Inhibition of A-SMase attenuates ATP-induced TF decryption and TF⁺MVs release in macrophages. (A-B) MDMs were treated with A-SMase inhibitors desipramine (Des) and imipramine (Imi) in SF-RPMI medium for 1 hour at 37°C. After that, MDMs were exposed to 200 μM BzATP in SF-RPMI at 37°C for 15 minutes, and cell surface TF activity (A), and MVs TF activity (B) were measured in FX activation assay using 10 nM FVIIa and 175 nM FX. (C) MDMs were subjected to mock transfection (transfection reagent alone) or transfected with scRNA or siRNA for A-SMase for 48 hours. Cell lysates were probed for A-SMase levels by immunoblot analysis. The same blot was also probed for β-actin as a loading control. (D) MDM cells transfected with a control scRNA or A-SMase siRNA were treated with a control vehicle or BzATP (200 µM) for 15 minutes. The cells were immunostained for A-SMase. (E) MDMs transfected with a mock transfection reagent, scRNA, and A-SMase siRNA were treated with a control vehicle or 200 μM BzATP for 15 minutes and immunostained for A-SMase. The fluorescence intensity of staining of A-SMase at the cell surface was quantified as described in the legend to Figure 4E. MDMs transfected with scRNA or siRNA for A-SMase were treated with a control vehicle or BzATP (200 µM) for 15 minutes, and TF activity at the cell surface (F) and MVs (G) were measured in FX activation assay. Mann-Whitney U test was used to determine the significance between control and experimental groups. ***P < .001. (H) MDMs pretreated with A-SMase inhibitors Des (10 µM) and Imi (5 µM) for 1 hour or transfected with scRNA or siRNA for A-SMase were treated with a control vehicle (−ATP) or BzATP (+ATP) for 15 minutes. The cells were stained with AF488–annexin V to evaluate PS exposure.