Peroxynitrite reduces endotoxin-induced tissue factor expression in human peripheral blood monocytes

Objectives

Tissue factor (TF) is considered to be the primary physiologic activator of the blood coagulation system. TF initiates the extrinsic pathway via factor VII and the intrinsic pathway via factor IX. Expression of TF by blood monocytes is an established trigger for intravascular coagulation in several pathologic conditions such as cancer, sepsis and inflammation, since activated macrophages, endothelial cells and type II epithelial cells synthesize nitric oxide (NO), superoxide anion (O₂) and other free radicals under such conditions, we examined their influence on endotoxin-induced TF expression in human peripheral blood monocytes.

Methods

Human peripheral blood monocytes were isolated by Ficoll-gradient centrifugation and incubated with LPS (0–10 μg/ml) for 4 h at 37°C (incubator). SIN1 (0–2000 μl) — an NO and O₂ releasing compound —, NOC18 (0–2000 μM) - a pure NO donor —, superoxide dismutase (SOD) (0–500 IE/ml), hypoxanthine (1 mM) with xanthine oxidase (0–1 U/ml) — an O^–₂ liberating system — and peroxynitrite (0–2000 μM) — a reaction product of NO with O^–₂ were added under different conditions. TF was assayed by flow cytometry using a monoclonal antibody against TF, by a one stage clotting assay and by PCR.

Results

SIN1, peroxynitrite and the combination of NOC18, hypoxanthine and xanthine oxidase reduce the LPS-induced TF expression in a dose and time dependent manner, NOC18 or hyroxanthine/xanthine oxidase alone had no effect, SOD reversed the SIN1 mediated TF production having a weak decreasing effect on its own. Low doses of SIN1 resulted in a slight increase in TF expression.

Conclusion

These data implicate that free radicals influence the TF expression of peripheral human blood monocytes and, that the balance of NO and O^-₂ plays a crucial role for that regulation.