Recycling of internalized FVIIa and EPCR, and the fate of internalized ligands. (A) CHO-EPCR cells were incubated with AF488-FVIIa (50 nM) for 1 hour at 37°C to allow the internalization of FVIIa. Thereafter, the cells were washed with the buffer containing 5 mM ethylenediaminetetraacetic acid to remove the cell surface bound FVIIa, and thereafter the cells were maintained in calcium-containing buffer (buffer B) at 37°C. The fate of internalized FVIIa was monitored by fixing the cells at various times. The fixed cells were permeabilized, stained with EPCR mAbs, and analyzed by confocal microscopy. (B-C) CHO-EPCR cells were exposed to ¹²⁵I-FVIIa (triangle) or ¹²⁵I-APC (square), 10 nM, for 2 hours at 37°C, and then the surface associated ¹²⁵I-labeled ligand was eluted by treating the cells with 0.1 M glycine, pH 2.3, for 3 minutes at the room temperature. The cells were washed with buffer B and allowed to stay at 37°C. At various time intervals, the overlying supernatant medium was removed and precipitated with 10% ice-cold TCA. The radioactivity present in both TCA-precipitable (B) and TCA-soluble fractions (C) was counted. (D) CHO-EPCR cells were incubated with a control buffer or the buffer containing unlabeled FVII, FVIIa, protein C, or APC (100 nM). After 2 hours at 37°C, the unbound ligand was removed, and the monolayers were washed with 0.1 M glycine, pH 2.3, to remove the bound ligand. After washing the monolayers with buffer B, the cells were chilled on ice and incubated with ¹²⁵I-EPCR mAbs (10 nM) at 4°C for 1 hour. After 1 hour, the unbound radioactivity was removed, the cells were washed, and the total cell lysate was counted for the radioactivity to determine the amount of EPCR mAbs bound to the cells.