were treated with HNE for 20 minutes, and ROS generation was analyzed by the fluorescence of H2DCFDA that results from its oxidation by ROS by confocal microscopy. (C) ROS generation was quantified by measuring the fluorescence intensity of the cells as described in “Materials and methods.” The inhibitors used were 2.5 µM rotenone (Rot), 0.5 mM 2-thenoyltrifluoroacetone (THF), 10 µM antimycin (Anti), 10 mM sodium azide (Azide), and 5 µM oligomycin (Oligo). (D-E) THP-1 cells were treated with electron transport chain inhibitors for 1 hour in concentrations indicated in Figure 1B and then 20 µM HNE for 4 hours. Following HNE treatment, cell surface TF (D) and prothrombinase (E) activity was analyzed. (F) THP-1 cells were treated with electron transport chain inhibitors and HNE as described in panels D-E, and PS exposure on the cell surface was analyzed by labeling the fixed cells with AF488-annexin V. 4′-6-Diamidino-2-phenylindole was used to stain nuclei. (G) Quantification of annexin V bound to THP-1 cells (from the fluorescence staining shown in panel F) treated with HNE with or without electron transport chain inhibitors. (H) To investigate the effect of electron transport chain inhibitors on HNE-induced p38 MAPK activation, THP-1 cells were treated with the inhibitors as described above and then treated with HNE for 15 minutes. Cell lysates were subjected to western blot analysis to probe for p38 MAPK phosphorylation and total MAPK. Intensities of phosphorylated and total p38 MAPK bands on the blots were quantified to obtain fold increase in p38 MAPK phosphorylation. *P < .05; **P < .01; ***P < .001; ns, not significant (compared with the values obtained in their respective controls or as indicated in the figures). Images in panels A-B,F were obtained at 63× magnification (oil immersion). Con, control (no treatments); CV, control vehicle; DIC, differential interference contrast; Mito, mitochondria. Shabbir A. Ansari et al. Blood Adv 2017;1:2399-2413 © 2017 by The American Society of Hematology

HNE inhibition of TrxR activity inversely correlates with MKK3/6 and p38 MAPK activation. HNE inhibition of TrxR activity inversely correlates with MKK3/6 and p38 MAPK activation. (A-B) THP-1 cells were treated with varying concentrations of HNE for 1 hour (A) or with 20 µM HNE for varying time periods (B). After treatment, cells were washed, resuspended in the assay buffer, and sonicated to lyse cells. Equal amounts of protein were taken to measure TrxR activity using the TrxR activity assay kit. (C) Cell lysates of THP-1 cells treated with 20 µM HNE for varying time periods were probed to analyze the phosphorylation status of MKK3/6 and p38 MAPK by western blot analysis. (D) The phosphorylation status of MKK3/6 and p38 MAPK upon HNE stimulation was quantified and plotted against HNE-induced inhibition of TrxR activity at identical time periods. (E) THP-1 cells were treated with the antioxidant NAC (3 mM) or the electron chain transport inhibitors antimycin (Anti; 10 µM) or oligomycin (Oligo; 5 µM) for 1 hour prior to HNE treatment for 4 hours. TrxR activity was analyzed as described above. ***P < .001. ns, not significant; TNB, 5-thio-2-nitrobenzoic acid. Shabbir A. Ansari et al. Blood Adv 2017;1:2399-2413 © 2017 by The American Society of Hematology

Pharmacological inhibition of TrxR enhances cell surface TF activity in THP-1 cells. Pharmacological inhibition of TrxR enhances cell surface TF activity in THP-1 cells. (A) THP-1 cells were treated with curcumin (25 µM) for varying time periods, and TrxR activity was measured. (B-C) THP-1 cells were treated with curcumin (25 µM) for varying time periods. Cell surface TF (B) and prothrombinase (C) activity was analyzed. (D) THP-1 cells were treated with curcumin (25 µM) for 2 hours and then fixed with 4% paraformaldehyde. After fixation, cells were washed and stained with AF488-annexin V and analyzed using confocal fluorescence microscopy, and the intensity of fluorescence was quantified. Images were obtained at 63× magnification (oil immersion). (E) THP-1 cells were treated with curcumin (25 µM) for 2 hours. After removing curcumin and washing cells once, cells were incubated with annexin V (400 nM) for 30 minutes to block cell surface PS before measuring TF activity. (F) THP-1 cells were treated with specific inhibitors of TrxR (auranofin or PMX-464) at varying concentrations for 2 hours, and cell surface TF activity was measured. (G) THP-1 cells were treated with 10 µM auranofin or 250 µM PMX-464 for 2 hours. Thereafter, a control vehicle or annexin V (400 nM) was added to cells for 30 minutes before measuring cell surface TF activity. THP-1 cells were treated with 25 µM curcumin (H), 10 µM auranofin (I), or 3 different doses of PMX-464 (50, 100, or 250 µM) (J) for a time period between 0 and 60 minutes, and the activation of MKK3/6 and p38 MAPK was analyzed by western blot analysis. (K) THP-1 cells were pretreated with the p38 MAPK activation inhibitor SB203580 (20 µM) for 1 hour, followed by PMX-464 (250 µM) or auranofin (10 µM) for 2 hours, and then cell surface TF activity was measured. *P < .05; **P < .01; ***P < .001; ns, not significant (compared with the values obtained in their respective controls or as indicated in the figures). DAPI, 4′-6-diamidino-2-phenylindole. Shabbir A. Ansari et al. Blood Adv 2017;1:2399-2413 © 2017 by The American Society of Hematology

Inhibition of Trx contributes to p38 MAPK activation– and PS-dependent TF decryption. 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. Shabbir A. Ansari et al. Blood Adv 2017;1:2399-2413 © 2017 by The American Society of Hematology

Inhibition of ASK-1 partly attenuates HNE-induced TF activation. 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. Shabbir A. Ansari et al. Blood Adv 2017;1:2399-2413 © 2017 by The American Society of Hematology

Effect of thiol blocker PAO and PDI inhibitors on HNE-induced PS exposure and TF activation. Effect of thiol blocker PAO and PDI inhibitors on HNE-induced PS exposure and TF activation. (A-B) THP-1 cells were treated with PAO (10 µM) for 15 minutes. After washing cells in serum-free medium, cells were treated with HNE (20 µM) for 4 hours, and cell surface TF activity (A) or prothrombinase (B) activity was measured. (C) THP-1 cells were treated with PAO and HNE as described for panels A-B, and fixed cells were stained with 4′-6-diamidino-2-phenylindole (DAPI) and AF488-annexin V. (D) THP-1 cells were treated with HNE (20 µM) for 4 hours; after washing with buffer A, cells were chilled on ice in buffer A containing Ca2+ for 5 minutes. Cells were then incubated with NBD-PS (2 µM) for 10 minutes on ice and washed twice with ice-cold buffer A. Cells were resuspended in buffer A containing Ca2+ prewarmed to 37°C, and fluorescence was read for a time course of 0 to 60 minutes with or without sodium dithionite to determine % NBD-PS internalized (see “Materials and methods”). (E) THP-1 cells, intact or permeabilized, were labeled with anti-PDI antibody and subjected to flow cytometry. Green, intact cells stained with the anti-PDI antibody; red, permeabilized cells stained with anti-PDI antibody; gray, intact cells stained with control immunoglobulin G. (F) Nonpermeabilized THP-1 cells were labeled with either anti-PDI antibody (red) or anti-TF antibody (green) and subjected to confocal microscopy. (G) Recombinant PDI was treated with various PDI inhibitors or anti-PDI inhibitory antibody for 1 hour at the concentrations indicated in the figure. PDI activity was measured using a fluorescence-based insulin reduction assay. (H) THP-1 cells were pretreated with PDI inhibitors or the inhibitory antibody for 1 hour, followed by HNE (20 µM) for 4 hours. Cell surface TF activity was measured in an FX activation assay. (I) THP-1 cells were treated with either HNE (20 µM) for 4 hours or HgCl2 (100 µM) for 5 minutes. After treatment, cells were washed and labeled with MPB (100 µM) for 30 minutes, and excess, unbound MPB was removed and quenched with reduced glutathione (200 µM) for 30 minutes. Cells were then lysed, and TF was pulled down using anti-TF antibodies. The sample was then run on sodium dodecyl sulfate-polyacrylamide gel electrophoresis and probed for MPB and TF using streptavidin and anti-TF antibodies, respectively. Quantification of thiol-labeled TF protein band by densitometry (right). The band intensity measured in control vehicle-treated cells was taken as 1. *P < .05; **P < .01; ***P < .001; ns, not significant (compared with the values obtained in their respective controls or as indicated in the figures). Images in panels C,F were obtained at 63× magnification (oil immersion). IB, immunoblot; IgG, immunoglobulin G. Shabbir A. Ansari et al. Blood Adv 2017;1:2399-2413 © 2017 by The American Society of Hematology

Schematic representation of the proposed signaling mechanisms involved in HNE-induced TF decryption. Schematic representation of the proposed signaling mechanisms involved in HNE-induced TF decryption. Oxidative stress causes lipid peroxidation and produces reactive aldehydes, such as HNE. HNE induces ROS generation in mitochondria, and ROS increase PS levels in the outer leaflet. Increased PS levels at the outer leaflet contribute to TF decryption. At present, the mechanism by which ROS increase PS levels is unknown. HNE, in addition to generating ROS, also inactivates the TrxR/Trx system by inhibiting its activity or forming adducts. Inhibition of Trx leads to dissociation of the Trx–ASK-1 complex, leading to ASK-1 activation. ASK-1 activation promotes the activation of MKK3/MKK6 and the upstream activation of p38 MAPK. p38 MAPK activation leads to inhibition of flippase activity and a resultant increase in PS levels at the outer leaflet, which contributes to TF activation. HNE-induced TF decryption can be inhibited using specific inhibitors that target different steps in the HNE-induced signaling pathway. Shabbir A. Ansari et al. Blood Adv 2017;1:2399-2413 © 2017 by The American Society of Hematology