Fgl2 Prothrombinase Research Articles

FGL2 prothrombinase contributes to the early stage of coronary microvascular obstruction through a fibrin-dependent pathway

BackgroundMembrane-associated fibrinogen-like protein 2 (FGL2 prothrombinase, pFGL2) is abundantly expressed in activated microvascular endothelial cells (MVECs) and plays a crucial role in microthrombus formation in microcirculatory vasculature. It has been widely reported that coronary microvascular obstruction (CMVO) contributes to adverse outcomes following myocardial ischemia/reperfusion. However, the role of pFGL2 in CMVO is poorly understood. Methods and resultsWe aimed to identify the effect of MVECs-pFGL2 in CMVO using FGL2 knockout mice. As results, the MVECs-pFGL2 expression progresses significantly over 3 days and then gradually decreases, which is positively correlated with the extent of CMVO as detected by HE staining in wild type mice. Furthermore, FGL2 deficiency is correlated with decreased areas of no-reflow and necrosis as detected by Evans Blue and TTC staining and that it ameliorates cardiac dysfunction detected by hemodynamics in the early stage of CMVO. Moreover, fibrin deposition in microvasculature is significantly reduced in FGL2-deficient mice as evidenced by immunohistochemistry, MSB and Carstairs staining, along with the down-regulation of leukocyte adhesion and infiltration. Additionally, we observed that the FGL2 deficiency decreases macrophage infiltration and shifts the macrophage phenotype from pro-inflammatory (M1,) to anti-inflammatory (M2,) pattern in the early stage of CMVO. ConclusionThese findings highlight the MVECs-pFGL2-fibrin pathway in the early stage of CMVO and provide insights into coagulation and inflammation for the coronary artery disease therapeutics.

Increased activity of cell membrane-associated prothrombinase, fibrinogen-like protein 2, in peripheral blood mononuclear cells of B-cell lymphoma patients.

Fibrinogen-like protein 2, FGL-2, was reported to be overexpressed in various cancer tissues, where it acts as a transmembrane prothrombinase. This study aims to determine the prothrombinase activity of FGL-2 in peripheral blood mononuclear cells (PBMC) of patients with B-cell lymphoma. FGL-2 activity was determined in patients with B-cell lymphoma (n = 53), and healthy controls (n = 145). FGL-2 activity in patients at diagnosis increased 3±0.3 fold (p<0.001). Sensitivity and specificity of the test was established at 73.6% and 80.7%, respectively, using a cutoff of 150% activity over control. Moreover, FGL-2 activity in 10 of 11 patients in remission decreased by 76%. In contrast, no significant difference was observed in expression levels of fgl-2 gene in patients and controls. Taken together, our study indicates that FGL-2 prothrombinase activity in PBMC of lymphoma patients is increased in active disease and normalizes during remission, thus being a potential marker for follow up of lymphoma patients.

Novel antibody against a glutamic acid-rich human fibrinogen-like protein 2-derived peptide near Ser91 inhibits hfgl2 prothrombinase activity.

Fibrinogen-like protein 2 (fgl2) is highly expressed in microvascular endothelial cells in diseases associated with microcirculatory disturbances and plays a crucial role in microthrombosis. Previous studies have demonstrated that the Ser89 residue is a critical site for mouse fgl2 prothrombinase activity. The aim of this study was to investigate the prothrombinase inhibitory ability of antibodies against an hfgl2-derived peptide. The peptide was termed NPG-12 because it is located at the N-terminus of membrane-bound hfgl2, contains 12 amino acid residues (corresponding to residues 76 to 87), and is rich in Glu. This peptide was selected as an antigenic determinant to produce antibodies in immunized rabbits using the DNAStar and HomoloGene software program. Abundant hfgl2 expression was induced in human umbilical vein endothelial cells through treatment with TNF-α. The generated anti-NPG-12 antibodies specifically recognize fgl2, as determined by ELISA, Western Blot and immunostaining. Moreover, one-stage clotting and thrombin generation tests provide evidence that the antibodies can reduce the hfgl2 prothrombinase activity without affecting the platelet-poor plasma prothrombin time (PT) or the activated partial thromboplastin time (APTT). In addition, the antibodies exerted undetectable influence on the proliferation or activation of bulk T cell populations. In conclusion, the selected peptide sequence NPG-12 may be a critical domain for hfgl2 prothrombinase activity, and the development of inhibitors against this sequence may be promising for research or management of hfgl2-associated microcirculatory disturbances.

TNF‐α Upregulates Fgl2 Expression in Rat Myocardial Ischemia/Reperfusion Injury

Proinflammatory cytokine TNF-α during MI/R injury has been studied extensively. However, how TNF-α induces microvascular dysfunction in MI/R is still unclear. This study investigates whether TNF-α regulates fibrinogen-like protein 2 (fgl2) expression, a procoagulant resulting in the formation of fibrin-rich microthrombus in MI/R injury. Microthrombosis, TNF-α and fgl2 expression were assessed in rats with MI/R injury. The effect of TNF-α on fgl2 expression and fgl2 prothrombinase activity was investigated in CMECs, then CMECs were pretreated with selective inhibitors of NF-κB and p38 MAPK pathways. TNF-α and fgl2 expression were both upregulated in MI/R group. When neutralization of TNF-α, fgl2 expression was decreased in vivo. Fgl2 expression was upregulated in CMECs exposed to TNF-α. Accordingly, the ability of thrombin generation was increased in CMECs. Besides, TNF-α-induced fgl2 expression in the cells was suppressed by NF-κB inhibitor PDTC and/or p38 MAPK inhibitor SB203580. TNF-α upregulates fgl2 expression via activation of NF-kB and p38 MAPK in CMECs. TNF-α-induced flg2 in CMECs mediates the formation of fibrin-rich microthrombus, which may be one of the mechanisms of microvascular dysfunction or obstruction due to MI/R injury.

Fgl2 Prothrombinase is involved in Severe Acute Pancreatitis-Associated Liver Injury

Severe acute pancreatitis (SAP)-associated liver injury is systematically one of main pathophysiological events due to SAP development. The aim of the study was to investigate whether fgl2 prothrombinase is involved in SAP-associated liver injury. Microthrombosis in the liver of rats with SAP was observed by Masson staining. Fgl2 prothrombinase expression in the liver of rats with SAP was analyzed by real-time PCR and immunohistochemistry methods. Fgl2 prothrombinase gene and protein expression in SAP group were significantly up-regulated compared to sham-operation (SO) group. Immunohistochemistry staining showed that fgl2 prothrombinase was localized speci?cally to the endothelial cells of intrahepatic veins and hepatic sinusoids. Furthermore, Masson staining demonstrated that the proportion of hepatic microthrombotic capillaries in SAP group were evidently increasing in comparison to SO group and closely correlated with fgl2 expression (r=0.948, p<0.01 ). In addition, there was a positive correlation between fgl2 expression and the severity of hepatocellular injury as indicated by hepatic pathological grade (r=0.704, p<0.01). Fgl2 prothrombinase may contribute to microthrombosis in SAP-associated liver injury, thus resulting in hepatic microcirculatory disturbance and measurement of fgl2 may be used as a helpful biomarker in the prognosis of the severity of hepatic pathological injury in SAP.

Expression and significance of fgl2 prothrombinase in cardiac microvascular endothelial cells of rats with type 2 diabetes

Microthrombosis may be involved in the pathogenesis of cardiac microangiopathy due to diabetes. Recent studies have shown that fibrinogen-like protein 2 (fgl2) plays a pivotal role in microthrombosis in viral hepatitis, acute vascular xenograft rejection and cytokine-induced fetal loss syndrome. The current study was designed to examine the expression of fgl2 in microvascular endothelial cells and investigate the effects of microthrombi due to fgl2 on cardiac function and structure in rats with type 2 diabetes. Following induction of type 2 diabetes, 24 rats were observed dynamically. Fgl2 expression and related cardiac microthrombosis were examined. Local or circulating TNF-α was measured. Coronary flow (CF) per min was calculated as an index of cardiac microcirculation. Cardiac function and morphology were evaluated. It was found that Fgl2 was highly expressed in cardiac microvascular endothelial cells of rats with type 2 diabetes, which was promoted by local or circulating TNF-α. The Fgl2 expression was associated with cardiac hyaline microthrombosis. In parallel with the fgl2 expression, CF per min, cardiac diastolic or systolic function and cardiac morphology were aggravated to some extent. It was concluded that in rats with type 2 diabetes, microthrombosis due to fgl2 contributes to the impairment of cardiac diastolic or systolic function and morphological changes.

Dual Interference with Novel Genesmfgl2andmTNFR1Ameliorates Murine Hepatitis Virus Type 3-Induced Fulminant Hepatitis in BALB/cJ Mice

Our studies and those of many others have implicated hepatocyte necrosis and apoptosis mediated by fibrinogen-like protein-2 (fgl2) prothrombinase and tumor necrosis factor receptor (TNFR) in the development of fulminant viral hepatitis, a disease with a mortality rate greater than 80% in cases lacking immediate organ transplantation. This study was designed to explore the efficacy of dual short hairpin RNA (shRNA) interference with fgl2 and TNFR1 in the treatment of murine hepatitis virus strain 3 (MHV-3)-induced fulminant hepatitis in mice. Plasmids p-mfgl2shRNA and p-mTNFR1shRNA, complementary to the sequences for mfgl2 and mTNFR1, were constructed. Plasmids pEGFP-mfgl2 and pEGFP-mTNFR1 expressing mfgl2-EGFP (enhanced green fluorescent protein) and mTNFR1-EGFP fusion proteins were also constructed to screen the inhibitory effect of p-mfgl2shRNA and p-mTNFR1shRNA on mfgl2 and mTNFR1 expression. Cotransfection of individual shRNA plasmids and pcDNA3.0-mfgl2 and pcDNA3.0-mTNFR1 expression constructs into Chinese hamster ovary (CHO) cells significantly inhibited mfgl2 and mTNFR1 gene expression, as evidenced by fluorescence microscopy, reverse transcription-polymerase chain reaction, and Western blotting. In vivo hydrodynamic delivery of dual-interference shRNA plasmids for mfgl2 and mTNFR1 significantly decreased mfgl2 and mTNFR1 expression; markedly ameliorated fibrin deposition, hepatocyte necrosis, and apoptosis; and prolonged survival against fulminant viral hepatitis induced by MHV-3 in BALB/cJ mice compared with mfgl2 or TNFR1 single-gene interference. These results indicate that in vivo interference with genes for more than one key target provides superior treatment efficacy compared with single-gene interference.

Establishment of pig acute liver failure model and the role of pig fibrinogen-like protein 2

To establish a pig model of fulminant hepatic failure for evaluating the pre-clinical efficacy of drug treatment on severe hepatitis, and to detect the expression of fibrinogen-like protein-2 (fgl2) prothrombinase in the model, so as to provide basis for gene therapy targeting to fgl2 for fulminant hepatic failure. D-galactosamine hydrochloride was used to induce pig model of fulminant hepatic failure, and the experiment animals were divided into model group (rapid injection of D-galactosamine hydrochloride by ear vein, a dose of 1.2 g/kg) and negative control group (5% Glucose). Clinical, biochemical and pathological changes of animals were observed. The expression of pigs fgl2 (pfgl2) mRNA in liver tissue was detected by real time RT-PCR, the expression of pfgl2 protein in liver tissue was detected by immunohistochemistry. A pig model of fulminant hepatic failure was successfully established using the D-galactose hydrochloride; Real time RT-PCR of liver fgl2 mRNA showed that fgl2 mRNA expression was increased significantly in liver tissue of fulminant hepatic failure pig model compared with the control group (P = 0.016); Immunohistochemical staining showed that there were fgl2 protein expression in liver tissue of fulminant hepatic failure pig model, mainly in the membrane and cytoplasm of hepatocytes, inflammatory cells, liver sinusoidal endothelial cells and vascular endothelial cells of liver cell necrosis region. However, there are no fgl2 positive staining on negative control. The pig model of fulminant hepatic failure induced by D-galactosamine hydrochloride is similar to human pathological process and can be used to evaluate the pre-clinical efficacy and safety of drug treatment on fulminant hepatic failure. Abnormal expression of pfgl2 at both mRNA level and protein level in the liver of fulminant hepatic failure pig model shows that pfgl2 induced coagulation pathway is also involved in the development of fulminant hepatic failure. Gene therapy targeting fgl2 genes for fulminant hepatic failure may provide a new means for the treatment of fulminant hepatic failure.

Hepatitis B Virus-induced hFGL2 Transcription Is Dependent on c-Ets-2 and MAPK Signal Pathway

Fibrinogen-like protein 2 (FGL2)/fibroleukin plays a pivotal role in the pathogenesis of experimental and human fulminant and chronic viral hepatitis. To define the transcription factor(s) and upstream signal transduction pathways involved in the transcription of human FGL2 (hFGL2) in response to hepatitis B (HB) virus, hepatitis B core (HBc), hepatitis B virus S protein (HBs), or hepatitis B virus X protein (HBx) protein, expression plasmids were cotransfected with an hFGL2 promoter luciferase reporter construct into Chinese hamster ovary and HepG2 cells, respectively. HBc and HBx proteins, but not HBs protein, enhanced hFGL2 transcription in both cell lines. A strong regulatory region from -712 to -568 (relative to the transcriptional starting site) was shown to be responsible for hFGL2 gene transcription in response to both HBc and HBx proteins. c-Ets-2 was shown to be translocated to the nucleus in association with hFGL2 expression in response to both HBc and HBx proteins. Short hairpin RNA (shRNA) interference of c-Ets-2 expression inhibited hFGL2 gene transcription by 64.8 and 60.0% in response to HBc and HBx, respectively. c-Ets-2 protein was highly expressed in peripheral blood mononuclear cells from patients with severe chronic hepatitis B (CHB) in contrast to patients with mild CHB. Increased phosphorylation of ERK and JNK was detected in peripheral blood mononuclear cells from patients with severe CHB. ERK inhibitor PD098059 or ERK shRNA abolished the nuclear c-Ets-2 DNA binding activity and hFGL2 induction in response to HBc, whereas JNK inhibitor SP600125 or JNK shRNA abolished the nuclear c-Ets-2 DNA binding activity and hFGL2 induction in response to HBx. In conclusion, HBc and HBx proteins enhance transcription of hFGL2 through c-Ets-2 dependent on MAPK signal pathways.

ORIGINAL ARTICLE: LPS‐Induced Murine Abortions Require C5 but not C3, and are Prevented by Upregulating Expression of the CD200 Tolerance Signaling Molecule

Problem Lipopolysaccharide (LPS) acts via tlr4 to promote Th1 cytokine secretion and abortions. LPS is an essential co‐factor in spontaneous abortion in the CBA × DBA/2 model and in stress‐triggered abortions. In the CBA × DBA/2 model, C3a, C5a, and fgl2 prothrombinase participate in triggering inflammation that terminates embryo viability. As fgl2 prothrombinase (via thrombin) can generate C5a, it was predicted that LPS‐driven abortions (which require fgl2) would be independent of C3. CD200Fc can prevent abortions in the CBA × DBA/2 model, but an action through Fc could not be excluded.Method of study C3−/− and C5−/− knock‐out mice on a B6 background were syngeneically mated and Salmonella enteritidis LPS was administered i.p. on day 6.5 or pregnancy along with 2 mg progesterone in sesame oil s.c. The total number of implants and the number of resorbing embryos were counted on day 13.5 of pregnancy. CD200‐rtTA double transgenic homozygous males (B6 background) mated with B6+/+ females were similarly treated. To up‐regulate CD200 expression in embryonic trophoblasts, doxycycline was added to the drinking water from the time of mating.Results The LPS boosted the abortion rate from 15.5% (control) to 42.0% in C3−/− mice (χ2 = 9.28, P &lt; 0.005). In C5−/− mice, there was no increase in abortion rate with LPS compared to progesterone‐treated controls (22.8%versus 26.3%, P = NS). LPS‐treated transgenic mice given LPS + progesterone had a 42.5% abortion rate, but when the mice were given doxycycline to induce expression of CD200 by the embryo, the abortion rate was only 8.3% (χ2 = 14.40, P &lt; 0.005, Fisher’s exact test P = 0.00007).Conclusion C5, but not C3, appears necessary for LPS‐driven abortions. Up‐regulation of CD200 can prevent LPS‐driven abortions, possibly by altering dendritic cells to promote Treg cell development or by a direct suppressive action on macrophages and mast cells that also express CD200 receptors.

The Nucleocapsid Protein of SARS-CoV Induces Transcription of hfgl2 Prothrombinase Gene Dependent on C/EBP Alpha

Fibrin deposition was universal in the lungs of SARS patients and fgl2 prothrombinase gene, a novel procoagulant, was demonstrated to express highly in a clinically relevant SARS model. To investigate whether and which structural protein of SARS-CoV induced transcription of hfgl2 prothrombinase gene, three eukaryotic expression plasmids expressing nucleocapsid protein (N), membrane protein (M) and spike protein 2 (S2) of SARS-CoV were co-transfected with hfgl2 promoter luciferase-reporter plasmids and β-galactosidase plasmid in CHO cells, respectively. M, N and S2 protein of SARS-CoV were detected by western blotting and immunohistochemistry analysis. Further assays demonstrated that expression of hfgl2 gene was related with N protein, but not with M or S2 protein in THP-1 cells and Vero cells. N protein significantly induced functional procoagulant activity in comparison with control group. Luciferase assay showed that N protein of SARS-CoV could activate the transcription of hfgl2 promoter compared with the pcDNA3.1 empty vector. Site-directed mutagenesis and EMSA assay further demonstrated that transcription factor C/EBP alpha band with its cognate cis-element in hfgl2 promoter. The results showed that N protein of SARS-CoV induced hfgl2 gene transcription dependent on the transcription factor C/EBP alpha, which maybe contribute to the development of thrombosis in SARS.

LPS‐Induced Occult Loss in Mice Requires FGL2

FGL2 prothrombinase is required for spontaneous abortion (resorptions) in the CBA x DBA/2 model, and for abortions in C57Bl/6 (B6) mice triggered by Salmonella enteritidis lipopolysaccharide (LPS). Unlike abortions, occult losses in B6 mice, which begin before gestation day 9.5 in mice, do not require the tumor necrosis factor-alpha receptor type 1, and may be triggered by either Salmonella enteritidis or Escherichia coli LPS. Heterozygous matings of fgl2+/-xfgl2+/- B6 mice also have a high spontaneous occult loss of fgl2-/- and to a lesser extent, fgl2+/- embryos caused by hemorrhage between trophoblast and Reichert's membrane. However, the frequency of such losses appears to vary among breeding periods and between laboratories. We tested the hypothesis that FGL2-deficient embryos were uniquely susceptible to LPS by treating B6 fgl2+/-xfgl2+/- heterozygous matings with 2 microg E. coli LPS intraperitoneally on day 6.5 of pregnancy. Progesterone 2 mg in 100 microL sesame oil was administered subcutaneously at the same time to ensure that the effects of the LPS were not because of suppression of ovarian hormone production. PCR DNA genotyping was performed on embryos at day 13.5 of pregnancy, or after parturition. Surprisingly, we found that LPS caused occult loss of fgl2+/+, and to a lesser extent fgl2+/- embryos, both at term and by PCR typing of embryos at gestation day 13.5, and these losses obscured the effect of fgl2 knockout. Spontaneous loss of fgl2-/- embryos may relate to adhesion effects, whereas fgl2+/+ embryos are susceptible to LPS-induced loss; fgl2+/- embryos may be affected by both mechanisms. Antagonizing FGL2 could prevent occult pregnancy loss in some human and animal situations.

Novelmfgl2Antisense Plasmid Inhibits Murinefgl2Expression and Ameliorates Murine Hepatitis Virus Type 3-Induced Fulminant Hepatitis in BALB/cJ Mice

Our previous reports, both experimental and human studies, have shown the importance of fibrinogen-like protein-2 (fgl2) prothrombinase in the development of fulminant viral hepatitis, a disease with a mortality of more than 80% in cases lacking immediate organ transplantation. To interfere with this potentially effective target, a 322-bp mouse fgl2 (mfgl2) antisense plasmid complementary to the exon 1 sequence of the gene, including the translation initiation site AUG, was successfully constructed. A dose-dependent inhibitory effect on mfgl2 expression by mfgl2 antisense plasmid was observed in interferon-gamma-treated RAW 264.7 cells. On hydrodynamic delivery, mfgl2 antisense plasmid significantly reduced mfgl2 expression in vivo; markedly ameliorated inflammatory cell infiltration, fibrin deposition, and hepatocyte necrosis; prolonged the survival time period; and elevated the survival rate among BALB/cJ mice with murine hepatitis virus type 3-induced fulminant hepatitis. This study may provide an effective way to interfere with the potential therapeutic target fgl2 gene for fulminant viral hepatitis and other diseases with similar pathological characteristics of microcirculation disorders, including acute rejection of xeno- or allograft transplantation and fetal loss syndrome, in which studies show fgl2 plays an important role.

Immunological Analogy Between Allograft Rejection, Recurrent Abortion and Pre-Eclampsia – the Same Basic Mechanism?

There are still controversies concerning the role of immunological mechanisms engaged both in recurrent abortions (RA) and pre-eclampsia (PE). According to some opinions, recurrent miscarriage is comparable to organ-specific autoimmune disease. Analysis of immune reactions shows that graft rejection shares many similar mechanisms with RA and PE. This fact allows us to conclude that rejection of transplanted alloantigenic organs and pregnancy loss have probably the same evolutionary origin. Subsets and functions of immunocompetent cells (T CD4, suppressor γδT, cytotoxic T CD8, Treg, Tr1, uterine NK cells), over-activation of innate immunity (activation of NK cytotoxic cells, macrophages, neutrophils and complement), changes of Th1/Th2 cytokine balance (IL-2, IL-12, IL-15, IL-18, IFNγ, TNFα vs. IL-4, IL-10, TGFβ), importance of HLA-G molecule, CD200/CD200R interaction, over-expression of adhesion molecules, fgl2 prothrombinase activation and stimulation of IDO and HO expression, all suggest that RA and PE are syndromes of fetal allograft rejection, and not organ-specific autoimmune diseases. According to that supposition, an analogy might exist between acute graft rejection and recurrent abortion, and between chronic graft rejection and pre-eclampsia.

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

Immune coagulation is a major contributor to the pathogenesis of xenograft rejection, viral-induced hepatocellular injury and cytokine-induced fetal loss syndrome. In this study, we investigated the contribution of the novel gene product, fibrinogen-like protein 2 (fgl2) prothrombinase, in mediating immune injury in experimental and human acute allograft rejection. Using a mouse heterotopic cardiac transplant model, mouse fgl2(mfgl2)/fibroleukin mRNA transcripts and protein were highly expressed in macrophages, CD4- and CD8-positive T lymphocytes, and endothelial cells in rejecting cardiac allografts in association with deposits of fibrin. Although mfgl2-deficient mice rejected allografts at similar rates to littermate controls, survival of grafts from mfgl2-deficient mice were prolonged and deposition of intravascular fibrin was diminished. Treatment of wild-type mice with a neutralizing anti-fgl2 Ab ameliorated histological evidence for allorejection and intravascular fibrin deposition, and resulted in an increase in graft survival. To address further the relevance of fgl2 in acute allograft rejection, we examined kidney biopsies from patients who had undergone renal transplantation. Human fgl2 mRNA transcripts and protein were markedly expressed mainly in renal tubule cells, infiltrating lymphoid cells including macrophages, CD8(+) T cells, mature B cells (plasma cells), and endothelial cells. Dual staining showed fibrin deposition was localized mainly to blood vessels, in the glomerulus and interstitium and the lumen of tubules, and occurred in association with human fgl2 expression. These data collectively suggest that fgl2 accounts for the fibrin deposition seen in both experimental and human allograft rejection and provide a rationale for targeting fgl2 as adjunctive therapy to treat allograft rejection.

Upregulation of fgl2 prothrombinase in hepatitis B transgenic mice which develop chronic liver disease: Camie W Chan Ms, Reginald M Gorczynski Dr, M J Phillips Dr, Univ of Toronto, Toronto, ON Canada; M A Feitelson Dr, Thomas Jefferson Univ, Philadelphia, PA; Gary A Levy Dr, Univ of Toronto, Toronto, ON Canada

Upregulation of fgl2 prothrombinase in hepatitis B transgenic mice which develop chronic liver disease: Camie W Chan Ms, Reginald M Gorczynski Dr, M J Phillips Dr, Univ of Toronto, Toronto, ON Canada; M A Feitelson Dr, Thomas Jefferson Univ, Philadelphia, PA; Gary A Levy Dr, Univ of Toronto, Toronto, ON Canada

The fgl2 prothrombinase/fibroleukin gene is required for lipopolysaccharide-triggered abortions and for normal mouse reproduction.

Increased fgl2 prothrombinase activity in maternal decidua and fetal trophoblasts may trigger abortions by proinflammatory cytokines induced by bacterial lipopolysaccharide (LPS) in mice and is implicated in human recurrent miscarriages and pre-eclampsia. Defining the physiological and pathological role of the fgl2/fibroleukin gene required an fgl2-knockout mouse and data on normal pattern of fgl2 expression during pregnancy. Expression of fgl2 protein was determined by immunostaining with specific antibody. Fgl2 knockout mice were generated and typed by PCR for presence of the altered gene. Immunostaining of timed CBAxDBA/2 mouse matings in a low-abortion-rate colony showed a distinct pattern of development of fgl2 protein expression in maternal decidua, and in embryonic tissues in early pregnancy. Outbred (mixed background) heterozygous fgl2 +/-x+/- matings with a similar low abortion rate showed selective occult loss of both +/- and, to a greater extent, -/- embryos prior to gestation day 11.5, in association with haemorrhage at the anti-mesometrial pole of fgl2-deficient embryo. LPS injected on day 6.5 caused classical abortions at mid-pregnancy in fgl2 +/+x+/+ matings, but not -/-x-/- matings. Physiological expression of fgl2 in fetal trophoblast may prevent occult loss in early pregnancy, along with other coagulation factors, but fgl2 expression is required for LPS to induce abortion pathology.

Regulation of interleukin-6 fetoplacental levels could be involved in the protective effect of low-molecular weight heparin treatment on murine spontaneous abortion.

CBA/J x DBA/2 abortion rate could be the consequence of a deficient local production of T helper (Th2) cytokines, which cause fetal wastage via fgl2 prothrombinase. Heparin reduces significantly the abortion rate in mice and recurrent spontaneous abortion (RSA) patients. We proposed to determine the effect of enoxaparin on the levels of local interleukin (IL)-6 during murine pregnancy. Recombinant human IL-6 (rhIL-6) or enoxaparin were inoculated in CBA/J x DBA/2 pregnant mice on days 6.5-12.5. IL-6 levels in sera as well as in culture supernatants of day 9.5 fetoplacental units of CBA/J x BALB/c control mice or CBA/J x DBA/2 abortion combination were determined by enzyme-linked immunosorbent assay (ELISA) test. CBA/J x DBA/2 fetoplacental units secreted significantly lower levels of IL-6 with regard to CBA/J x BALB/c normal units. rhIL-6h and enoxaparin treatments decreased the resorption rate and regulated IL-6 fetoplacental levels. This study suggests that regulation of IL-6 fetoplacental levels could be involved in heparin-mediated anticoagulation protection against abortion.

Induction of Prothrombinase fgl2 by the Nucleocapsid Protein of Virulent Mouse Hepatitis Virus Is Dependent on Host Hepatic Nuclear Factor-4α

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.

FGL2 prothrombinase messenger RNA expression in gravid and nongravid human myometrium

Objective: Thrombin is a potent uterotonic agent that may play an important role in human parturition. FGL2 is a prothrombinase that cleaves prothrombin to thrombin. The purpose of this study was to determine FGL2 messenger RNA expression in human myometrium and to assess its relationship to the expression of EP3-6 prostaglandin receptor and inducible nitric oxide synthase. Study Design: Myometrium was obtained from gravid women who underwent cesarean delivery (n = 19), from premenopausal (n = 10) and menopausal (n = 10) women who underwent hysterectomy, and from nonpregnant women who received 200 mg of misoprostol vaginally 12 hours before hysterectomy (n = 8). FGL2 messenger RNA expression was determined with the use of semiquantitative polymerase chain reaction and was compared with previously determined messenger RNA expression levels for EP3-6 and inducible nitric oxide synthase for the same samples. Statistical analysis was performed with the use of independent t test, Fisher exact test, analysis of variance, and linear regression, where appropriate. Results: All samples that were analyzed contained demonstrable quantities of FGL2 messenger RNA. The highest FGL2 expression was found in gravid women, followed by women who were treated with misoprostol, by premenopausal nonpregnant women, and by menopausal women (P <.0001, analysis of variance). Significantly higher levels of FGL2 expression were found in the gravid women compared with the women who were treated with misoprostol (P <.001), the premenopausal nonpregnant women (P <.001), and the menopausal women (P <.0001). FGL2 messenger RNA expression correlated with EP3-6 and inducible nitric oxide synthase expression in all samples combined (r = 0.55, P <.0001; and r = 0.67, P <.001, respectively). Conclusion: The expression of FGL2 messenger RNA is increased in gravid myometrium compared with nonpregnant samples. Its expression significantly correlates to EP3-6 and inducible nitric oxide synthase expression. FGL2 prothrombinase expression may mediate the role of thrombin in human parturition. (Am J Obstet Gynecol 2003;188:1057-62.)