ATP stimulation enhances cell surface TF activity of macrophages and TF⁺MVs release. (A) MDMs were treated with benzoyl-ATP (BzATP) (200 µM) for varying time periods, supernatants were removed, and cells were washed once. Cell surface TF activity was measured by adding FVIIa (10 nM) and FX (175 nM) and determining the rate of FXa generation in a chromogenic assay. (B) MDMs were treated with control vehicle or BzATP (200 µM, 15 minutes). MDMs were labeled with control IgG (“C”) or rabbit anti-human TF IgG and subjected to FACS analysis. As a positive control, MDMs were stimulated with LPS for 4 hours. (C) MDMs were treated with BzATP as described in panel A, and the cell supernatants were collected. MVs were isolated, and TF activity in MVs was measured in FX activation as described in panel A. (D) MDMs were treated with BzATP as described in panel A, and prothrombinase activity on the cell surface was measured by adding FVa (10 nM), FXa (1 nM), and prothrombin (1.4 µM). (E) MDMs treated with BzATP (200 µM) for varying times were stained with AF488–annexin V and subjected to fluorescence microscopy. In control, MDMs were incubated with a control vehicle for 60 minutes, and this panel was labeled as “0” ATP. (F-G) MDMs were pretreated with annexin V (400 nM) for 30 minutes and then stimulated with a control vehicle or BzATP (200 µM) for 10 minutes. Cell surface TF (F) and prothrombinase (G) activities were measured as described previously. *P < .05; **P < .01; ***P < .001 compared with the values obtained in MDMs treated with a control vehicle or as specified by bars in the figures.