Molecular interactions of circulating fibrinogen as a risk factor for cardiovascular and cerebrovascular pathologies

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Impact of γ’γ’ fibrinogen interaction with red blood cells on fibrin clots

Publication . Guedes, Ana Filipa; Carvalho, Filomena Almeida; Domingues, Marco; Macrae, Fraser L.; McPherson, Helen R.; Sabban, Aliaa; Martins, Ivo C.; Duval, Cédric; Santos, Nuno C.; Ariёns, Robert A.S.

AIM: γ' fibrinogen has been associated with thrombosis. Here the interactions between γ'γ' or γAγA fibrinogen and red blood cells (RBCs), and their role on fibrin clot properties were studied. MATERIALS & METHODS: Atomic Force microscopy (AFM)-based force spectroscopy, rheological, electron and confocal microscopy, and computational approaches were conducted for both fibrinogen variants. RESULTS & CONCLUSION: AFM shows that the recombinant human (rh)γ'γ' fibrinogen increases the binding force and the frequency of the binding to RBCs compared with rhγAγA, promoting cell aggregation. Structural changes in rhγ'γ' fibrin clots, displaying a nonuniform fibrin network were shown by microscopy approaches. The presence of RBCs decreases the fibrinolysis rate and increases viscosity of rhγ'γ' fibrin clots. The full length of the γ' chain structure, revealed by computational analysis, occupies a much wider surface and is more flexible, allowing an increase of the binding between γ' fibers, and eventually with RBCs.

2018Journal article

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Essential arterial hypertension patients present higher cell adhesion forces, contributing to fibrinogen-dependent cardiovascular risk

Publication . Guedes, Ana F.; Carvalho, Filomena A.; Moreira, Carlos; Nogueira, José B.; Santos, Nuno C.

The increase of erythrocyte aggregation by high fibrinogen levels may be an indicator of cardiovascular risk. γ' fibrinogen variant has been considered as a possible player in enhancing aggregation. Here, we assessed, at the single-cell level, the influence of fibrinogen on erythrocyte aggregation in essential arterial hypertension. We also aimed at understanding how γ' fibrinogen is altered in this disease. Using atomic force microscopy (AFM), we show that the work and force necessary for erythrocyte-erythrocyte detachment is higher for patients than for healthy donors, with these parameters further increasing in both groups when higher fibrinogen concentrations are present. This can be associated with changes in blood flow, due to transient bridging of two erythrocytes by fibrinogen, representing an important cardiovascular risk factor. γ' fibrinogen can influence the increased risk in essential arterial hypertension, as we demonstrate that its levels are significantly increased in these patients' blood. Nevertheless, this cannot be the only cause for the changes observed in the AFM data.

2017Journal article

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Sensing adhesion forces between erythrocytes and γ’ fibrinogen, modulating fibrin clot architecture and function

Publication . Guedes, Ana Filipa; Carvalho, Filomena Almeida; Domingues, Marco; Macrae, Fraser L.; McPherson, Helen R.; Santos, Nuno C.; Ariёns, Robert A.S.

Plasma fibrinogen includes an alternatively spliced γ-chain variant (γ'), which mainly exists as a heterodimer (γAγ') and has been associated with thrombosis. We tested γAγ' fibrinogen-red blood cells (RBCs) interaction using atomic force microscopy-based force spectroscopy, magnetic tweezers, fibrin clot permeability, scanning electron microscopy and laser scanning confocal microscopy. Data reveal higher work necessary for RBC-RBC detachment in the presence of γAγ' rather than γAγA fibrinogen. γAγ' fibrinogen-RBCs interaction is followed by changes in fibrin network structure, which forms an heterogeneous clot structure with areas of denser and highly branched fibrin fibers. The presence of RBCs also increased the stiffness of γAγ' fibrin clots, which are less permeable and more resistant to lysis than γAγA clots. The modifications on clots promoted by RBCs-γAγ' fibrinogen interaction could alter the risk of thrombotic disorders.

2018Journal article

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AFM imaging-force spectroscopy combination for molecular recognition at the single-cell level

Publication . Carvalho, Filomena Almeida; Santos, Nuno

Molecular recognition at the single-cell level is an increasingly important issue in Biomedical Sciences. With atomic force microscopy, cell surface receptors may be recognized through the interaction with their ligands, inclusively for the identification of cell-cell adhesion proteins. The spatial location of a specific interaction can be determined by adhesion force mapping, which combines topographic images with local force spectroscopy measurements. Another valuable possibility is to simultaneously record topographic and recognition images (TREC imaging) of cells, enabling the mapping of specific binding events on cells in real time. This review is focused on recent developments on these molecular recognition approaches, presenting examples of different biological and biomedical applications.

2018Book part

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The 95RGD97 sequence on the Aα chain of fibrinogen is essential for binding to its erythrocyte receptor

Publication . Carvalho, Filomena Almeida; Guedes, Ana Filipa; Duval, Cedric; Macrae, Fraser L.; Swithenbank, Luke; Farrell, David H.; Ariëns, Robert A. S.; Santos, Nuno C.

BACKGROUND: Erythrocyte aggregation, a cardiovascular risk factor, is increased by high plasma fibrinogen levels. Here, the effect of different fibrinogen mutations on binding to its human erythrocyte receptor was assessed in order to identify the interaction sites. METHODS: Three fibrinogen variants were tested, specifically mutated in their putative integrin recognition sites on the Aα chain (mutants D97E, D574E and D97E/D574E) and compared with wild-type fibrinogen. RESULTS: Atomic force microscopy-based force spectroscopy measurements showed a significant decrease both on the fibrinogen-erythrocyte binding force and on its frequency for fibrinogen with the D97E mutation, indicating that the corresponding arginine-glycine-aspartate sequence (residues 95-97) is involved in this interaction, and supporting that the fibrinogen receptor on erythrocytes has a β3 subunit. Changes in the fibrin clot network structure obtained with the D97E mutant were observed by scanning electron microscopy. CONCLUSION: These findings may lead to innovative perspectives on the development of new therapeutic approaches to overcome the risks of fibrinogen-driven erythrocyte hyperaggregation.

2018Journal article

Open access