Molecular and Functional Analysis of the Human Prothrombinase Gene (HFGL2) and Its Role in Viral Hepatitis
2000; Elsevier BV; Volume: 156; Issue: 4 Linguagem: Inglês
10.1016/s0002-9440(10)64992-9
ISSN1525-2191
AutoresGary Levy, Mingfeng Liu, Jinwen Ding, Shankary Yuwaraj, Julian L. Leibowitz, Philip A. Marsden, Qin Ning, Ana Kovalinka, M. James Phillips,
Tópico(s)Cancer-related gene regulation
ResumoIn 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 full-length 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. 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 full-length 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.1O'Grady JG Portmann B Williams R Fulminant hepatic failure.in: Schiff L Schiff ER Diseases of the Liver. ed 7. Lippincott, Philadelphia1993: 1077-1090Google Scholar 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.2Parr RL Fung L Reneker J Myers-Mason N Leibowitz JL Levy GA Association of mouse fibrinogen-like protein with murine hepatitis virus-induced prothrombinase activity.J Virol. 1995; 69: 5033-5038Crossref PubMed Google Scholar 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 125I-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).3Yuwaraj S Cattral M Pope M Levy GA Murine hepatitis virus: molecular biology and pathogenesis.Viral Hepatitis Rev. 1996; 2: 125-142Google Scholar 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.4Ding JW Ning Q Liu M Lai A Leibowitz J Peltekian KM Cole EH Fung LS Holloway C Marsden PA Yeger H Phillips MJ Levy GA Fulminant hepatic failure in murine hepatitis virus strain 3 infection: tissue-specific expression of a novel fgl2 prothrombinase.J Virol. 1997; 71: 9223-9230Crossref PubMed Google Scholar The infusion of high-titered monoclonal antibodies to fgl2 prevented the coagulation disturbance, the hepatic necrosis, and mortality associated with MHV-3 infection.5Li C Fung LS Crow A Myers-Mason N Leibowitz J Cole E Levy G Monoclonal antiprothrombinase (3D4.3) prevents mortality from murine hepatitis virus (MHV-3) infection.J Exp Med. 1992; 176: 689-697Crossref PubMed Scopus (65) Google Scholar Here we report the isolation and characterization of the HFGL2 gene, which encodes for a potent prothrombinase and demonstrate mRNA transcripts of this gene in the earliest hepatic lesions of fulminant viral hepatitis. This study defines distinctive aspects of the cellular and molecular pathology of fulminant viral hepatitis and implicates viral-induced up-regulation of the potent human fgl2 prothrombinase gene in this lethal disease. Therapies directed toward this prothrombinase offer the potential for attenuation of disease in man, similar to exciting recent findings in a murine model of fulminant viral hepatitis. Primers specific to a recently reported cDNA sequence6Ruegg C Pytela R Sequence of a human transcript expressed in T-lymphocytes and encoding a fibrinogen-like protein.Gene. 1995; 160: 257-262Crossref PubMed Scopus (39) Google Scholar which correspond to exon 2 of mouse fgl2 were used to amplify the genomic DNA from human liver; the sense primer CAA AAG AAG CAG TGA TAC CTA CA (huflp7) at position 693 and the antisense primer TTA TCT GGA GTG AAA AAC TT (huflp8) at position 1115 of the reported human cDNA generated a probe of 423 nucleotides which was used to screen the human PAC library (Genome Systems, St. Louis, MO) and 3 clones were isolated. Restriction mapping of three PAC clones was performed with the frequently cutting enzymes EcoRI, HindIII, PstI, and PvuII and the rare cutting enzymes NotI, SalI, and SmaI. A plasmid sublibrary was prepared using partial Sau3A digestion and subcloned into the BamHI site of PBluescript 1 (Sk−; Stratagene, La Jolla, CA) as previously described.7Hall AV Antoniou H Wang Y Cheung AH Arbus AM Olson SL Lu WC Kau CL Marsden PA Structural organization of the human neuronal nitric oxide synthase gene (NOS1).J Biol Chem. 1994; 269: 33082-33090Abstract Full Text PDF PubMed Google Scholar The HFGL2 cDNA coding region was amplified by polymerase chain reaction (PCR) from human small intestine total RNA using the forward primer TGA GCA GCA CTG TAA AGA TG 17 bp upstream of the translation start codon ATG and the reverse primer GTG GCT TAA AGT GCT TGG GT starting 6 bp upstream of the stop codon TAA to 11 bp downstream of TAA. The PCR product was first cloned into PCR II cloning vector (Invitrogen), sequenced, and subcloned into pcDNA 3.1 his mammalian expression vector inframe with the his-tag. Transfection of HFGL2 into CHO cells was performed using the lipofectamine reagent (Gibco, Mississauga, Ontario). The method of extraction of total RNA from tissue has been described elsewhere.8Ribaudo R, Gilman RE, Kingston P, Chomczynski P, Sacchi N: Single-step RNA isolation from cultured cells or tissues. Current Protocols in Immunology, supplement 3. Edited by JE Coligan, AM Kruisbeek, DH Margulies, EM Shevach, W Strober. New York, 1991, pp 10.11.7–10.11.14Google Scholar In brief, 100 mg of tissue was homogenized in a denaturing solution containing 4 mol/L guanidine isothiocyanate, 25 mmol/L sodium citrate, 0.1 mol/L 2-mercaptoethanol, and 0.5% N-lauroylsarcosine. The homogenate was then mixed with 2 mol/L sodium acetate (pH 4.0), water-saturated phenol, and choloroform-isoamyl alcohol (49:1) and incubated on ice for 15 minutes. The mixture was centrifuged at 15,800 × g at 4°C for 20 minutes. The upper aqueous phase was transferred to a fresh tube and then mixed with an equal volume of 100% isopropanol and precipitated at −20°C for 20 minutes. RNA was pelleted by centrifugation at 15,800 × g at 4°C for 30 minutes and then dissolved in the denaturing solution, to which an equal volume of 100. isopropanol was added for reprecipitation of RNA at −20°C. After centrifugation, the RNA pellet was washed with 75% ethanol twice, dried in vacuum, and resuspended in DEPC-treated water. Total cellular RNA (20 μg) from each tissue was first quantitated spectroscopically and then electrophoresed in a 1% denaturing agarose gel with 1.7% formaldehyde and then transferred to nitrocellulose membranes (Schleicher & Schuell, Keene, NH) in 20 × SSC (1 × SSC in 0.15 mol/L NaCl plus 0.015 mol/L sodium citrate). RNA was immobilized by baking the membrane at 80°C for 2 hours in vacuum and was hybridized in 50% formamide-5 × SSPE (1 × SSPE is 0.18 mol/L NaC1, 10 mm NaH2PO4, and 1 mm ethylenediaminetetraacetic acid [pH 7.1])−5 × Denhardt's solution-0.5% sodium dodecyl sulfate-100 μg of denatured salmon sperm DNA per ml at 42°C for 18 hours with an [α-32P]dCTP-labeled 539-bp DNA probe of human fgl2 cDNA (7 × 108 cpm/μg) encompassing nucleotides 100 (GCAAACAAT…) to 639 (… ATACAGTCA). Labeled human glyceraldehyde-3-phosphate dehydrogenase cDNA was used to ensure the integrity of the RNA in each tissue. The membrane was washed with 1 × SSPE and 0.1% sodium dodecyl sulfate at room temperature for 15 minutes and then autoradiographed by exposure to Kodak film (X-OMAT. Eastman Kodak Company, Rochester, NY). Forty-eight hours after transfection, CHO cells were washed twice with phosphate-buffered saline before addition of 200 μl of lysis buffer (10 mmol/L Tris-HCI, pH 7.5, 150 nmol/L NaC1, 1 mmol/L ethylenediaminetetraacetic acid, 0.25% NP-40, 0.2 mg of phenylmethylsulfonyl fluoride per ml) at 106 cells/100 μl for 20 minutes at 4°C. Lysates were clarified by centrifugation, and 20 μl of each was separated by 10% sodium dodecyl sulfate-polyacrylamide gel electrophoresis and transferred to a Hybond-N nitrocellulose membrane (Amersham, Mississauga, Ontario). Membranes were probed with monoclonal anti-His antibody (Invitrogen). Bound antibodies were visualized with an enhanced chemiluminescence detection system (Amersham). Transfected CHO cells were harvested after 48 hours of transfection and suspended in unsupplemented RPMI 1640 at a concentration of 2 × 106 cells/ml. The cells were then subjected to three cycles of freeze-thawing to obtain maximal total cellular procoagulant activity. Samples were assayed for the ability to shorten the spontaneous clotting time of normal citrated human platelet-poor plasma.7Hall AV Antoniou H Wang Y Cheung AH Arbus AM Olson SL Lu WC Kau CL Marsden PA Structural organization of the human neuronal nitric oxide synthase gene (NOS1).J Biol Chem. 1994; 269: 33082-33090Abstract Full Text PDF PubMed Google Scholar Milliunits of procoagulant activity were assigned by reference to a standard curve generated with serial log dilutions of a standard rabbit brain thromboplastin (Sigma Chemical Co., St. Louis, MO) as previously described.9Abecassis M Falk JA Makowka L Dinzans VJ Levy GA 16,16-dimethyl prostaglandin E2 prevents the development of fulminant hepatitis, and blocks the induction of monocyte/macrophage procoagulant activity after murine hepatitis virus strain 3 infection.J Clin Invest. 1987; 80: 881-889Crossref PubMed Scopus (111) Google Scholar Media and reagents were without activity. Factor X and prothrombin were isolated from Cohn fraction III.10Schwartz BS Levy GA Fair DS Edgington TS Murine lymphoid procoagulant activity induced by bacterial lipopolysaccharide and immune complexes is a monocyte prothrombinase.J Exp Med. 1982; 155: 1464-1479Crossref PubMed Scopus (67) Google Scholar For assay of cleavage, prothrombin was radioiodinated enzymatically with immobilized lactoperoxidase and glucose oxidase (Enzymobeads; Biorad, Richmond, CA) to a specific activity of 6.2 μCi/μg. To 25 μl of cellular homogenate, 10 μl of 125I-prothrombin and 10 μl of 25 mmol/L CaC12 were added. The reaction was allowed to proceed at 37°C for 30 minutes. Human factor Xa (0.5 μg/ml), generated by incubating 0.6 ng human factor X with 0.06 ng of Russell's viper venom as previously described, in the presence of homogenates of CHO cells was used as a positive control for 125I-prothrombin cleavage. Each reaction mixture was electrophoresed on 10% polyacrylamide gel. After electrophoresis, the gels were fixed, dried, and subjected to autoradiography. Eight cases of fulminant viral hepatitis were examined (virus types: hepatitis B, 3; syncytial giant cell hepatitis with paramyxoviral like particles, 1; hepatitis virus non-A non-B non-C, 4). The micrographs shown are from one of the latter cases which are representative of all cases presented. Histological sections were stained with hematoxylin and eosin. Immunoperoxidase staining (dark brown) used the avidin-biotin complex method. For fibrin detection, a rabbit-anti-human fibrinogen antibody (DAKO, Carpenteria, CA), known to react with fibrinogen and fibrin in human tissues was used. The CD68 antibody (DAKO) was used to detect macrophages (histiocytes). A 216-bp DNA fragment from the conserved carboxyl end of the hfgl2 coding region was used as a template, to synthesize, with T3 and T7 RNA polymerases, digoxigenin-11-UTP (Boehringer Mannheim, Laval, PQ)-labeled sense and antisense cRNA probes for the in situ hybridization studies.4Ding JW Ning Q Liu M Lai A Leibowitz J Peltekian KM Cole EH Fung LS Holloway C Marsden PA Yeger H Phillips MJ Levy GA Fulminant hepatic failure in murine hepatitis virus strain 3 infection: tissue-specific expression of a novel fgl2 prothrombinase.J Virol. 1997; 71: 9223-9230Crossref PubMed Google Scholar Alkaline phosphatase (dark blue. served as a marker of the reaction product. Tissue for electron microscopy was prepared by routine methods,11Tsukada N Azuma T Phillips MJ Isolation of the hepatic pericanalicular contractile apparatus: actin filaments and myosin II motor with the surrounding intermediate filament sheath.Proc Natl Acad Sci USA. 1994; 91: 6919-6923Crossref PubMed Scopus (18) Google Scholar ultra-thin epon embedded sections were stained with lead citrate and examined in a Philips 400 electron microscope (Einthoven, The Netherlands). The HFGL2 gene was isolated using three genomic clones from a human PAC library (Genome Systems Inc., St Louis, MO. using a PCR-based approach (Figure 1). The HFGL2 gene was localized to the short arm of chromosome 7 by fluorescence in situ hybridization and radiation hybrid mapping (data not shown). The HFGL2 gene is approximately 7 kb in length. This size was determined by the known mRNA sequence that contains exon I and II (4.4 kb) plus an intron sequence of 2.2 kb. Exon II encodes for the last 235 amino acids of the HFGL2 protein and contains the 3′ UTR. The putative promoter region contains cis element consensus sequences such as a TATA box, an AP1 site, a C/EBP binding site (CAAT), and multiple Ets sites which suggest the inducibility of HFGL2. Of interest, the C/EBP binding sites have been shown to play a pivotal role in the induction of acute phase response genes of the liver and cytokine genes of macrophages.12Akira S Kishimoto T IL-6 and NF-IL6 in acute-phase response and viral infection.Immunol Rev. 1992; 127: 25-50Crossref PubMed Scopus (498) Google Scholar, 13Woodgett JR Avruch J Kyriakis JM Regulation of nuclear transcription factors by stress signals.Clin Exp Pharmacol Physiol. 1995; 22: 281-283Crossref PubMed Scopus (33) Google Scholar Two messages of size 4.4 kb and 1.5 kb were detected in the Northern blot studies. The polyadenylation (polyA) site identified at position 1476 most likely gives rise to transcription termination that leads to the shorter 1.5-kb message. A 282-nucleotide long Alu element, starting at position 1655, has technically challenged our cloning of the distal end of the 3′ UTR of HFGL2. A possible polyA signal further downstream in the yet to be cloned region of the HFGL2, probably gives rise to the 4.4-kb mRNA. Our Northern blot studies show that the presence of the two mRNA species is tissue dependent (Figure 2, A and B). For example, in tissues such as the colon, small intestine, stomach, and prostate the 4.4-kb mRNA predominates. However, in tissues such as the ovary, the 1.5-kb mRNA transcript is expressed predominantly, whereas in the spleen, uterus, skeletal muscle, and peripheral blood leukocytes, the two mRNA transcripts are present in near equal amounts (Figure 2, A and B). The data provided in this report do not reflect quantitative comparisons between tissues but do demonstrate qualitatively, relative expression of the different sized mRNAs within each tissue (organ). The HFGL2 nucleotide sequence predicts a 439-amino acid protein (Figure 3A). The overall amino acid identity between murine fgl2 and HFGL2 proteins is over 70%. The 225 amino acids at the carboxyl end corresponds to the well-conserved fibrinogen-related domain.2Parr RL Fung L Reneker J Myers-Mason N Leibowitz JL Levy GA Association of mouse fibrinogen-like protein with murine hepatitis virus-induced prothrombinase activity.J Virol. 1995; 69: 5033-5038Crossref PubMed Google Scholar The five potential glycosylation sites are conserved in the mouse and human proteins. A cDNA of HFGL2, encompassing the entire coding region, was cloned into pcDNA3.1-His and expressed as a fusion protein (phfgl2his) in CHO cells (Figure 3B). Three hfgl2 specific proteins were detected (200, 140, and 62 kd) by immunoblotting, suggesting that the encoded protein is a dimer or trimer composed of 65-kd subunits joined by disulfide bonds. These results are consistent with a previous report.14Fung LS Neil G Leibowitz J Cole EH Levy GA Monoclonal antibody analysis of a unique macrophage procoagulant activity induced by murine hepatitis virus strain 3 infection.J Biol Chem. 1991; 266: 1789-1795Abstract Full Text PDF PubMed Google Scholar Transient transfection of CHO cells with a full-length cDNA of the HFGL2 coding region resulted in the expression of a procoagulant activity which accelerated the clotting time of recalcified plasma (Table 1). The high level of procoagulant activity coincided with the expression of a prothrombinase which directly cleaved prothrombin to thrombin (Figure 4). This data strongly supports the hypothesis that like its murine counterpart, the HFGL2 gene encodes a prothrombinase activity.Table 1Procoagulant Activity (PCA) of HGFL2 Expressed in CHO CellsSamplesMean PCA (mU/106 Cells) ± SDCHO cells alone1.25 ± 0.5CHO cells with pcDNA3.1his4.75 ± 1.44CHO cells with phfg12his233 ± 152Plasmids encoding fg12 were transfected into CHO cells and analyzed for prothrombinase activity (see Refs. 5Li C Fung LS Crow A Myers-Mason N Leibowitz J Cole E Levy G Monoclonal antiprothrombinase (3D4.3) prevents mortality from murine hepatitis virus (MHV-3) infection.J Exp Med. 1992; 176: 689-697Crossref PubMed Scopus (65) Google Scholar and 6Ruegg C Pytela R Sequence of a human transcript expressed in T-lymphocytes and encoding a fibrinogen-like protein.Gene. 1995; 160: 257-262Crossref PubMed Scopus (39) Google Scholar). Open table in a new tab Figure 4Characterization of hfgl2 prothrombinase by the prothrombin cleavage assay. Lane 1, prothrombin; lane 2, factor Xa + prothrombin; lane 3, prothrombin plus CHO cells alone; lane 4, prothrombin plus CHO cells with phfgl2his; lane 5, prothrombin plus CHO cells with pcDNA3.1his.View Large Image Figure ViewerDownload Hi-res image Download (PPT) Plasmids encoding fg12 were transfected into CHO cells and analyzed for prothrombinase activity (see Refs. 5Li C Fung LS Crow A Myers-Mason N Leibowitz J Cole E Levy G Monoclonal antiprothrombinase (3D4.3) prevents mortality from murine hepatitis virus (MHV-3) infection.J Exp Med. 1992; 176: 689-697Crossref PubMed Scopus (65) Google Scholar and 6Ruegg C Pytela R Sequence of a human transcript expressed in T-lymphocytes and encoding a fibrinogen-like protein.Gene. 1995; 160: 257-262Crossref PubMed Scopus (39) Google Scholar). The eight instances of fulminant viral hepatitis examined all had classical pathological features. The patients ranged in age from 0.5 to 53 years; 6 were females and 2 were males (Table 2). The livers were removed at transplantation for fulminant hepatic failure and showed widespread (range, 60% to 90%) hepatocellular necrosis (Table 3). In six instances, there were surviving areas of parenchyma comprising less than 40% of the liver. These areas typically showed earlier stages of active hepatitis with focal, confluent, or bridging necrosis (Figure 5). In two instances, the entire liver showed end-stage liver disease with total panlobular necrosis. All were examined by in situ hybridization for the presence of HFGL2 prothrombinase mRNA transcripts and by immunohistochemical methods for CD68-positive macrophages or histiocytes as well as fibrin. In the four cases in which surviving parenchyma was seen, fgl2 prothrombinase RNA was detected in macrophages in association with fibrin deposits in vascular sinusoids located selectively in the areas of acute focal, confluent, or bridging necrosis but was infrequent or absent in the more advanced areas of panlobular necrosis in which only the consequences of the active hepatocellular necrotizing process remained. Fibrin deposits were observed by immunoperoxidase staining with an antibody specific for fibrinogen and fibrin, and fibrin thrombi in sinusoids were confirmed by electron microscopy. Panels A to E in Figure 5 are all from areas of residual parenchyma to show regions of bridging necrosis and for comparison, a large area of complete hepatic necrosis and collapse. Panels D and E are deeper sections of the same block that are matched to show equivalent areas of acute bridging necrosis. The positive cells (dark blue stained cells) in the in situ micrograph (E. correspond to the large macrophages (CD68-positive cells) in micrograph (D). Panel C (electron micrograph) is also from an area of acute bridging necrosis.Table 2Patients StudiedPatient no.Age (years)SexDiagnosisEtiology: virus typeOutcome115FFHFHepatitis BTransplant216FFHFSyncytial giant cellTransplant33MFHFNANBNCTransplant42FFHFNANBNCTransplant51FFHFNANBNCTransplant6 90−−−6>90−−−7>80+++8>90+++Abbreviations: +, positive; −, negative.* Representative histology presented (see Figure 5) is from this case. Open table in a new tab Abbreviations: F, female; M, male; FHF, fulminant hepatic failure; NANBNC, non-A, non-B, non-C. Abbreviations: +, positive; −, negative. Two important results are reported. First is the isolation and characterization of the HFGL2 prothrombinase gene that encodes for a prothrombinase having procoagulant activity with the ability to directly cleave prothrombin to thrombin. The second is the co-localization of the HFGL2 prothrombinase gene expression in macrophages and fibrin deposition in sinusoids with microvascular thrombosis and hepatocellular necrosis in the most hepatitic lesions in fulminant viral hepatitis. These observations are key to the pathogenesis of the rapidly progressive necrosis in this disease. The fact that HFGL2 and fibrin are not seen in the more advanced lesions may be attributed to the transitory nature of the immune coagulation response and the rapidity of the hepatocellular necrotizing process that rapidly results in end-stage necrosis of the liver. Of particular relevance to this study, are recent studies of murine viral hepatitis caused by mouse Coronavirus (murine hepatitis virus type 3, MHV-3) in susceptible strains which is an excellent animal model for studying the pathogenesis of fulminant viral hepatitis.3Yuwaraj S Cattral M Pope M Levy GA Murine hepatitis virus: molecular biology and pathogenesis.Viral Hepatitis Rev. 1996; 2: 125-142Google Scholar Especially important is that MHV-3 infection in susceptible BALB/cJ mice causes the de novo synthesis of a unique procoagulant fgl2 prothrombinase by macrophages.2Parr RL Fung L Reneker J Myers-Mason N Leibowitz JL Levy GA Association of mouse fibrinogen-like protein with murine hepatitis virus-induced prothrombinase activity.J Virol. 1995; 69: 5033-5038Crossref PubMed Google Scholar The murine fgl2 gene has been cloned, sequenced, and characterized in this laboratory.2Parr RL Fung L Reneker J Myers-Mason N Leibowitz JL Levy GA Association of mouse fibrinogen-like protein with murine hepatitis virus-induced prothrombinase activity.J Virol. 1995; 69: 5033-5038Crossref PubMed Google Scholar, 4Ding JW Ning Q Liu M Lai A Leibowitz J Peltekian KM Cole EH Fung LS Holloway C Marsden PA Yeger H Phillips MJ Levy GA Fulminant hepatic failure in murine hepatitis virus strain 3 infection: tissue-specific expression of a novel fgl2 prothrombinase.J Virol. 1997; 71: 9223-9230Crossref PubMed Google Scholar, 14Fung LS Neil G Leibowitz J Cole EH Levy GA Monoclonal antibody analysis of a unique macrophage procoagulant activity induced by murine hepatitis virus strain 3 infection.J Biol Chem. 1991; 266: 1789-1795Abstract Full Text PDF PubMed Google Scholar, 15Ning Q Brown D Parodo J Cattral M Gorczynski R Cole EH Fung LS Ding JW Liu MF Rotstein O Phillips MJ Levy GA Ribavirin inhibits viral-induced macrophage production of TNF, IL-1, the procoagulant fgl2 prothrombinase and preserves Th1 cytokine production but inhibits Th2 cytokine response.J Immunol. 1998; 160: 3487-3493PubMed Google Scholar Recent sequential studies showed that the development of fulminant viral hepatitis always followed the same pattern: initiation by viral-induced up-regulation of the fgl2 prothrombinase gene with focal deposits of fibrin in sinusoids and accumulation of inflammatory cells with a predominance of neutrophils and macrophages and focal individual liver cell necrosis. Progression ensued by further fibrin deposition and arrest of sinusoidal blood flow leading to the rapid development of confluent multicellular hepatic necrosis resulting in fulminant hepatic failure and death in 4 days.4Ding JW Ning Q Liu M Lai A Leibowitz J Peltekian KM Cole EH Fung LS Holloway C Marsden PA Yeger H Phillips MJ Levy GA Fulminant hepatic failure in murine hepatitis virus strain 3 infection: tissue-specific expression of a novel fgl2 prothrombinase.J Virol. 1997; 71: 9223-9230Crossref PubMed Google Scholar There is strong evidence for implicating fgl2 prothrombinase as pivotal in the pathogenesis of this disease in the mouse model: levels of this prothrombinase activity correlate with the severity of the disease;16Levy GA MacPhee PJ Fung LS Fisher MM Rappaport AM The effect of mouse hepatitis virus infection on the microcirculation of the liver.Hepatology. 1983; 3: 964-973Crossref PubMed Scopus (40) Google Scholar, 17MacPhee PJ Dindzans VJ Fung LS Levy GA Acute and chronic changes in the microcirculation of the liver in inbred strains of mice following infection with mouse hepatitis virus type 3.Hepatology. 1985; 5: 649-660Crossref PubMed Scopus (52) Google Scholar and there is concordance between expression of fgl2 prothrombinase in the liver with fibrin deposition; and neutralizing antibodies attenuate the pathological and clinical manifestations.5Li C Fung LS Crow A Myers-Mason N Leibowitz J Cole E Levy G Monoclonal antiprothrombinase (3D4.3) prevents mortality from murine hepatitis virus (MHV-3) infection.J Exp Med. 1992; 176: 689-697Crossref PubMed Scopus (65) Google Scholar Indeed, it is not the viral load that determines the occurrence of massive necrosis but the viral induced up-regulation of the fgl2 gene which initiates the pathological process.5Li C Fung LS Crow A Myers-Mason N Leibowitz J Cole E Levy G Monoclonal antiprothrombinase (3D4.3) prevents mortality from murine hepatitis virus (MHV-3) infection.J Exp Med. 1992; 176: 689-697Crossref PubMed Scopus (65) Google Scholar, 18Pope M Rotstein O Cole E Sinclair S Parr R Cruz B Fingerote R Chung SW Gorccynski R Fung L Leibowitz J Rao YS Levy GA Pattern of disease after murine hepatitis strain 3 infection correlates with macrophage activation and not viral replication.J Virol. 1995; 69: 5252-5260Crossref PubMed Google Scholar Although, treatment with neutralizing antibodies to the fgl2 gene abrogates the disease, a h
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