Antithrombin inhibits lipopolysaccharide-induced tumor necrosis factor-alpha production by monocytes in vitro through inhibition of Egr-1 expression.
BACKGROUND
Antithrombin (AT) improves the outcome of septic patients with intravascular coagulation. However, the mechanisms underlying the therapeutic benefits of AT are not fully understood. Tumor necrosis factor-alpha (TNF-alpha) plays a critical role in the development of organ failure and intravascular coagulation in sepsis.


OBJECTIVE
This study aimed to elucidate a molecular mechanism by which AT inhibits TNF-alpha production.


METHODS
Human peripheral monocyte was stimulated by lipopolysaccharide (LPS) and TNF-alpha concentration in media was measured. Levels of phosphorylation of extracellular signal-regulated protein kinases (ERK) 1/2 and early growth response factor-1 (Egr-1) were estimated by western blotting or by electrophoretic mobility shift assay.


RESULTS
Antithrombin (3 U mL(-1)) inhibited TNF-alpha production by monocytes stimulated with LPS. Conversely, chemically modified AT that lacks affinity for heparin did not. AT inhibited the phosphorylation of ERK 1/2 and decreased the expression of Egr-1 in LPS-stimulated monocytes. However, it did not affect the activation of either nuclear factor-kappaB or activator protein-1. Pretreatment with KT5720, a protein kinase A inhibitor, reversed the inhibitory effect of AT on the LPS-induced phosphorylation of ERK1/2. Although 2 U mL(-1) AT slightly inhibited TNF-alpha production by LPS-stimulated monocytes, it significantly inhibited TNF-alpha production in the presence of a low concentration of beraprost, a stable derivative of prostacyclin.


CONCLUSIONS
These observations suggest that AT might inhibit LPS-induced production of TNF-alpha by inhibiting the increase in Egr-1 expression in monocytes via interaction with heparin-like substances expressed on the cell surface.
Komura
H
H
Department of Anaesthesiology, Nagoya City University Graduate School of Medical Sciences, Nagoya, Japan.
Uchiba
M
M
Mizuochi
Y
Y
Arai
M
M
Harada
N
N
Katsuya
H
H
Okajima
K
K
D000991:Antithrombins / Q000378:metabolism*; D002227:Carbazoles / Q000494:pharmacology; D002470:Cell Survival; D051766:Early Growth Response Protein 1 / Q000037:antagonists & inhibitors* / Q000096:biosynthesis*; D011464:Epoprostenol / Q000031:analogs & derivatives / Q000494:pharmacology; D048049:Extracellular Signal-Regulated MAP Kinases / Q000378:metabolism; D006493:Heparin / Q000737:chemistry; D006801:Humans; D007211:Indoles / Q000494:pharmacology; D008070:Lipopolysaccharides / Q000378:metabolism*; D008954:Models, Biological; D009000:Monocytes / Q000378:metabolism*; D010766:Phosphorylation; D010975:Platelet Aggregation Inhibitors / Q000494:pharmacology; D011758:Pyrroles / Q000494:pharmacology; D014409:Tumor Necrosis Factor-alpha / Q000096:biosynthesis*
D000991:Antithrombins; D002227:Carbazoles; C497325:EGR1 protein, human; D051766:Early Growth Response Protein 1; D007211:Indoles; D008070:Lipopolysaccharides; D010975:Platelet Aggregation Inhibitors; D011758:Pyrroles; D014409:Tumor Necrosis Factor-alpha; C048081:beraprost; C057416:KT 5720; D006493:Heparin; D011464:Epoprostenol; D048049:Extracellular Signal-Regulated MAP Kinases