Intrathrombus NETosis Defines Prognostically Distinct Phenotypes in Large-Vessel Occlusion Stroke
- 1Instituto Investigación Hospital 12 Octubre (imas12). Complutense Medical School.
Abstract
BackgroundImmunothrombosis is increasingly recognized in large-vessel-occlusion acute ischemic stroke (LVO-AIS), yet most biomarker studies infer thrombus biology from circulating neutrophil extracellular trap (NET) markers or from pre-defined thrombus subtypes. We prospectively studied 160 patients with anterior-circulation LVO-AIS treated with mechanical thrombectomy to determine whether intrathrombus NETosis markers identify thrombus phenotypes with distinct clinical trajectories, and whether plasma NETosis biomarkers reflect the molecular composition of thrombi.
MethodsThrombi underwent quantitative histopathology and immunohistochemistry for citrullinated histone H3 (CitH3), neutrophil elastase (NE) and CD61. Thrombi were analyzed without prior etiological or histological stratification, and intrathrombus markers were dichotomized at cohort medians (CitH3 6.62%, NE 3.95%, CD61 23.09%) to define internally standardized candidate phenotypes. Associations with thrombus composition, collateral circulation (Tan 2-3), 3-month functional independence (modified Rankin Scale 2 or less) and long-term mortality were assessed by correlation analyses and progressively adjusted multivariable models, with cross-compartment correlations corrected by the Benjamini-Hochberg false discovery rate (FDR).
ResultsIntrathrombus markers defined prognostically distinct phenotypes: CD61 tracked functional recovery, NE preserved collateral circulation and CitH3 long-term survival, each remaining significant after multivariable adjustment. Circulating NE, myeloperoxidase and leukocyte count instead tracked the macroscopic red-blood-cell-fibrin composition of retrieved thrombi. Cross-compartment concordance was limited: after FDR correction the only significant plasma-thrombus association was an inverse correlation between plasma cell-free DNA and intrathrombus NE, and no intrathrombus marker correlated positively with its circulating counterpart.
ConclusionsLocal immunothrombotic signatures therefore provide clinically relevant information not captured by plasma markers, and median-defined intrathrombus thresholds represent hypothesis-generating cutoffs for external validation and NET-targeted treatment stratification.
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BibTeX
@misc{diaz2026intrathrombus,
title = {{Intrathrombus NETosis Defines Prognostically Distinct Phenotypes in Large-Vessel Occlusion Stroke}},
author = {B. Diaz and P. Calleja and F. Ostos and A. Ruiz and L. Alzamora and A. MARTINEZ-SALIO and M. Gutierrez-Sanchez and F. Ballenilla and J. Alvarez-Cienfuegos and C. Gubern-Merida and Y. Silva and A. Garcia-Culebras and M. A. Moro and A. Moraga and I. Lizasoain},
howpublished = {medRxiv},
year = {2026},
month = oct,
doi = {10.64898/2026.10.04.26364710},
url = {https://www.medrxiv.org/content/10.64898/2026.10.04.26364710v1},
note = {CC BY-NC-ND},
}
RIS
TY - UNPB
TI - Intrathrombus NETosis Defines Prognostically Distinct Phenotypes in Large-Vessel Occlusion Stroke
AU - B. Diaz
AU - P. Calleja
AU - F. Ostos
AU - A. Ruiz
AU - L. Alzamora
AU - A. MARTINEZ-SALIO
AU - M. Gutierrez-Sanchez
AU - F. Ballenilla
AU - J. Alvarez-Cienfuegos
AU - C. Gubern-Merida
AU - Y. Silva
AU - A. Garcia-Culebras
AU - M. A. Moro
AU - A. Moraga
AU - I. Lizasoain
PY - 2026
DA - 2026/10/04
PB - medRxiv
DO - 10.64898/2026.10.04.26364710
UR - https://www.medrxiv.org/content/10.64898/2026.10.04.26364710v1
AB - BackgroundImmunothrombosis is increasingly recognized in large-vessel-occlusion acute ischemic stroke (LVO-AIS), yet most biomarker studies infer thrombus biology from circulating neutrophil extracellular trap (NET) markers or from pre-defined thrombus subtypes. We prospectively studied 160 patients with anterior-circulation LVO-AIS treated with mechanical thrombectomy to determine whether intrathrombus NETosis markers identify thrombus phenotypes with distinct clinical trajectories, and whether plasma NETosis biomarkers reflect the molecular composition of thrombi. MethodsThrombi underwent quantitative histopathology and immunohistochemistry for citrullinated histone H3 (CitH3), neutrophil elastase (NE) and CD61. Thrombi were analyzed without prior etiological or histological stratification, and intrathrombus markers were dichotomized at cohort medians (CitH3 6.62%, NE 3.95%, CD61 23.09%) to define internally standardized candidate phenotypes. Associations with thrombus composition, collateral circulation (Tan 2-3), 3-month functional independence (modified Rankin Scale 2 or less) and long-term mortality were assessed by correlation analyses and progressively adjusted multivariable models, with cross-compartment correlations corrected by the Benjamini-Hochberg false discovery rate (FDR). ResultsIntrathrombus markers defined prognostically distinct phenotypes: CD61 tracked functional recovery, NE preserved collateral circulation and CitH3 long-term survival, each remaining significant after multivariable adjustment. Circulating NE, myeloperoxidase and leukocyte count instead tracked the macroscopic red-blood-cell-fibrin composition of retrieved thrombi. Cross-compartment concordance was limited: after FDR correction the only significant plasma-thrombus association was an inverse correlation between plasma cell-free DNA and intrathrombus NE, and no intrathrombus marker correlated positively with its circulating counterpart. ConclusionsLocal immunothrombotic signatures therefore provide clinically relevant information not captured by plasma markers, and median-defined intrathrombus thresholds represent hypothesis-generating cutoffs for external validation and NET-targeted treatment stratification.
ER -