PB0781 - Involvement of peptidylarginine deiminase 4 in eosinophil extracellular trap formation and contribution to citrullinated histone signal in thrombi

Monday, June 26, 2023

18:30 – 19:30 ET

Room: Exhibition

Presenting Author(s)

Kimberly Martinod, PhD

Assistant Professor
Center of Molecular and Vascular Biology, Department of Cardiovascular Sciences, KU Leuven, Leuven, Belgium
Leuven, Vlaams-Brabant, Belgium

Co-Author(s)

SM

Severien Meyers

Researcher
Center for Molecular and Vascular Biology, Department of Cardiovascular Sciences, KU Leuven, Leuven, Belgium
Leuven, Vlaams-Brabant, Belgium
MC

Marilena Crescente, PhD

Senior Lecturer
Manchester Metropolitan University
Manchester, England, United Kingdom
Stijn Van Bruggen, MA, Ir. (he/him/his)

PhD candidate
Center of Molecular and Vascular Biology, Department of Cardiovascular Sciences, KU Leuven, Leuven, Belgium
Leuven, Vlaams-Brabant, Belgium
MS

Mathias Stroobants

Lab Technician
Center of Molecular and Vascular Biology, Department of Cardiovascular Sciences, KU Leuven, Leuven, Belgium
Leuven, Vlaams-Brabant, Belgium
PS

Patrick M. Siegel

Resident Physician
Department of Cardiology and Angiology, University Heart Center Freiburg - Bad Krozingen, Faculty of Medicine, University of Freiburg, Freiburg, Germany
Freiburg, Baden-Wurttemberg, Germany
KG

Katharina Glatz

Pathologist
Institute of Pathology, University Hospital Basel, Basel, Switzerland
Basel, Basel-Stadt, Switzerland
TW

Thilo Witsch

Attending Physician, Interventional Cardiology
Department of Cardiology and Angiology, University Heart Center Freiburg - Bad Krozingen, Faculty of Medicine, University of Freiburg, Freiburg, Germany
Freiburg, Baden-Wurttemberg, Germany

Background: Extracellular traps (ETs) 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 but the contribution of PAD4 to EET formation is not known.

Aims: To characterize the role of PAD4 in EET formation and the relevance in thrombosis.

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. Moreover, 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(s): 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.