Platelet-Mediated NET Formation Exacerbates Ischemic Stroke Brain Injury
Authors: Denorme Frederik , Portier Irina , Cody Mark , Grandhi Ramesh , Neal Matthew D , Majersik Jennifer J , Yost Christian , Campbell Robert A. 
Published: November 2021

Abstract

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 and improved stroke outcomes. Combined, these results imply a causative role for platelet-derived HMGB1 in mediating detrimental NET formation after ischemic stroke.
Finally, as NETs appeared injurious in ischemic stroke, we investigated the therapeutic potential of a recently discovered neonatal NET inhibitory factor (nNIF). nNIF is a cleavage fragment of alpha-1-antitrypsin and specifically blocks NET formation in human and murine neutrophils without affecting other critical neutrophil functions such as chemotaxis or phagocytosis. Mice treated with nNIF had reduced brain and plasma NETs after stroke while cerebral neutrophil recruitment remained unaffected. The reduction in NET formation after stroke was associated with reduced neuronal apoptosis and smaller brain infarcts. Furthermore, nNIF treated mice had improved neurological and motor function and enhanced 7-day survival after ischemic stroke.
Conclusions: These results support a pathological role for NETs in acute ischemic stroke and warrant further investigation into nNIF as a therapeutic to improve stroke outcomes.

Disclosures
Neal: Instrumentation Laboratories: Research Funding; Janssen Pharmaceuticals: Membership on an entity's Board of Directors or advisory committees, Research Funding; Haima Therapeutics: Membership on an entity's Board of Directors or advisory committees; Haemonetics: Honoraria, Research Funding.