NORMAL GENOMIC

• Matsuda M, Sugo T
• Hereditary disorders of fibrinogen.
• Ann N Y Acad Sci. 2001; 936: 65-88
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• Fibrinogen, a 340-kDa plasma protein, is composed of two identical molecular halves each consisting of three non-identical A alpha-, B beta- and gamma-chain subunits held together by multiple disulfide bonds. Fibrinogen is shown to have a trinodular structure; that is, one central nodule, the E domain, and two identical outer nodules, the D-domains, linked by two coiled-coil regions. After activation with thrombin, a pair of binding sites comprising Gly-Pro-Arg is exposed in the central nodule and combines with its complementary binding site a in the outer nodule of another molecules. By using crystallographic analysis, the alpha-amino group of alpha Gly-1 is shown to be juxtaposed between gamma Asp-364 and gamma Asp-330, and guanidino group of alpha Arg-3 between the carboxyl group of gamma Asp-364 and gamma Gln-329 in the a site. Half molecule-staggered, double-stranded protofibrils are thus formed. Upon abutment of two adjacent D domains on the same strand, D-D self association takes place involving Arg-275, Tyr-280, and Ser-300 of the gamma-chain on the surface of the abutting two D domains. Thereafter, carboxyl-terminal regions of the alpha-chains are untethered and interact with those of other protofibrils leading to the formation of thick fibrin bundles and networks. Although many enigmas still remain concerning the exact mechanisms of these molecular interactions, fibrin assembly proceeds in a highly ordered fashion. In this review, these molecular interactions of fibrinogen and fibrin are discussed on the basis of the data provided by hereditary dysfibrinogens on introducing representative molecules at each step of fibrin clot formation.

• Doolittle RF, Yang Z, Mochalkin I
• Crystal structure studies on fibrinogen and fibrin.
• Ann N Y Acad Sci. 2001; 936: 31-43
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• X-ray crystallography studies on fragments D and double-D from human fibrinogen and fibrin have revealed the details of knob-hole interactions between fibrin units, as well as the nature of the association at their ends. More recently, a lower-resolution structure of native chicken fibrinogen has provided details about the structure of the central domain, and particularly the arrangement of disulfide bonds. Parts of the fibrinogen molecule are so flexible that they have not been visualized in electron density maps. The elusive regions include the alpha C domain, the amino-terminal segments of the alpha and beta chains, and the carboxyl-terminal segments of the gamma chains. Nonetheless, when all the structural data are considered together, it is possible to construct a realistic model not only of a fibrinogen molecule but also of a fibrin protofibril.

• Brown JH, Volkmann N, Jun G, Henschen-Edman AH, Cohen C
• The crystal structure of modified bovine fibrinogen.
• Proc Natl Acad Sci U S A. 2000; 97: 85-90
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• Here we report the crystal structure at approximately 4-A resolution of a selectively proteolyzed bovine fibrinogen. This key component in hemostasis is an elongated 340-kDa glycoprotein in the plasma that upon activation by thrombin self-assembles to form the fibrin clot. The crystals are unusual because they are made up of end-to-end bonded molecules that form flexible filaments. We have visualized the entire coiled-coil region of the molecule, which has a planar sigmoidal shape. The primary polymerization receptor pockets at the ends of the molecule face the same way throughout the end-to-end bonded filaments, and based on this conformation, we have developed an improved model of the two-stranded protofibril that is the basic building block in fibrin. Near the middle of the coiled-coil region, the plasmin-sensitive segment is a hinge about which the molecule adopts different conformations. This segment also includes the boundary between the three- and four-stranded portions of the coiled coil, indicating the location on the backbone that anchors the extended flexible Aalpha arm. We suggest that a flexible branch point in the molecule may help accommodate variability in the structure of the fibrin clot.

• Everse SJ, Spraggon G, Veerapandian L, Doolittle RF
• Conformational changes in fragments D and double-D from human fibrin(ogen) upon binding the peptide ligand Gly-His-Arg-Pro-amide.
• Biochemistry. 1999; 38: 2941-6
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• The structure of fragment double-D from human fibrin has been solved in the presence and absence of the peptide ligands that simulate the two knobs exposed by the removal of fibrinopeptides A and B, respectively. All told, six crystal structures have been determined, three of which are reported here for the first time: namely, fragments D and double-D with the peptide GHRPam alone and double-D in the absence of any peptide ligand. Comparison of the structures has revealed a series of conformational changes that are brought about by the various knob-hole interactions. Of greatest interest is a moveable "flap" of two negatively charged amino acids (Glubeta397 and Aspbeta398) whose side chains are pinned back to the coiled coil with a calcium atom bridge until GHRPam occupies the beta-chain pocket. Additionally, in the absence of the peptide ligand GPRPam, GHRPam binds to the gamma-chain pocket, a new calcium-binding site being formed concomitantly.

• Doolittle RF, Spraggon G, Everse SJ
• Three-dimensional structural studies on fragments of fibrinogen and fibrin.
• Curr Opin Struct Biol. 1998; 8: 792-8
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• Fibrinogen is a 340 kDa glycoprotein found in the blood plasma of all vertebrates. It is transformed into a fibrin clot by the action of thrombin. Recent X-ray structures of core fragments of both fibrinogen and fibrin have revealed many details about this polymerization event. These include structures of a 30 kDa recombinant gammaC domain, an 86 kDa fragment D from human fibrinogen and a cross-linked double-D fragment from fibrin.

• Everse SJ, Spraggon G, Veerapandian L, Riley M, Doolittle RF
• Crystal structure of fragment double-D from human fibrin with two different bound ligands.
• Biochemistry. 1998; 37: 8637-42
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• Factor XIII-cross-linked fragment D (double-D) from human fibrin was crystallized in the presence of two different peptide ligands and the X-ray structure determined at 2.3 A. The peptide Gly-Pro-Arg-Pro-amide, which is an analogue of the knob exposed by the thrombin-catalyzed removal of fibrinopeptide A, was found to reside in the gamma-chain holes, and the peptide Gly-His-Arg-Pro-amide, which corresponds to the beta-chain knob, was found in the homologous beta-chain holes. The structure shows for the first time that the beta-chain knob does indeed bind to a homologous hole on the beta-chain. The gamma- and beta-chain holes are structurally very similar, and it is remarkable they are able to distinguish between these two peptides that differ by a single amino acid. Additionally, we have found that the beta-chain domain, like its gamma-chain counterpart, binds calcium.

• Gerloff DL, Cohen FE, Benner SA
• A predicted consensus structure for the C terminus of the beta and gamma chains of fibrinogen.
• Proteins. 1997; 27: 279-89
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• A secondary structure has been predicted for the C termini of the fibrinogen beta and gamma chains from an aligned set of homologous protein sequences using a transparent method that extracts conformational information from patterns of variation and conservation, parsing strings, and patterns of amphiphilicity. The structure is modeled to form two domains, the first having a core parallel sheet flanked on one side by at least two helices and on the other by an antiparallel amphiphilic sheet, with an additional helix connecting the two sheets. The second domain is built entirely from beta strands.

• Mosesson MW, Siebenlist KR, DiOrio JP, Matsuda M, Hainfeld JF, Wall JS
• The role of fibrinogen D domain intermolecular association sites in the polymerization of fibrin and fibrinogen Tokyo II (gamma 275 Arg-->Cys).
• J Clin Invest. 1995; 96: 1053-8
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• Intermolecular end-to-middle domain pairing between a thrombin-exposed 'A' polymerization site in the central 'E' domain of fibrin, and a constitutive complementary 'a' site in each outer 'D' domain ('D:E'), is necessary but not alone sufficient for normal fibrin assembly, as judged from previous studies of a congenital dysfibrinogen, Tokyo II (gamma 275 arg-->cys), which showed defective fibrin clot assembly and a normal D:E interaction (Matsuda, M., M. Baba, K. Morimoto, and C. Nakamikawa, 1983. J. Clin. Invest. 72:1034-1041). In addition to the 'a' polymerization site, two other constitutive intermolecular association sites on fibrinogen D domains have been defined: between gamma chain regions containing the carboxy-terminal factor XIIIa crosslinking site ('gamma XL:gamma XL'); and between sites located at the outer ends of each molecule ('D:D') (Mosesson, M. W., K. R. Siebenlist, J. F. Hainfeld, and J. S. Wall, manuscript submitted for publication). We evaluated the function of these sites in Tokyo II fibrinogen, and confirmed that there was a normal fibrin D:E interaction, as determined from a normal fibrin crosslinking rate in the presence of factor XIIIa. We also found a normal gamma XL: gamma XL interaction, as assessed by a normal fibrinogen crosslinking rate. Judging from electron microscopic images, factor XIIIa-crosslinked Tokyo II fibrinogen failed to form elongated double-stranded fibrils like normal fibrinogen. Instead, it formed aggregated disordered collections of molecules, with occasional short fibrillar segments. In addition, Tokyo II fibrin formed an abnormal, extensively branched clot network containing many tapered terminating fibers. These findings indicate that the Tokyo II fibrinogen defect results in a functionally abnormal D:D self-association site, and that a normal D:D site interaction is required, in addition to D:E, for normal fibrin or fibrinogen assembly.

• Veklich YI, Gorkun OV, Medved LV, Nieuwenhuizen W, Weisel JW
• Carboxyl-terminal portions of the alpha chains of fibrinogen and fibrin. Localization by electron microscopy and the effects of isolated alpha C fragments on polymerization.
• J Biol Chem. 1993; 268: 13577-85
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• The locations of the carboxyl-terminal two thirds of the A alpha chains, or the alpha C domains, were determined for fibrinogen and some of its derivatives by electron microscopy of rotary-shadowed preparations. A monoclonal antibody, G8, to the carboxyl-terminal 150 amino acids of the A alpha chain, binds near the central region of fibrinogen, indicating that the alpha C domains of most molecules are not normally visible because they are on or near the amino-terminal disulfide knot. At pH 3.5, fibrinogen and fibrin monomers appear to be similar, with a projection terminating in a small globular domain from each end of most molecules. In contrast, fragment X monomers, produced by cleavage of the alpha C domains from fibrinogen with plasmin, show no such projections. When fibrin monomer is brought to neutral pH under conditions where polymerization is delayed, individual molecules are still visible showing the alpha C domains as a single additional nodule near the central region. Moreover, analysis of clusters of molecules reveals some intermolecular associations via the alpha C domains. A 40-kDa fragment comprising the alpha C domain has been isolated from a plasmin digest of fibrinogen and characterized by SDS-polyacrylamide gel electrophoresis and determination of amino-terminal amino acid sequences. Electron microscopy of alpha C fragments reveals individual globular structures, as well as oligomeric aggregates. The addition of alpha C fragments to fibrin monomer followed by dilution to neutral pH to initiate polymerization results in lower turbidity, longer lag period, and slower maximum rate of turbidity increase. Also, electron microscopy reveals complexes of alpha C fragments with fibrin monomer at neutral pH. It appears that the free alpha C fragments can bind to the alpha C domains of fibrin, competing with the normal alpha C domain interactions involved in polymerization.

• Haidaris PJ, Francis CW, Sporn LA, Arvan DS, Collichio FA, Marder VJ
• Megakaryocyte and hepatocyte origins of human fibrinogen biosynthesis exhibit hepatocyte-specific expression of gamma chain-variant polypeptides.
• Blood. 1989; 74: 743-50
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• The gamma chain of human fibrinogen is heterogeneous in length at the C-terminus due to differential RNA processing of the gamma chain-gene primary transcript. We have produced two specific monoclonal antibodies (MoAbs) against the gamma-chain epitopes generated by this alternative processing event: anti-gamma 57.5(408-416) (L2B), which reacts with gamma 57.5 and gamma 55 chains, and anti-gamma 50(337-411) (H9B7), which reacts preferentially with gamma 50 chains. Using these MoAbs we have studied the expression of gamma-chain polypeptides by immunofluorescence microscopy in the tissues of fibrinogen biosynthesis and have determined that gamma 57.5 polypeptide is expressed in hepatocytes but is absent or present in significantly reduced amounts in megakaryocytes. Therefore the gamma 50 chain is found in plasma, platelet, and megakaryocyte fibrinogens, but the gamma 57.5 chain is found only in plasma fibrinogen. The C-terminal amino acid sequence of gamma 55 includes the L2B epitope 57.5(408-416). Using MoAb L2B we have determined that gamma 55, which is a post-translationally modified gamma 57.5 chain, is found only in plasma fibrinogen and is absent or present in markedly reduced amounts in platelet or megakaryocyte fibrinogen. In addition, the conformation of the L2B epitope is preserved in gamma 55, as determined by Western blot analysis. The hepatocyte-specific expression of the gamma 57.5-chain polypeptide and the post-translational modification to gamma 55 result in a compartmentalization of gamma-chain polypeptide expression. This is suggestive of different mechanisms regulating human fibrinogen gamma-chain gene expression in hepatocytes v megakaryocytes that may operate in a tissue-specific manner at the level of 3' RNA processing events.

• Budzynski AZ
• Fibrinogen and fibrin: biochemistry and pathophysiology.
• Crit Rev Oncol Hematol. 1986; 6: 97-146
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• Fibrinogen is a thrombin-coagulable glycoprotein occurring in the blood of vertebrates. The primary structure of the alpha, beta, and gamma polypeptide chains of human fibrinogen is known from amino acid and nucleic acid sequencing. The intact molecule has a trinodular, dimeric structure and is functionally bivalent. Thrombin cleaves short peptides from the amino termini of the alpha and beta chains exposing polymerization sites that are responsible for the formation of fibrin fibers and appearance of a clot. The major physiological function of fibrinogen is the formation of fibrin that binds together platelets and some plasma proteins in a hemostatic plug. In pathological situations, the network entraps large numbers of erythrocytes and leukocytes forming a thrombus that may occlude a blood vessel. Fibrinogen and fibrin are multifunctional proteins. Fibrinogen is indispensable for platelet aggregation; it also binds to several plasma proteins, however, the biological function of this interaction is not completely understood. Fibrin is an essential matrix for regulation of fibrinolysis and for facilitation of cell attachment in wound healing.

• Weisel JW, Stauffacher CV, Bullitt E, Cohen C
• A model for fibrinogen: domains and sequence.
• Science. 1985; 230: 1388-91
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• Electron microscopy of rotary-shadowed fibrinogen demonstrates that the molecules modified for crystallization by limited cleavage with a bacterial protease retain the major features of the native structure. This evidence, together with image processing and x-ray analysis of the crystals and of fibrin, has been used to develop a three-dimensional low resolution model for the molecule. The data indicate that the two large end domains of the molecule would be composed of the carboxyl-terminus of the B beta chain (proximal) and gamma chain (distal), respectively; the carboxyl-terminus of the A alpha chain would fold back to form an additional central domain. On this basis, the carboxyl-terminal region of each of the three chains of fibrinogen is folded independently into a globular domain.

• Budzynski AZ, Olexa SA, Pandya BV
• Fibrin polymerization sites in fibrinogen and fibrin fragments.
• Ann N Y Acad Sci. 1983; 408: 301-14

• Laudano AP, Doolittle RF
• Influence of calcium ion on the binding of fibrin amino terminal peptides to fibrinogen.
• Science. 1981; 212: 457-9
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• The affinity of the amino terminal tetrapeptide of the beta chain of fibrin, Gly-His-Arg-Pro, for fibrinogen dramatically increases in the presence of 2 millimolar calcium ion. In contrast, there is no significant increase in the affinity of peptides beginning with the amino terminal sequence of the fibrin alpha chain, Gly-Pro-Arg, in the presence of calcium ions, although the number of binding sites increases. In the latter case, the increased number of sites is due to the alpha chain analogs binding to the site ordinarily occupied by the beta chain analogs. These results indicate that structures at the amino terminus of the fibrin beta chain play a more important role in fibrin polymerization when calcium ions are present.

• Mosesson MW, Feldmann G, Menache D
• Electron microscopy of fibrin Paris I.
• Blood. 1980; 56: 80-3
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• Fibrinogen Paris I, a congenital fibrinogen abnormality, is characterized by delayed fibrin aggregation and poor clot retraction owing to the replacement of normal gamma-chains by mutant gamma-chains, which are termed gamma-Paris I. Available evidence indicates that the structural abnormality involves the amino acid sequence near the COOH-terminus of the mutant chain and probably includes the region containing the normal gamma-chain crosslinking site. Electron microscopy was carried out on Paris I fibrin. In place of the normally interwoven network of branching cross-striated fibers, negatively or positively contrasted Paris I fibrin was characterized by nonfibrous clumps of material connected by distince fibrous strands tending to be thinner and more irregular in width than normal fibrin. Most Paris I fibrin fibers tended to the aperiodic, although cross-striations were observed occasionally in negatively contrasted specimens and rarely in positively contrasted specimens. In addition, Paris I fibrin frequently showed relatively short, abruptly terminating fibers. The gross ultrastructural differences between normal and Paris I fibrin suggest that for fibrin assembly to take place normally, a region(s) in the fibrin molecule near to or possibly overlapping the COOH-terminal gamma-chain crosslinking site must be preserved or at least not sterically hindered.

• Cuddigan BJ, Kay D
• Structure of fibrinogen and fibrin.
• Br J Haematol. 1978; 40: 165-8

• Doolittle RF
• The evolution of vertebrate fibrinogen.
• Fed Proc. 1976; 35: 2145-9
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• The origins of the vertebrate fibrinogen molecule and its constituents chains have been considered, first by examining certain features of the molecule as it exists in a primitive vertebrate (the lamprey), and then by comparing the amino acid sequences of certain portions of the three nonidentical chains as they exist in the human molecule. Although the lamprey protein is distinctly homologous to mammalian fibrinogens, at the present stage of characterization its three nonidentical chains appear to be even more different one from another than are the three mammalian chain types. On the other hand, certain sequence resemblances in the three human chains clearly indicate a common ancestry for the alpha-, beta-, and gamma-chains. It is concluded that the ancient fibrinogen molecule was composed of all identical chains and that its differentiation into three chain types occurred long before the divergence of lampreys and higher vertebrates.

• Pozdniakova TM, Lugovskou EV, Demchenko AP, Musialkovskaia AA
• [New data on the structure of fibrinogen]
• Ukr Biokhim Zh. 1975; 47: 247-61
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• The results of physical and chemical investigations show the closeness of fibrinogen to globular proteins, but some differences from globular proteins in certain characteristic features are found resulted from high asymmetry or high hydration of the molecule. The results of electron microscopy cannot be interpreted in a simple way, but nevertheless they show the presence of conformational mobility and existence of configurational isomers. The amino acid sequence of the most important parts of a fibrinogen molecule is known now: "the N-terminal disulphide knot" including peptides A and B being splitted by thrombin, and the part of the gamma-chain participating in covalent binding of fibrin. The study of plasmin and CNBr split products is a fruitful approach to the study of fibrinogen structure and the chemical models of fibrinogen are based on them. In the latest models the N-terminal parts of all 6 polypeptide chains of fibrinogen are located in the centre of a molecule, so the earlier concepts on monomeric fibrin polymerization in the end-to-end way must be reconsidered. None of the existing models produces a definite description of the functional properties of fibrinogen; the appearance of such a model is expected in the nearest future.

• Velican D
• Structural organization of the fibrinogen macromolecule related to the process of fibrin formation.
• Rev Roum Med Intern. 1972; 9: 419-36

• Okude M, Iwanaga S
• Carboxyl-terminal residues of mammalian fibrinogen and fibrin.
• Biochim Biophys Acta. 1971; 251: 185-96
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• The carboxyl-terminal residues of mammalian fibrinogens of six different species and the chain peptides, alpha(A), beta(B) and gamma, isolated from these fibrinogens were determined by hydrazinolysis, digestion with carboxypeptidases and selective tritium labelling. The C-terminal ends of bovine fibrinogen and fibrin were identified as proline and valine, in the molar ratio of approximately 1:2. Proline was identified as the C-terminus of the alpha(A)-chain, and C-terminal valine was found on both the beta(B)- and gamma-chains. On hydrazinolysis after selective tritium labelling of fibrinogen, radioactive C-terminal valine was also identified. The same C-terminal ends as those of bovine fibrinogen were found on the corresponding chain peptides isolated from sheep fibrinogen. The C-terminal residues of all the chain peptides of human and horse fibrinogens, however, were valine. In hog and dog fibrinogens, proline was identified at the C-termini of the alpha(A)-chains, and C-terminal valine and isoleucine were found on the beta(B)- and gamma-chains, respectively. Thus, the C-terminal amino acid residues of the fibrinogens of all mammalian species tested were very similar. It should be noted that hydrophobic amino acids, like isoleucine, valine and proline, are mainly located in the C-terminal ends of all three chain peptides in the fibrinogen molecule.