Involvement of peptidylarginine deiminase 4 in eosinophil extracellular trap formation and contribution to citrullinated histone signal in thrombi

Kimberly Martinod; Frederik Denorme; Severien Meyers; Marilena Crescente; Stijn Van Bruggen; Mathias Stroobants; Patrick M Siegel; Ramesh Grandhi; Katharina Glatz; Thilo Witsch

doi:10.1016/j.jtha.2024.02.010

Involvement of peptidylarginine deiminase 4 in eosinophil extracellular trap formation and contribution to citrullinated histone signal in thrombi

J Thromb Haemost. 2024 Feb 22:S1538-7836(24)00112-0. doi: 10.1016/j.jtha.2024.02.010. Online ahead of print.

Affiliations

¹ Center for Molecular and Vascular Biology, Department of Cardiovascular Sciences, KU Leuven, Leuven, Belgium. Electronic address: kim.martinod@kuleuven.be.
² Division of Vascular Neurology, Department of Neurology, University of Utah, Salt Lake City, Utah, USA.
³ Center for Molecular and Vascular Biology, Department of Cardiovascular Sciences, KU Leuven, Leuven, Belgium.
⁴ Department of Cardiology and Angiology I, University Heart Center Freiburg-Bad Krozingen, Faculty of Medicine, University of Freiburg, Freiburg, Germany.
⁵ Department of Neurosurgery, Clinical Neurosciences Center, University of Utah, Salt Lake City, Utah, USA.
⁶ Institute of Pathology, University Hospital Basel, Basel, Switzerland.
⁷ Department of Cardiology and Angiology I, University Heart Center Freiburg-Bad Krozingen, Faculty of Medicine, University of Freiburg, Freiburg, Germany; Department of Cardiology, University Hospital Basel, University of Basel, Basel, Switzerland. Electronic address: thilo.witsch@uniklinik-freiburg.de.

PMID: 38395360
DOI: 10.1016/j.jtha.2024.02.010

Abstract

Background: Extracellular traps formed by neutrophils (NETs) and eosinophils (EETs) have been described in coronary thrombi, contributing to thrombus stability. A key mechanism during NET formation is histone modification by the enzyme PAD4. Citrullinated histones, the product of PAD4 activity, are often attributed to neutrophils. Eosinophils also express high levels of PAD4.

Objectives: We aimed to explore the contribution of PAD4 to EET formation.

Methods: We performed immunohistological analyses on thrombi, including a large, intact, and eosinophil-containing thrombus retrieved from the right coronary artery using an aspiration catheter and stroke thrombi from thrombectomy retrieval. We studied eosinophils for their capability to form PAD4-dependent EETs in response to strong ET-inducing agonists as well as activated platelets and bacteria.

Results: Histopathology and immunofluorescence microscopy identified a coronary thrombus rich in platelets and neutrophils, with distinct areas containing von Willebrand factor and citrullinated histone H3 (H3Cit). Eosinophils were also identified in leukocyte-rich areas. The majority of the H3Cit+ signal colocalized with myeloperoxidase, but some colocalized with eosinophil peroxidase, indicating EETs. Eosinophils isolated from healthy volunteers produced H3Cit+ EETs, indicating an involvement of PAD4 activity. The selective PAD4 inhibitor GSK484 blocked this process, supporting PAD4 dependence of H3Cit+ EET release. Citrullinated histones were also present in EETs produced in response to live Staphylococci. However, limited evidence for EETs was found in mouse models of venous thrombosis or infective endocarditis.

Conclusion: As in NETosis, PAD4 can catalyze the formation of EETs. Inhibition of PAD4 decreases EET formation, supporting the future utility of PAD4 inhibitors as possible antithrombotic agents.

Keywords: citrullination; eosinophils; extracellular traps; myocardial infarction; stroke; thrombosis.