Platelet-Mediated Neutrophil Extracellular Traps Regulate Ischemic Stroke Injury

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.

Grant Number: 7R01HL163019-04
NIH Institute/Center: NIH
Principal Investigator: Robert Campbell