Cleavage of PAR3 by APC and thrombin on endothelial cells. (A) Schematic representation of the PAR3 cleavage site sequence with the epitopes for the cleavage sensitive PAR3 antibodies H103 and Mab19b as indicated. (B) Peptide mapping of the cleavage site-sensitive epitopes of anti-PAR3 antibodies H103 and Mab19b. Peptides represent the N-terminal (NT-P3K) or C-terminal fragment (P3K) of PAR3 after cleavage at Lys38 or the N-terminal (NT-P3R) or C-terminal fragment (P3R) of PAR3 after cleavage at Arg41 or the entire cleavage site region of the PAR3 N-terminal tail (P3). (C) On Cell Western of anti-PAR3 antibody H103 on SEAP-PAR3 (open symbols) and SEAP-PAR3/wt-EPCR (closed symbols) HEK293 cells after incubation with APC (●, ○) or thrombin (IIa) (■, □) for 2 hours. (D) EA.hy926 endothelial cells after incubation with control buffer, APC (100 nM), or thrombin (0.25 nM) for 3 hours. The minus and plus refer to the presence or absence of thrombin and APC, respectively. Cell bound anti-PAR3 antibodies were detected using the On Cell Western assays with PAR3 cleavage site sensitive H103 and Mab19b antibodies and the cleavage-insensitive antibody M14 directed against the PAR3 extracellular loop 3. (B) Representative experiment in triplicates. (C-D) Data points represent the mean ± SEM (n ≥ 3). ns, not significant.