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The metabolic and mechanistic regulators of NET formation in relation to platelet activation during ischemic stroke remain poorly understood. In the present study, using multiple animal and clinical models of stroke, we examined the role of homocysteine and its biochemical modifications on NET formation and concomitant neuronal damage, platelet aggregation, motor and gait functions. Phosphoproteomics analysis of neutrophils in response to homocysteine revealed an enrichment of kinases and phosphoproteins associated with thrombosis. Homocysteinylated albumin induced significant NET formation via Erk1\/2, Akt, ATM, and PAD4\u2010dependent pathways, independent of reactive oxygen species. Hyperhomocysteinemic mice fed a methionine\u2010rich diet exhibited elevated NETs components (neutrophil elastase, citrullinated histones, cell\u2010free DNA (cfDNA)), platelet activation, neuronal damage, and impaired motor, balance, and learning functions. UCCAO\u2010induced ischemia exacerbated neuronal damage, motor dysfunction, and platelet activation in hyperhomocysteinemic mice, which were reversed by disrupting NETs with N\u2010acetyl cysteine and DNase. In stroke patients, homocysteine, neutrophil elastase, and cfDNA levels were significantly elevated, independent of comorbidities (e.g., hypertension, type 2 diabetes), etiology (TOAST classification), or stroke severity. Additionally, stroke patients generated autoantibodies against homocysteinylated albumin, which positively correlated with neutrophil elastase levels. This study identifies homocysteine and its modifications as key metabolic regulators of NETosis in stroke, linking NETs formation to platelet activation and neuronal damage. These findings highlight potential therapeutic targets for mitigating stroke pathogenesis through the modulation of NETs.<\/jats:p>","DOI":"10.1096\/fj.202500844rr","type":"journal-article","created":{"date-parts":[[2025,11,13]],"date-time":"2025-11-13T10:38:37Z","timestamp":1763030317000},"update-policy":"https:\/\/doi.org\/10.1002\/crossmark_policy","source":"Crossref","is-referenced-by-count":3,"title":["Biochemical Modifications of Homocysteine Drive Neutrophil Extracellular Trap Formation in Ischemic Stroke"],"prefix":"10.1096","volume":"39","author":[{"given":"Aswathy S.","family":"Nair","sequence":"first","affiliation":[{"name":"Department of Ageing Research, Manipal School of Life Sciences Manipal Academy of Higher Education  Manipal India"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Pooja Yedehalli","family":"Thimmappa","sequence":"additional","affiliation":[{"name":"Department of Ageing Research, Manipal School of Life Sciences Manipal Academy of Higher 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