Synergistic effects of tumor necrosis factor-alpha and thrombin in increasing endothelial permeability

Because activation of the coagulation cascade and the generation of thrombi n coexist with sepsis and the release of tumor necrosis factor (TNF)-alpha, we determined the effects of TNF-alpha on the mechanism of thrombin-induce d increase in endothelial permeability. We assessed Ca2+ signaling in human umbilical vein endothelial cells. In human umbilical vein endothelial cell s exposed to TNF-alpha for 2 h, thrombin produced a rise in the intracellul ar Ca2+ concentration ([Ca2+](i)) lasting up to 10 min. In contrast, thromb in alone produced a rise in [Ca2+](i) lasting for 3 min, whereas TNF-alpha alone had no effect on [Ca2+](i). Thrombin-induced inositol 1,4,5-trisphosp hate generation was not different between control and TNF-alpha -exposed ce lls. In the absence of extracellular Ca2+, thrombin produced similar increa ses in [Ca2+](i) in both control and TNF-alpha -exposed cells. In TNF-alpha -exposed cells, the thrombin-induced Ca2+ influx after intracellular Ca2store depletion was significantly greater and prolonged compared with contr ol cells. Increased Ca2+ entry was associated with an approximately fourfol d increase in Src activity and was sensitive to the Src kinase inhibitor PP 1. After TNF-alpha exposure, thrombin caused increased tyrosine phosphoryla tion of junctional proteins and actin stress fiber formation as well as aug mented endothelial permeability. These results suggest that TNT-alpha stimu lation of endothelial cells results in amplification of the thrombin-induce d Ca2+ influx by an Src-dependent mechanism, thereby promoting loss of endo thelial barrier function.