Citrullinated fibrinogen forms densely packed clots with decreased permeability

• Published in: Journal of thrombosis and haemostasis Vol. 20; no. 12; pp. 2862 - 2872
• Main Authors: Varjú, Imre, Tóth, Erzsébet, Farkas, Ádám Z., Farkas, Veronika J., Komorowicz, Erzsébet, Feller, Tímea, Kiss, Balázs, Kellermayer, Miklós Z., Szabó, László, Wacha, András, Bóta, Attila, Longstaff, Colin, Kolev, Krasimir
• Format: Journal Article
• Language: English
• Published: England Elsevier Limited 01.12.2022; John Wiley and Sons Inc
• Subjects: Animals; Arginine deiminase; Atomic force microscopy; citrullination; Citrulline; Confocal microscopy; Fibrin; Fibrin - chemistry; Fibrinogen; Fibrinogen - chemistry; Hemostatics; Humans; Leukocytes (neutrophilic); Mice; Microscopy, Electron, Scanning; Monomers; Original; Periodicity; Permeability; Porosity; Protein-arginine deiminase; protein–arginine deiminases; Scanning electron microscopy; Scattering, Small Angle; small‐angle X‐ray scattering; Thrombin; THROMBOSIS; X-Ray Diffraction; protein-arginine deiminases; atomic force microscopy; fibrin; small-angle X-ray scattering; citrullination
• ISSN: 1538-7933; 1538-7836; 1538-7836
• DOI: 10.1111/jth.15875

Background Fibrin, the main scaffold of thrombi, is susceptible to citrullination by PAD (peptidyl arginine deiminase) 4, secreted from neutrophils during the formation of neutrophil extracellular traps. Citrullinated fibrinogen (citFg) has been detected in human plasma as well as in murine venous thrombi, and it decreases the lysability and mechanical resistance of fibrin clots. Objective To investigate the effect of fibrinogen citrullination on the structure of fibrin clots. Methods Fibrinogen was citrullinated with PAD4 and clotted with thrombin. Scanning electron microscopy (SEM) and atomic force microscopy (AFM) were used to measure fiber thickness, fiber height/width ratio, and fiber persistence length in clots containing citFg. Fiber density was measured with laser scanning microscopy (LSM) and permeability measurements were carried out to estimate the porosity of the clots. The intra‐fiber structure of fibrin was analyzed with small‐angle X‐ray scattering (SAXS). Results SEM images revealed a decrease in the median fiber diameter that correlated with the fraction of citFg in the clot, while the fiber width/length ratio remained unchanged according to AFM. With SAXS we observed that citrullination resulted in the formation of denser clots in line with increased fiber density shown by LSM. The permeability constant of citrullinated fibrin decreased more than 3‐fold indicating significantly decreased porosity. SAXS also showed largely preserved periodicity in the longitudinal assembly of fibrin monomers. Conclusion The current observations of thin fibers combined with dense packing and low porosity in the presence of citFg can provide a structural framework for the mechanical fragility and lytic resistance of citrullinated fibrin.