APC glycosylation at Asn-329 specifically restricts PAR1 proteolysis and signaling activity. 293T cells transfected with EPCR and either (A) PAR1-AP, (B) PAR1^R41Q-AP, (C) PAR1^R46Q-AP were treated with APC (black circles), APC^PNG (red squares) or APC^N329Q (green triangles) (all 6.25-50 nM) for 3 hours, before AP activity was measured using an AP substrate. An extended period of incubation with the AP substrate was required to observe any AP activity in cell supernatants from APC-treated EPCR/PAR1^R46Q-AP–transfected 293T cells. Murine macrophages (RAW264.7) were incubated with APC (black bars) or APC^PNG (red bars) (5-20 nM) for 3 hours before stimulation with LPS (50 ng/mL) for 18 hours. (D) TNFα and (E) IL-6 secretion were measured by ELISA. Similarly, APC (black bars) and APC^N329Q (yellow bars; 10-20 nM) were incubated with RAW264.7 macrophages for 3 hours before LPS (50 ng/mL) incubation for 18 hours. The resultant supernatants were assessed for the presence of (F) TNFα and (G) IL-6 as before. 293T-transfected cells with (H) PAR1-AP alone (black), EPCR/PAR1-AP (red), or α_Mβ₂/PAR1-AP (green) were treated with thrombin (5 nM), wild-type APC, or APC^N329Q (both 50 nM) for 3 hours before AP activity was assessed. All experiments were performed at least in triplicate and results are presented as the mean ± standard error of the mean; *P < .05, **P < .01 by Student t test.