The effect of calcium ionophore A23187 on tissue factor activity and mRNA in endothelial cells

Tissue factor (TF) is an integral membrane glycoprotein that serves as a cofactor for blood coagulation factor VIIa. The induction of TF on the surface of endothelial cells is initiated by various kinds of stimuli including lipopolysaccharide (LPS), interleukin-1 beta (IL-1 beta), and tumor necrosis factor alpha (TNF alpha). The mechanisms leading to induction of TF are largely unknown and the present study explores the influence of calcium influx on TF induction in LPS-stimulated human umbilical vein endothelial cells. TF cofactor activity was measured on cell surfaces and in lysates by a two-stage chromogenic assay after the cells were incubated under a variety of conditions. TF activity of cell surfaces increased 3.3-fold above control values after LPS stimulation (100 ng/ml, 4 h). Addition of 20 microM A23187, a calcium ionophore, to the LPS-stimulated cells just before the TF assay, resulted in an additional 8.8-fold enhancement. TF activity of lysed cells increased 10.5-fold above control values after LPS stimulation (100 ng/ml). Incubation with lower concentrations of A23187 and 100 ng/ml-1 micrograms/ml LPS for 4 h resulted in activity twice that of LPS stimulation alone. The TF mRNA signal of LPS plus 1 microM A23187-treated cells was also increased in addition to LPS treated cells.