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

Abstract

Because activation of the coagulation cascade and the generation of thrombin coexist with sepsis and the release of tumor necrosis factor (TNF)-α, we determined the effects of TNF-α on the mechanism of thrombin-induced increase in endothelial permeability. We assessed Ca 2+ signaling in human umbilical vein endothelial cells. In human umbilical vein endothelial cells exposed to TNF-α for 2 h, thrombin produced a rise in the intracellular Ca 2+ concentration ([Ca 2+ ] i ) lasting up to 10 min. In contrast, thrombin alone produced a rise in [Ca 2+ ] i lasting for 3 min, whereas TNF-α alone had no effect on [Ca 2+ ] i. Thrombin-induced inositol 1,4,5-trisphosphate generation was not different between control and TNF-α-exposed cells. In the absence of extracellular Ca 2+ , thrombin produced similar increases in [Ca 2+ ] i in both control and TNF-α-exposed cells. In TNF-α-exposed cells, the thrombin-induced Ca 2+ influx after intracellular Ca 2+ store depletion was significantly greater and prolonged compared with control cells. Increased Ca 2+ entry was associated with an approximately fourfold increase in Src activity and was sensitive to the Src kinase inhibitor PP1. After TNF-α exposure, thrombin caused increased tyrosine phosphorylation of junctional proteins and actin stress fiber formation as well as augmented endothelial permeability. These results suggest that TNF-α stimulation of endothelial cells results in amplification of the thrombin-induced Ca 2+ influx by an Src-dependent mechanism, thereby promoting loss of endothelial barrier function.