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 platelet function, whereas direct effects on plasmatic coagulation are rarely described. In sepsis and inflammation, when synthesis of NO, oxygen radicals and toxic metabolites is crucial, the expression of tissue factor (TF) on monocytes stimulated by lipopolysaccharides (LPS) induces intravascular coagulation. This study was performed to examine the influence of NO and the NO-dependent metabolite peroxynitrite on LPS-induced TF expression and activity in human monocytes.


METHODS
Experimental study.


METHODS
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 cytometry, and its activity by a clotting assay. TF-mRNA was measured by reverse transcriptase polymerase chain reaction (RT-PCR-ELISA).


RESULTS
Whereas NOC18, a pure NO donor, had no effect, SIN1, releasing both NO and superoxide (O2-), reduced TF expression and activity in a dose- and time-dependent manner; superoxide dismutase (SOD) reversed the SIN1-mediated effect. Adding the O2(-)-deliberating system hypoxanthin/xanthin oxidase (which had no significant effect per se) to NOC18, or using the NO and O2- reaction product peroxynitrite resulted in a reduction of TF expression. RT-PCR-ELISA indicated upregulation of TF-mRNA by SIN1 with a peak at 500 microM; higher doses had less effect.


CONCLUSIONS
These data demonstrate an influence of NO on LPS-induced TF expression in monocytes by prior formation of peroxynitrite; furthermore, the balance between NO and O2- seems to play a crucial role.
Gerlach
M
M
Department of Anesthesiology and Critical Care Medicine, Charité-Virchow-Clinic, Humboldt University, Berlin, Germany.
Keh
D
D
Bezold
G
G
Spielmann
S
S
Kürer
I
I
Peter
R U
RU
Falke
K J
KJ
Gerlach
H
H
D004305:Dose-Response Relationship, Drug; D004353:Drug Evaluation, Preclinical; D005434:Flow Cytometry; D016166:Free Radical Scavengers / Q000378:metabolism / Q000494:pharmacology*; D005786:Gene Expression Regulation / Q000187:drug effects*; D006801:Humans; D008070:Lipopolysaccharides; D009000:Monocytes / Q000187:drug effects* / Q000276:immunology / Q000378:metabolism; D009566:Nitrates / Q000494:pharmacology*; D009569:Nitric Oxide / Q000378:metabolism / Q000494:pharmacology*; D016877:Oxidants / Q000494:pharmacology*; D013925:Thromboplastin / Q000032:analysis / Q000187:drug effects* / Q000235:genetics* / Q000378:metabolism
D016166:Free Radical Scavengers; D008070:Lipopolysaccharides; D009566:Nitrates; D016877:Oxidants; C039642:peroxynitric acid; D009569:Nitric Oxide; D013925:Thromboplastin