thromboplastin and Hepatitis-B

Topics: Adolescent; Adult; Aged; Blood Coagulation; Blood Platelets; Blood Preservation; Blood Transfusion, Autologous; Blood Viscosity; Blood Volume; Cardiac Surgical Procedures; Child; Child, Preschool; Extracorporeal Circulation; Female; Fibrinogen; Hematocrit; Hepatitis B; Humans; Infant; Male; Middle Aged; Prothrombin Time; Thromboplastin

Antiviral resistance mutations in the hepatitis B virus (HBV) polymerase (pol) gene have been demonstrated to play an important role in the progression of liver disease and the development of hepatocellular carcinoma. The HBV pol gene overlaps the S gene encoding surface antigen (HBsAg). Previous studies from our laboratory have shown that HBV core protein (HBc) and X protein (HBx), but not HBV S protein (HBs), promote hfgl2 prothrombinase transcription. To investigate whether the nucleotide (nucleoside)-induced resistant mutations of HBs potentiate transcription of hfgl2 prothrombinase gene, we generated two mutant HB expression constructs harboring rtM204V/sI195M or rtM204I/sW196L mutations. Two mutant expression plasmids were co-transfected with hfgl2 promoter luciferase-reporter plasmids and β-galactosidase plasmid in CHO cells and HepG2 cells, respectively. Luciferase assay showed that the rtM204I/V mutant HBs could activate the transcription of hfgl2 promoter compared with the wild type HBs. Site-directed mutagenesis and further experiment (co-transfection) demonstrated that transcription factor Ets translocated to its cognate cis-element in the hfgl2 promoter. The results show that mutated HBs caused by antiviral drug resistance induce transcription of the hfgl2 gene dependent on the transcription factor Ets.

Topics: Animals; Antiviral Agents; Base Sequence; Cell Line; Drug Resistance, Multiple, Viral; Fibrinogen; Hepatitis B; Hepatitis B Surface Antigens; Hepatitis B virus; Humans; Molecular Sequence Data; Mutation; Promoter Regions, Genetic; Thromboplastin; Transcription, Genetic

To evaluate the expression of fibrinogen-like protein 2 (fgl2) and its correlation with disease progression in both mice and patients with severe viral hepatitis.. Balb/cJ or A/J mice were infected intraperitoneally (ip) with 100 PFU of murine hepatitis virus type 3 (MHV-3), liver and serum were harvested at 24, 48, and 72 h post infection for further use. Liver tissues were obtained from 23 patients with severe acute chronic (AOC) hepatitis B and 13 patients with mild chronic hepatitis B. Fourteen patients with mild chronic hepatitis B with cirrhosis and 4 liver donors served as normal controls. In addition, peripheral blood mononuclear cells (PBMC) were isolated from 30 patients (unpaired) with severe AOC hepatitis B and 10 healthy volunteers as controls. Procoagulant activity representing functional prothrombinase activity in PBMC and white blood cells was also assayed. A polyclonal antibody against fgl2 was used to detect the expression of both mouse and human fgl2 protein in liver samples as well as in PBMC by immunohistochemistry staining in a separate set of studies. Alanine aminotransferase (ALT) and total bilirubin (TBil) in serum were measured to assess the severity of liver injury.. Histological changes were found in liver sections 12-24 h post MHV-3 infection in Balb/cJ mice. In association with changes in liver histology, marked elevations in serum ALT and TBil were observed. Mouse fgl2 (mfgl2) protein was detected in the endothelium of intrahepatic veins and hepatic sinusoids within the liver 24 h after MHV-3 infection. Liver tissues from the patients with severe AOC hepatitis B had classical pathological features of acute necroinflammation. Human fgl2 (hfgl2) was detected in 21 of 23 patients (91.30%) with severe AOC hepatitis B, while only 1 of 13 patients (7.69%) with mild chronic hepatitis B and cirrhosis had hfgl2 mRNA or protein expression. Twenty-eight of thirty patients (93.33%) with severe AOC hepatitis B and 1 of 10 with mild chronic hepatitis B had detectable hfgl2 expression in PBMC. No hfgl2 expression was found either in the liver tissue or in the PBMC from normal donors. There was a positive correlation between hfgl2 expression and the severity of the liver disease as indicated by the levels of TBil. PCA significantly increased in PBMC in patients with severe AOC hepatitis B.. The molecular and cellular results reported here in both mice and patients with severe viral hepatitis suggest that virus-induced hfgl2 prothrombinase/fibroleukin expression and the coagulation activity associated with the encoded fgl2 protein play a pivotal role in initiating severe hepatitis. The measurement of hfgl2/fibroleukin expression in PBMC may serve as a useful marker to monitor the severity of AOC hepatitis B and a target for therapeutic intervention.

Topics: Animals; Disease Models, Animal; Disease Progression; Female; Fibrinogen; Hepatitis B; Hepatitis, Viral, Animal; Humans; Liver; Mice; Mice, Inbred BALB C; Murine hepatitis virus; Thromboplastin

Fibrinogen-like protein 2/fibroleukin (Fgl2) plays a pivotal role in the pathogenesis of both experimental and human fulminant hepatic failure. We have reported recently that the nucleocapsid (N) protein from strains of murine hepatitis virus (MHV-3, MHV-A59), which cause massive hepatocellular necrosis but not from strains (MHV-JHM, MHV-2) which do not produce serious liver disease, induces transcription of fgl2. The purpose of the present study was to characterize both viral and host factor(s) necessary for viral induced transcription of fgl2. Mutation of residues Gly-12, Pro-38, Asn-40, Gln-41, and Asn-42 within domain 1 of the N protein of MHV-A59 to their corresponding residues found in MHV-2 abrogated fgl2 transcription, whereas mutation of other N protein domains, including a protein expressed from an internal reading frame (I protein), did not affect fgl2 gene transcription. We then examined the -372 to -306 sequence within the 1.3-kb fgl2 promoter region upstream from the transcription start site that was previously identified as necessary for N protein-induced gene transcription. We demonstrated that the -331/-325 HNF4 cis-element and its cognate transcription factor, HNF4alpha, are necessary for virus-induced fgl2 gene transcription. In uninfected macrophages and macrophages infected with MHV-2, an unidentified protein occupies the HNF4 cis-element. Following stimulation with MHV-A59, it was shown by electrophoretic mobility shift assay that HNF4alpha binds the HNF4 cis-element in the fgl2 promoter. We further report the unprecedented presence of HNF4alpha in peritoneal macrophages. Collectively, the results of this study define both viral and host factors necessary for induction of fgl2 prothrombinase gene transcription in MHV infection and may provide an explanation for the hepatotrophic nature of MHV-induced fulminant hepatic failure.

Topics: Amino Acid Sequence; Animals; Basic Helix-Loop-Helix Leucine Zipper Transcription Factors; CHO Cells; Cricetinae; DNA-Binding Proteins; Female; Fibrinogen; Gene Expression Regulation; Hepatitis B; Hepatitis C; Hepatocyte Nuclear Factor 4; Mice; Mice, Inbred BALB C; Molecular Sequence Data; Murine hepatitis virus; Nucleocapsid Proteins; Phosphoproteins; Promoter Regions, Genetic; Thromboplastin; Transcription Factors; Transcription, Genetic

Topics: Animals; Fibrinogen; Hepatitis B; Humans; Mice; Severe Acute Respiratory Syndrome; Thromboplastin

Topics: Animals; Blood Coagulation; Cell Differentiation; Hepatitis B; Hepatitis, Chronic; Humans; Lymphocyte Cooperation; Lymphocytes; Mice; Models, Biological; Monocytes; Phenotype; Plasminogen Activators; Rats; Receptors, Immunologic; Thromboplastin

Topics: Blood Coagulation Tests; Blood Donors; Carrier State; Hepatitis B; Humans; Liver Function Tests; Mass Screening; Thromboplastin