FVIIa or APC binding to EPCR leads to internalization of both the ligand and the receptor. CHO-EPCR cells were incubated with ¹²⁵I-labeled FVIIa (□) or APC (Δ; 10 nM) for various time intervals at 37°C, and the amount of ¹²⁵I-labeled proteins associated with the cell surface (A) and internalized (B) was measured. Data are mean ± SEM (n = 4-5). (C) CHO-EPCR cells were surface-labeled with sulfo-NHS-SS-biotin at 4°C and then incubated at 37°C for various time periods with a control buffer or the buffer containing FVIIa or APC (10 nM). The cells were then treated with the reducing agent to remove biotin label from the cell surface, lysed, and immunoprecipitated with anti-EPCR antibody. The immunoprecipitated samples were subjected to SDS-PAGE followed by immunoblot analysis with antibiotin antibodies to detect the internalized EPCR. Biotinylated EPCR signal was quantified by densitometry. Biotinylated EPCR signal detected in the cells immediately after the biotinylation was taken as 100%. The values shown in the figure represent mean ± SEM (n = 3).