We have provided a structural foundation for fibrin mechanics at the molecular scale (~10–100 nm) and at different levels of complexity varying from the isolated structural elements (γ-nodule, D region, and D-dimer) to the full-length fibrin(ogen) monomer and oligomers. This information is important to understand fibrin(ogen) nanomechanics and has far reaching implications for the (patho)physiology of fibrin clots and thrombi, the key contributors to life-threatening hemostatic and thrombotic disorders. Using single-molecule AFM unfolding along with a Cα-based SOP model and Langevin simulations accelerated ~200-fold on graphics processors, we were able to directly compare the theoretical and experimental data, generated under the same force-load, to unambiguously interpret the experimental protein unfolding patterns.
The central result of our studies is that fibrin(ogen) forced unfolding is a collective process, which involves a number of mechanically coupled structural elements of the fibrin(ogen) molecule (). Mainly, the process involves reversible extension-contraction of the α-helical coiled-coils, connecting the distal γ-nodules and the central nodule, and sequential dissociation and unraveling of the compact globular structural elements in the γ-nodules. In each γ-nodule, first, the C-terminal β strand (residues γ380–392) peels off from the five-stranded antiparallel β sheet in the central domain. This is followed by unraveling of residues γ234–311, leading to the separation of the γ-nodule into two globular parts, the C-terminal portion (residues γ311–380) and the N-terminal part (residues γ139–234). Each of these domains might unfold second or third with an equal probability (). These consecutive transitions of type 1, 2, and 3 summarized in Table S3
correspond to the force signals observed in the simulated force spectra at an average fibrin(ogen) elongation of ~33, ~28, and ~25 nm (). It is especially important that the experimental unfolding force spectra (; Figure S1
) and simulated force-extension profiles (), indeed, look very similar and that the experimental and theoretical estimates of the average peak-to-peak distance of ~31 and ~28 nm agree quite well (). It is noteworthy that fibrin(ogen) and its smaller and larger derivatives invariably followed the same reproducible unfolding patterns, indicating that the overall unfolding mechanism is robust (deterministic) rather than variable (stochastic).
Detachment of the C
-terminal β strand and its structural consequences observed in simulations confirm the “pull-out hypothesis” (Yakovlev et al., 2000a
; Yakovlev et al., 2001
), stating that the β strand (residues γ380–392) inserted into the central domain of the γ-nodule, can be removed (“pulled-out”) without destroying its compact structure; yet, such structure without the β strand insert is significantly destabilized. Our results show that the applied force first pulls out the β strand, after which the γ-nodule becomes unstable and falls apart (type 1 transition).
There is a good agreement between the results of thermal and force-driven unfolding of the γ-nodule. The enthalpy of melting of fibrinogen molecules ΔHm
= 1365 kcal/mol measured by (Privalov and Medved, 1982
) at pH 8.5 is relatively close to the combined enthalpy of forced unfolding of two D regions, 1384 kcal/mol, but is smaller than the enthalpy of mechanical unfolding of the full-length fibrin(ogen) monomer, ΔHFg
= 1520 kcal/mol, determined in this study (). Our estimates are relatively close to the value of ΔHm
= 1452 kcal/mol observed at pH = 6.0 by (Mihalyi and Donovan, 1985
). The small disagreement is likely due to the difference between the mechanisms of thermal denaturation and of mechanical elongation and unfolding.
The α-helical coiled-coils in fibrin(ogen) act as highly elastic molecular capacitors. Reversible unfolding-refolding transitions enable the coiled-coils to store the mechanical energy and smooth out the effect of external perturbation. The synergy between the coiled-coil connectors and γ-nodules makes coiled-coils able to gradually accumulate mechanical tension before and reduce tension after an unfolding transition (of types 1–3) has occurred in the γ-nodules. The dynamic signature of this coupling is large, 5–7 nm stochastic fluctuations in the length of coiled-coils, which accompany unfolding transitions in the γ-nodules (Figure S2B
), and large standard deviations of the peak-to-peak distances especially for the transitions of type 2 and 3 (; Table S3
). In contrast with this new notion on the mechanical role of the coiled-coil-connectors, it has been suggested previously (Brown et al., 2007
; Lim et al., 2008
) that this region is fully unfolded and is primarily responsible for force-induced elongation of fibrin(ogen). However, this hypothesis seems to be unjustified for the following reasons. First, as discussed above, the complexity of fibrinogen requires that unfolding of domains of the γ-nodule also be considered. Second, the experimental AFM traces reported in one study (Lim et al., 2008
) displayed unrealistic nanoNewton pulling forces. Third, in MD simulations (Lim et al., 2008
), the pulling force was applied to the end of the coiled-coil region, and hence, no information about possible unfolding in the γ-nodule could be gathered. Fourth, the force-loading rate used in these simulations (Lim et al., 2008
) was ~8 orders of magnitude higher than in their AFM experiments, making experimental and simulation results difficult to compare.
In addition to the major roles played by the γ-nodules and the coiled-coil connectors, the β-nodule, which remains unaffected by mechanical tension, was found to provide intramolecular stabilization to the γ-nodule through noncovalent interdomain interactions, which agrees with earlier findings by Yakovlev et al. (2000b)
and Medved et al. (2001)
. This stabilizing effect is reflected in the emergence of an additional force signal in the force-extension curves for the (γ-nodule):(β-nodule) complex (). The central nodule in the E region is the most mechanically stable portion of the fibrin(ogen) molecule as it yields last at >300 pN pulling force, long after the γ-nodules are fully unfolded and the coiled-coil connectors are maximally stretched. Because of the presence of disulfide rings (), forced elongation of the central nodule leads to a small ~7nm extension of the fibrin(ogen) molecule (). There were missing parts of the human fibrinogen molecule (i.e., at the N- and C-terminal portions of the Aα,Bβ, and γ chains), which were not resolved in the crystal structure (3GHG) and hence not included in the simulations. However, these parts do not seem to directly affect the fibrin(ogen) unfolding mechanism because they do not possess compact structures detectable by X-ray crystallography. Hence, these parts are not expected to generate force signals, although, potentially, they might affect unfolding scenarios indirectly.
The αC region has been suggested to be involved in fibrin unfolding and there is considerable experimental evidence that the αC regions play an important role in determining the mechanical properties of fibrin clots (Collet et al., 2005
; Houser et al., 2010
; Tsurupa et al., 2009
; Doolittle and Kollman, 2006
; Falvo et al., 2008
). Clots made from α251 fibrinogen are considerably less stiff than those made from normal fibrinogen and show more plastic deformation (Collet et al., 2005
). Furthermore, the mechanical properties of individual fibers made from fibrinogen of different animal species are strongly dependent on the lengthof the αC regions of their fibrin (Falvo et al., 2008
). Finally, several models for the mechanical properties of fibrin suggest a role for the αC regions in the observed macroscopic stiffness (Falvo et al., 2010
; Purohit et al., 2011
). We tested the potential importance of this portion of the molecule by probing monomeric Fg α251 and oligomeric Fg I-9, fibrinogen derivatives without the αC region (Collet et al., 2005
; Mosesson and Sherry, 1966
). The AFM unfolding patterns of both truncated fibrinogen variants were indistinguishable from the full-length Fg, indicating that the αC regions are not involved in the unfolding of fibrinogen monomers or single-chain oligomers. However, there is no conflict between the literature and experiments reported here, because the effects of this part of the molecule are most likely to be mediated through αC-αC interactions, which are present in the fibers/clots but not in Fg or Fgn
. In addition to the αC-αC interaction, there are at least two more important features of a fibrin oligomer not considered in this study, which are the A:a and B:b knob-hole interactions. Further work is in progress with more complex molecular models, such as double-stranded fibrin oligomers containing the knob-hole bonds and the αC-polymers.
The distance x‡ between the minimum (folded state) and the transition state barrier (maximum) in the free energy landscape is an important molecular characteristic of mechanical unfolding of proteins. We estimated x‡ for fibrinogen unfolding by analyzing transient structures of the D region (γ-nodule-β-nodule complex) for the folded state and the transition state for each transition of type 1–3. We found that these transitions were characterized by a long x‡ ≈ 1 nm. We tested whether intermolecular interactions in oligomeric and polymeric fibrin could serve as an additional source of mechanical stability. Because the D-D interface was found to open first at an average force of ~40 pN, which resulted in an average extension of ~5nm (), this transition does not seem to alter the mechanics and kinetic pathways of unfolding of fibrin(ogen) monomers and oligomers. Hence, most likely, individual fibrin(ogen) molecules forming fibrin(ogen) polymers unfold independently.
Our results are directly comparable to the parameters of fibrin stretching observed in the experiments on single fibrin fibers, in which the estimated mechanical force exerted on fibrin(ogen) monomer was found to be about 140 pN (Liu et al., 2010
). This is a force large enough to unfold the γ-nodules and partially stretch the coiled-coils, as inferred from our AFM experiments and pulling simulations ( and ). According to our data, at tensile forces of ~125-165 pN the fibrin(ogen) monomer is extended by ~160-170 nm (), which is 360%–380% extension of the initial 45 nm length of the molecule. Hence, taking into account the half-staggered geometry of fibrin protofibrils forming a single fibrin fiber, we find that in a fibrin fiber at 100% strain, either a fraction (~26%–28%) of fibrin monomers forming fibrin protofibrils are fully unfolded, or all fibrin units are partially stretched on average by ~45 nm (100%-elongation), or elongation of fibrin fibers is a combination of these states.
Distinguishing between these possibilities precisely is challenging, but previous small angle X-ray scattering measurements on stretched fibrin clots (Brown et al., 2009
) show that even at strains up to 1.0, there remains a population of molecules with the characteristic 22.5 nm spacing of folded half-staggered fibrin. This observation is inconsistent with significant gradual extension of the coiled-coils because this would lead to an increase in this spacing and therefore a shift in the X-ray diffraction peak. Although there was no shift in the diffraction pattern, there was an increase in the peak width indicative of an increase in sample disorder. This was attributed to a two-state like unfolding of the coiled-coils, but our new results suggest the alternative interpretation that the stretching and increased disorder are primarily due to unfolding of domains within the γ-nodules. Because coiled-coils in other systems have previously been shown to refold rapidly (Schwaiger et al., 2002
), the unfolding of globular domains in the γ-nodule might also explain the slow refolding observed after fibrin clot relaxation (Brown et al., 2009
These results are essential for understanding the mechanical properties of blood clots, where it has been demonstrated that unfolding is necessary to account for fibrin’s unusual properties. According to a recent constitutive model for the mechanical properties of fibrin, initially fibrin stiffness is small because early stages of tensile deformation are accommodated primarily by alignment of fibers along the direction of strain (Brown et al., 2009
; Purohit et al., 2011
). However, significant amounts of unfolding start at strains of about 0.15–0.2, and strain stiffening occurs at strains around 1.0 because of chain stretching. Direct experimental evidence for unfolding of fibrin in clots comes from both observation of changes in X-ray fiber diffraction from a pattern consistent with an α-helical coiled-coil to β sheet transition (Bailey et al., 1943
) and measurements of changes in staining by Congo Red, specific for β sheet structure (Purohit et al., 2011
). All of these results are entirely consistent with the new results on unfolding of fibrin(ogen) presented here. The general features of the simulated atomic force microscopy unfolding experiment derived from a discrete version of the model (Purohit et al., 2011
) are qualitatively in agreement with these results, but the peak forces are smaller, because the calculations assume equilibrium with no rate dependence, and the distances are slightly different because of the model parameters.
The mechanical unfolding of the D region and full-length fibrinogen monomer is mostly an enthalpy driven process (). The enthalpy required to disrupt the network of native contacts stabilizing the D region is ΔHD ≈ 692 kcal/mol, which is the sum of ΔH1 ≈ 257 kcal/mol (type 1 transition), ΔH2 ≈ 218 kcal/mol kcal/mol (type 2 transition), and ΔH3 ≈ 217 kcal/mol (type 3 transition). The enthalpy change associated with unraveling of the D regions is roughly half the value for the full-length fibrinogen monomer (ΔHFg = 1520 kcal/mol). We found that ΔH accounts for ~90% of the change in Gibbs free energy (ΔG) for the D-dimer and Fg, whereas the entropic contribution (TΔS) is only ~10% (). The entropy change for forced elongation of the coiled-coils accounts for ~25% of ΔG. Hence, most of the energy change is due to unraveling of the D region, and only ~10% is due to elongation of the coiled-coils.
To conclude, we have provided important information and structural characteristics of the dynamic mechanical behavior of fibrin(ogen) at different spatial scales. The molecular mechanism of fibrin(ogen) elongation is shown to be based on the stepwise unraveling of the γ-nodules concomitant with partial stretching and contraction of the coiled-coil connectors. The adjacent β-nodule provides stabilization for the γ-nodule, and, because interdomain interactions at the D-D junction are weak, individualfibrin(ogen) monomers undergo independent unfolding transitions in fibrin(ogen) oligomers and polymers. The GPU-based computational acceleration makes direct comparison of the experimental and simulation results of dynamic force measurements possible. Hence, dynamic signatures for unfolding transitions observed in silico can be used to provide meaningful interpretation and modeling of the force peaks obtained in vitro and to unmask unfolding mechanisms.