RELATIONSHIP BETWEEN ENDOTHELIAL TISSUE FACTOR AND THROMBOGENESIS UNDER BLOOD-FLOW CONDITIONS

We have evaluated the relationship between the level of tissue factor (TF) expression by stimulated endothelial cells and thrombus formation under blood flow conditions. Cultures of human umbilical venous endot helial cells (HUVECs) were treated in order to express different level s of TF activity. They were stimulated for 4 h with either I) lipopoly saccharides (LPS, 10 mu g/ml), II) recombinant interleukin 1 beta (IL1 beta, 50 UI/ml) or III) simultaneously with LPS and IL1 beta (LPS + I L1 beta). TF activity was low on confluent HUVECs or on the correspond ing extracellular-matrix (ECM prepared by exposure of HUVECs to 0.1 N NH4OH). In contrast, it was high when HUVECs were stimulated with LPS or IL1 beta, and significantly higher (p <0.05) with LPS + IL1 beta. T he TF activity associated with the stimulated ECM was 2-fold higher (p <0.05) than that expressed on the luminal surface of the stimulated H UVECs, irrespective of the agonist or combination of agonists used. Th ese surfaces were exposed to non-anticoagulated human blood at a venou s (50 s(-1)) and an arterial (650 s(-1)) wall shear rate in parallel-p late perfusion chambers for 5 min. Thrombus formation was morphologica lly quantified by measuring the deposition of platelets and fibrin. Fi brin deposition was also immunologically quantified. Fibrin deposition was related to the level of TF expression. Non-stimulated HUVECs and corresponding ECMs were not thrombogenic. The luminal surface of HUVEC s stimulated with LPS or IL1 beta alone expressed low levels of TF act ivity and was a poor inducer of platelet deposition and fibrin deposit ion (<15%) at 50 s(-1). In contrast, fibrin deposition increased to 80 % when the cells were stimulated with LPS and IL1 beta simultaneously. This fibrin deposition was comparable to that found on the correspond ing ECM, despite a two-fold lower TF activity. However, at 650 s(-1), platelet and fibrin deposition on HUVECs stimulated with LPS + IL1 bet a were significantly lower than that observed on the corresponding ECM . In all circumstances, the thrombogenicity was TF-dependent, since fi brin deposition was totally blocked by anti-TF antibodies. Thus, it ap pears that the level of TF activity expressed on endothelial cells gov erns thrombus formation. However, the impact of TF expression on throm bus formation is also affected by the blood flow.