Cooperation between VEGF and TNF-alpha is necessary for exposure of activetissue factor on the surface of human endothelial cells

This study was undertaken to characterize tissue factor (TF) induction, loc alization, and functional activity in cultured human umbilical vein endothe lial cells (HUVECs) exposed to recombinant vascular endothelial growth fact or (rVEGF) and recombinant tumor necrosis factor-alpha (rTNF-alpha). rVEGF (1 nmol/L) and rTNF-alpha (500 U/mL) synergistically increased TF mRNA, pro tein, and total activity, as measured in cell lysates. To examine surface T F expression, living cells were treated with antibody to TF and examined mi croscopically. Almost no staining was seen in control cells or cells treate d with a single agent. In contrast, cells treated with both agonists showed intense membrane staining with surface patches, appearing as buds by confo cal microscopy. To determine surface TF activity, studies were performed us ing a parallel-plate flow chamber, which allows detection of factor Xa gene ration on living cells. rVEGF and rTNF-alpha induced little surface TF acti vity (0.032+/-0.008 and 0.014+/-0.008 fmol/cm(2), respectively). In combina tion, they significantly increased TF expression on the cell surface (0.429 +/-0.094 fmol/cm(2), P<0.05). These data indicate that the synergistic effe ct of rVEGF and rTNF-alpha is necessary to generate functional TF on the su rface of endothelial cells. The requirement for multiple agonists to expose active TF may serve to protect endothelial cells from acting as a procoagu lant surface, even under conditions of cell perturbation.