Fibrin Formation, Structure and Properties

Abstract

Fibrinogen and fibrin are essential for hemostasis and are major factors in thrombosis, wound healing, and several other biological functions and pathological conditions. The X-ray crystallographic structure of major parts of fibrin(ogen), together with computational reconstructions of missing portions and numerous biochemical and biophysical studies, have provided a wealth of data to interpret molecular mechanisms of fibrin formation, its organization, and properties. On cleavage of fibrinopeptides by thrombin, fibrinogen is converted to fibrin monomers, which interact via knobs exposed by fibrinopeptide removal in the central region, with holes always exposed at the ends of the molecules. The resulting half-staggered, double-stranded oligomers lengthen into protofibrils, which aggregate laterally to make fibers, which then branch to yield a three-dimensional network. Much is now known about the structural origins of clot mechanical properties, including changes in fiber orientation, stretching and buckling, and forced unfolding of molecular domains. Studies of congenital fibrinogen variants and post-translational modifications have increased our understanding of the structure and functions of fibrin(ogen). The fibrinolytic system, with the zymogen plasminogen binding to fibrin together with tissue-type plasminogen activator to promote activation to the active proteolytic enzyme, plasmin, results in digestion of fibrin at specific lysine residues. In spite of a great increase in our knowledge of all these interconnected processes, much about the molecular mechanisms of the biological functions of fibrin(ogen) remains unknown, including some basic aspects of clotting, fibrinolysis, and molecular origins of fibrin mechanical properties. Even less is known concerning more complex (patho)physiological implications of fibrinogen and fibrin.

Keywords: Blood clot; Clot mechanical properties; Fibrin formation; Fibrin polymerization; Fibrin properties; Fibrin structure; Fibrinogen composition; Modulation of clot structure; Molecular mechanisms of fibrinolysis; α-Helical coiled-coil.

Figures

Fig. 13.1

Basic scheme of fibrin clot formation and fibrinolysis and the balance between these processes. The clot is formed via a cascade of enzymatic reactions that activates prothrombin to the proteolytic enzyme thrombin, which converts soluble fibrinogen to make insoluble fibrin, the process referred to as blood clotting. The fibrin clot is dissolved through fibrinolysis or cleavage by the proteolytic enzyme plasmin, resulting in fibrin degradation products (FDPs). Plasmin is formed on the fibrin surface from the zymogen plasminogen by plasminogen activators. There is a balance between clotting and fibrinolysis such that excess clotting can lead to thrombosis, while excess fibrinolysis can lead to bleeding

Fig. 13.2

Fibrinogen structure. (a) The atomic resolution structure of about two-thirds of the fibrinogen molecule has been determined by X-ray crystallography (PDB Entry: 3GHG). Fibrinogen and its parts are shown with addition of portions missing from the crystal structure reconstructed computationally, namely the amino terminal ends of the Aα and Bβ chains with FpA and FpB in the central nodule and the beginning of the αC regions. (b) Schematic diagram of the polypeptide chains of fibrinogen. The Aα, Bβ and γ chains are represented by lines with lengths proportional to the number of amino acid residues in each chain and various structural regions are labeled (Zhmurov et al. 2011, with permission from Elsevier Ltd.)

Fig. 13.3

Schematic diagram of fibrin polymerization. Fibrinopeptides in the central nodule cover knobs that are complementary to holes that are always exposed at the ends of the protein. When the fibrinopeptides are removed by thrombin, knob-hole interactions occur, giving rise to oligomers (a trimer is shown), which elongate to produce the two-stranded protofibrils made up of half-staggered molecules. The protofibrils aggregate laterally to make fibers, a process enhanced by interactions of the αC regions and formation of the αC-polymers. The fiber has a 22.5 nm periodicity as a result of half-staggering of 45-nm molecules. At the bottom of the diagram, branch points have been initiated by the divergence of two protofibrils (right) and splitting of each strand of a single protofibril (left) (Weisel and Litvinov 2013; Weisel and Dempfle 2013)

Fig. 13.4

Complementary binding sites or knob-hole interactions in fibrin polymerization. Top. Schematic diagram of knob-hole interactions. Knobs ‘A’ and ‘B’ in the central domain of a fibrin monomer are complementary to holes ‘a’ and ‘b’ that are always exposed at the ends of the protein. When the fibrinopeptides are removed by thrombin, exposing the knobs, knob-hole interactions occur, giving rise to the trimer shown and eventually to the two-stranded protofibril made up of half-staggered molecules. Bottom. Atomic resolution structure of the knob-hole interactions. The γ- and β-nodules near the ends of the molecule contain the holes ‘a’ and ‘b’, respectively, that are complementary to the knobs ‘A’ and ‘B’ in the central nodule. Most of these structures were derived from X-ray crystallographic data, although the disordered and/or flexible N-terminal regions of the α and β chains were derived from computational modeling (with permission from Elsevier Ltd.)

Fig. 13.5

Atomic force microscopy images of fibrinogen, fibrin oligomers, and protofibrils and reconstruction of a protofibril model. (a–i). Images by high-resolution atomic force microscopy (Published with permission and thanks to Drs. Anna D. Protopopova, Nikolay Barinov, Dmitry Klinov). All magnification bars = 30 nm. (a–c). Fibrin monomer with visible αC regions. (d). Fibrin dimer. (e). Fibrin trimer. (f). Fibrin tetramer. (g–h). Fibrin protofibrils. (i). Two protofibrils aggregating laterally. On the left, the two protofibrils are diverging, creating a branch-point. (j). Reconstruction of a twisted fibrin protofibril based on the X-ray crystallographic structure of fibrinogen (PDB Entry: 3GHG). The molecules are shown with addition of missing parts of the crystal structure reconstructed from molecular dynamics simulations, including the full-length αC regions. ‘A-a’ knob-hole bonds that are the major basis of fibrin polymerization are as in Fig. 13.4 (Published with permission and thanks to Dr. Artem Zhmurov)

Fig. 13.6

Fibrin clot network. A 3-dimensional reconstruction of a hydrated fibrin gel obtained using fluorescent confocal microscopy. Fibers are very straight under tension and branch to form a network. Fibrin(ogen) was fluorescently labeled with Alexa 488 (Brown et al. 2009)

Fig. 13.7

Formation of isopeptide bond catalyzed by Factor XIIIa. The chemical reaction catalyzed by Factor XIIIa, yielding insoluble fibrin crosslinked by ε-(γ-glutamyl)-lysine bonds

Fig. 13.8

Stress-strain curve of a fibrin clot. Representative stress-strain curve of a cylindrical fibrin clot reaching greater than a two-fold longitudinal stretch. As the strain increases, the stress on the clot increases linearly until a strain of ~80 % is reached, at which point the sample hardens and enters a new regime with a steeper slope, corresponding to increased stiffness or strain hardening or stiffening. Insets show photographs of the initial clot and stretched clot

Fig. 13.9

Unfolding of fibrin(ogen). A schematic representation of the fibrin(ogen) molecule in the naturally folded (b) and fully unfolded states ((a) and (c)). The molecule is constrained at the C-terminal part of one γ chain, and mechanical force is applied to the C-terminal part of the other γ chain. (a) Shows full unfolding without much detail, while the structural details are given in (c), showing schematically the lengths of the central nodule, γ-nodules, β-nodules, coiled-coils, taking into account the disulfide bonds. Dimensions are shown in the compact crystal structure (b), and the contour lengths of various structural elements are shown in the fully unfolded state (c), assuming a contour length per residue of 0.38 nm (Zhmurov et al. 2011, with permission of Elsevier Ltd.)

Fig. 13.10

Unfolding of the coiled-coils of fibrin. (a). scanning electron micrograph of a fibrin clot, with a box enclosing part of a fiber. (b). A transmission electron micrograph of a negatively contrasted fibrin fiber showing the ultrastructure, with the 22.5 nm repeat arising from the half-staggering of 45-nm molecules. (c). A fibrin trimer from X-ray crystallographic data and molecular dynamics simulations of regions not present in the crystal structure. (d). Fibrin α-helical coiled coils undergoing a forced transition from α-helix to β-sheet. The mechanical transition from α-helical coiled coils to β-sheets in the fibrin(ogen) molecule was characterized using molecular dynamics simulations of their forced elongation and theoretical modeling (Adapted with permission from Zhmurov et al. 2012. Copyright 2012 American Chemical Society)

Fig. 13.11

Schematic diagram of fibrinolysis on the fibrin clot surface and in the liquid phase. Schematic representation of the major reactions of fibrinolysis and their regulation on a fibrin clot surface and in the surrounding plasma milieu. The grey highlighted area represents a fibrin clot surface (solid phase) surrounded by the blood plasma (liquid phase). Black arrows show the biochemical conversions involving proteolytic cleavage. T-like symbols indicate inhibitory effects. Abbreviations and functions of molecules: Lys-Fibrin C-terminal lysine residues on fibrin to which Plg and t-PA bind selectively, Plg plasminogen, bound to the C-terminal lysine residues on fibrin and free in plasma, Pn plasmin, formed on fibrin (by the action of t-PA) and in plasma (by the action of tcu-PA) from Plg. Pn cleaves fibrin and fibrinogen, activates scu-PA and TAFI, t-PA tissue-type Plg activator, fibrin-selective Plg activator, bound to fibrin via the C-terminal lysine residues on fibrin, scu-PA single-chain urokinase-type Plg activator (inactive), tcu-PA two-chain u-PA (active), non-fibrin-selective Plg activator, PAI-1 plasminogen activator inhibitor-1, blocks both t-PA and tcu-PA, TAFIa thrombin-activatable fibrinolysis inhibitor (enzymatically active form) that splits off the C-terminal lysine residues from fibrin, thus preventing binding of Plg and t-PA to fibrin, α2-AP α2-antiplasmin, direct Pn inhibitor, forms circulating Pn-α2-AP complexes, FDP fibrin(ogen) degradation products, resulting from cleavage of fibrin or fibrinogen by Pn, D-dimer a proteolytic fragment (degradation product) that is formed by Pn only from crosslinked fibrin