Inhibition of ASMase reduces TNFα+IL-1β–induced TF procoagulant activity in endothelial cells. HUVECs were treated with saline (Con), desipramine (Des; 10 µM), or imipramine (Imi; 10 µM) for 1 hour and then stimulated with TNF-α+IL-1β (10 ng/mL each) for 6 hours. Cell surface TF activity (A) and prothrombinase activity (B) were measured. (C) Fixed HUVECs were stained for expression of TF and ASMase by incubating cells with mouse anti-human TF mAb (9C3; 10 µg/mL) or rabbit anti-human ASMase antibody (2 µg/mL) overnight, followed by secondary antibodies conjugated with AF488 or AF546 fluorophore, and subjected to immunofluorescence confocal microscopy. Arrowheads point to expression of ASMase in the plasma membrane. The fluorescence intensity of TF and ASMase staining in the plasma membrane was quantified (right panel). (D) Representative image of staining of SM by lysenin binding (left panel). Quantification of fluorescence intensity of lysenin staining (right panel). Original magnification ×63 for panels C and D. *P < .05; **P < .01, ***P < .001.