FGL -2 (Fibrinogen-like protein 2)/Fibroleukin

               The fibrinogen-like protein 2 prothrombinase may be of importance in the development of fibrosis.  The following material is taken from a book on thrombin which is in process.   It should be noted that there are other mechanisms for the generation of thrombin in the extravascular space which also do not involve the canonical prothrombinase complex.
               A gene encoding a protein of  approximate Mr of 42 kDa with homology to the β- and γ-chains of fibrinogen was identified in T-lymphocytes (1).  The term fibroleukin was advanced in 1998 by Marazzi and coworkers to describe a fibrinogen-like protein secreted by CD4+ and CD+ lymphocytes (84). The term fibroleukin was developed to show the relation of the protein to the extracellular matrix and leukocytes.  Fibroleukin mRNA was preferentially expressed in memory T cells.  Production by PBMC was lost in culture but restored by interferon-gamma (INF-γ).  The protein was shown to undergo self-association to form a complex with molecular weight of 350-400 kDa.  The monomer form has an Mr­­ of 70 kDa on reducing SDS gel.  Despite the disparity in the observed molecular weight, it is likely that this material is soluble fibrinogen-like protein 2 (sFLG2).  This protein was subsequently characterized as soluble fibrinogen-like protein 2 (sFLG2), a molecule with immunomodulatory activity (3,4). There has been considerable work on the immunomodulatory effect of sFLG2 (5-7).   There is a study which has shown that the mononmeric sFLG2 has increased immunosuppressive activity (8)
 The serum level of sFLG-2 has been suggested as a biomarker for traumatic brain injury (9).  This is a limited selection of citations described the role of sFGL-2 is immunomodulation as this function is not the focus of this discussion.
               The identification of a macrophage membrane protein which could activate prothrombin to thrombin was suggested by work in 1980 (10) and more clearly defined in 1982 as a monocyte prothrombinase  by the same group in La Jolla (11). This latter work showed the advantage, at that time, of having specific factor-deficient plasma for the characterization of procoagulant activity (PCA).  The PCA did clot prothrombin-deficient plasma but did clot plasmas deficient in factors  V,VII,VIII, X, and XII as well as normal plasma.  The action of PCA on prothrombin did require the presence of calcium ions but no other cofactors.   The PCA is suggested to be a serine protease based on inhibition by DFP..  The activity was not detected in freshly isolated PBMC but increased on exposure to LPS or soluble antigen-antibody complexes.  The expression of FGL-2 prothrombinase has been observed in epithelial cells, endothelial cells, dendritic cells, monocytes, and macrophages (12,13).  The characterization and functional expression of the FGL-2 gene has been reported (14). The expressed MGl-2 protein is a 439-residue type II membrane protein (type II membrane protein contains a hydrophobic transmembrane segment which is not cleaved).  mFGL-2 protein has a carboxyl-terminal fibrinogen-related domain (FRED) which does appear in other proteins 15,16).  Inhibition by DFP and mutagenesis studies (17) suggest the presence of a catalytically important serine residue (Ser91); it was suggested that a clan SE serine protease (α-lytic endopeptidase)(18-21). A search for FGL-2 in the MEROPS database was unsuccessful; there may not be sufficient information to include FGL-2 in the MEROPS database.
               There are several studies characterizing FLG-2 as a peptidase (11,17,22). The most complete study (17) studied the FGL-2 protein as expressed in baculovirus and as truncated/mutant forms in CHO cells.  The purified full-length protein did not have an PCA activity; Reconstitution of the expressed protein in a phosphatidyl choline-phosphatidyl serine vesicle (75% PC/25%PS) yield an active procoagulant   The addition of Factor Va further increased the rate of prothrombin activation.  As noted above, FLG-2 is inactivated by DFP.  It was observed that PMSF did not inactivate FLG-2.  This is a bit unusual, since both reagents react with the active site serine of trypsin  and thrombin at similar rates (23).104  The active site triad (asp-his-ser) present in canonical serine proteases is absent in FLG-2, perhaps providing an explanation.   I would note that there are regulatory serine proteases such as factor IXa that are not inhibited by DFP (24,25).  It is of interest that a monoclonal antibody elicited from a segment close to Ser91 (residues 76-87, EEVFKEVQNLKE) inhibited FLG-2 prothrombinase activity (26).  There have been no further studies on the characterization of this interesting enzyme.  There have been a number of studies on potential role of FLG-2 in various pathologies (27-31).
1.  Koyama, T., Hall, L.R., Haser, W.G., Tonegawa, S., and Saito, H., Structure of a cytotoxic T-lymphocyte-specific gene shows a strong homology to fibrinogen β and γ chains, Proc.Natl.Acad.Sci.USA 84, 1609-1613, 1987.
2.  Marazzi, S.,Blum, S., Hartmann,R., et al., Characterization of human fibroleukin, a fibrinogen-like protein secreted by T lymphocytes, J.Immunol. 161, 138-147, 1998.
3. Yang, G. and Hooper, W.C., Physiological functions and clinical implications of fibrinogen-like protein 2: a review, World J.Clin.Infect.Dis. 3, 37-46, 2013.
4. Liu, X.-g., Liu, Y., and Chen, F. Soluble fibrinogen-like protein 2, the novel effector molecule for immnoregulation, Oncotarget 8, 3711-3723, 2017.
5.  Shrivastavi, S.,  Moley, J.H., and Darling, A., The interface between coagulation and immunity, Am.J.Transplant. 7, 499-506, 2007.
6. Liu, H., Shalev, I., Manuel, J., et al. The FGL-2 Fcγ RIIB pathway: A novel mechanism leading to immunosuppression, Eur.J.Immunol. 38, 3114-3126, 2008.
7. Shalev, I., Liu, H., Koscik, C., et al., Targeted deletion of fgl2 leads to imparted regulatory T cell activity and development of autoimmune glomerulonephritis, J.Immunol. 180, 249-260, 2008.
8.  Liu, H., Yang, P.S., Zhu, T., et al., Characterization of fibrinogen-like protein 2 (FGL2): Monomeric FGL2 has enhanced immunosuppressive activity in comparison to oligomeric FGL2, Int.J.BIochem.Cell Biol. 45, 408-418, 2013
9. Chen, T.-J., Ji, M.-X., Tao, Z.-Q., et al., The relationship between serum fibrinogen-like protein 2 concentrations and 30-day mortality of patients with traumatic brain injury, Clin.Chim.Acta 489, 53-57, 2019.
10.   Levy, G.A. and Edginton, T.S., Lymphocyte cooperation is required for amplification of macrophage procoagulant activity, J.Expt.Med. 151, 1232-1244, 1980.
11. Schwartz, B.S., Levy, G.A., Fair, D.S., and Edgington, T.S., Murine lymphoid procoagulant activity induced by bacterial lipopolysaccharide and immune complexes is a monocyte prothrombinase, J.Expt.Med. 155, 1464-1479, 1982.
12. Parr, R.L., Fung, L., Reneker, J., et al., Association of mouse fibrinogen-like protein with murine fibrinogen-like protein with murine hepatitis virus-induced prothrombinase activity, J.Virol. 69, 5033-5038, 1995.
13.  Yang, Y. and Hooper, W.C., Physiological function and clinical implications of fibrinogen-like 2: A review, World J.Clin.Infect.Dis. 3, 37-46, 2013.
14. Yuwaraj, S., Ding, J., Liu, M., Marsden, P.A., and Levy, G.A., Genomic characterization, localization, and functional expression of FGL2, the human gene encoding fibroleukin: a novel human procoagulant, Genomics 71, 330-338, 2001.
15. Gordy, M.A., Pila, E.A., and Harrington, P.C., The role of fibrinogen-related protein in the gastropod immune response,  Fish Shellfish Immunol. 46, 39-49, 2015.
16.  Thomsen, T., Schlosser, A., Holmskov, V. and Sorenson, G.L, Ficolins and FIBCD1: soluble and membrane bound pattern recognition molecules with acetyl group selectivity, Mol.Immunol. 48, 369-381, 2011
17.  Chan, C.W.Y., Chan, M.W.C., Liu, M., et al., Kinetic analysis of a unique direct prothrombinase, fgl2, and identification of a serine residue critical for the prothrombinase activity, J.Immunol. 168, 5170-5117, 2002.
18. Barrett, A.J. and Rawlings, N.D., Families and clans of serine peptidases, Arch.Biochem.Biophys. 318, 247-250, 1995.
19.  Barrett, A.J., Rawlings, N.D., and O’Briens, E.A., the  MEROPS database of proteolytic enzymes as a protease information system, J.Struct.Biol. 134, 95-102, 2001.
20. https://www.ebi.ac.uk/merops
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22.  Fung, L.S., Neil, G., Leibowitz, J., et al., Monoclonal antibody analysis of a unique macrophage procoagulase activity induced by murine hepatitis virus strain 3 infection, J.Biol.Chem. 286, 1789-1795, 1990.
23.  Fahrney, D.E. and Gold, A.M., Sulfonyl fluorides as inhibitor of  esterases I.  Rates of reaction with acetylcholinesterase, α-chymotrypsin, and trypsin, J.Amer.Chem.Soc. 85, 997-2006, 1965.
24.  Lundblad, R.L. and Davie, E.W., The activation of antihemophilic factor (Factor 8) by activated Christmas factor (activated factor 9), Biochemistry 3, 1720-1725, 1964,
25.  Di Scipio, R.G., Kurachi, K., and Davie, E.W., Activation of human factor IX (Christmas factor), J.Clin.Invest. 61, 1528-1538, 1978.
26.  Li, W.-Z., Wang, J., Long, R., et al., Novel antibody against a glutamic acid-rich human fibrinogen-like protein 2-derived peptide near Ser91 inhibits hfgl2 prothrombinase activity, PLoS One 9(4):e94551, 2014.
27.  O’Brien, M., Morrison,  J.J., and Smith, T.J., Expression of prothrombin and protease activated receptors in human myometrium during pregnancy and labor, Biol.Reprod. 78, 20-26, 2008.
28. Su, K., Chen, F., Yan, W.-H., et al., Fibrinogen-like protein 2/fibroleukin prothrombinase contributes to tumor hypercoagulability via IL-2 and IFN-γ, World J.Gastroenterol. 14, 5980-5989, 2008.
29.  Melnyk, M.C., Shalev, J., Zhang, J., et al., The prothrombinase of FLG2 contributes to the pathogenesis of experimental arthritis, Scand.J.Rheumatol. 40, 269-278, 2011
30. Liu, Y., Li, X., Zeng, Q., et al., Downregulation of FGL2/prothrombinase delays HCCLM6 xenograft tumour growth and decreases tumour angiogenesis, Liver Int. 32, 1585-1595, 2012.
31. Rabizadeh, E., Cherny, I., Wolach, O., et al., Increased activity of cell membrane-associated prothrombinase, fibrinogen-like protein 2, in peripheral blood mononuclear cells of B-cell lymphoma patients, PLoS One 9(10):e101371, 2014.
32,  Liu, J., Tan, Y., Zhang, J., et al., C5aR, TNF-α, and FGL2 contribute to coagulant and complement activation in virus-induced fulminant hepatitis, J.Hepatol. 62, 354-362, 2015.
33.  Rabizadeh, E., Cherny, I., Lederfein, D., et al., The cell-membrane prothrombinase, fibrinogen-like protein 2, promotes angiogenesis and tumor development, Thromb.Res. 136, 118-124, 2015.
34.  Fan, C., Wang, J., Mao, C., et al., The Fgl2 prothrombinase contributes to the pathological process of experimental hypertension, J.Appl.Physiol., in press (doi.1152/japplphysiol.00396.2019, 2019.