Molecular and Functional Analysis of the Human Prothrombinase Gene (HFGL2) and Its Role in Viral Hepatitis

Abstract

In the present studies, we report the cloning and structural characterization of the HFGL2 gene and its functional role in human fulminant hepatitis. The HFGL2 gene is approximately 7 kb in length with 2 exons. The putative promoter contains cis element consensus sequences that strongly suggest the inducibility of its expression. From the nucleotide sequence of the human gene, a 439-amino acid long protein is predicted. The overall identity between the murine fgl2 and hfgl2 coded proteins is over 70%. About 225 amino acids at the carboxyl end of these molecules are almost 90% identical, and correspond to a well-conserved fibrinogen-related domain. Both HFGL2 and FGL2 encode a type II transmembrane protein with a predicted catalytic domain toward the amino terminus of the protein. Transient transfection of Chinese hamster ovary (CHO) cells with a fulllength cDNA of HFGL2 coding region resulted in high levels of prothrombinase activity. Livers from 8 patients transplanted for fulminant viral hepatitis were examined for extent of necrosis, inflammation, fibrin deposition, and HFGL2 induction. In situ hybridization showed positive staining of macrophages in areas of active hepatocellular necrosis. Fibrin stained positively in these areas and was confirmed by electron microscopy. These studies define a unique prothrombinase gene (HFGL2) and implicate its importance in the pathogenesis of fulminant viral hepatitis. The majority of individuals who develop acute viral hepatitis recover completely, and only a small fraction, less than one tenth of 1%, develop fulminant hepatic failure; why this occurs is not known.1 The fulminant form of the disease occurs at all ages of life and is not specific for any one viral type. The hallmark of the condition is the extreme rapidity of the necroinflammatory process resulting in widespread or total hepatocellular necrosis in weeks or even days; any satisfactory explanation must explain this rapid progression.A novel murine cDNA fgl2, encoding a protein with prothrombinase-like activity, was previously cloned in our laboratory.2 The sequence of the cDNA was essentially identical to a previously described sequence corresponding to a gene encoding a mouse fibrinogen-like protein, originally described as a cytotoxic T-cell-specific gene. When the cDNA containing the entire coding region was expressed in RAW 264.7 cells, a prothrombinase activity was detected by both a one-stage clotting assay and cleavage of 125 I-labeled prothrombin. Using a model of fulminant viral hepatitis, we demonstrated a causal relationship between the induction of fgl2 prothrombinase and the mortality of murine hepatitis virus infection (MHV-3). 3 We demonstrated that after MHV-3 infection in susceptible mice, mRNA transcripts of fgl2 were seen in macrophages and endothelial cells in the liver followed by fibrin deposition and liver necrosis. 4 The infusion of high-titered monoclonal antibodies to fgl2 prevented the coagulation disturbance, the hepatic necrosis, and mortality associated with MHV-3 infection. 5H...

The novel CD4+CD25+ regulatory T cell effector molecule fibrinogen-like protein 2 contributes to the outcome of murine fulminant viral hepatitis

“…21,35,36 The results of this study clearly demonstrate that FGL2 is an important effector cytokine of Tregs that contributes to host susceptibility to MHV3-induced FH. Because we have shown that patients with FH and chronic HBV and hepatitis C virus infection have increased levels of FGL2, 30,37 the data presented here in concert with human studies suggest that measurement of FGL2 levels in the plasma may be useful in predicting the outcome of both experimental and human viral hepatitis and may provide a rationale for targeting FGL2 for the treatment of patients with acute and chronic viral hepatitis.…”

Intact type 1 immunity and immune-associated coagulative responses in mice lacking IFNγ-inducible fibrinogen-like protein 2

“…Given that Fgl2 is IFN␥-inducible (Figs. 1 and 2) and putatively contributes to fibrin formation (5,7,8), we evaluated T. gondii infection in Fgl2-deficient mice. In contrast to IFN␥-or fibrin-deficient mice, which succumb within 2 weeks of infection (12,27), Fgl2-deficient mice survived acute infection by strain ME49 T. gondii.…”

“…Thus, despite its IFN␥ inducibility (Figs. 1 and 2) and putative procoagulant functions (5,7,8), Fgl2 expression is not required to survive the acute phase of T. gondii infection in mice, a model in which both IFN␥ and coagulation play critical protective roles (12,27).…”

“…Functional studies using neutralizing antisera suggested that Fgl2 may also contribute to coagulant activity associated with spontaneous abortion in mice (6). These observations led to a series of publications describing the direct coagulant activity of Fgl2 (5,7,8). In addition, recombinant Fgl2 was recently shown to suppress T cell proliferation and dendritic cell maturation in vitro (9).…”

Role of Fibrinogen-Like Protein 2 Prothrombinase/Fibroleukin in Experimental and Human Allograft Rejection

“…Mouse fgl2 (mfgl2) and human fgl2 (hfgl2) have been localized to chromosomes 5 and 7, respectively (1-3). fgl2 prothrombinase has been shown previously to have the attributes of a serine protease capable of directly cleaving prothrombin to thrombin leading to fibrin deposition (2). Several studies indicate that mfgl2 is involved in experimental xenograft rejection by mediating "immune coagulation," fibrin deposition, and microthrombus formation, leading to classical pathological changes of acute vascular rejection (4).…”