Antithrombin inhibits lipopolysaccharide-induced tumor necrosis factor-α production by monocytes in vitro through inhibition of Egr-1 expression

Abstract

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-α (TNF-α) plays a critical role in the development of organ failure and intravascular coagulation in sepsis. Aim: This study aimed to elucidate a molecular mechanism by which AT inhibits TNF-α production Methods: Human peripheral monocyte was stimulated by lipopolysaccharide (LPS) and TNF-α 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-α 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-κB 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-α production by LPS-stimulated monocytes, it significantly inhibited TNF-α 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-α by inhibiting the increase in Egr-1 expression in monocytes via interaction with heparin-like substances expressed on the cell surface. © 2008 International Society on Thrombosis and Haemostasis.