Increased TF procoagulant activity associated with HgCl₂ treatment in HL60 cells depended on PS exposure at the cell surface. (A) HL60 cells (1 × 10⁶/mL) were stimulated with PMA (1μM) in serum-free medium for 6 hours at 37°C to induce TF expression. PMA-stimulated HL60 cells (2 × 10⁵ cells in 200 μL) were treated with HgCl₂ (100μM) for 30 seconds and 3 minutes at 37°C in the presence or absence of annexin V (400nM). At the end of HgCl₂ treatment, FVIIa (10nM) and FX (175nM) were added to the cells, and the amount of FXa generated at the end of the 2-minute activation period was measured in a chromogenic assay (n = 3; mean ± SEM). *P < .05; §P < .001. (B) HL60 cells were stimulated with PMA and treated with HgCl₂ as described above, and TF activity was measured in a clotting assay. TF activity is shown in arbitrary units. (C) HL60 cells stimulated with PMA and treated with HgCl₂ as described in panel A were stained for cell-surface expression of PS (Alexa Fluor 488 [AF488]–annexin V staining) and TF. Control and treated cells were washed with buffer A and then incubated with AF488–annexin V in the binding buffer at room temperature for 15 minutes. The cells were then washed, fixed, and immunostained with anti-TF antibodies followed by Rhodamine Red–conjugated secondary antibodies. The immunofluorescence was analyzed by confocal microscopy (Axio Observer Z1 microscope, Plan-APOCHROMAT 63×/1.4 NA oil objective lens, Carl Zeiss LSM 510 Meta confocal system). (D) PMA-stimulated HL60 cells were incubated with 400nM annexin V for 30 minutes at 37°C and then the unbound annexin V was removed, and the cells were washed once with buffer B before they were treated with either control buffer or HgCl₂ (100μM) for 3 minutes. In one set, annexin V (400nM) was added to cells again at the time of the HgCl₂ treatment. TF activity was measured in FX activation assay as described for panel A.