Abstract 116: Platelet-Induced Neutrophil Extracellular Traps Drive Ischemic Stroke Brain Injury

Abstract

Ischemic stroke prompts a strong inflammatory response which is associated with exacerbated stroke outcomes. However, classic anti-inflammatory strategies have been unsuccessful in stroke patients implying other unknown mechanisms contribute to injurious inflammation in stroke. Increasing evidence suggests immunothrombosis, a process involving coagulation, neutrophil and platelet activation, and neutrophil extracellular trap (NET) formation, is an important contributor to cardiovascular diseases. However, mechanistic regulators of immunothrombosis and their role in ischemic stroke remain unclear. We examined markers of immunothrombosis in ischemic stroke patients and matched healthy donors. Stroke patients had significantly increased levels of D-Dimers, platelet factor 4, neutrophil calprotectin, citrullinated histone H3 (H3cit) and MPO-DNA complexes, markers of NET formation. In particular, H3cit and MPO-DNA complexes positively correlated with long-term stroke outcomes. Mechanistically, we observed increased plasma and platelet HMGB1 in stroke patients, which significantly correlated with plasma NETs, indicating a role for platelet HMGB1 in NET formation. To directly examine the role of platelet HMGB1, we employed a transient ischemic stroke mouse model. Depleting platelets significantly reduced plasma HMGB1 levels, inhibited NET formation and improved stroke outcomes. Correspondingly, administrating a HMGB1 inhibitor reduced NET formation and improved stroke outcomes, implying a causative role for platelet HMGB1 in mediating NET formation after stroke. As NETs appeared detrimental in ischemic stroke, we investigated the therapeutic potential of an endogenous NET inhibitory factor (NIF), recently discovered in neonates. Mice pretreated with NIF had reduced brain injury, improved neurological and motor function and enhanced survival after stroke. Importantly, NIF specifically blocked NET formation after stroke without affecting brain neutrophil recruitment. Critically, NIF still improved stroke outcomes when administered after stroke onset. These results support a pathological role for NETs in stroke brain injury and indicate the use of NIF as a therapeutic strategy to target immunothrombosis in stroke.