Effect of antithrombin III (AT) on lipopolysaccharide (LPS)-induced production of tissue factor and interleukin-6 (IL-6) by human umbilical vein endothelial cells (HUVECs), mononuclear cells (MNCs) and whole blood

During disseminated intravascular coagulation (DIC), the extrinsic tissue factor (TF)-dependent pathway has been implicated as the dominant route to thrombin generation and the production of IL-6 has been shown to correlate positively with the severity of sepsis-induced DIC. Pharmacological doses of AT have been shown to reduce mortality and morbidity in patients with DIC and there is increasing evidence to suggest that AT possesses anti-inflammatory properties in addition to its anticoagulant properties. In the present study, we have investigated the effect of AT on LPS-induced TF and IL-6 production in three different in vitro systems. Citrated whole blood. HUVECs and MNCs were stimulated with LPS for 4–6 h in the presence or absence of AT. TF activity was estimated by a TF-dependent clotting or chromogenic assay and IL-6 was measured by ELISA. Our results show a dose-dependent inhibition of TF and IL-6 production by AT, EC50 — ~36 and 20-35 iu/ml respectively in MNCs and HUVECs, but ~14 and <10 iu/ml in whole blood. Immuopurification experiments confirmed that the inhibitory activity was attributable to the AT and not to components that may have co-purified with the clinical product. In addition, up to 40 μM of hirudin, specific thrombin inhibitor, did not inhibit the production of TF and IL-6 in either of the three cell systems, suggesting that the observed inhibition by AT was not due solely to the inhibition of thrombin. Our investigation has shown that, apart from the inhibition of thrombin and other activated clotting factors, AT may also down-regulate the cellular expression of proinflammatory cytokines. Consequently, AT concentrates may have value in the treatment of sepsis-induced DIC.