Background: Citrullination, the conversion of peptidyl-arginine into peptidyl-citrulline is catalyzed by PAD (peptidyl-arginyl-deiminase) enzymes. Fibrin, the main scaffold of thrombi is susceptible to PAD2/4 released during NETosis by neutrophils, which are abundant in pathological thrombi. However, no study has investigated the presence and significance of citrullinated fibrin(ogen) in thrombi formed in vivo.

Aims: We hypothesized that citrullinated fibrin(ogen) is present in venous thrombi, altering the structural, mechanical and fibrinolytic properties of the clots.

Methods: Thrombi were induced in a mouse inferior vena cava stenosis model. Citrullinated fibrin(ogen) was detected with a specifically designed western blot-based protocol. In vitro citrullination of fibrinogen was performed with purified PAD4. The structure and biomechanical properties of fibrin formed from citrullinated fibrinogen were examined with a broad range of methods (scanning-electron/laser-scanning/atomic-force microscopy-SEM/LSM/AFM, rheometry, turbidimetry, permeability measurements, small-angle X-ray scattering-SAXS, nano-thromboelastography).

Results: Citrullinated fibrinogen was abundant in thrombi formed 1-48 h post ligation, but not in concurrent plasma samples (Fig. 1). In pure fibrin clots, SEM confirmed a 34% decrease in median fiber diameters after extensive citrullination, while the width/length ratios and fractal geometry of fibers remained preserved according to AFM and SAXS. LSM showed increased fiber density (Fig. 2A), and accordingly clot permeability declined by >50% even after milder citrullination. Mechanical stability of citrullinated clots was compromised as evidenced by a 50% decrease in maximal cantilever pulling force in nano-thromboelastography (Fig. 2B) and a 30% decrease in the critical shear stress needed to disassemble clots in rheometry. Both plasmin- and tPA-mediated fibrinolysis were hindered in pure fibrin and plasma clots containing citrullinated fibrinogen.

Conclusions: For the first time, we provide evidence for the presence of citrullinated fibrin(ogen) in venous thrombi. Furthermore, we show that citrullination contributes to a clot structure that is lysis-resistant because of higher compactness and lower porosity, but mechanically vulnerable and consequently more prone to embolization.

[Figure 1. Citrullinated fibrin(ogen) is abundant in murine thrombi, but not in plasma.]

[Figure 2. Structure and biomechanical properties of citrullinated fibrin.]

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ISTH Congress Abstracts - https://abstracts.isth.org/abstract/fibrinogen-is-citrullinated-in-venous-thrombi-and-forms-fragile-clots-with-increased-resistance-to-lysis/