- Atomic structures of fibrin oligomers and protofibrils are reconstructed in silico
- Structural models show good agreement with high-resolution AFM images
- Fibrin protofibrils are double-stranded twisted oligomers that can bend and kink
- Hydrodynamic parameters of fibrin oligomers and protofibrils are calculated
The space-filling fibrin network is a major part of clots and thrombi formed in blood. Fibrin polymerization starts when fibrinogen, a plasma protein, is proteolytically converted to fibrin, which self-assembles to form double-stranded protofibrils. When reaching a critical length, these intermediate species aggregate laterally to transform into fibers arranged into branched fibrin network. We combined multiscale modeling in silico with atomic force microscopy (AFM) imaging to reconstruct complete atomic models of double-stranded fibrin protofibrils with γ-γ crosslinking, A:a and B:b knob-hole bonds, and αC regions—all important structural determinants not resolved crystallographically. Structures of fibrin oligomers and protofibrils containing up to 19 monomers were successfully validated by quantitative comparison with high-resolution AFM images. We characterized the protofibril twisting, bending, kinking, and reversibility of A:a knob-hole bonds, and calculated hydrodynamic parameters of fibrin oligomers. Atomic structures of protofibrils provide a basis to understand mechanisms of early stages of fibrin polymerization.
Fibrin is an end product of blood clotting that forms the scaffold of hemostatic clots and obstructive thrombi in blood vessels. Fibrin is also a major component of the extracellular matrix and is involved in a broad range of cellular processes, including cell adhesion, migration, proliferation and differentiation, wound healing, angiogenesis, and inflammation (Weisel and Litvinov, 2017, Litvinov and Weisel, 2017). Fibrin is widely used as a versatile biomaterial in a variety of applications, such as hemostatic sealants, tissue engineering, as a delivery vehicle for cells, drugs, growth factors, and genes, and matrices for cell culturing (Janmey et al., 2009, Radosevich et al., 1997). Because of the fundamental biological and medical importance, molecular mechanisms of fibrin formation as well as fibrin structure and properties continue to be major areas of research (Weisel and Litvinov, 2013, Weisel and Litvinov, 2017, Litvinov and Weisel, 2016).
Fibrin formation is initiated by the cleavage of fibrinopeptides A and B from the N termini of Aα and Bβ chains of fibrinogen, respectively, to produce fibrin monomer. The release of fibrinopeptides A exposes an N-terminal α-chain motif GPR, called knob “A”, which binds to constitutively exposed hole “a” in the γ nodule of another fibrin molecule (Everse et al., 1998, Kostelansky et al., 2002), resulting in the formation of an A-a knob-hole non-covalent bond (Litvinov et al., 2005). Exposure of knobs “A” is necessary and sufficient to form fibrin through the interaction with holes “a.” The release of fibrinopeptides B exposes an N-terminal β-chain motif GHRP, called knob “B”, which is complementary to hole “b” located in the β nodule of another fibrin molecule.
Fibrin polymerization begins when two monomeric fibrin molecules interact in a half-staggered fashion through the A-a knob-hole interaction. The addition of a third molecule is accompanied by an end-to-end association where, in addition to the A-a knob-hole interactions, the globular D regions of two adjacent molecules form the D:D interface. The D:D interface provides a junction between the monomers in one of the two strands in a fibrin trimer. Furthermore, fibrin monomers add longitudinally via the inter-strand A-a knob-hole bond formation and intra-strand D-D interactions to form fibrin oligomers. This growth continues until the fibrin oligomers reach the critical length of protofibrils: oligomers made of ∼20–25 fibrin monomers. Fibrin protofibrils self-associate laterally to form twisted fibers of variable thickness. These branches form a three-dimensional fibrin network called a clot (Weisel and Litvinov, 2017).
Figure 1 | From Fibrin Oligomer to Fibrin Protofibril Based on Computational Crystallomics
The monomeric fibrin is essentially identical in structure and composition to fibrinogen except for small fibrinopeptides A and B, which are cleaved when fibrinogen is converted to fibrin, and αC domains, which are bound to the central nodule in fibrinogen but detached in fibrin (Medved et al., 2001). Therefore, fibrin oligomers and protofibrils can be reconstructed using resolved crystal structures of the human fibrinogen molecule and parts of fibrinogen and fibrin molecules, including the fibrinogen fragment D and the double-D fragment from crosslinked fibrin (see Table S1). Yet using the crystal structures of fibrinogen or fibrin [together denoted as fibrin(ogen)] is challenging. First, the crystallographic data available are incomplete. There are several flexible unstructured portions that are not resolved crystallographically yet are essential for fibrin formation, including residues 1–26 and 1–57 at the N-termini of the Aα and Bβ chains, respectively, and residues 201–610, 459–461, and 395–411 at the C-termini of the Aα, Bβ, and γ chains, respectively (Kollman et al., 2009). Second, a manifold of possible spatial arrangements of fibrin monomers when forming a protofibril makes in silico reconstruction of fibrin protofibril difficult. Third, the large system size requires using vast computational resources: a 0.5- to 0.6-μm-long protofibril made of 20 fibrin monomers contains ∼60,000 amino acids, which corresponds to ∼106 atoms.
Determination of atomic structures of fibrin oligomers cannot be accomplished by X-ray crystallography and/or electron microscopy, owing to the unstable nature of these heterogeneous intermediate supramolecular assemblies and their characteristic elongated shape. Yet atomic-level information about these structures is necessary to elucidate the mechanisms of formation and properties of fibrin polymers, which provide the three-dimensional scaffold necessary to maintain the integrity and viscoelasticity of blood clots and thrombi. Here we employed multiscale modeling to computationally reconstitute the atomic structures of double-stranded fibrin oligomers of varying length. The atomic structural models were successfully validated using high-resolution atomic force microscopy (AFM) imaging of fibrin oligomers.
We employed the molecular dynamics (MD) simulations of atomic structural models (Brooks et al., 2009, Zhmurov et al., 2012) and Cα-based self-organized polymer (SOP) models of fibrin(ogen) and its fragments (Hyeon et al., 2006), accelerated on graphics processing units (GPUs) (Zhmurov et al., 2010a, Zhmurov et al., 2010b, Zhmurov et al., 2011, Alekseenko et al., 2016), to perform a step-by-step reconstruction of a complete atomic structure of a 19-monomer-long fibrin protofibril using the recently published structure of a short fibrin oligomer (Zhmurov et al., 2016). The protofibril structure has interesting properties, such as twisting, bending, and kinking, and the presence of free knobs “B” necessary for formation of additional intra- and inter-protofibril bonds. The models obtained enabled us to explore the dynamic structural transitions in fibrin protofibrils and to predict experimentally unavailable dynamic characteristics of fibrin oligomers and protofibrils, including density, radius of gyration, diffusion coefficient, and intrinsic viscosity.
Stepwise Reconstruction of Fibrin Oligomers and Protofibril
As a building block, we used the structure of short double-stranded fibrin oligomer FO2/3 with two fibrin monomers in the first and three monomers in the second strands (Figure 1A). Using this structure, we performed stepwise elongation to create longer oligomers up to the length of a protofibril. The full-atomic model of FO2/3 was constructed computationally in our previous study (Zhmurov et al., 2016) using all 27 relevant crystal structures of fibrinogen and its fragments resolved to date (Table S1). The structure of FO2/3 showed good agreement with high-resolution AFM images (Zhmurov et al., 2016). In this work, we took the next step to elongate several-fold the known structure of FO2/3 in order to reconstruct longer fibrin oligomers FOm/n (m/n is the number of fibrin monomers in the first/second strand). This enabled us to recreate short fibrin oligomers from FO2/3 to FO5/6. A mere replication of the FO2/3 structure along the longitudinal axis has resulted in formation of elongated oligomers and protofibrils that do not show any twisting detected in experimental AFM and electron microscopy images (Weisel et al., 1987, Medved et al., 1990, Protopopova et al., 2015, Huang et al., 2014). To overcome this problem, we designed an approach that uses two main crystal structures of the D:D junction. These structures correspond to the straight conformation (PDB: 1N86) of the D:D interface, used to recreate shorter fibrin oligomers, and the bent conformation (PDB: 1FZG) of the D:D interface, which we used to recreate longer oligomers (and a protofibril). To recreate twisted structures, we align two FO2/3constructs using the straight conformation of D-D junctions and then gradually switch to the bent conformation of the D:D junction (STAR Methods). This builds in the desired twist in the fibrin strands, in full agreement with AFM and electron microscopy experiments (Protopopova et al., 2015, Weisel et al., 1987).
A step-by-step reconstruction of short fibrin oligomers (FOm/n) is illustrated in Figure 1B (elongation step with the straight conformation of D:D interface). Reconstruction of longer fibrin oligomers and protofibril (FP9/10) is illustrated in Figure 1C (a procedure to introduce a twist; bent conformation of D:D interface; see also STAR Methods for more details). In step A of the twisting procedure, coarse-graining of FO2/3 is performed (diamonds in Figure 1B) and Langevin simulations of the Cα-based representation of FO2/3 is carried out to switch from the straight to the bent configuration of the D:D interfaces. In step B of the twisting procedure, the obtained conformation of FO2/3 is back-mapped and energy-minimized using the all-atom solvent-accessible surface area model of implicit solvation. Next, we perform the elongation procedure. The atomic model of FO2/3 with knobs “A” and “B” is replicated to reconstruct fibrin oligomers (FOn/m) of the desired length. In the last structure-addition step, the αC regions are incorporated into each fibrin monomer, and the covalent γ-γ crosslinks between residues γ398 and γ406 of abutted fibrin monomers are introduced (Rosenfeld et al., 2015). The final structures of double-stranded fibrin polymers from the structure-addition step (Table 1) were energy-minimized to exclude possible steric clashes.
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