Binding of protein C and PC-Gla/meizothrombin to soluble EPCR and the cleavage of PAR-1 exodomain by the proteases. (A) An ELISA-based assay using the monoclonal antibody HPC4 (1 μg/mL) was used to measure the affinity of either protein C (○) or PC-Gla/meizothrombin (■) for interaction with soluble EPCR (0.5 μmol/L). (B) EA.hy926 cells were transiently transfected with ALP-PAR-1 reporter plasmid and incubated with increasing concentrations of thrombin (○), meizothrombin (□), PC-Gla/meizothrombin (■) and APC (▵) for 1 hour. The activity of soluble alkaline phosphatase (ALP) was measured as described under “PAR-1 cleavage assay.” The activity of 10 nmol/L thrombin is presented as 100%. OD, optical density. Error bars represent SD.