Abstract Number: OC 02.4
Meeting: ISTH 2020 Congress
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.
To cite this abstract in AMA style:Varju I, Sorvillo N, Cherpokova D, Farkas VJ, Farkas AZ, Komorowicz E, Feller T, Kiss B, Kellermayer MS, Szabo L, Wacha A, Bota A, Longstaff C, Wagner DD, Kolev K. Fibrinogen Is Citrullinated in Venous Thrombi and Forms Fragile Clots with Increased Resistance to Lysis [abstract]. Res Pract Thromb Haemost. 2020; 4 (Suppl 1). https://abstracts.isth.org/abstract/fibrinogen-is-citrullinated-in-venous-thrombi-and-forms-fragile-clots-with-increased-resistance-to-lysis/. Accessed May 6, 2021.
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