Russsell F Doolittle

Center for Molecular Genetics, University of California, San Diego, La Jolla, California

Fibrinogen is an extracellular protein found in significant concentrations in the blood plasmas of all vertebrate animals. It is a large, multi-domained protein, some portions of which share common ancestry with lectins and other cyto-tactic proteins found throughout the animal kingdom [1]. Although the principal role of fibrinogen has to do with its polymerization into fibrin clots, the protein also interacts with a number of other extracellular proteins, blood platelets, and a variety of cells. Directly or indirectly, the fibrinogen-fibrin system is involved in hemostasis, inflammation, wound healing, and angiogenesis. Fibrinogen also interacts with various bacteria, especially certain strains of Staphylococcus.

Fibrinogen is a covalent dimer composed of two sets of three nonidentical chains (a2P2y2). The P and y chains are homologous over their full lengths, but the a chain homol-ogy is limited to its amino-terminal third. The molecular weights of vertebrate fibrinogens range from 320,000 to 400,000, the variation invariably being due to differences in the a chains, the carboxyl terminal two-thirds of which are extremely variable from species to species. In contrast, the carboxyl-terminal halves of the P and y chains are globular and conserved. Together, they constitute bi-lobed macro domains at the extremeties of the extended molecule, being connected to a small central domain by three-strands coiled coils made up of all three chains (Fig. 1).

Several regions of the fibrinogen molecule are highly mobile and are not resolvable in crystallographic electron density maps, even at moderately high resolution [2]. The flexible parts include the entire a-chain carboxyl region, which can contain from 300 to 500 amino acid residues, depending on the species (region I in Fig. 1). Additionally, the last 15 residues at the carboxyl terminus of the y chain have not been pinned down with any precision [3,4], nor have the amino-terminal segments corresponding to approximately the first 30 residues of the a chain and the first 60 of the P chain (numbering varies slightly from species to species; the numbering here is based on human fibrinogen). Several of these mobile regions figure prominently in interactions with other proteins and with cells.

Apart from the mobile and highly variable carboxyl-terminal domains of the a chains, the general framework of all vertebrate fibrinogens is highly conserved, as evidenced by the ready superposition of the chicken fibrinogen crystal structure on that of a modified bovine fibrinogen [5,6]. The length of the protein is about 45 nm.

The conversion of fibrinogen to fibrin is initiated by thrombin-removing short peptide regions, called fibrinopep-tides, from the amino-terminal ends of the a and P chains. The consequence of these narrowly specific proteolytic events is the exposure of sets of A and B "knobs" on the a and P chains, respectively, that fit into holes on the terminal globular domains of neighboring fibrinogen molecules. The initial knob-hole interactions position a pair of A knobs (Gly-Pro-Arg is the sequence at the newly exposed a-chain site, residues 17-19) so as to pin together two neighboring molecules by fitting into holes on their y-chain carboxyl domains. Further propagation results in a noncovalently associated, two-molecule-thick, half-staggered protofibril. Interactions involving the B knob (Gly-His-Arg is the sequence at the newly exposed P-chain site) can fill holes in the P-chain carboxyl domains and, directly or indirectly, lead to lateral growth of the fibrin network. Meanwhile, thrombin-activated factor XIII reinforces the fibrin polymer by introducing y-glutamyl-e-amino crosslinks, initially between the

Figure 1 Ribbon model of those portions of fibrinogen for which high-resolution X-ray structures are available. Highly mobile regions of the molecule are not shown fully (broken lines), including the carboxyl-terminal domain of a chains (I) and the last 15 residues of Y chains (A), as well as the amino-terminal segments of a and P chains (G, F). The molecule is a covalent dimer with a pseudo-axis of symmetry running through the central domain. Key structural features are labeled on the right half, and some reported recognition sites are designated on the left half. A, Y-chain carboxyl terminal (platelets, fibroblasts, staphylococcal clumping factors); B, Y-chain 383-395 (aMP2); C, Y-chain 195-202 (aMP2); D, Y-chain 117-133 (ICAM-1); E, a-chain 151-158 (t-PA stimulator); F, P-chain 15-42 (angio-genesis, heparin-binding); G, a-chain 17-20 (aMP2); H, a-chain 15-44 and P-chain 61-72 (thrombin); I, a-chain 240-610 (aMP2).(Adapted from Yang, Z. et al., Biochemistry, 40, 12515-12523, 2001.)

Figure 1 Ribbon model of those portions of fibrinogen for which high-resolution X-ray structures are available. Highly mobile regions of the molecule are not shown fully (broken lines), including the carboxyl-terminal domain of a chains (I) and the last 15 residues of Y chains (A), as well as the amino-terminal segments of a and P chains (G, F). The molecule is a covalent dimer with a pseudo-axis of symmetry running through the central domain. Key structural features are labeled on the right half, and some reported recognition sites are designated on the left half. A, Y-chain carboxyl terminal (platelets, fibroblasts, staphylococcal clumping factors); B, Y-chain 383-395 (aMP2); C, Y-chain 195-202 (aMP2); D, Y-chain 117-133 (ICAM-1); E, a-chain 151-158 (t-PA stimulator); F, P-chain 15-42 (angio-genesis, heparin-binding); G, a-chain 17-20 (aMP2); H, a-chain 15-44 and P-chain 61-72 (thrombin); I, a-chain 240-610 (aMP2).(Adapted from Yang, Z. et al., Biochemistry, 40, 12515-12523, 2001.)

carboxyl-terminal segments of abutting y chains, but eventually also between carboxyl domains of a chains.

Fibrin can be distinguished from fibrinogen by many recognition systems. Quite apart from sites lost with the removal of the fibrinopeptides and the coincident appearance of the A and B knobs, the mere act of polymerization can mask certain sites. Additionally, conformational changes occur, some of which have been observed in crystal structures of fibrin(ogen) fragments complexed with synthetic knobs [7]. Other more subtle changes may occur during the later stages of polymerization. For example, there is a region of the a chain that has been implicated in the stimulation of tissue plasminogen activator [8] that is wholly inaccessible to solvent in fibrinogen but which somehow becomes accessible as a result of the polymerization process.

Over the years, there have been numerous reports describing regions of fibrinogen or fibrin responsible for binding various macromolecules or cells. The availability of X-ray structures now provides a backdrop for visualizing some of these at atomic resolution (Fig. 1). Among the cells and particles known to bind fibrin(ogen) are platelets, endothelial cells, monocytes, lymphocytes, neutrophils, and fibroblasts, all of which are actively involved in hemostasis, wound healing, inflammation, or angiogenesis. For the most part, studies have utilized fragments of fibrin(ogen), antibodies directed against localized features, site-directed mutagene-sis of recombinant fibrinogens, or synthetic peptides corresponding to specific regions.

Some parts of fibrinogen have been implicated in several different events. The carboxyl-terminal segments of Y chains bind platelets [9] and fibroblasts [10]; the same sites bind to certain strains of Staphylococcus aureus [11]. The locations of these sites at the tips of the dimeric fibrinogen molecule are well disposed for bridging and clumping cells or platelets. The crosslinking of these segments in fibrin by factor XIII must render the sites inaccessible. Similarly, certain regions of the a-chain carboxyl domain have been implicated in binding to platelets and various leucocytes, and these must also be compromised by becoming crosslinked in the final stages of clot formation.

The flexible amino-terminal segment of the P chain is another targeted region. The bacterium Staphylococcal epidermis binds to a peptide segment that includes a bond cleaved by thrombin [12]. Other entities bind in the region of P-chain residues 15 to 42, exposed after thrombin attack, including certain cadherins [13] and heparin [14]. Angiogenesis is also stimulated by this general region [15].

In the main, two kinds of cell surface proteins have been associated with fibrinogen binding: (a) members of the immunoglobulin family such as cadherins [13] and ICAM-1 [16], and (b) heterodimeric integrins. The most commonly implicated integrins are aMp2 and a%p2 [17]. Some findings about the sites of interaction remain uncertain in that the same integrins have been reported to interact with widely differing regions of the fibrinogen molecule for which there are no apparent structural similarities. Final resolution may have to await crystal structures of complexes of fibrin(ogen) fragments with specific integrins or other interactants.

References

1. Doolittle, R. F., Spraggon, G., and Everse, S. J. (1997). Evolution of vertebrate fibrin formation, and the process of its dissolution. In Plasminogen-Related Growth Factors, John Wiley & Sons, Chichester.

2. Yang, Z., Kollman, J. M., Pandi, L., and Doolittle, R. F. (2001). Crystal structure of native chicken fibrinogen at 2.7 Â resolution. Biochemistry 40, 12515-12523.

3. Yee, V. C., Pratt, K. P., Cote, H. C.,LeTrong, I., Chung, D., Davie, E. W., Stenkamp, R. E., and Teller, D. C. (1997). Crystal structure of a 30 kDa C-terminal fragment from the g chain of human fibrinogen. Structure 5, 125-138.

4. Spraggon, G., Everse, S. J., and Doolittle, R. F. (1997). Crystal structures of fragment D from human fibrinogen, and its crosslinked counterpart from fibrin. Nature 389, 455-462.

5. Brown, J. H., Volkmann, N., Jun, G., Henschen-Edman, A. H., and Cohen, C. (2000). Crystal structure of a modified bovine fibrinogen. Proc. Natl. Acad. Sci. USA 97, 85-90.

6. Yang, Z., Mochalkin, I., Veerapandian, L., Riley, M., and Doolittle, R. F. (2000). Crystal structure of native chicken fibrinogen at 5.5 Â resolution. Proc. Natl. Acad. Sci. USA 97, 3907-3912.

7. Everse, S. J., Spraggon, G., Veerapandian, L., and Doolittle, R. F. (1999). Conformational changes in fragments D, and double-D from human fibrin(ogen) upon binding the peptide ligand Gly-His-Arg-Pro-amide. Biochemistry 38, 2941-2946.

8. Schielen, W. J. G., Adams, H. P. H. M., Voskuilen, M., Tesser, G. J., and Nieuwenhuizen, W. (1991). Structural requirements of position Aa-157 in fibrinogen for the fibrin-induced rate enhancement of the activation of plasminogen by tissue-type plasminogen activator. Biochem. J. 276, 655-659.

9. Hawiger, J., Timmons, S., Kloczewiak, M., Strong, D., and Doolittle, R. F. (1982). y and a chains of human fibrinogen possess sites reactive with human platelet receptors. Proc. Natl. Acad. Sci. USA 98, 2068-2071.

10. Farrell, D. H. and Al-Mondhiry, H. A. (1997). Human fibroblast adhesion to fibrinogen. Biochemistry 36, 1123-1128.

11. Strong, D. D., Laudano, A. P., Hawiger, J., and Doolittle, R. F. (1982). Isolation, characterization, and synthesis of peptides from human fib-rinogen that block the staphylococcal clumping reaction, and construction of a synthetic clumping particle. Biochemistry 21, 1214-1420.

12. Davis, S. L., Gurusiddappa, S., McCrea, K. W., Perkins, S., and Hook, M. (2001). A fibrinogen-binding bacterial adhesin of the microbial surface components recognizing adhesive matrix molecules subfamily from Staphylococcus epidermis. J. Biol. Chem. 276, 27799-2805.

13. Martinez, J., Ferber, A., Bach, T. I., and Yaen, C. H. (2001). Interaction of fibrin, and VE-cadherin. Ann. N.Y. Acad. Sci. 936, 386-405.

14. Odrijin, T. M., Shainoff, J. R., Lawrence, S. O., and Simpson-Haideris, P. J. (1996). Thrombin cleavage enhances exposure of a heparin binding domain in the N-terminus of the fibrin P chain. Blood 88, 2050-2061.

15. Thompson, W. D., Smith, E. B., Stirk, C. M., Marshall, F. I., Stout, A. J., and Kocchar, A. (1992). Angiogenic activity of fibrin degradation products is located in fibrin fragment E. J. Pathol. 168, 47-53.

16. Altieri, D. C., Duperray, A., Plescia, J., Thornton, G. B., and Languino, L. R. (1995). Structural recognition of a novel fibrinogen Y chain sequence (117-133) by intercellular adhesion molecule-1 mediates leukocyte-endothelium interaction. J. Biol. Chem. 270, 696-699.

17. Ugarova, T. P. and Yakubenko, V. P. (2001). Recognition of fibrinogen by leukocyte integrins. Ann. N.Y. Acad. Sci. 936, 368-385.

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