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.

Keywords: cirrhosis; fibrin structure; fibrinogen oxidation; stimulated emission depletion (STED) microscopy; thrombus.

Figure 1

Plasma protein carbonyl content (A), plasma lipid peroxidation (B), plasma total antioxidant capacity (C) and fibrinogen oxidation in purified fibrinogen fractions (D) in patients with liver cirrhosis at different disease severity (n = 5 for each Child–Turcotte–Pugh category) and controls (n = 20). All experiments were performed in triplicate. Values are represented as median with interquartile range. * Significant difference vs. controls at the p < 0.05 level. # Significant difference vs. CTP A at the p < 0.05 level. § Significant difference vs. CTP B at the p < 0.05 level.

Figure 2

Fibrin resistance to plasmin-induced lysis experiments. (A) Representative gel of fibrin degradation after 0, and 6 h of plasmin digestion using fibrinogen purified from patients with liver cirrhosis at different disease severity (n = 5 for each Child–Turcotte–Pugh category) and controls (n = 20). (B) Residual fibrin β chain intensity after 6 h of plasmin digestion in fibrinogen purified from patients with liver cirrhosis at different stages (n = 5) and controls (n = 20). All experiments were performed in triplicate. Values are represented as median with interquartile range. * Significant difference vs. controls at the p < 0.05 level. # Significant difference vs. CTP A at the p < 0.05 level. § Significant difference vs. CTP B at the p < 0.05 level.

Figure 3

Fibrinogen polymerization experiments. (A) Representative curves of thrombin-catalyzed fibrinogen polymerization and corresponding (B) Max Absorbance, (C) Vmax, and (D) lag time in fibrinogen purified from patients with liver cirrhosis at different disease severity (n = 5 for each Child–Turcotte–Pugh category) and controls (n = 20). Values are represented as median with interquartile range. * Significant difference vs. controls at the p < 0.05 level. # Significant difference vs. CTP A at the p < 0.05 level. § Significant difference vs. CTP B at the p < 0.05 level.

Figure 4

Fibrinogen structure. (A) Representative far-UV circular dichroism spectra of fibrinogen purified from patients with liver cirrhosis at different disease severity and controls. The two negative peaks observed in controls at 208 and 222 nm (arrows) are typical of protein α-helix structure. Fibrinogen purified from cirrhotic patients displayed an altered CD spectrum consisting mainly of a decrease in the negative peak in the 215–225 nm region, suggesting a decrease in alpha-helical content. This secondary structure alteration is associated with disease severity. (B) To demonstrate whether oxidation could induce fibrinogen secondary structure alterations, in vitro fibrinogen oxidation experiments were performed. After fibrinogen oxidation, the fibrinogen CD spectrum showed an increased ellipticity in an oxidation-dependent manner. Moreover, the antioxidant Trolox was able to prevent these fibrinogen structural changes, demonstrating the key role of oxidation in fibrinogen secondary structure modification. (C) Fibrinogen tertiary structure was investigated by intrinsic emission fluorescence spectroscopy. Tryptophan residues that are buried in the core of the protein show high intrinsic fluorescence, as in fibrinogen purified from controls. On the contrary, fibrinogen purified from patients with liver cirrhosis exhibited lower intrinsic fluorescence intensity, indicating changes in protein tertiary structure. (D) In line with the CD spectra experiments, in vitro fibrinogen oxidation demonstrated the direct role of tertiary structure alterations. Once again, Trolox treatment prevented these structural changes, demonstrating the pivotal role of oxidation in fibrinogen tertiary structure alterations. The arrows indicate the two negative peaks at 208 and 222 nm, typical of protein α-helix structure.

Figure 5

Confocal microscopy analysis of fibrin gels of fibrinogen purified from patients with liver cirrhosis at different stages and controls. Three-dimensional confocal microscopy images clearly show control fibrin gel characterized by large pores and tick fibers when compared to fibrin from cirrhotic patients which are much dense, with narrow pores and thin fibers. In particular, fibrin gels from fibrinogen purified from CTP B and CTP C patients exhibited a dramatic gel rearrangement: fibers were so closely packed in the bulk of gel, obscuring the individual fibers and producing thin sheets with small pores. Surface plot and histogram values are referred to in the corresponding confocal image (first column).

Figure 6

STED super-resolution microscopy analysis of fibrin gels of fibrinogen purified from patients with liver cirrhosis at different disease severity (n = 5 for each Child–Turcotte–Pugh category) and controls. STED super-resolved microscopy revealed a marked increase in fiber density and clot porosity in fibrin from cirrhotic patient when compared to controls. Leica Application Suite X Software analysis demonstrated that fibrin fibers from fibrinogen purified from cirrhotic patients showed a significant decrease in fiber diameter and clot porosity when compared to controls, and this became more marked with disease progression. Values are represented as mean ± SD. * Significant difference vs. controls at the p < 0.05 level. # Significant difference vs. CTP A at the p < 0.05 level. § Significant difference vs. CTP B at the p < 0.05 level.