EPCR-dependent PAR1 and PAR3 cleavage by FXa. FXa-mediated cleavage of PAR1 and PAR3 was analyzed by the proteolytic release of SEAP from SEAP-PAR1 and SEAP-PAR3 fusion protein expressed in HEK-293 cells in the absence or presence of wild-type (wt)-EPCR coexpression. (A,C) Dose response of PAR1 (A) and PAR3 (C) cleavage by FXa in the presence (□) or absence (▪) of EPCR. PAR1 and PAR3 cleavage was expressed as a percentage of the total available SEAP-PAR1 or SEAP-PAR3 on the cells. (B,D) Specificity controls of PAR1 and PAR3 cleavage by FXa on SEAP-PAR1/wt-EPCR (B) or SEAP-PAR3/wt-EPCR (D) cells. Inhibitors used were hirudin (20 U/mL) directed against thrombin and rivaroxaban (5 µM), blocking (5051) and nonblocking (5050) antibodies directed against FXa, and blocking antibody (C1) against APC (all 20 µg/mL). FXa, γ-carboxyglutamic acid (Gla)-domainless βFXa, or βFXa were used at 25 nM. (B, D) PAR1 and PAR3 cleavage was expressed relative to the cleavage in the absence of inhibitors or antibody. Data points represent mean ± standard error of the mean (SEM) of at least 3 independent experiments with 2 to 3 replicates per experiment.