Antithrombin inhibits lipopolysaccharide-induced tissue factor and interleukin-6 production by mononuclear cells, human umbilical vein endothelial cells, and whole blood

Objective: To investigate the effects of antithrombin on lipopolysaccharide (LPS)-induced tissue factor and interleukin-6 (IL-6) production in three different in vitro cellular systems: whole blood, human umbilical vein endothelial cells, and mononuclear cells. Design and Setting: Laboratory in vitro study of the effects of antithrombin on procoagulant activity and cytokine release by LPS-stimulated endothelial and peripheral blood cells. Subjects: In vitro whole blood, isolated human umbilical vein endothelial cell, and mononuclear cell cultures. Interventions: Addition of antithrombin to LPS-treated whole blood, human umbilical vein endothelial cells, and mononuclear cells. Measurement and Main Results: Citrated whole blood, isolated human umbilical vein endothelial cells, or mononuclear cells were stimulated with LPS for 4-6 hrs in the presence or absence of antithrombin. Tissue factor activity was estimated by a tissue factor-dependent clotting or chromogenic assay and IL-6 was measured by specific ELISA. Antithrombin was found to inhibit tissue factor and IL-6 production in all three systems in a dose-dependent manner (0-40 IU/mL). Flow-through fractions of immunoadsorbed antithrombin concentrate were found to be ineffective. Five different batches of the same antithrombin concentrate were tested and the inhibitory activity was found to be consistent throughout all batches, Up to 40 muM of recombinant hirudin, a specific thrombin inhibitor, did not inhibit the production of tissue factor or IL-6 in either of the three cell systems, suggesting that the observed inhibition by antithrombin was not due solely to its ability to inhibit thrombin. Conclusions: Apart from the inhibition of thrombin and other activated clotting factors, antithrombin may also down-regulate the cellular expression of proinflammatory cytokines, Consequently, antithrombin concentrates may have value in the treatment of sepsis-induced disseminated intravascular coagulation.