44.05 HMGB1 Released by Platelets Leads to Microvascular Thrombosis Following Trauma and Hemorrhage

M. D. Neal¹, J. Markel¹, B. S. Zuckerbraun¹, J. L. Sperry¹, P. Loughran¹, T. R. Billiar^{1  1}University Of Pittsburgh,Department Of Surgery,Pittsburgh, PA, USA

Introduction:  Platelet dysfunction has been identified as a major contributor to the pathogenesis of trauma and hemorrhage.  Recently, high-mobility group protein 1 (HMGB1), an endogenous danger signal and mediator of sterile inflammation has been shown to be released from platelets.  However, the response of platelets to HMGB1 and the specific function of platelet derived HMGB1 in sterile injury remain unexplored.   

Methods:  HMGB1 in human and murine platelets was identified using ELISA, flow cytometry, and immunostaining.  Mice were treated with recombinant HMGB1 (rHMGB1) to study the exogenous effect of HMGB1 on platelets and platelet function was measured by aggregometry and thromboelastography (TEG).  Ferric chloride induced thrombosis was used to study the specific role of HMGB1 in thrombosis.  Using cre-loxp technology, we generated mice specifically lacking HMGB1 on platelets (HMGB1^Pf4-cre).  HMGB1^Pf4-cre and wild-type (WT) mice were subjected to a model of trauma and hemorrhagic shock consisting of laparotomy, liver crush injury, and hemorrhage followed by resuscitation (THS-R) with tissue harvest for analysis of injury.  Aggregometry was performed on isolated platelets after trauma.  

Results:  Administration of rHMGB1 to mice led to increased platelet aggregation, microvascular thrombosis, and hypercoagulability as measured by TEG.  THS-R resulted in similar platelet hyper-aggregation and sequestration of platelets into lung and liver in WT mice.  Platelets released HMGB1 following activation and sterile injury.  Strikingly, selective deletion of HMGB1 from platelets (HMGB1^Pf4-cre) resulted in platelets that were resistant to hyperaggregation and sequestration with reduced adherence to microvascular endothelium.  HMGB1^Pf4-cre mice had markedly reduced liver injury compared to WT as measured by serum AST (850 ± 33.2 IU/L vs 505 ± 61.7 IU/L, p=0.02).   Additionally, HMGB1^Pf4-cre mice were protected from lung and liver injury as measured by histologic scoring, with markedly reduced microvascular thrombosis vs WT (22.5 ±2.2 vs 5.1 ±1.4, p<0.01).  Analysis of thrombi from WT and HMGB1^Pf4-cre mice revealed that platelets are the major source of HMGB1 in clot.

Conclusion:  Platelet derived HMGB1 is an essential mediator of platelet aggregation following trauma and hemorrhage.  Deletion of HMGB1 from platelets nearly eliminated microvascular thrombosis and decreased organ injury following THS-R.  Modulating HMGB1 release from platelets may represent a novel therapeutic target for preventing organ injury after trauma.