Oxidative stress-induced fibrinogen modifications in liver transplant recipients: unraveling a novel potential mechanism for cardiovascular risk

Abstract

Background: Cardiovascular events represent a major cause of non-graft-related death after liver transplant. Evidence suggest that chronic inflammation associated with a remarkable oxidative stress in the presence of endothelial dysfunction and procoagulant environment plays a major role in the promotion of thrombosis. However, the underlying molecular mechanisms are not completely understood.

Objectives: In order to elucidate the mechanisms of posttransplant thrombosis, the aim of the present study was to investigate the role of oxidation-induced structural and functional fibrinogen modifications in liver transplant recipients.

Methods: A case-control study was conducted on 40 clinically stable liver transplant recipients and 40 age-matched, sex-matched, and risk factor-matched controls. Leukocyte reactive oxygen species (ROS) production, lipid peroxidation, glutathione content, plasma antioxidant capacity, fibrinogen oxidation, and fibrinogen structural and functional features were compared between patients and controls.

Results: Patients displayed enhanced leukocyte ROS production and an increased plasma lipid peroxidation with a reduced total antioxidant capacity compared with controls. This systemic oxidative stress was associated with fibrinogen oxidation with fibrinogen structural alterations. Thrombin-catalyzed fibrin polymerization and fibrin resistance to plasmin-induced lysis were significantly altered in patients compared with controls. Moreover, steatotic graft and smoking habit were associated with high fibrin degradation rate.

Conclusion: ROS-induced fibrinogen structural changes might increase the risk of thrombosis in liver transplant recipients.

Keywords: cardiovascular risk; fibrinogen; lipid peroxidation; liver transplant; oxidative stress.

Figure 1

Increased leukocyte reactive oxygen species (ROS) production and decreased leukocyte glutathione (GSH) content in liver transplant (LT) patients compared with controls. Flow cytometry analysis shows significant alteration in (A–C) lymphocyte-, monocyte-, and neutrophil-derived ROS and (D–F) lymphocyte, monocyte, and neutrophil GSH content in LT patients vs controls. ∗Statistical significance (P < .001). RFU, relative fluorescence units.

Figure 2

Signs of oxidative stress in plasma from liver transplant (LT) patients. (A, B) Plasma lipid peroxidation and total antioxidant capacity in LT patients and controls. (C, D) Nitrate/nitrite and glutathione (GSH) levels in LT patients vs controls. ∗Statistical significance (P < .001). MDA, malondialdehyde.

Figure 3

Fibrinogen from liver transplant (LT) patients displays structural alterations and oxidation with respect to controls. Fibrinogen secondary structure was evaluated by circular dichroism spectroscopy. (A) Representative circular dichroism spectra of fibrinogen oxidation, (B) fibrinogen tertiary structure by intrinsic fibrinogen fluorescence, and (C) fibrinogen oxidation analysis via dityrosine content assessment in LT patients and controls. ∗Statistical significance (P < .001). RFU, relative fluorescence units.

Figure 4

Fibrin clot from liver transplant (LT) patients is denser than that from controls. Three-dimensional (3D) confocal microscopy analysis (630× magnification) of fibrin clot from purified fibrinogen purified from LT patients and controls. The fibrin clot from LT patient is noticeably denser with closely packed fibers (A). The surface plot of the LT sample is more uniform and peaks sharply, suggesting a denser and more uniform fibrin deposition. This may contribute to a stiffer clot, which could be less effective at accommodating physical stress or strain. The fibrin fibers in the control are thicker than those in the LT sample (B). Thicker fibers typically indicate a more robust and elastic network. Consistent with the single stack analysis, LT fibrin clot shows a significant decrease in pore diameter compared with the control (C). This aligns with the observations of a denser, potentially more rigid network in LT patients. Single stacks are 184.6 μm × 184.6 μm. Values are represented as median and IQR. ∗Statistical significance (P < .001).

Figure 5

Fibrinogen from liver transplant (LT) patients displays functional alterations with respect to controls. (A) The ability of fibrinogen to polymerize into fibrin in LT patients and controls. (B–D) Variation in lag phase, maximum slope (Vmax), and maximum (Max) absorbance (Abs) of fibrinogen polymerization curves in LT patients vs controls. These alterations are related to a different fibrinogen structure in LT fibrinogen with respect to controls. ∗Statistical significance (P < .001).

Figure 6

Fibrin from liver transplant (LT) patients is resistant to plasmin-induced lysis. (A) Representative gel of fibrin degradation after 0, 3, and 6 hours of plasmin digestion using fibrinogen purified from LT patients and controls. (B) Degradation rate of the fibrin β chain. ∗Statistical significance (P < .001).

Supplementary Figure 1

3D confocal laser scanning microscopy analysis of fibrin gels from liver transplant (LT) patients and controls. The figure shows representative 3D reconstructions of fibrin gels from three different controls (A-C) and three different LT patients (D-F). Fibrin gels from controls (A-C) show a well-defined and interconnected fibrin network with large pores and thicker fibers. Fibrin gels from LT patients (D-F) display a denser fibrin structure with smaller pores and thinner fibers compared to controls.

Supplementary Figure 2

Thrombin-catalyzed fibrinogen polymerization curves in purified human fibrinogen (Sigma, Milan, Italy) incubated with increasing concentrations of the peroxyl radical generator 2,2′-azobis(2-amidinopropane) dihydrochloride (AAPH) (A). Lag phase (B), max velocity (C) and max absorbance (D) of thrombin-catalyzed fibrin polymerization curves in (A). ∗ p<0.05 vs Control; ° p<0.05 vs AAPH 1mM+TROLOX; § p<0.05 vs AAPH 0.5 mM.