Effect of PDI inhibition on ATP- and b-SMase-induced TF decryption, TF⁺MVs release, and SM hydrolysis. (A) Cell lysates of MDMs transfected with mock, scRNA, or siRNA specific for PDI were probed for PDI expression levels by western blot analysis. (B-C) MDMs transfected with mock, scRNA, or PDI siRNA were treated with a control vehicle or BzATP (200 µM) for 15 minutes. TF activity on the cell surface (B) or MVs (C) was measured in FX activation assay. (D-E) MDMs transfected with mock, scRNA, or PDI siRNA were treated with a control vehicle or b-SMase (1 U/mL) for 1 hour. Following the treatment, cell surface TF activity (D) or TF activity in MVs (E) was measured in FX activation assay. (F-G) MDMs were first treated with a control vehicle or PDI inhibitor PACMA31 (10 µM) for 1 hour, and then treated with control vehicle, BzATP (200 µM, 15 minutes), or b-SMase (1 U/mL, 1 hour). Cell surface TF activity (F) or MVs TF activity (G) was measured in FX activation assay. (H) MDMs transfected with mock, scRNA, or PDI siRNA were metabolically labeled with [methyl-¹⁴C]-choline chloride (0.2 μCi/mL) for 48 hours. Labeled MDMs were treated with a control vehicle, BzATP (200 µM, for 15 minutes), or b-SMase (1 U/mL, for 1 hour). Cell supernatants were collected and centrifuged to remove cell debris and MVs before they were counted for the radioactivity to determine the release of [¹⁴C]-phosphorylcholine as an index for SM hydrolysis.