Super-Resolution Microscopy Reveals an Altered Fibrin Network in Cirrhosis: The Key Role of Oxidative Stress in Fibrinogen Structural Modifications

Abstract

Cirrhotic patients show a reduced synthesis of both pro- and anti-coagulant factors. Recent reports indicate that they are characterized by a higher risk of thrombotic rather than hemorrhagic complications, but the mechanisms conferring this risk are not fully elucidated. Oxidative-mediated fibrinogen modifications may explain, at least in part, a prothrombotic profile. The aim of the present pilot study was to investigate the alterations in fibrinogen structure and function in patients with cirrhosis of various severity and to correlate these findings with the mechanisms of thrombus formation. We assessed in plasma specific oxidative stress markers and measured oxidative modifications, functional and structural parameters in purified fibrinogen fractions obtained from cirrhotic patients and control subjects. We enrolled 15 cirrhotic patients (5 patients belonging to each of the three Child–Turcotte–Pugh classes) and 20 age- and sex-matched healthy controls. Plasma redox status, fibrinogen oxidative modifications, thrombin-catalyzed fibrin polymerization and fibrin resistance to plasmin-induced lysis were significantly altered in cirrhotic patients and were associated to disease severity. Importantly, clot structure obtained by stimulated emission depletion (STED) super-resolution microscopy indicated modifications in fiber diameter and in clot porosity in cirrhotic patients. Fibrin fiber diameter significantly decreased in cirrhotic patients when compared to controls, and this difference became more marked with disease progression. In parallel, fibrin pore size progressively decreased along with disease severity. In cirrhotic patients, fibrinogen clot analysis and oxidative-dependent changes reveal novel structural and functional fibrinogen modifications which may favor thrombotic complications in cirrhosis.

The Applicability of Current Turbidimetric Approaches for Analyzing Fibrin Fibers and Other Filamentous Networks

“…Fibrin clots at identical concentrations were also prepared for STORM imaging, and fiber diameters were calculated from reconstructed STORM images and were used as a standard for evaluating the accuracy of the diameter values obtained using the turbidimetric approaches. STORM imaging and Simulated Emission Depletion Microscopy (STED), another super-resolution microscopy technique, have been previously used to determine fiber diameter from plasma [ 49 , 50 , 51 ] and from purified fibrinogen [ 52 ], providing fiber diameters over a range of 100–400 nm, matching those previously reported from SEM imaging [ 34 ]. However, super-resolution fluorescence imaging, unlike SEM imaging, does not require drying of the samples, which could cause changes in the morphology of the fibers.…”

Quality Attributes of Plasma Derived Human Fibrinogen (Fibrogen-I®)

“…Fibrogen-I® predominantly exists in a β-sheet secondary structure, which is in accordance with the three-dimensional structure of human Fibrinogen according to far-UV CD spectroscopy findings. 26 Both structurally and functionally, the human Fibrinogen product matches the market comparator.…”

Extension of the Human Fibrinogen Database with Detailed Clinical Information—The αC-Connector Segment

“…Changes in a subject’s fibrinogen characteristics may originate not only from changed levels of fibrinogen but also from altered fibrinogen properties, caused, e.g., by post-translational modifications (PTMs) [ 70 , 71 , 72 , 73 , 74 , 75 ]. A certain level of PTMs is necessary for the proper function of fibrinogen.…”