Despite advancements, about 700,000 people in the U.S. still experience an ischemic stroke annually. Current treatments for acute ischemic stroke are largely restricted to thrombolysis or thrombectomy, for which many stroke patients are ineligible for. Therefore, developing novel therapies for stroke is a significant public health need. Understanding the cellular mechanisms underlying stroke is critical for the development of new stroke therapies. Neutrophils and platelets are critical regulators of ischemic stroke injury. In humans and mice, platelet-neutrophil aggregates increase after ischemic stroke as well do neutrophil extracellular traps (NETs), a marker of neutrophil activation. NETs are critical during inflammation and infection and are released by neutrophils to trap pathogens. While NETs help fight infection, excessive NET formation can be detrimental to the host by promoting thrombosis. However, the pathological role of NET release has not been studied in ischemic stroke injury. Furthermore, the molecular regulators that trigger NET formation during stroke remain unclear. Finally, if targeting NET release during ischemic stroke injury improves outcomes is completely unknown. Here, we will test the innovative hypothesis that platelets are a primary driver of NET release during ischemic stroke and targeting NET formation with a novel, endogenous NET- inhibitory factor (nNIF), will improve stroke outcomes. We will employ complementary clinical, in vitro, and in vivo approaches, along with state-of-the-art techniques and models to rigorously test this hypothesis. Specific Aim 1 will determine if NETs are released after ischemic stroke and if they are present in the brains of human stroke patients and mice after experimental stroke. Furthermore, we will examine if neutrophils are primed to release NETs in ischemic stroke patients. Specific Aim 2 will establish whether platelet high mobility group box 1 (HMGB1), a danger associated molecular pattern released by platelets after activation, regulates NET formation during ischemic stroke injury. Specific Aim 3 will determine if therapeutic nNIF administration blocks NET formation during experimental stroke and improves acute and long-term stroke outcomes, including motor and neurological function. Successful completion of these aims will 1) determine if NETs are present in ischemic stroke injury including intravascular and extravascular locations; (2) establish whether neutrophils and platelets are primed to participate in NET formation during ischemic stroke, (3) determine whether platelet HMGB1 is a critical regulator of platelet-mediated NETosis during ischemic stroke; and (4) determine if NET inhibition improves stroke outcomes and the therapeutic window associated with pathological NET formation. Data generated in this proposal will significantly increase our understanding of how platelets contribute to pathological NET formation during ischemic stroke and associated neurological injury.