EFFECTS OF LIPOPOLYSACCHARIDE ON THE EXPRESSION OF FIBRINOLYTIC FACTORS IN AN ESTABLISHED CELL-LINE FROM HUMAN ENDOTHELIAL-CELLS

Human endothelial cells express antithrombotic properties by producing prostacyclin, heparan sulphate and plasminogen activator (PA). Bacter ial extract, such as lipopolysaccharide (LPS), damaged the blood vesse ls and destroyed the balance between the antithrombotic and thrombotic functions of endothelial cells. The fibrinolytic system is involved i n antithrombotic functions. The TKM-33 cell line was established from human endothelial cells. In order to determine whether TKM-33 is a goo d fibrinolytic system endothelial cell expression model, the expressio n of fibrinolytic factors in TKM-33 cells treated with or without LPS was studied. The endothelial cells which had not been treated with LPS produced and secreted a large amount of urokinase-type PA (u-PA), and small amounts of tissue-type PA (t-PA) and PA inhibitor-1 (PAI-1), wh ich were identified immunohistochemically and by electrophoretic enzym ography. Diisopropylfluorophosphate-treated I-125-u-PA bound specifica lly to acid-treated monolayered endothelial cells with a K-d of 2.83 /- 0.61 nM, and B-max of (0.11 +/- 0.01) x 10(6) sites/cell. u-PAR exp ression was detected in endothelial cells by Northern blot analysis. T hus, endothelial cells was shown to express u-PAR which binds u-PA spe cifically. In the binding assay, the stimulation of endothelial cells with 0.1, 1.0 and 10 mu g/ml of LPS altered the K-d values to 6.04 +/- 0.71, 7.03 +/- 1.55 and 7.38 +/- 1.03 nM, respectively. However the B -max values did not change significantly. Although LPS treatment incre ased u-PAR expression in endothelial cells in a dose-dependent manner, the expression of u-PA and t-PA mRNAs was not altered significantly. LPS stimulation (10 mu g/ml) increased the expression of PAI-1 mRNA, s ignificantly. The PA activity recovered from the cell surface fraction was not affected by LPS stimulation, but the PAI-1 activity was incre ased. These findings suggest that the established endothelial cell lin e, TKM-33, possesses the characteristics of endothelial cells and they express u-PAR on their cell surface, which is occupied by intrinsic u -PA secreted from the cells, and that treatment of endothelial cells w ith LPS changes the cell surface characteristics and inhibited the u-P AR expression thus promoting the prothrombotic function concomitantly with increased PAI-1 activity.