thromboplastin and peroxynitric-acid

Peroxynitrite, a reactive oxidising species resulting from a reaction between nitric oxide and the superoxide anion, modifies proteins by nitration of certain amino acids such as tyrosine. Tissue factor (TF), a transmembrane protein, is expressed on cells under inflammatory conditions and initiates the coagulation cascade. The extracellular domain of TF is rich in tyrosine. Exposure of recombinant TF and cellular TF to peroxynitrite was associated with a reduction in procoagulant activity. This was accompanied by an elevated level of nitrotyrosine residues. Peroxynitrite may have a protective role by attenuation of the thrombotic properties of TF.

Topics: Coagulants; Membrane Proteins; Monocytes; Nitrates; Oxidants; Recombinant Proteins; Thromboplastin; Tyrosine

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.. Experimental study.. Laboratory for cell biology.. 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).. 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.. 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.

Topics: Dose-Response Relationship, Drug; Drug Evaluation, Preclinical; Flow Cytometry; Free Radical Scavengers; Gene Expression Regulation; Humans; Lipopolysaccharides; Monocytes; Nitrates; Nitric Oxide; Oxidants; Thromboplastin