Internalization of FVIIa bound to EPCR. (A) HUVECs were exposed to AF488-FVIIa (50 nM) for 15 minutes, 30 minutes, or 1 hour at 37°C, and then fixed and processed for EPCR immunostaining. (Left) EPCR staining. (Middle) AF488-FVIIa fluorescence. (Right) The overlay image of left and middle panels. Arrows represent the accumulation of AF488-FVIIa with EPCR in the REC. (B) CHO-EPCR cells were exposed to AF488-FVIIa (50 nM) for 1 hour at 4°C. At the end of 1 hour, the supernatant was removed, and the cells were washed quickly with calcium-containing buffer to remove the unbound ligands and then transferred to 37°C to allow internalization of the surface-bound ligands. Various times at 37°C, the cells were fixed, permeabilized, and immunostained with nonblocking EPCR mAb. (Left) AF488-FVIIa. (Middle) EPCR staining. (Right) The merged image of AF488-FVIIa and EPCR. Because the REC could locate at apical, basal, or lateral position to the nucleus, it may not be visible in all sections of the cell. (C) Ligand-induced EPCR accumulation in the REC. CHO-EPCR cells were first incubated with FVIIa (50 nM) at 4°C for 1 hour and then transferred to 37°C. At various times, the cells were fixed, permeabilized, and stained with EPCR mAbs. The pixel density of the fluorescence of EPCR staining in the REC at different time periods was measured using Image suite software (PerkinElmer Life and Analytical Sciences; n = 15 cells or more).