J. Goswami1, P. Varley1, D. Van Der Windt1, H. Waring1, P. Loughran3, D. Geller1, H. Huang1,2, A. Tsung1 1University Of Pittsburgh Medical Center,Surgery,Pittsburgh, PA, USA 2Huazhong University Of Science And Technology,Surgery,Wuhan, Hubei, China 3Center For Biologic Imaging,Cell Biology,Pittsburgh, PA, USA
Introduction: We have recently shown that liver ischemia/reperfusion (I/R), an unavoidable consequence of liver resection, releases neutrophil extracellular traps (NETs). However, the mechanisms by which liver I/R leads to remote organ injury are as yet unknown. We now hypothesize that NET formation after liver I/R stimulates a pro-coagulant state with resultant distal organ injury.
Methods: Wild-type and wild-type treated with DNase (to inhibit NETs) were subjected to 70% one-hour hepatic ischemia with 1-24 hours reperfusion. Immunofluorescence and hematoxylin & eosin staining of liver, lung, and kidney were performed to assess for platelet microthrombi. Platelet aggregometry was assessed with Chronolog-Lumi aggregometer. Flow cytometry for platelet activation (CD62P, P-selectin) and circulating platelet neutrophil aggregates (CD41, Ly6G) was performed.
Results: In wild-type mice, platelet-rich microthrombi and neutrophil infiltration were identified in liver, lung, and kidney microvasculature within 6 hours after reperfusion. This correlated with elevated sALT and serum cystatin C (a marker of kidney injury) levels at 6 hours. Platelet aggregation increased significantly within 1 hour after reperfusion and returned to baseline by 24 hours. Platelet surface CD62P and circulating platelet-neutrophil aggregates were increased, most prominently at 6 hours, both indicative of increased platelet activation. In DNase-treated mice, platelet aggregates were not seen in remote organ microcirculation, and platelet function studies did not reveal significant changes. DNase treatment was protective against both local and distal organ injury as measured by sALT and cystatin C levels, suggesting that NET formation is necessary for induction of systemic hypercoagulability and organ injury.
Conclusion: NET formation after liver I/R induces a systemic pro-coagulant state with resultant remote organ injury.