FVIIa activation of p44/42 MAPK in endothelial cells. (A-B) Confluent monolayers of HUVECs were treated with FVIIa (10nM) or control vehicle for varying times at 37°C. Cell lysates were subjected to immunoblot analysis with phospho-specific or total p44/42 MAPK antibodies. Panel A shows an immunoblot from a representative experiment and panel B shows quantified data of FVIIa-induced p44/42 MAPK activation in HUVECs (n = 4-7). (C) HUVECs were treated with a control vehicle, nonblocking (NB) EPCR mAb, or blocking (B) EPCR mAb for 30 minutes at room temperature before being treated with FVIIa (10nM) for 20 minutes. (D) HUVECs, transfected with control siRNA or EPCR siRNA, were treated with a control vehicle or FVIIa (10nM) for 20 minutes. Cell lysates were analyzed for p44/42 MAPK activation by immunoblot analysis. (E) HUVECs were transfected with control siRNA, PAR1 siRNA, or PAR2 siRNA, and the transfected cells were treated with control vehicle, FVIIa (10nM), PAR1 agonist peptide (50μM), or PAR2 agonist peptide (50μM) for 20 minutes. Cell lysates were analyzed for p44/42 MAPK activation by immunoblot analysis and the band intensities were quantified by densitometry (n = 2-5).