Combined Immunohistochemical and Proteomic Description of Neutrophil Extracellular Trap (NET) Burden in Acute Ischemic Stroke Thrombi

Abstract

Background: Neutrophil extracellular traps (NETs) are important in host defense but implicated in pathological thrombosis in acute ischemic stroke (AIS). NET quantification is typically done with immunostaining for NET-specific markers, but molecular techniques like mass spectrometry can provide another angle into thrombo-physiology.

Objectives: This study leverages both histological analysis of NETs, and molecular insights from proteomics, to validate using proteomics for evaluating stroke thrombus NET burden.

Patients/methods: Sections of 30 AIS thrombi were stained for myeloperoxidase (MPO), citrullinated histone H3 (H3Cit), and DNA. ImageJ measured average staining area. Mass spectrometry of sample thrombi fragments detected 5,784 proteins. Spearman correlations and Mann Whitney U-tests compared NET burden from staining to proteomic composition. Dimensional reduction of H3Cit and MPO staining created a single NET marker variable (PC1), used to compare thrombus composition between thrombi with NET marker greater (NET-rich) or lesser (NET-poor) than median.

Results: Percent coverage for the three stains was interrelated (Spearman's ρ > 0.6, p < 5E-4 for all). 531 neutrophil degranulation proteins were positively associated with all stains. PC1 explained ∼92% of the variance. Ubiquitin-proteasome degradation pathways were negatively correlated with the NET markers (q-val < 0.05 for all relevant pathways). Older patient age (median, IQR: 79, 73-85 vs 61, 57-73; Mann Whitney p = 0.03) and atrial fibrillation-associated stroke (Fisher's Exact p = 0.03) thrombi were NET-rich.

Conclusions: This study marries immunostaining with proteomics for quantifying NET burden, pointing to molecular phenotypes within NET-rich and NET-poor thrombi that may underpin NET formation and maintenance in stroke thrombi.

Keywords: Acute Ischemic Stroke; Immunohistochemistry; Mass Spectrometry; Primary: Neutrophil Extracellular Traps; Proteomics; Secondary: Myeloperoxidase; thrombectomy.