The interaction between antithrombin and endothelial heparan sulfate mitigates pulmonary thromboinflammation after trauma and hemorrhagic shock.

Abstract

Introduction: Trauma and hemorrhagic shock (T/HS) are associated with multiple organ injury. Antithrombin (AT) has anti-inflammatory and organ protective activity through its interaction with endothelial heparan sulfate containing a 3-O-sulfate modification. Our objective was to examine the effects of T/HS on 3-O-sulfated (3-OS) heparan sulfate expression and determine whether AT-heparan sulfate interactions are necessary for its anti-inflammatory properties.

Methods: Male Sprague Dawley rats underwent laparotomy, gut distension and fixed-pressure hemorrhagic shock (HS) and resuscitation. Liquid chromatography-coupled mass spectrometry analyses were performed to measure pulmonary and plasma heparan sulfate di/tetrasaccharides. Pulmonary mRNA levels were assessed by nCounter panel. Rats were treated with vehicle or surfen (1 mg/kg), a small molecule heparan sulfate antagonist, to block the interaction between AT and endothelial cells prior to T/HS and resuscitated with fresh frozen plasma (FFP), lactated Ringer's (LR), or AT-supplemented LR. Lung injury was assessed histologically for injury and fibrin deposition and immunostained for myeloperoxidase (MPO). Plasma was assessed for circulating inflammatory biomarkers.

Results: T/HS significantly reduced pulmonary expression of 6-O and 3-O sulfated heparan sulfate, which was associated with reduced pulmonary 6-O- and 3-O-sulfotransferase mRNA levels. Surfen increased fibrin deposition and inflammatory cell infiltration into pulmonary tissue in T/HS rats resuscitated with FFP but had no effect in LR resuscitated rats. Although T/HS and LR resuscitation worsened histologic lung injury compared to sham, regardless of surfen treatment, lung injury was notably improved in FFP resuscitated rodents pre-treated with vehicle but not surfen. Surfen abrogated the anti-inflammatory effects of FFP, indicated by notable increases in circulating levels of multiple pro-inflammatory mediators compared to rats pre-treated with vehicle. Finally, we observed significant increases in pulmonary fibrin and MPO staining in rats pre-treated with surfen followed by resuscitation with LR supplemented with AT compared to vehicle, which was associated with notable increases in lung injury scores.

Conclusions: T/HS causes pronounced reductions in pulmonary expression of 3-OS heparan sulfate, which is essential to AT's anti-thrombotic and anti-inflammatory activity. Blocking the interaction between AT and the endothelium attenuates the anti-thromboinflammatory and organ protective properties of FFP, suggesting that AT-endothelial anticoagulant function and anti-inflammatory signaling is important for organ protection during T/HS.