APC enhances TF procoagulant activity on endothelial cells. EAhy926 cells were treated in all experiments with TNFα (5nM) for 5 hours to induce TF expression, unless otherwise noted. (A) Different concentrations of APC or active site–blocked APC (APC blocked with dansyl-glutamyl-glycyl-arginyl-chloromethylketone [DEGR-APC]) were added for the final 3 hours of TNFα incubation. Procoagulant TF activity was analyzed as described in “Xa generation assay.” Activity of generated factor Xa relative to only TNFα-stimulated controls is shown. (B) APC (20nM) was added for the indicated period at the end of the TNFα incubation. (C) Cells were incubated for 5 hours in the presence or absence of TNFα, and different concentrations of thrombin and APC (10nM) were added for the final 3 hours as indicated. (D) APC (20nM) and/or the PI3K inhibitor wortmannin (100nM) was added for the final 3 hours of TNFα incubation. Means ± SEM are shown; n = 5 (A, B), 6 (C), or 3 (D). *P < .05; **P < .005, compared with corresponding sample without APC.