APC-cleaved PAR1 is retained on the cell surface even in the presence of thrombin. (A) Nonfixed cells were incubated for 3 hours with the indicated agonists and cell surface–expressed PAR1 was quantified by analysis of ATAP2 and WEDE15 binding. (B) After incubation with the indicated agonists for 3 hours, surface proteins were biotinylated and isolated with streptavidin agarose. PAR1 was detected by Western blotting using anti-PAR1 WEDE15. A representative experiment is shown in the top panel. Optical density of immunoreactive bands was measured in 3 independent experiments and means plus or minus SEM are shown in the bottom panel. Coincubation with APC led to detection of more surface PAR1 at all thrombin concentrations, a finding that was borderline significant at lower thrombin concentrations but significant at 360 pM thrombin. (C) As indicated, cells were induced with TNFα for 2 hours and thrombin and/or APC added for an additional 3 hours followed by quantification of ATAP2 and WEDE15 binding. Means plus or minus SEM are shown in panels A,C (n = 15 in panel A and 7 in panel C, *P < .05, **P < .005, comparing results without and with APC in panel A).