Effect of APC on thrombin signaling pathway in epithelial cells. (a) Activated protein C (APC) reverses thrombin-induced extracellular-regulated kinase 1/2 (ERK) and cytosolic Phospholipase A2 (cPLA2) activation in A549 cells. Immunoblotting detection of activated (phosphorylated) ERK and cPLA2. A549 cells were incubated with serum-free medium, thrombin for 15 minutes, APC for 60 minutes or APC for 60 minutes followed by thrombin for 15 minutes. APC alone induced minimal ERK and cPLA2 activation. In contrast, thrombin addition was associated with increased activation of ERK and cPLA2, both of which were abrogated in the presence of APC (Representative of five experiments). (b) ERK phosphorylates cPLA2 downstream of the thrombin receptor. ERK activation was blocked by MEK inhibitor UO126 in A549 cells challenged with thrombin. Inhibition of ERK activation led to abrogated cPLA2 phosphorylation (representative of three experiments).