THE INTERACTION BETWEEN ANTITHROMBIN AND ENDOTHELIAL HEPARAN SULFATE MITIGATES PULMONARY THROMBOINFLAMMATION AFTER TRAUMA AND HEMORRHAGIC SHOCK

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 pretreated with vehicle but not surfen. Surfen abrogated the anti-inflammatory effects of FFP, indicated by notable increases in circulating levels of multiple proinflammatory mediators compared to rats pretreated with vehicle. Finally, we observed significant increases in pulmonary fibrin and MPO staining in rats pretreated 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 antithrombotic and anti-inflammatory activity. Blocking the interaction between AT and the endothelium attenuates the antithromboinflammatory and organ protective properties of FFP, suggesting that AT-endothelial anticoagulant function and anti-inflammatory signaling is important for organ protection during T/HS.