Proteolysis of PAR3 cleavage site mutants by APC and thrombin on cells. Cleavage of SEAP-PAR3 mutants by APC and thrombin (IIa) in the absence (A,C,E,G) and presence (B,D,F,H) of wt-EPCR. (A-B) Lys32Gln-SEAP-PAR3. (C-D) Lys38Gln-SEAP-PAR3. (E-F) Arg41Gln-SEAP-PAR3. (G-H) Lys38Gln/Arg41Gln-SEAP-PAR3. SEAP released in the media was determined 1 hour after the addition of APC or thrombin and corrected for background and spontaneous SEAP release in the absence of protease (<2%). The activity of released SEAP was expressed as a percentage of total available SEAP activity on the cells determined in the same volume. No SEAP activity was detected on HEK-293 or wt-EPCR HEK-293 cells in the absence of SEAP-PAR3. Data points represent the mean ± SEM (n ≥ 3). Single amino acid abbreviations denote K, Lys; R, Arg; and Q, Gln.