Atomic Structural Models of Fibrin Oligomers

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 C_α-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.