Lipopolysaccharide induced procoagulant activity and cytokine production: influence of antithrombin

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Sepsis-induced disseminated intravascular coagulation is usually caused by endotoxin evoked production of cellular procoagulant tissue factor (TF) and pro-inflammatory cytokines, which further perpetuate the generation of TF. Large doses of antithrombin (AT) reduce mortality and morbidity in septic patients and there is increasing evidence to suggest that AT has anti-inflammatory properties in addition to its anticoagulant properties. In the present study, we investigated the effect of AT in three in vitro cellular systems: citrated whole blood, human umbilical vein endothelial cells (HUVECs) and mononuclear cells (MNCs). The cells were stimulated with lipopolysaccharide (LPS) for 4–6 h in the presence and absence of AT. Procoagulant tissue factor (TF) activity was estimated by a TF-dependent clotting or chromogenic assay and interleukin-6 (IL-6) was measured by ELISA. In all three systems, 5-40 IU/ml AT was found to inhibit TF and IL-6 production in a dose-dependent manner. This inhibitory effect was not attributable to excipients or co-purified components of AT. Experiments with chemically modified AT and a low heparin binding fraction of AT indicated that binding to heparin and/or cell surface glycosaminoglycans is important for the inhibitory activity. Up to 40 μM of a specific thrombin inhibitor, r-hirudin, did not inhibit the production of TF or IL-6 in either of three cellular systems, suggesting that inhibition of thrombin might not be the main mechanism by which AT prevents the production of TF and IL-6. The results of this study have shown that, apart from the inhibition of thrombin and other activated coagulation factors, AT may also downregulate the cellular expression of pro-inflammatory responses and therefore may have an added value in the treatment of sepsis-induced DIC.