Factor VIIa mobilizes tissue factor from the Golgi. (A) Fibroblasts were exposed to FVIIa (10 nM) for different time intervals (0 to 120 minutes). Cells were fixed, permeabilized, and immunostained with rabbit polyclonal anti–human TF and monoclonal anti–human golgin-97 antibodies, followed by Rhodamine Red–labeled anti–rabbit IgG and Oregon Green–labeled anti–mouse IgG as secondary reporter antibodies. (B) Factor VIIa's protease activity and the binding to cell-surface tissue factor are essential for the trafficking of tissue factor from the Golgi. WI-38 cells were exposed to FVIIa (10 nM) or FFR-FVIIa (10 nM) alone for 2 hours at 37°C or first incubated with 20-fold molar excess of FFR-FVIIa or anti-TF IgG (10 μg/mL) for 30 minutes before FVIIa (10 nM) was added to the cells. The cells were stained with anti-TF and anti–golgin-97 antibodies and analyzed as described in panel A. Left panel images represent TF staining, middle panel images represent the Golgi staining, and right panel images represent the overlay of TF and the Golgi staining (colocalization). Insets in panel A show a magnified view of TF localization in the Golgi.