Abstract 713: S 18886 Inhibits Thromboxane A2 Receptor-dependent Tissue Factor Expression, Procoagulant Activity And NADP(H) Oxidase Activation In Stimulated Endothelial Cells
Background Tissue factor (TF) expression and surface exposure are key events in thrombosis, likely contributing to clinical events in vascular disease. Thromboxane (TX)A2, an unstable metabolite of arachidonic acid released from vaious cell types, is known for its pro-aggregating and vasoconstrictor properties. Cellular effects of TXA2 are effected through the TP (TX-prostaglandin endoperoxide) receptor, also expressed in endothelial cells (EC). The TP receptor antagonist S 18886 (Terutroban) demonstrated antithrombotic and antiatherogenic effects in activated EC. As the underlying molecular mechanisms are largely unexplored, we studied the effects of TP agonism and of antagonism on TF expression and procoagulant activity in human umbilical vein endothelial cells (HUVEC), and signal transduction pathways involved.
Methods and Results HUVEC ± 30 min pretreatment with the TP antagonist S 18886 were stimulated with the TP receptor agonist U 46619 or TNF-α for 6 hours. TF total expression and surface exposure were assessed by enzyme immunoassays, and TF-dependent procoagulant activity by the generation of Factor Xa. HUVEC exposed to U 46619 featured a concentration-dependent increase in TF total expression and surface exposure. These were associated with enhanced procoagulant activity. S 18886 (1 μmol/L) significantly reduced U 46619 (1 μM)-induced TF expression (−20% ± 7%, P<0.05) and procoagulant activity (−32% ± 11%, P<0.05). Interestingly, S 18886 (1 μmol/L) prevented the increase of TF expression after TNF-α (20 ng/mL) stimulation (−25% ± 9%, P<0.05). Both U 46619- and TNF-α-induced TF expression were mediated by the increase of intracellular reactive oxygen species (ROS), and this was inhibited by S 18886 (−44% ± 6% and −24% ± 5% P<0.05, respectively). S 18886 decreased the membrane association of p47-phox component of NADP(H) oxidase, accounting for the reduced production of ROS.
Conclusions Our results show that endothelial TP receptor mediates TF expression, surface exposure and activity stimulated both by TP agonists and by TNF-α. This occurs through NADP(H) oxidase activation and the consequent generation of ROS. These procoagulant and oxidant pathways are inhibited by the TP receptor antagonist S 18886.