Factor VIIa–induced internalization and recycling of tissue factor. (A) Fibroblasts were surface-labeled with sulfo NHS-SS-biotin at 4°C and then incubated at 37°C for varying times with a control buffer or the buffer containing FVIIa or FFR-FVIIa (10 nM). The cells were then treated with the reducing agent, lysed, and immunoprecipitated with anti-TF beads. The immunoprecipitated samples were analyzed for the biotin to identify the internalized TF. TF biotin signal detected in cells that were treated with the reducing agent immediately following the biotinylation was taken as 100%. To show the extent of cell-surface TF biotinylation, immunoprecipitates of cells that were not treated with the reducing agent were analyzed for the biotin label. *Values significantly differ from the TF internalized in the absence of FVIIa (P < .02). (B) To measure the recycling of the internalized cell-surface TF, biotin-labeled cells were first incubated with FVIIa at 37°C for 60 minutes to allow TF internalization. Cells were washed with PBS, and the internalized receptors were chased by reincubation at 37°C for various time periods in duplicate samples. After the incubation, only 1 of 2 samples was reduced again. The cells were lysed and subjected to TF immunoprecipitation, followed by immunoblotting. Differences between the chased cells that were treated or not treated with the reducing agent represent the amount of TF recycled back to the cell surface. NS denotes no statistically significant difference. The data shown in the figure represent mean ± SEM (n = 3 to 6 for A, n = 3 for B).