Up-regulation of tissue factor mRNA in human umbilical vein endothelial cells by calmodulin inhibitor W7

Tissue factor (TF) is an integral membrane glycoprotein that serves as a cofactor for the blood coagulation factor VIIa. The induction of TF synthesis and activity on the surface of endothelial cell membrane is initiated by lipopolysaccharide (LPS), phorbol 12-myristate 13-O-acetate (PMA), and inflammatory factors such as interleukin-1 beta (IL-1 beta) and tumor necrosis factor-alpha (TNF alpha). Treatment of cells with 1 micrograms/ml LPS induced an 8.7-fold increase in total TF activity compared with nontreated cells. Co-incubation with 1 micrograms/ml LPS and 30 microM W7, a potent calmodulin inhibitor, resulted an additional 2.0 fold increase in total TF activity. A similar tendency was observed after treatment with either TNF alpha plus W7, or IL-1 beta plus W7. The effect of W7 appeared to be synergistic since incubation with 30 microM W7 alone increased TF activity levels to only 1.5-fold that of control cells. Northern blot analysis showed that W7 and LPS-treated endothelial cells expressed about three times higher levels of TF mRNA compared to LPS-treated cells. Treatment with W7 and LPS resulted in a slow but large calcium influx into endothelial cells. This result suggest that the contribution of W7 may be dependent mainly on calcium influx by unknown mechanisms rather than direct inhibition of calmodulin, because calcium ionophore treatment also showed a synergistic effect on TF mRNA and activity expression.