Effect of poly I:C on TF/TM mRNA, protein, and activity level in EC as a function of time and concentration. (A) EC were stimulated for the indicated times with 10 μg/mL poly I:C and mRNA levels for TF, TM, and β-actin were determined by RT-PCR. The size of the 3 PCR products was 427, 208, and 170 bp, respectively, and 34, 30, and 29 cycles were used. (B) EC were stimulated with various concentrations of poly I:C, and mRNA levels for TF (after 2 hours), TM (5 hours), and β-actin (2 and 5 hours) were determined by RT-PCR. (C) EC were stimulated with various concentrations of poly I:C or 25 ng/mL TNF-α for 16 hours, and the protein levels for TF, TM, and actin were determined by Western blot analysis using the respective antibodies. (D) Poly I:C (10 μg/mL) or 25 ng/mL TNF-α were used to stimulate EC for the indicated times, and the activities of TF and TM were determined using chromogenic substrate assays for FXa generation and APC generation, respectively. Various concentrations of poly I:C or poly dI:dC were used to stimulate EC for 16 hours, and the activities of TF and TM were determined. Results are presented as relative enzyme activity; mean plus SEM of triplicate wells. (E) Poly I:C (10 μg/mL) or 25 ng/mL TNF-α were used to stimulate EC for the indicated times, and the clotting time was determined. Various concentrations of poly I:C or poly dI:dC were used to stimulate EC for 16 hours, and then plasma clotting time was measured (in seconds). Results are expressed as mean plus SEM of triplicate wells.