Thrombin augments poly(I:C)-induced TF and CXCL8 mRNA induction by activating PAR1/2 heterodimers. (A) Cell surface Thbd expression level in pool-sorted and single-cell clone Thbd-deficient EA.hy926 cells by flow cytometry. Shaded, nonimmune isotype control; black, parent (Thbd-expressing) cells; and red, Thbd-deficient cells. The numbers in the plots indicate the percentage of estimated maximum contamination, with cells expressing Thbd from 1 residual allele. (B) TM-deficient, pool-sorted cells and single-cell clones were stimulated with poly(I:C) (12.5 μg/mL) and thrombin (5 nM) for 3 hours. TF and CXCL8 mRNA abundance relative to GAPDH was measured by RT-PCR (n = 3-6; in duplicate). (C) EA.hy926 cells were pretreated with PAR1 (WEDE15, 10 μg/mL; ATAP2 10 μg/mL) and/or PAR2 (SAM-11, 10 μg/mL) cleavage-blocking antibodies, followed by treatment with poly(I:C) (12.5 μg/mL) and/or thrombin (5 nM) for 3 hours. TF and CXCL8 mRNA abundance relative to GAPDH was measured by RT-PCR (n = 3-12; in duplicate). (D) EA.hy926 cells were pretreated with PAR-specific inhibitors (PAR1, vorapaxar, 1 μM; PAR2, GB83, 25 μM) followed by stimulation with poly(I:C) (12.5 μg/mL) and PAR1/2 agonist peptides. TF and CXCL8 mRNA abundance relative to GAPDH mRNA was measured by RT-PCR (n = 3-9; in duplicate). Data are expressed as the mean ± standard deviation. The statistical significance of differences between groups was analyzed by ANOVA followed by the multiple-comparison test. *P < .05; **P < .01; ***P < .001; ****P < .0001. ns, nonsignificant.