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.
