Nitric oxide inhibits tissue factor synthesis, expression and activity in human monocytes by prior formation of peroxynitrite

Objective: Nitric oxide (NO) has antithrombotic properties by regulating pl atelet function, whereas direct effects on plasmatic coagulation are rarely described. In sepsis and inflammation, When synthesis of NO, oxygen radica ls and toxic metabolites is crucial, the expression of tissue factor (TF) o n monocytes stimulated by lipopolysaccharides (LPS) induces intravascular c oagulation. This study was performed to examine the influence of NO and the NO-dependent metabolite peroxynitrite on LPS-induced TF expression and act ivity in human monocytes. Design: Experimental study. Setting: Laboratory for cell biology. Methods. Human peripheral blood mononuclear cells were isolated from buffy coats by gradient centrifugation. The NO-releasing compounds SIN1 and NOC18 were used under different conditions. TF antigen was assayed by flow cytom etry, and its activity by a clotting assay. TF-mRNA was measured by reverse transcriptase polymerase chain reaction (RT-PCR-ELISA). Measurements and results: Whereas NOC18, a pure NO donor, had no effect, SI N1, releasing both NO and superoxide (O-2(-)) reduced TF expression and act ivity in a dose- and time-dependent manner; superoxide dismutase (SOD) reve rsed the SIN1-mediated effect. Adding the O-2(-)-deliberating system hypoxa nthin/xanthin oxidase (which had no significant effect per se) to NOC18, or using the NO and O-2(-) reaction product peroxynitrite resulted in a reduc tion of TF expression. RT-PCR-ELISA indicated upregulation of TF-mRNA by SI N1 with a peak at 500 mu M; higher doses had less effect. Conclusion: These data demonstrate an influence of NO on LPS-induced TF exp ression in monocytes by prior formation of peroxynitrite; furthermore, the balance between NO and O-2(-) seems to play a crucial role.