EPCR-mediated FVIIa transcytosis. (A) CHO-EPCR cells cultured in transwells were treated with control vehicle or EPCR blocking mAb (10 μg/mL) for 30 minutes. Thereafter, ¹²⁵I-FVIIa (10 nM) and BSA coupled to Evans Blue (0.67 mg/mL) were added to the upper chamber, and the cells were allowed to stay in the CO₂ incubator. At various times, a small aliquot was removed from the bottom chamber and counted for the radioactivity and measured absorbance at 650 nm to monitor the transfer of FVIIa and BSA, respectively, to the bottom chamber. The data shown in the figure represent EPCR-specific FVIIa transport (n = 3). BSA transfer in CHO-EPCR cells treated with control vehicle or EPCR blocking mAbs is very similar. (B) C57BL/6 mice were injected with saline control (100 μL), AF488-FVII, or AF488-protein C (10 μg/mice in 100 μL saline) via tail vein. One hour after the injection, the mice were exsanguinated, and various tissues were collected into Excel fixative. Bone-joint tissue was sectioned (5-μm thickness) and immunostained with anti-AF488 antibodies to localize the administered FVII and protein C. [] represents endothelial lining; [], adventitia and extravascular tissue.