Abstract 688: Endothelial Anticoagulant Heparan Sulfate Mediates Anti-inflammatory Effects of Antithrombin in Sepsis.
Introduction: Anticoagulant heparan sulfate (HSAT+) is a form of heparan sulfate (HS) that, in vitro, catalyzes antithrombin’s (AT) anticoagulant activity. However, Hs3st1−/− mice exhibit large reductions in HSAT+ but show normal hemostasis. Perhaps HSAT+ regulates alternative activities. AT elicits anti- and pro-inflammatory signaling in vitro. The net in vivo effect is anti-inflammatory; AT reduces mortality of septic animals. AT signaling requires cell surface HS but involvement of HSAT+ is unknown. Clinical importance is highlighted by the Kybersept trial of AT in sepsis, where only patients not treated with heparin had enhanced survival.
Hypothesis: Anti-inflammatory effects of AT require endothelial HSAT+.
Methods: AT effects on leukocyte-endothelial (LE) interactions (rolling flux fraction, firm adhesion, adhesion efficiency) were measured by intravital microscopy of cremaster muscle venules. Human AT (0.25 U/g), or vehicle, was given 2 h before i) exteriorization of the cremaster muscle, ii) an intrascrotal injection of tumor necrosis factor-α (TNFα), or iii) an intraperitoneal injection of lipopolysaccharide (LPS). AT effects were first characterized in C57BL/6 mice, then examined in congenic Hs3st1+/+ and Hs3st1−/− mice. Endothelial and leukocyte HSAT+ were evaluated using fluorophore-labeled AT.
Major Results: In C57BL/6 mice, AT reduced select LE interactions induced by cremaster exteriorization and LPS, but enhanced TNFα induced interactions. LPS treated Hs3st1−/−, compared to Hs3st1+/+, mice trended towards enhanced mortality (P = 0.17) and select leukocyte-endothelial interactions were enhanced (P < 0.01). Most importantly, AT treatment produced opposite effects on LPS induced leukocyte adhesion efficiency. Hs3st1+/+ mice exhibited a 7-fold reduction (P < 0.001) but Hs3st1−/− mice had a 3.7-fold elevation (P < 0.02). In Hs3st1−/− mice, HSAT+ levels were normal on leukocytes but reduced to undetectable on cremaster endothelial cells.
Conclusions: AT’s inhibition of LE interactions is highly context dependent and likely involves a balance of AT’s pro- and anti-inflammatory activities. Endothelial HSAT+ is one component that mediates AT’s anti-inflammatory activity.