Both exogenous and endogenously generated APC support additional PAR1 cleavage in the presence of thrombin. (A) Nonfixed or PFA-fixed cells were incubated for 3 hours with different concentrations of thrombin in the absence or presence of APC as indicated, followed by analysis of ATAP2 binding. (B) Cells were incubated with 20 nM thrombin for 10 minutes followed by quenching of the protease with 50 nM hirudin. Recovery of ATAP2 binding upon incubation at 37°C over 3 hours is plotted. Where indicated, the cells were PFA-fixed or pretreated with brefeldin A (1 μM, 10 minutes) before the addition of thrombin. A representative experiment (of 3) is shown. (C) Brefeldin A–treated cells were incubated for 3 hours as indicated followed by analysis of ATAP2 binding. (D) Nonfixed cells were incubated with the indicated concentrations of thrombin in the absence or presence of 80 nM PC. After 3 hours the APC concentration in the conditioned medium was determined by chromogenic assay and ATAP2 binding was analyzed. Means plus or minus SEM are shown (n = 9 in panels A,C,D. *P < .05, **P < .005).