Role of thrombin and coagulation factors VII and X in the augmentation of poly(I:C)-induced responses EC. (A) HUVECs and EA.hy926 cells were stimulated with poly(I:C) (12.5 μg/mL) for 6 hours and the functionally active cell-surface TF was determined by FXa generation assays (n = 4-8). (B) EA.hy926 cells were stimulated with poly(I:C) (12.5 μg/mL) and candidate physiological PAR2 agonists, such as fXa (100 nM), fVIIa (FVIIa-100 nM), fVIIa plus fX (fVIIa, 500 pM; fX, 150 nM; enabling ternary TF-VIIa-Xa complex formation), or thrombin (5 nM) for 3 hours. Relative TF and CXCL8 mRNA abundance was determined by RT-PCR (n = 5-12 per condition). (C) EA.hy926 cells were pretreated with poly(I:C) (12.5 μg/mL) and thrombin (0.5-10 nM) for 6 hours, and the functionally active cell-surface TF was determined by an fXa-generation assay (n = 4). (D) Baseline abundance of TLR3, PAR1, and PAR2 mRNA relative to GAPDH mRNA in EA.hy926 cells and HUVECs. Target gene/GAPDH ratios in EA.hy926 cells were arbitrarily set to 1. (E) Changes in the abundance of PAR1, PAR2, and TLR3 mRNA relative to GAPDH mRNA in response to thrombin and/or poly(I:C) (n = 6) in EA.hy926 and HUVECs stimulated for 3 hours with poly(I:C) (12.5 μg/mL) and/or thrombin (5 nM). Baseline levels were arbitrarily set to 1. (F) TF and CXCL8 mRNA abundance in HUVECs and EA.hy926 cells treated for 3 hours with poly(I:C) (12.5 μg/mL) and/or thrombin (5 nM; n = 6/condition). All data represent the mean ± standard deviation of the indicated number of replicates and were generated in at least 2 biological replicates. The statistical significance of the differences between 2 groups was analyzed by Student t test. A comparison of more than 2 groups was conducted by ANOVA followed by a multiple-comparison test. *P < .05; **P < .01; ***P < .001; ****P < .0001. ns, nonsignificant.