Coagulation proteases increase uPA and PAI-1 expression in the culture supernatant of 4T1 and 67NR breast cancer cell lines. (A-B) Real-time PCR analysis of TF, PAR-1, PAR-2, uPA, and PAI-1 mRNA expression in 4T1 cells (A) and 67NR cells (B). Cells were grown to confluence and starved overnight. Results are shown as mean ± SEM of at least 3 independent experiments. (C-E) Serum starved confluent cell monolayers were incubated for 24 hours with the following coagulation factors: mFVIIa (10nM), FX (130nM), mFVIIa (10nM) and FX (130nM), FXa (125nM), or thrombin (FIIa; 20nM). Levels of uPA in treated 4T1 cells (C) and 67NR cells (D) were determined by ELISA. The amount of PAI-1 released from the treated 4T1 (E) and 67NR (F) cell lines was measured by a PAI-1 ELISA. Results are shown as mean ± SEM of at least 5 independent experiments. *P ≤ .05 and **P ≤ .001 (control versus protease treated).