Citrullinated fibrinogen forms densely packed clots with decreased permeability

Abstract

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.

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“…Following laser irradiation of the NETS the supernatant was collected, and the extent of methylene blue released was recorded from the absorbance at 540 nm. (d) Scanning electron microscopy (SEM): the change in surface morphology of the brin clot and thrombus-depleted areas, as well as diameter of brin bres was recorded using an analytical SEM 37,38 as mentioned in the ESI section. † (e) Confocal laser scanning microscopy (CLSM): the ber density and thrombus-depleted areas in the uorescently labelled clots were analysed employing confocal microscopy 37,38 (details in the ESI section †).…”

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“…Citrullinated Fgn (citFgn) is converted to citrullinated F (citF) and is present in the structure of murine model of venous thrombi [ 5 ]. The citF is composed of thinner fibers and larger pores [ 6 ]. The modified structure of citF results in impaired mechanical stability and enhanced lytic resistance compared with noncitrullinated F [ 5 ].…”

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“…Surprisingly, such effects of PADs have so far been demonstrated only in a few ECM proteins. For example, citrullination of fibrinogen/fibrin has been well documented and shown to alter the property of blood clots [33,34]. Interestingly, fibrinogen/ fibrin is also an endogenous ligand of toll-like receptor 4 (TLR4), and cit-fibrinogen/fibrin appears to be more potent in activating TLR4 than its native counterpart [35,36] (figure 5).…”