Platelet-Mediated NET Formation Exacerbates Ischemic Stroke Brain Injury

• Published in: Blood 2021-11, Vol.138 (Supplement 1), p.437-437
• Main Authors: Denorme, Frederik, Portier, Irina, Cody, Mark, Grandhi, Ramesh, Neal, Matthew D, Majersik, Jennifer J, Yost, Christian, Campbell, Robert A.
• Format: Article
• Language: English
• ISSN: 0006-4971; 1528-0020
• DOI: 10.1182/blood-2021-151423

▪ Background: Ischemic stroke provokes a strong inflammatory response which has been shown to exacerbate ischemic stroke brain injury in mice and is associated with worse outcomes in ischemic stroke patients. Classic anti-inflammatory strategies have been unsuccessful in clinical trials for ischemic stroke, implying other mechanisms contribute to injurious inflammation in ischemic stroke. Immunothrombosis is the interplay between coagulation, platelet activation and the innate immune system leading to thrombosis. A critical component of immunothrombosis is the formation of neutrophil extracellular traps (NETs). In this study, we investigated mechanistic regulators of NET formation in stroke and if they contribute to ischemic stroke outcomes. Methods: Markers of immunothrombosis were assessed in plasma from ischemic stroke patients and healthy matched donors. Flow cytometry was used to characterize platelet and neutrophil function. For murine studies, we used male and female C57Bl/6 mice that were subjected to transient middle cerebral artery occlusion. Stroke outcomes were assessed 24 hours or 7 days after stroke using neurological and motor function testing as well as brain infarct size analysis. Results: Ischemic stroke patients had significantly increased plasma biomarkers of immunothrombosis including D-Dimers (coagulation), platelet factor 4 (platelet activation) and calprotectin (neutrophil activation). Moreover, specific markers of NET formation including citrullinated histone H3 (H3cit) and MPO-DNA complexes were significantly elevated in ischemic stroke patients. Interestingly, H3cit and MPO-DNA complexes positively correlated with worse stroke outcomes at discharge while they did not correlate with stroke severity at admission. Next, we observed increased plasma and platelet surface expressed high mobility group box 1 (HMGB1) in ischemic stroke patients compared to matched healthy controls. NETs were found in platelet-rich areas in ischemic stroke thrombi, and HMGB1 colocalized with platelets in ischemic stroke thrombi. Blocking platelet-derived HMGB1 in vitro prevented platelet-induced NET formation. Mechanistically, depleting platelets in mice reduced plasma HMGB1 levels as well as NET formation and improved outcomes after ischemic stroke. In contrast, depleting neutrophils did not affect plasma HMGB1 levels, but reduced plasma NETs and improved stroke outcomes. Treatment of mice with a competitive HMGB1 inhibitor (BoxA) reduced NET formation a