Effect of PAR activation on tissue factor endocytosis. (A) Fibroblasts were surface-labeled with sulfo NHS-SS-biotin at 4°C and then incubated at 37°C for 1 hour with a control buffer or the buffer containing FVIIa (10 nM), thrombin (10 nM), PAR1 agonist peptide (50 μM), and PAR2 agonist peptide (50 μM). The cells were then treated with glutathione (50 mM) to remove the surface biotin, 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 (labeled as −GSH). The top panel shows a representative blot and the bottom panel shows quantified data obtained by measuring the band intensity by densitometry (n = 3). (B) Mock, PAR1 siRNA–, or PAR2 siRNA–transfected cells were labeled with sulfo NHS-SS-biotin at 4°C and treated with a control vehicle or FVIIa (10 nM) for 1 hour at 37°C, and the cells were processed as in panel A. * denotes the values significantly differ (P < .05) from the TF internalized in the absence of FVIIa. The data shown in the figure represent mean ± SEM (n = 3 to 4 experiments).