Abstract 2217

A new field of biomedical research, biomechanics of hemostasis and thrombosis, has been quickly developing over the past few years. The mechanical properties of fibrin are essential in vivo for the ability of clots to stop bleeding in flowing blood but also determine the likelihood of obstructive thrombi that cause heart attack and stroke. Despite such critical importance, the structural basis of clot mechanics is not well understood. The structural changes underlying deformation of fibrin polymer occur at different spatial scales from macroscopic to submolecular, including molecular unfolding, about which relatively little is known. In this work, fibrin mechanics was studied with respect to molecular structural changes during fibrin deformation. The results of atomic force microscopy-induced unfolding of fibrinogen monomers and oligomers were correlated with force-extension curves obtained using Molecular Dynamics simulations. The mechanical unraveling of fibrin(ogen) was shown to be determined by molecular transitions that couple reversible extension-contraction of the α-helical coiled-coil regions with unfolding of the terminal γ-nodules. The coiled-coils act as molecular springs to buffer external mechanical perturbations, transmitting and distributing force as the γ-nodules unfold. Unfolding of the γ-nodules, stabilized by strong inter-domain interactions with the neighboring β-nodules, was characterized by an average force of ∼90 pN and peak-to-peak distance of ∼25 nm. All-atom Molecular Dynamics simulations further showed a transition from α-helix to β-sheet at higher extensions. To reveal the force-induced α-helix to β-sheet transition in fibrin experimentally, we used Fourier Transform infrared spectroscopy of hydrated fibrin clots made from human blood plasma. When extended or compressed, fibrin showed a shift of absorbance intensity mainly in the amide I band but also in the amide II and III bands, demonstrating an increase of the β-sheets and a corresponding reduction of the α-helices. These structural conversions correlated directly with the strain or pressure and were partially reversible at the conditions applied. The spectra characteristic of the nascent inter-chain β-sheets were consistent with protein aggregation and fiber bundling during clot deformation observed using scanning electron microscopy. Additional information on the mechanically induced α-helix to β-sheet transition in fibrin was obtained from computational studies of the forced elongation of the entire fibrin molecule and its α-helical coiled-coil portions. We found that upon force application, the coiled-coils undergo ∼5–50 nm extension and 360-degree unwinding. The force-extension curves for the coiled-coils showed three distinct regimes: the linear elastic regime, the constant-force plastic regime, and the non-linear regime. In the linear regime, the coiled-coils unwind but not unfold. In the plastic regime, the triple α-helical segments rewind and re-unwind while undergoing a non-cooperative phase transition to form parallel β-sheets. We conclude that under extension and/or compression an α-helix to β-sheet conversion of the coiled-coils occurs in the fibrin clot as a part of forced protein unfolding. These regimes of forced elongation of fibrin provide important qualitative and quantitative characteristics of the molecular mechanisms underlying fibrin mechanical properties at the microscopic and macroscopic scales. Furthermore, these structural characteristics of the dynamic mechanical behavior of fibrin at the nanometer scale determine whether or not clots have the strength to stanch bleeding and if thrombi become obstructive or embolize. Finally, this knowledge of the functional significance of different domains of fibrin(ogen) suggests new approaches for modulation of these properties as potential therapeutic interventions.


No relevant conflicts of interest to declare.

Author notes


Asterisk with author names denotes non-ASH members.