Fibrinogen and Fibrin

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Abstract

Fibrinogen is a large, complex, fibrous glycoprotein with three pairs of polypeptide chains linked together by 29 disulfide bonds. It is 45 nm in length, with globular domains at each end and in the middle connected by α-helical coiled-coil rods. Both strongly and weakly bound calcium ions are important for maintenance of fibrinogen's structure and functions. The fibrinopeptides, which are in the central region, are cleaved by thrombin to convert soluble fibrinogen to insoluble fibrin polymer, via intermolecular interactions of the “knobs” exposed by fibrinopeptide removal with “holes” always exposed at the ends of the molecules. Fibrin monomers polymerize via these specific and tightly controlled binding interactions to make half-staggered oligomers that lengthen into protofibrils. The protofibrils aggregate laterally to make fibers, which then branch to yield a three-dimensional network—the fibrin clot—essential for hemostasis. X-ray crystallographic structures of portions of fibrinogen have provided some details on how these interactions occur. Finally, the transglutaminase, Factor XIIIa, covalently binds specific glutamine residues in one fibrin molecule to lysine residues in another via isopeptide bonds, stabilizing the clot against mechanical, chemical, and proteolytic insults. The gene regulation of fibrinogen synthesis and its assembly into multichain complexes proceed via a series of well-defined steps. Alternate splicing of two of the chains yields common variant molecular isoforms. The mechanical properties of clots, which can be quite variable, are essential to fibrin's functions in hemostasis and wound healing. The fibrinolytic system, with the zymogen plasminogen binding to fibrin together with tissue-type plasminogen activator to promote activation to the active enzyme plasmin, results in digestion of fibrin at specific lysine residues. Fibrin(ogen) also specifically binds a variety of other proteins, including fibronectin, albumin, thrombospondin, von Willebrand factor, fibulin, fibroblast growth factor-2, vascular endothelial growth factor, and interleukin-1. Studies of naturally occurring dysfibrinogenemias and variant molecules have increased our understanding of fibrinogen's functions. Fibrinogen binds to activated αIIbβ3 integrin on the platelet surface, forming bridges responsible for platelet aggregation in hemostasis, and also has important adhesive and inflammatory functions through specific interactions with other cells. Fibrinogen-like domains originated early in evolution, and it is likely that their specific and tightly controlled intermolecular interactions are involved in other aspects of cellular function and developmental biology.

Introduction

Fibrinogen is a fibrous protein that was first classified with keratin, myosin, and epidermin based on its 5.1 Å repeat in wide-angle X-ray diffraction patterns (Bailey et al., 1943), which was later discovered to be associated with the α-helical coiled-coil structure. It is a glycoprotein normally present in human blood plasma at a concentration of about 2.5 g⧸L and is essential for hemostasis, wound healing, inflammation, angiogenesis, and other biological functions. It is a soluble macromolecule, but forms a clot or insoluble gel on conversion to fibrin by the action of the serine proteolytic enzyme thrombin (Fig. 1), which itself is activated by a cascade of enzymatic reactions triggered by injury or a foreign surface. A mechanically stable, protease-resistant clot is necessary to prevent blood loss and promote wound healing.

Fibrinogen is also necessary for the aggregation of blood platelets, an initial step in hemostasis. Each end of a fibrinogen molecule can bind with high affinity to the integrin receptor on activated platelets, αIIbβ3, so that the bifunctional fibrinogen molecules act as bridges to link platelets. In its various functions as a clotting and adhesive protein, the fibrinogen molecule is involved in many intermolecular interactions and has specific binding sites for several proteins and cells.

Fibrin clots are dissolved by another series of enzymatic reactions termed the fibrinolytic system (Fig. 1). The proenzyme plasminogen is activated to plasmin by a specific proteolytic enzyme, typically tissue-type plasminogen activator or urokinase-type plasminogen activator. Plasmin then cleaves fibrin at certain unique locations to dissolve the clot. The activation of the fibrinolytic system is greatly enhanced by taking place on the surface of fibrin. Thus, these reactions are highly specific for cleavage of the insoluble fibrin clot, rather than circulating fibrinogen.

There is a dynamic equilibrium between clotting and fibrinolysis, so that the conversion of fibrinogen to fibrin and the dissolution of the clot must be carefully regulated. Any imbalance can result in either loss of blood from hemorrhage or blockage of the flow of blood through a vessel from thrombosis. Thrombosis, often accompanying atherosclerosis or other pathological processes, is the most common cause of myocardial infarction and stroke. As a consequence, various fibrinolytics that activate plasminogen are now commonly used clinically to treat these conditions.

The clinical significance of fibrin was first recognized by Virchow. In 1859, Deni de Commercy proposed the existence of a precursor of fibrin that he called fibrinogen (Blombäck, 2001). Although Hammarsten first purified fibrinogen from horse plasma in 1876 (Blombäck, 2001), human fibrinogen was isolated in large quantities only fifty years ago (Cohn et al., 1946) and has been extensively studied since then. Fibrinogen and the other components of the clotting system are commonly isolated from human blood plasma, using purification methods based on fibrinogen's low solubility in various solvents or its isoelectric point (Blombäck 1956, Cohn 1946, Laki 1951).

Fibrinogen was last reviewed in this series in 1973 (Doolittle, 1973). However, many other reviews have appeared in the meantime, which have summarized various aspects of the biology and biochemistry of fibrinogen and fibrin (Blombäck 1991, Budzynski 1986, Doolittle 1984, Greenberg 2003, Hantgan 2000, Henschen 1986, Mosesson 2001, Shafer 1988, Tooney 1979, Weisel 1997). There has been an impressive amount of research on all aspects of fibrinogen and fibrin in the past 30 years. This Chapter will provide an outline of some of this body of work with selected references to the relevant literature.

As one indication of the progress, there have been generally accepted resolutions of the three fundamental controversies cited by Doolittle (1973), although of course not everyone agrees with all of these conclusions: (1) while there was then “no general accord on the shape of the fibrinogen molecule,” first electron microscopy and then X-ray crystallography have been used for the determination of the detailed structure of fibrinogen; (2) while “the nature of the forces holding subunit portions of fibrinogen together” was not known, biochemical and structural studies showed the bonds involved; and (3) while “the general location of the fibrinopeptides in the parent molecule” was undecided at that time, now we know where they are in the molecule, although they have not been resolved at atomic resolution. Moreover, we now know a great deal about the process of fibrin polymerization, fibrinogen synthesis, and interactions with plasma proteins and cells.

Section snippets

Physico-Chemical Properties, Amino Acid Sequence, and Disulfide Bonding

The physicochemical characteristics of fibrinogen reflect its nature as a fibrous protein (Table I). Fibrinogen is a large glycoprotein made up of three pairs of polypeptide chains, designated Aα, Bβ, and γ, with molecular masses of 66,500, 52,000, and 46,500 Da, respectively (Fig. 2). The posttranslational addition of asparagine-linked carbohydrate to the Bβ and γ chains brings the total molecular mass to about 340,000 Da. The nomenclature for fibrinogen, (Aα, Bβ, γ)2, arises from the

Biosynthesis and Metabolism of Fibrinogen

Human fibrinogen is the product of three closely linked genes, each specifying the primary structure of one of the three polypeptide chains (Chung 1983, Chung 1990, Crabtree 1987). The ability to express human fibrinogen in mammalian cells provides a means to study the synthesis and secretion of fibrinogen. Site-directed mutagenesis of fibrinogen has established the sequence of steps in fibrinogen assembly. There is a progression from single chains to two-chain complexes to trimeric half

Conversion of Fibrinogen to Fibrin

Fibrin polymerization is initiated by the enzymatic cleavage of the fibrinopeptides, converting fibrinogen to fibrin monomer (Fig. 1). Then, several nonenzymatic reactions yield an orderly sequence of macromolecular assembly steps. Several other plasma proteins bind specifically to the resulting fibrin network. The clot is stabilized by covalent ligation or crosslinking of specific amino acids by a transglutaminase, Factor XIIIa.

Fibronectin and Albumin

Some proteins, such as plasma fibronectin and albumin, interact with fibrin to alter clot structure and properties, although the former becomes crosslinked to fibrin while the latter does not. As a result of these and other interactions, fibrin clots formed in plasma have very different properties than those made with purified proteins (Blombäck 1994, Carr 1988, Shah 1987). Albumin has significant effects on the extent of lateral aggregation, yielding either thicker or thinner fibers depending

Fibrinolysis

The clot is meant to be a temporary plug for hemostasis or wound healing, so there are natural mechanisms in the body for the efficient removal of fibrin. Various proteolytic enzymes and cells can dissolve fibrin depending on the circumstances, but the most specific mechanism involves the fibrinolytic system. The dissolution of fibrin clots under physiological conditions involves the binding of circulating plasminogen to fibrin, and the activation of plasminogen to the active protease, plasmin,

Fibrinogen Binding to Integrins in Platelet Aggregation and Other Cellular Interactions

Fibrin(ogen) binds specifically to integrin receptors on platelets, endothelial cells, and many other cells and plays a vital role in platelet aggregation and other aspects of cellular adhesion. Clot formation is a normal part of the wound healing process, sealing the injury and preventing bleeding. The fibrin clot serves as a scaffold for the migration of various cells, but less is known about this process than about earlier stages of healing. Fibroblasts migrate through the gel and deposit

Dysfibrinogenemias, Variants, and Heterogeneity of Fibrinogen

Dysfibrinogenemias are characterized by structural changes in the fibrinogen molecule that result in detectable alterations in clotting or other properties of the molecule. Traditionally, they are named after the place of their discovery or where the patient lives. Most congenital defects are rare but offer the opportunity to study the effects of these molecular changes on fibrinogen function. These mutations are listed and described in several reviews (Galanakis 1992, Galanakis 1993, Matsuda

Evolution of Fibrinogen and Fibrinogen-Like Domains

The thrombin-catalyzed clotting of fibrinogen to form a fibrin gel is common to all extant vertebrates. Because clots are necessary to stop bleeding, but can also cause thrombosis if not dissolved in a timely fashion, an effective scheme for fibrinolysis evolved concomitantly. The amino acid sequences of the Aα, Bβ, and γ chains are homologous, indicating that they have evolved from a common ancestor (Doolittle 1997, Henschen 1982). This homology even extends to the genomic sequences, with two

Conclusions

There is now much structural information about fibrinogen, including its primary sequence, the chain connectivity via disulfide bonds, and organization into domains. There are X-ray crystallographic structures of large parts of the molecule, but some key regions have not yet been visualized. Some aspects of calcium binding are beginning to be discovered, but less is known of the low affinity sites.

Tremendous strides have been taken in understanding the metabolism and biosynthesis of fibrinogen,

Acknowledgements

This work was supported by NIH grant HL30954. The author thanks Rustem Litvinov, Dennis Galanakis, Leonid Medved, Robert Ariëns, Oleg Gorkun, and Leona Mášová for providing many helpful suggestions and comments on this review, and Igor Pechik for generating Fig. 4.

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