Combination of antithrombin and recombinant thrombomodulin modulates neutrophil cell-death and decreases circulating DAMPs levels in endotoxemic rats.

The activation of coagulation is recognized as a universal event in severe sepsis. Both antithrombin and thrombomodulin play pivotal roles as suppressors of coagulation. Since the levels of both anticoagulants decrease significantly, we hypothesized that a combination therapy would be beneficial. A sepsis model was established using the intravenous infusion of lipopolysaccharide (LPS). Either 125 IU/kg of antithrombin, 0.25mg/kg of recombinant thrombomodulin, or a combination of both agents was injected immediately after LPS infusion (n=7 each), while only a physiological saline was injected in the control group (n=7). Blood samples were obtained at eight hours after LPS infusion, and organ damage markers and the plasma levels of damage-associated molecular patterns (DAMPs), such as histone H3 and cell-free DNA (cf-DNA), were measured. In another series, the leukocytes harvested from normal rats were cultured in plasma obtained from each group (n=7). Eight hours later, the leukocytes were stained with green fluorescent protein, Annexin V and 7-AAD, and the proportion of alive+apoptic/necrotic cells was calculated. Organ damage markers such as ALT and BUN were maintained best in the combination group (P<0.05). The circulating levels of histone H3 and cf-DNA were both significantly lower in the combination therapy group (P<0.01, 0.05, respectively). The proportion of alive+apoptic/necrotic cells was significantly higher in the combination therapy group (P<0.05). The coadministration of antithrombin and recombinant thrombomodulin can modulate cell death and decrease the circulating levels of histone H3 and cf-DNA, leading to protection against organ damage in a rat model of sepsis.