PhD Scientific Days 2025

Molecular Medicine II.

The effect of neutrophil extracellular traps on fibrinolysis and fibrin structure in vivo and ex vivo

Name of the presenter

Csikós Petra Metta

Institute/workplace of the presenter

Department of Biochemistry

Authors

Petra Metta Csikós¹, Dr. Barbara Baráth¹, Kata Balog Virág¹, Dr. Alexandra Raska^1,2, Dr. Nikolett Wohner^1,2, Prof. Dr. Kraszimir Kolev¹

1: Department of Biochemistry
2: South-Pest Central Hospital - National Institute of Hematology and Infectology Szent Laszlo Hospital

Text of the abstract

Introduction - Neutrophil extracellular traps (NETs) are mesh-like structures consist of DNA, histone, granular and cytoplasmic proteins secreted by neutrophil granulocytes and are also found in the fibrin matrix of arterial and venous thrombi. The enzyme peptidyl-arginine deiminase-4 (PAD4) citrullinates histones, thereby decondensing chromatin and enhancing NET formation. Neutrophil serine proteases, such as the neutrophil elastase inhibitor SERPINB1, reduce NET formation.

Aims - Study the effects of NETs on fibrinolysis and fibrin structure in vivo and ex vivo

Method - Deep venous thrombosis was modeled in mice with different NET-forming capacity (wild type (WT), PAD4KO, SERPINB1KO) using inferior vena cava stenosis surgery. Thrombi were removed after 1-4 days and the fibrin structure was examined by scanning electron microscopy (SEM). Whole peripheral blood from mice of different genotypes and isolated neutrophil granulocyte-containing clots were examined using a viscoelastic assay (ClotPro) and a laser scanning microscope. H3 histones were citrullinated with PAD4 and plasminogen activation was observed in the presence of citrullinated and non-citrullinated histones and white blood cells from mouse lines with different NET-forming capacity.

Results - The odds of thrombus formation were 61% lower in PAD4KO mice compared to WT (OR 0.39; 95% CI 0.17-0.94), but there was no significant difference between SERPINB1KO and WT mice. The thrombus mass reached a maximum on day 3 and thrombolysis was initiated on day 4. PAD4KO thrombus mass was significantly lower on days 3-4 compared to WT thrombus mass. Neutrophil activation decelerated the lysis of WT and SERPINB1KO clots in ClotPro measurements (from 34.2±8.0 minutes to 56.4±18.8 minutes and from 36.7±11.1 minutes to 55.5±17.1 minutes, n=6), but not the PAD4KO clots. Citrullination of histones inhibited plasminogen activation. SEM measurements on day 1 showed that PAD4KO mice had significantly thinner fibrin fibers than WT and SERPINB1KO mice, it was reversed on day 4.

Conclusion - The presence of NETs increases the likelihood of thrombus formation and thrombus size in a mouse model of venous thrombosis and slows down fibrinolytic processes ex vivo.

Funding - Egyetemi Kutatói Ösztöndíj Program (EKÖP), National Academy of Scientist Education, Hungarian Centre of Excellence for Molecular Medicine (HCEMM)