Fibrin oligomers and protofibrils are important intermediate products formed early during fibrin polymerization. Resolution of atomic structures of fibrin oligomers and protofibrils is needed to illuminate the mechanisms of the early stages of fibrin formation, including lateral aggregation of protofibrils, and to characterize the remarkable extensibility and viscoelasticity of fibrin fibers (Liu et al., 2010, Litvinov and Weisel, 2017). Yet experimental determination of the structure and characterization of the properties of double-stranded fibrin polymers is difficult due to their elongated shape and highly unstable nature. We employed a powerful combination of the state-of-the-art experimental AFM imaging technique and theoretical approaches to multiscale modeling accelerated on a GPU to gather the atomic-level information about the structure and properties of fibrin oligomers and protofibrils. Using the full-atomic structure of a short fibrin oligomer FO2/3 (Figure 1; Zhmurov et al., 2016), here we have reconstructed the atomic structures of longer fibrin oligomers FO3/4–FO7/8 up to a 19-mer protofibril FP9/10 (Figures 2 and 3; Table 1). These constructs involve γ-γ crosslinking, A:a and B:b knob-hole bonds, and αC regions—all important functional elements of fibrin—and carbohydrates. The double-stranded fibrin structures were successfully validated through the direct comparison with high-resolution AFM images of oligomers and protofibrils (Figures 2 and 3; Data S1 and S2).
Early products of fibrin polymerization are two-stranded oligomers (Fowler et al., 1981, Chernysh et al., 2011, Huang et al., 2014). A common feature of fibrin oligomers visualized with transmission electron microscopy is their twisted helical shape, although reported parameters of the oligomers’ helicity are highly variable. Medved et al. (1990) showed that some protofibrils form twisted structures with a helical pitch of ∼100 nm, whereas other protofibrils are nearly straight. In agreement with this study, we found that the helical pitch decreases from 3,300 nm to 400 nm (Figure 3) following the transition from the straight conformation (PDB: 1N86) to the bent conformation (PDB: 1FZG) of the D:D interface (Figures 3A, 3B, and S2), and the helical radius decreases from 650 nm to 5 nm. This points to the important role played by the D:D interfacial flexibility in early stages of fibrin polymerization. We did not observe a helical pitch <400 nm, which might be due to the experimental conditions used in previous studies (Medved et al., 1990) or to crystal packing forces in the atomic structures used. In most experiments, fibrin protofibrils are straight and thin, which agrees with our results. After reaching a certain length, fibrin protofibrils aggregate laterally to form thick twisted fibers. Fibrin fibers have a 20- to 60-nm helical radius and ∼2,000-nm helical pitch (Weisel et al., 1987), and continue to twist as they grow.
The reconstructed double-stranded fibrin oligomers and protofibrils correspond to the known ultrastructures, but surpass the available experimental data in spatial resolution. We are aware of other models of fibrin protofibrils (Yang et al., 2000, Pechik et al., 2006, Huang et al., 2014). The model proposed by Yang et al. (2000)captures the main structural features of fibrin oligomers, including the half-staggered molecular overlay formed by two fibrin strands. However, this model is ad hoc rather than systematic, and lacks a detailed analysis of the crystal forms. This model provides a static view of fibrin structure with missing γ-γ crosslinking, A:a and B:b knob-hole bonds, and αC regions. Another model (Huang et al., 2014) captures the half-staggered molecular arrangement of fibrin strands, and has a helical pitch of 90 nm. Yet a closer look reveals major steric clashes in the DED regions, which make the A-a bond formation unlikely. Also, this model is lacking γ-γ crosslinking, knob-hole bonds, and αC regions, and is based on the chicken fibrinogen structure (PDB: 1M1J).
We employed equilibrium MD simulations of fibrin protofibril FP9/10 to explore the dynamic structural transitions that occur in double-stranded fibrin polymers. Fibrin protofibrils behave like other double-stranded biopolymers, such as double-stranded DNA, but with a longer 320-nm persistence length, and show a high degree of bending. A theoretical probability density curve shows that due to bending, the end-to-end distance in FP9/10 decreases from ∼400 nm (contour length) to ∼330 nm (Figure 4F). This might be important for the fiber formation and fiber branching (Figure 4). Experimental AFM images reveal more bending flexibility than the in silico structures, and the protofibril’s bending becomes more pronounced with increasing length. We also observe reversible kinking of the protofibril backbone with the 80° to 140° kinking angle range and an average kinking angle of 115° (Figure 4G). Protofibril bending and kinking could be one of the mechanisms of initiation of branch points in growing fibrin fibers.
In the simulations, we observed the dissociation of A:a knob-hole non-covalent bonds, which was followed by the disruption of the D:E:D interface. This suggests a secondary role played by the D:D interface in fibrin polymerization, and also potentially supports the so-called Y-ladder model of fibrin fiber growth (Rocco et al., 2014). According to this model, the D:E:D interface becomes stable only after both knobs “A” are bound to their corresponding holes “a” (Rocco et al., 2014). Since upon the disruption of D:E:D interface and D-region rotation one A:a knob-hole bond is completely dissociated, a knob “A” and a hole “a” might become available for inter-protofibril cross-coupling. Another fibrin monomer from another protofibril could then bind, thus initiating a branch point. Formation of branch points is visible in some of the protofibril images (Figure 4E). We did not observe formation of new A:a knob-hole bonds in the millisecond timescale of simulations, hence this transition occurs in a longer timescale.
The length of αC connectors is very important for mechanical properties of fibrin fibers (Falvo et al., 2008). Since the αC domains are capable of interacting with each other (Tsurupa et al., 2011, Tsurupa et al., 2012, Litvinov et al., 2007) and with the globular parts of fibrin molecules (Tsurupa et al., 2009), it is important that they have an optimal length. When the αC connectors are long (as in human fibrinogen) their αC domains tend to form non-covalent bonds with other αC domains within the same protofibril and between the protofibrils. When the αC connectors are short (as in chicken fibrinogen), the αC domains hardly form binding contacts between protofibrils but only within the protofibril. We see in the simulations and in AFM images that the span of the αC regions is long enough so that the αC domains can form the αC-αC contacts within the protofibril and between protofibrils (Figure 5). The experimental histogram of the lengths of αC region shows the 10- to 35-nm range and an average length of 17.3 nm; the theoretical probability density curve reveals a smaller 10- to 20-nm range and a similar average length of 14.7 nm. This large variability also explains why in AFM images were on average 1.6 αC regions per fibrin monomer (<2). Our results imply that the conformational dynamics of αC regions plays a role in defining the thickness of fibrin fibers (number of protofibrils in a fiber).
The physiological role of B:b knob-hole bonds is not yet fully understood (Weisel and Litvinov, 2017). Although knobs “B” are long enough to reach and bind to the corresponding holes “b” in the same protofibril, our simulations of FP9/10 with free knobs “B” show that they have a limited span due to thermal fluctuations (Figure 5E). Hence, formation of intra-protofibril B:b contacts is possible only when Nβ regions are close to the globular parts of fibrin, interacting with γ and β nodules of adjacent molecules (Moskowitz and Budzynski, 1994). These interactions can guide the knob “B” to the hole “b” or/and to the thrombin active-site cleft (Pechik et al., 2006). Upon formation of B:b knob-hole bonds, the β nodule dissociates from the α-helical coiled coil, which results in the exposure of the tissue plasminogen activator and plasminogen binding cites in the coiled coil (Medved et al., 2001). This transition might help to bring holes “b” of adjacent protofibril closer to knobs “B,” thus facilitating the inter-protofibril contacts’ formation. Our simulations for protofibril FP9/10 suggest that the intra-protofibril B:b contacts are less probable than the inter-protofibril B:b contacts, which also explains why the formation of fibrin fibers occurs even in the absence of knobs “B” (Moskowitz and Budzynski, 1994, Weisel, 1986).
We calculated the molecular hydrodynamic parameters for double-stranded fibrin oligomers and protofibrils, which are not available experimentally (Table 1), and extracted the scaling laws for Rg, D, η, and ρ as functions of their size N (number of fibrin monomers; Figure 6). The protein density ρ was found to depend on Nexponentially (Figure 6C) in full agreement with the predictions made by Fischer et al. (2004). The profile of Rg shows a linear increase with N starting from 5 to 7 monomers (Figure 6A), because at larger N fibrin oligomers are pseudo-one-dimensional with size growing with N. This also explains why the intrinsic viscosity ηincreases quadratically with N. According to the Flory theory, η = ΦRg3/M, where Φis a universal constant and M is the molar mass (Doi and Edwards, 1986). Indeed, for a linear polymer Rg ∼ N and M ∼ N, and so η ∼ N2 (Figure 6D).
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