THE EFFECT OF CALCIUM IONOPHORE A23187 ON TISSUE FACTOR ACTIVITY AND MESSENGER-RNA 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-1beta (IL-1beta), and tumor necrosis factor alpha (TNFalpha). The mechanisms leading to induction of TF are largely un-known 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 muM 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 mug/ml LPS for 4 h resulted in activity twice that of LPS stimulation alone. The TF mRNA signal of LPS plus 1 muM A23187-treated cells was also increased in addition to LPS treated cells.