“Using the tip of an atomic force microscope (AFM), we have found that the mechanical force required to break the bond between fibrinogen and erythrocytes is higher in patients with chronic heart failure than in healthy subjects,” explains team leader Nuno C Santos from iMM and the Faculty of Medicine at the University of Lisbon. “Erythrocytes in chronic heart failure patients also show changes in their elasticity and behaviour while in the blood stream.”

During a subsequent one-year follow up of the study undertaken by the clinical members of our team, patients in which a higher force was initially required to release the fibrinogen from erythrocytes were also more likely to be hospitalized for cardiovascular-related complications than the other subjects, adds Santos.

Binding forces between fibrinogen and erythrocytes

Cardiovascular diseases are the leading cause of death worldwide, accounting for around one third of all deaths. The number of biomarkers for assessing cardiovascular risk is limited, but researchers have identified fibrinogen, a protein essential for blood clotting, as a relevant risk factor for heart disease. Indeed, high concentrations of fibrinogen in blood plasma appear to make erythrocytes aggregate together more.

Now, Santos and colleagues have used atomic force microscopy to measure the binding forces between fibrinogen and erythrocytes. They did this by allowing a fibrinogen molecule attached to the tip of the AFM to bind to its specific erythrocyte membrane receptor. They then calculated the forces needed to break fibrinogen molecules and erythrocytes apart.

In a separate experiment, the researchers also determined the stiffness of erythrocytes by measuring how deeply the AFM tip was able to penetrate into these cells.

Fibrinogen-erythrocytes modified in heart failure patients

“Using an AFM in ‘force spectroscopy’ mode, we in fact used a single fibrinogen molecule as a ‘bait’ to ‘fish’ its receptor on the erythrocytes’ surface,” says Santos. “We measured the strongest fibrinogen-erythrocyte interactions for ischaemic chronic heart failure patients. Weaker forces were measured for non-ischaemic chronic heart failure patients and the weakest in the healthy control subjects.

“As for the elasticity studies, erythrocytes from non-ischaemic patients had a higher average stiffness than those of the other two groups. Interestingly, we observed that the AFM tip penetrated much more deeply into the cells of ischaemic chronic heart failure patients at the same applied force.”

The work highlights the importance of fibrinogen-erythrocyte interactions in patients with chronic heart disease, and our technique can be used to identify patients at higher risk for this disease,” he tells nanotechweb.org. “We have shown that these interactions, as characterized by AFM-based force spectroscopy, are modified in heart failure patients and that they could also predict a patient’s health in the future. The proof: those presenting a higher AFM-measured fibrinogen-erythrocyte binding force had a higher probability of being admitted to hospital with cardiovascular-related complications in the 12 months following our AFM assessment.”

The researchers, reporting their work in Nature Nanotechnology doi:10.1038/nnano.2016.52, say they now need to extend their study to a larger number of patients (only 45 were followed in this pilot experiment) so they can “translate their findings into clinical applications”.