A NETs-centric multi-omics framework prioritizes PGLYRP1 and MMP9 as subtype-associated thromboinflammatory biomarker candidates and putative therapeutic hypotheses in ischemic stroke.

Neutrophil extracellular traps are implicated in immunothrombosis and neuroinflammation in ischemic stroke, but blood-based markers that distinguish subtype-specific thromboinflammatory patterns remain limited. We applied an integrated multi-omics strategy combining multi-cohort peripheral-blood transcriptomics, machine-learning feature selection, Mendelian randomization, and single-cell RNA sequencing, followed by in silico perturbation analyses and compound prioritization with molecular docking, molecular dynamics simulation, and cellular thermal shift assays. Plasma citrullinated histone H3 levels were elevated across ischemic stroke subtypes and were highest in cardioembolic stroke. Integrative transcriptomic analysis identified a 7-gene neutrophil extracellular trap-related diagnostic signature comprising PADI4, C5AR1, MMP9, LRG1, NFIL3, TREM1, and PGLYRP1, with good cross-cohort diagnostic performance (area under the curve 0.789-0.834). Among these genes, MMP9 showed a broad association across ischemic stroke cohorts, whereas PGLYRP1 showed a cardioembolic stroke-enriched signal and a putative causal association with cardioembolic stroke in Mendelian randomization analyses. Two-step mediation analyses did not support a significant mediating role for the tested systemic cytokines, consistent with a more localized thromboinflammatory context. Single-cell computational perturbation suggested that Pglyrp1 may influence neutrophil-associated programs and intercellular communication. Molecular dynamics simulations prioritized Naringin as a candidate MMP9-binding compound, and cellular thermal shift assays supported cellular target engagement for the MMP9-Naringin pair. These findings provide a neutrophil extracellular trap-centered framework for biologically informed stratification of ischemic stroke and nominate MMP9 and PGLYRP1 as candidate biomarkers and therapeutic hypotheses for further validation.