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.
Introduction:The composition of thrombi determines their structure, mechanical stability, susceptibility to lysis, and consequently, the clinical outcome in coronary artery disease (CAD), acute ischemic stroke (AIS), and peripheral artery disease (PAD). Fibrin forms the primary matrix of thrombi intertwined with DNA, derived from neutrophil extracellular traps (NETs), and von Willebrand factor (VWF) bridging DNA and platelets. Here we examined the relative content of fibrin, DNA and VWF in thrombi and analyzed their interrelations and quantitative associations with systemic biomarkers of inflammation and clinical characteristics of the patients. Patients, methods: Thrombi extracted from AIS (n = 17), CAD (n = 18) or PAD (n = 19) patients were processed for scanning electron microscopy, (immune)stained for fibrin, VWF and extracellular DNA. Fibrin fiber diameter, cellular components, fibrin/DNA and fibrin/VWF ratios were measured. Results: Patients' age presented as a strong explanatory factor for a linear decline trend of the VWF content relative to fibrin in thrombi from CAD (adjusted-R 2 = 0.43) and male AIS (adjusted-R 2 = 0.66) patients. In a subgroup of CAD and PAD patients with dyslipidemia and high (above 80%) prevalence of atherothrombosis a significant correlation was observed between the VWF and DNA content in thrombi (adjusted-R 2 = 0.40), whereas a 3.7-fold lower linear regression coefficient was seen in AIS patients, in whom the fraction of thrombi of atherosclerotic origin was 57%. Independently of anatomical location, in patients with atherosclerosis the VWF in thrombi correlated with the plasma C-reactive protein levels. Conclusions: The observed interrelations between thrombus constituents and systemic inflammatory biomarkers suggest an intricate interplay along the VWF/NET/fibrin axis in arterial thrombosis.
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