1225
LIVER
Οriginal Paper
Fgl2 Prothrombinase is
Involved in Severe Acute
Pancreatitis-Associated Liver Injury
Tanzhou Chen*, Xiaohua Ye*, Zhiming Huang, Xin Chen, Yong Du, Renpin Chen and Xiaoju Zhuge
*These authors contributed equally
Department of Gastroenterology and Hepatology,
The First Affiliated Hospital of Wenzhou Medical College, Wenzhou, China
Corresponding author: Zhiming Huang, Department of Gastroenterology and Hepatology, the First Affiliated Hospital of
Wenzhou Medical College, Wenzhou, China; Tel.: +577-88069257; E-mail: [email protected]
ABSTRACT
Background/Aims: 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. Methodology: 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. Results: Fgl2 prothrombinase gene and
protein expression in SAP group were significantly upregulated compared to sham-operation (SO) group. Immunohistochemistry staining showed that fgl2 prothrombinase was localized specifically to the endotheINTRODUCTION
Death in most patients with severe acute pancreatitis
(SAP) turns out to be specifically related to multiple organ dysfunction syndrome (MODS). Research suggests
that the highest mortality of SAP patients was complicated by liver (83%) failure (1).
Microcirculatory disturbance and coagulation imbalance such as microthrombosis have long been considered
as characteristic of severe necrotizing form of SAP (2,3)
and moreover, it is not only a local manifestation confined
to the damaged pancreas but also component of systematic inflammation to pancreatic injury (2). In addition to
pancreatic injury due to continuous microcirculatory dysfunction, as a consequence of hypovolemia and systemic inflammatory reaction, similar changes were also observed to be detrimental in liver, which tended to be vulnerable to serious strike (2,4-6). The hepatic microcirculatory disadvantages are involved in the process of liver
injury and functional attenuation (7). Research demonstrates that various vasoactive and inflammatory mediators released in the course of SAP positively participate in
the development of microcirculatory failure in extrapancreatic organs such as liver, as well as contribute to the
inflammatory reactions (8,9), indicating microcirculatory disturbance and inflammation are associated during
the course of SAP (10). Therapies with anti-coagulants
also could ameliorate hepatic microcirculatory perfusion
in the rat model of SAP-associated liver injury (4). But as
yet, the exact pathophysiological mechanism of SAP-associated liver injury remains to be obscure.
Fibrinogen-like protein 2 (fgl2/fibroleukin) is a novHepato-Gastroenterology 2012; 59:1225-1229 doi 10.5754/hge12117
© H.G.E. Update Medical Publishing S.A., Athens
Key Words:
fgl2 prothrombinase; Severe acute
pancreatitis; Liver
injury; Microthrombosis.
lial 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 Abbreviations:
Fibrinogen-like
(r=0.948, p<0.01 ). In addition, there was a positive cor- Protein 2 (fgl2);
relation between fgl2 expression and the severity of he- Severe Acute
patocellular injury as indicated by hepatic pathological Pancreatitis (SAP);
grade (r=0.704, p<0.01). Conclusions: Fgl2 prothrom- Sprague-Dawley
(SD); Sham-Operabinase may contribute to microthrombosis in SAP-as- tion (SO); Alanine
sociated liver injury, thus resulting in hepatic microcir- Aminotransferase
culatory disturbance and measurement of fgl2 may be (ALT); Aspartate
used as a helpful biomarker in the prognosis of the se- Transaminase
(AST); Multiple
verity of hepatic pathological injury in SAP.
Organ Dysfunc-
tion Syndrome
(MODS); Polymerase Chain
el prothrombinase with a highly concerned domain, Reaction (PCR);
Immunohistowhich can directly generate thrombin from prothrom- chemistry
(IHC);
bin in an independent way without a classical prothrom- Diaminobenzidine
binase complex and subsequently result in fibrin depo- (DAB); Real-time
sition (11,12). Induced by certain proinflammatory cyto- Quantitative
Chain
kines, fgl2 prothrombinase plays a pivotal role in micro- Polymerase
Reaction (RT-PCR).
thrombosis and leads to microvascular disturbance and
pathological injuries by mediating “immune coagulation”
in different inflammatory diseases like viral-induced fulminant hepatitis (13), allograft/xenograft rejection (14)
and cytokine-induced fetal loss syndrome (15). However,
it is still not yet understood whether fgl2 prothrombinase
contributes to liver injury due to SAP.
In this study, the rat model of SAP was induced by retrograde injection of 4% sodium taurocholate. Then, we
examined the expression of fgl2 prothrombinase in the
hepatic endothelial cells of intrahepatic veins and hepatic
sinusoids, and moreover we evaluated the expression of
fgl2 prothrombinase and its correlation with severity of
hepatic pathological injury and microthrombi in rats with
SAP, expecting that it could be a new view on the pathogenesis of SAP-associated liver injury.
METHODOLOGY
Animals
A total of forty-eight healthy adult male Sprague-Dawley (SD) rats, weighing 200-250g, were purchased from
the Experimental Animal Center of Wenzhou Medical College, Wenzhou, China. All the animals were housed at a
constant room temperature of 25°C with an alternative
12-hour day/night cycle and had free access to water
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and standard rat chow. All the animals were studied after one week of acclimatization. All procedures were performed in accordance with the Guidelines for Animal Experiments of Wenzhou Medical College, Wenzhou, China.
Animals were randomly divided into the following
groups: SAP group and sham operation (SO) group. Each
experimental group consisted of 24 individuals.
Animal model for SAP
All SD rats were subjected to intraperitoneal injection
of 10% chloraldurate (Solarbio, Beijing, China) (2mL/kg
body weight) for anesthesia. A laparotomy was carried
out through a midline incision in SAP group and the biliopancreatic duct was cannulated transduodenally with
a segmental epidural catheter followed by retrograde
injection of 1mL/kg body weight of 4% sodium taurocholate (Sigma, St. Louis, USA) into it via a microinjection pump at a speed of 0.2mL/min. The hepatic portion
of the biliopancreatic duct was clipped to prevent reflux
before injection of 4% sodium taurocholate. In the SO
group, rats underwent operations but without infusion
of 4% sodium taurocholate. After the operations, the abdomens of rats in each group were closed in two layers.
The entire procedures were carried out by using sterile techniques.
All the rats were sacrificed at 1, 4 and 8h time-points
(n=8 per time-point) since operations by exsanguinations. Blood was collected by post cava puncture and
the serum separated was stored at -20°C. The levels of
serum amylase, alanine aminotransferase (ALT) and aspartate transaminase (AST) were determined by a fully
automatic biochemical analyzer (Hitachi, Tokyo, Japan).
Parts of the liver were fixed in 4% paraformaldehyde
for histopathological analysis, while the remainig parts
were removed and stored in liquid nitrogen until use.
A
T Chen, X Ye, Z Huang, et al.
Hepato-Gastroenterology 59 (2012)
Histopathological analysis
For histopathological analysis, samples were dehydrated and embedded in paraffin. Four µm thick sections were cut and stained with hematoxylin and eosin
(HE staining) for light microscopic examination. Blinded analysis was applied for all histopathological studies.
The pathological grade was as follows: 0: vessels, cells
and intercellular substances in the liver are normal; I:
congestion with few cellular infiltration; II: microthrombosis, cellular swelling of hepatocyte and inflammation
around portal vein; III: diffuse hepatocellular necrosis
with massive inflammatory cells infiltration.
The sections were also performed with Masson staining in order to observe microvascular microthrombosis.
One hundred microvessels for each section were randomly selected and the proportion of microthrombi positive vessels was calculated.
B
Real-time polymerase chain reaction
Total RNA was extracted from each sample with Trizol
reagent (Invitrogen, Carlsbad, USA) and then cDNA was
synthetized by reverse transcription (MBI Fermentas, Burlington, Canada) according to the manufacturer’s protocols. The cDNA was subsequently amplified by
polymerase chain reaction (PCR) with primers to detect
the levels of fgl2 mRNA using ABI 7500 Sequence Detection System (Applied Biosystems Inc., Carlsbad, USA). The
sequences of the primers (Generay, Shanghai, China) for
fgl2 were as follows: (sense) 5’-cctggagattgtggtttcgt-3’ and (antisense) 5’-ggggtgttgaaggtctcaaa-3’;
β-actin: (sense) 5’-TGTCACCAACTGGGACGATA-3’ and
(antisense) 5’-GGGGTGTTGAAGGTCTCAAA-3’. cDNA was
denatured at 95°C for 5min and amplified over 40 cycles
of 95°C (15s), 60°C (45s), 72°C (60s) and a final extension at 72°C (5min). All samples were detected in triplicate and levels of fgl2 gene expression were calculated by
the 2-ΔΔCT method.
Immunohistochemistry staining
Immunohistochemistry staining was applied to semiquantified fgl2 prothrombinase expression in the liver
using EnVision technique. Paraffin sections were routinely prepared. To activate antigens, microwave antigen retrieval was used for 20min in citrate buffer (pH 6.0) before
endogenous peroxidase depleting in 0.3% H2O2 for 10min.
The sections were incubated with a 1:100 dilution of rabbit anti-rat fgl2 polyclonal antibodies (Biosynthesis Biotechnology, Beijing, China) for 2 hours at 37°C. With the
same washing procedure again, sections were re-incubated with EnVision reagents (Dako, Glostrup, Denmark) for
30min at 37°C. Finally, all sections were developed with
diaminobenzidine (DAB) and counterstained with hematoxylin for microscopy. Negative control was set in the experiment. For the evaluation of fgl2 prothrombinase expression, a total of 10 random selected fields across each
section were analyzed at a magnification of x200.
Statistical analysis
All data were expressed as mean ±SD. Statistical analysis was conducted with SPSS 15.0 software. Differences
between SAP group and SO group were analyzed by t-test.
ANOVA analysis was applied to check for statistical significance among 3 time-points in the same group. A value of
p<0.05 was considered to be statistically significant.
Results
Levels of serum biochemical parameters elevated in
SAP group
The serum amylase is most commonly used as a biochemical indicator for evaluating acute pancreatitis, while
ALT/AST is clinically detected for assessment of hepato-
C
FIGURE 1. Levels of serum amylase (A), ALT (B) and AST (C) in both groups at each time-point. Each time-point after operation consisted of 8 individuals. There were no significant differences (p>0.05) among 3 time-points in SO groups of all the biochemical parameters. Data are expressed as mean ±SD. #p<0.01 vs. SO group.
Fgl2 is involved in SAP-Associated Liver Injury
cellular injury and the change of liver function. Levels of
all the biochemical parameters were evidently elevated in
SAP group than those in SO group at each time-point (n=8
per group) (p<0.01). There were no significant differences (p>0.05) for the levels of all the parameters among 3
time-points in SO group (Figure 1).
Histopathological examinations
Histopathological changes in the hepatic tissues of rats
with SAP were discovered to be evident of severe liver injury. By 1h time-point post induction of SAP, isolated and
non-zonal foci of necrosis with scattered polymorphonuclear leukocyte infiltration were observed (Figure 2A).
The lesions were enlarged and deteriorated at 4h timepoint (Figure 2B) and thereafter confluent necrosis was
apparent at 8h time-point (Figure 2C). Nevertheless, the
rats in SO group tended to be normal morphologically at
8h time-point (Figure 2D).
The observation of hepatic microthrombosis was preceded by Masson staining and was shown to be a highly bright red area under microscope. Microthrombi were
determined to be connected tightly with microvascular
endothelium of hepatic sinusoids and occasional branches of intrahepatic veins that randomly distributed as discrete micro-foci throughout the liver, suggesting being microthrombi in situ comprised of fibrin. Masson staining
positive microvessel proportion of liver in SAP group was
markedly increased (p<0.01) compared to that in normal
ones and displayed an elevating tendency (p<0.01) (Figure 3).
Fgl2 prothrombinase expression
We determined significant up-regulated levels of fgl2
prothrombinase expression in SAP group compared to
SO group by using real-time PCR and immunochemistry analysis (p<0.01). Increasing fgl2 expression (p<0.01)
was detected in the liver with the progression of SAP (Figure 4A,B). Immunohistochemistry staining demonstrated that fgl2 prothrombinase was strongly expressed in
endothelial cells of intrahepatic veins and hepatic sinusoids of rats with SAP (Figure 4C1-C3) while little was
found in SO group (Figure 4C4). Pearson’s correlation coefficient (r) analysis concluded that fgl2 expression indicated by mean absorbance value was evidently correlated
with the proportion of Masson staining positive microvessels (r=0.948, p<0.01).
Hepato-Gastroenterology 59 (2012)
1227
Correlations between fgl2 expression and serum
ALT/AST levels
Spearman grade correlation analysis was conducted
to calculate the strength between hepatic fgl2 expression
and the severity of hepatic pathological injury revealed by
hepatic pathological grade. Significant direct correlations
were found between hepatic pathological grade and fgl2
gene expression (r=0.703, p<0.01).
Discussion
During the initial phase of SAP, hemodynamic shock
is an extremely serious attack of a series of pathological
processes, resulting in persistent microcirculatory dysfunction not only limited to pancreas, but also in various
extra-pancreatic organs (1,2,4,5). SAP-associated liver injury is systematically one of the main pathophysiological
events due to SAP development, during which aggressive
microcirculatory disturbances could possibly worsen the
severity of SAP. To our knowledge, cytokines generated
in SAP are related to dysfunction of microcirculation besides activating inflammatory process (10), implying the
interaction between these two facts and moreover, not
FIGURE 2. Hematoxylin-eosin (HE) staining (×200) of liver tissue 1h (A), 4h (B) and 8h (C)
after induction of SAP and 8h (D) in SO group.
FIGURE 3. Masson staining (×200) of hepatic microthrombosis of rats. (A-C) Microthrombosis in situ in microvascular endothelium of hepatic sinusoids
at 1h (A), 4h (B) and 8h (C) in SAP group (arrows), respectively; (D) Few microthrombi were determined in SO group (arrow); (E) Proportion of Masson staining positive micro-vessels (%). Each time-point after operation consisted of 8 individuals. Data are expressed as mean ±SD. #p<0.01 vs. SO group.
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Hepato-Gastroenterology 59 (2012)
A
T Chen, X Ye, Z Huang, et al.
B
FIGURE 4. Fgl2 determined by real-time PCR and immunohistochemistry (×200 in C1-C4). (A-B) Expression of fgl2 gene and protein expression. (C1C3) Fgl2 prothrombinase was strongly expressed in endothelial cells (arrows) of intrahepatic veins and hepatic sinusoids of rats with SAP at 1h (C1), 4h
(C2) and 8h (C3) time-point, respectively; (C4) No fgl2 prothrombinase was found in SO group at 8h time-point. Each time-point after operation consisted of 8 individuals. Data are expressed as mean ±SD. #p<0.01 vs. SO group.
only in the local tissues, studies suggested that both cytokines and microcirculatory dysfunction are involved in
the pathogenesis of SAP associated liver injury (5-7,8-10).
Fgl2/fibroleukin is one of the members of the fibrinogen-related protein superfamily, which is a novel procoagulant that could eventually be pernicious to microcirculation due to the microthrombi it synthesizes (11,16). Different from the common procoagulants, fgl2 prothrombinase serves its role independent of the old-fashioned
ways and furthermore, generates hyaline microthrombi
(12). The present research has proved for the first time,
that fgl2, which could result in microcirculatory disturbance, plays a pivotal role in SAP-associated liver injury. Our data have shown that microthrombi produced
by over expressed fgl2 led to pathological deterioration
and hepatocellular injuries of the liver and moreover, it is
demonstrated that fgl2 expression serves a role in hepatic microthrombosis of rats with SAP to some degree. In
addition, we also discovered that fgl2 was expressed positively as a source of procoagulant activity in the hepatic
endothelial cells of intrahepatic veins and hepatic sinusoids in rats with SAP and its expression was tightly correlated to the severity of hepatocellular injuries.
Fgl2 functions via “immune coagulation” (11), which
signifies that fgl2 is induced and up-regulated by related proinflammatory cytokines like IL-2, TNF-α as stimuli
(15,17-19) and positively expressed in microvascular endothelial cells, macrophages and other immunocytes. Investigation based on cytokine-induced hepatic apoptosis
suggested that fgl2 activated in macrophages by mediation of IFN-γ (18), whereas TNF-α was required in induction of fgl2 in endothelial cells (18,20). Clark et al. demonstrated that TNF-α induced abortion in CBA×DBA/2
mice by promoting fgl2 prothrombinase expression in
both trophoblast and in deciduas (21-23). In our present
study, fgl2 was observed to be obviously up-regulated and
localized in the inflammatory and necrotic areas, so we
proposed fgl2 as an effector molecule that may contribute to SAP-associated liver injury by initiating and irritating microthrombosis through the induction of proinflammatory cytokines like TNF-α as accelerators at the onset
of this disease; thus contributing to microcirculatory disturbance.
We noticed that fgl2 gene as well as protein expression levels in the liver were determined to be obviously
elevated in rats with SAP and gradually increased in parallel with degeneration of SAP course. Fgl2 prothrombinase expression was significantly associated with microthrombi formation and microthrombosis in situ due to
fgl2 prothrombinase may be disadvantageous to the hepatic tissues and liver function. Our study considered that
in the way fgl2 prothrombinase serves in other diverse
diseases (13-15,24-26), microthrombosis due to fgl2 in
liver causes microcirculatory disturbance and consequent pathological injury of liver in rats with SAP as well
as related liver function.
For the purpose of assessing the connection between
fgl2 expression and extent of hepatic pathological injury indicated by hepatic pathological grade, a Spearman
grade correlation analysis was applied and determination
of fgl2 gene expression levels in the liver revealed a correlation with the extent of hepatic pathological injury. We
may be convinced of the potential of fgl2 prothrombinase
determination which could be possibly used as a helpful
biomarker for the prognosis of the severity of SAP-associated liver injury in the early phase.
The importance of fgl2 prothrombinase in diseases is
supported by injection of neutralizing antibody against
fgl2, which attenuates fibrin deposition as well as the
pathological injury and preventing mice with MHV-3-induced fulminant hepatitis from death (27). Thus, we
would push in-depth investigation to see whether the inhibition of fgl2 prothrombinase or application of antibodies against fgl2 prothrombinase could delay or ameliorate
Fgl2 is involved in SAP-Associated Liver Injury
Hepato-Gastroenterology 59 (2012)
the course of SAP-associated liver injury.
In conclusion, fgl2, serving as a novel prothrombinase,
could generate coagulation reaction and finally result in
microthrombosis in endothelial cells of intrahepatic veins
and hepatic sinusoids in experimental model of rats with
SAP and consequently result in hepatocellular injury as
well as necrosis and dysfunction of the liver. Fgl2 pro-
thrombinase expression is closely correlated with the severity of hepatocellular injury and thus it may used as a
beneficial biomarker in predicting SAP-associated liver
injury at the onset of disease. Whether inhibition of fgl2 or
application of antibodies against fgl2 could delay or ameliorate the process of SAP and protect against SAP-associated liver injury are warranted for further investigation.
1. 16. Doolittle RF: The structure and evolution of vertebrate fibrinogen. Ann NY Acad Sci 1983; 408:13-27.
17. Su K, Chen F, Yan WM, et al.: Fibrinogen-like protein 2/fibroleukin prothrombinase contributes to tumor hypercoagulability via IL-2 and IFN-γ. World J Gastroenterol 2008; 14:59805989.
18. Liu M, Mendicino M, Ning Q, et al.: Cytokine-induced hepatic apoptosis is dependent on FGL2/fibroleukin: the role of Sp1/
Sp3 and STAT1/PU.1 composite cis elements. J Immunol 2006;
176:7028-7038.
19. Ning Q, Brown D, Parodo J, et al.: Ribavirin inhibits viral-induced macrophage production of TNF-α, IL-1, the procoagulant fgl2 prothrombinase and preserves Th1 cytokine production but inhibits Th2 cytokine response. J Immunol 1998;
160(7):3487-3493.
20. Clark DA, Foerster K, Fung L, et al.: The fgl2 prothrombinase/
fibroleukin gene is required for lipopolysaccharide-triggered
abortions and for normal mouse reproduction. Mol Hum Reprod 2004; 10(2):99-108.
21. Clark DA, Ding JW, Chaouat G, et al.: The emerging role of immuno-regulation of fibrinogen-related pro-coagulant Fgl2 in
the success or spontaneous abortion of early pregnancy in mice
and humans. Am J Reprod Immunol 1999; 42(1):37-43.
22. Clark DA, Chaouat G, Arck PC, et al.: Cutting edge: cytokine -dependent abortion in CBA × DBA/2 mice is mediated
by the pro-coagulant fgl2 prothrombinase. J Immunol 1998;
160(2):545-549.
23. Clark DA, Ding JW, Yu G, et al.: Fgl2 prothrombinase expression in mouse trophoblast and decidua triggers abortion but
may be countered by OX-2. Mol Hum Reprod 2001; 7(2):185194.
24. Ding YP, Liu K, Wang Y, et al.: Expression and significance of
fgl2 prothrombinase in cardiac microvascular endothelial cells
of rats with type 2 diabetes. J Huazhong Univ Sci Technol 2010;
30(5):575-581.
25. Su GH, Liu K, Wang Y, et al.: Fibrinogen-like protein 2 expression correlates with microthrombosis in rats with type 2 diabetic nephropathy. J Biomed Res 2011; 25:120-127.
26. Melnyk MC, Shalev I, Zhang J, et al.: The prothrombinase activity of FGL2 contributes to the pathogenesis of experimental
arthritis. Scand J Rheumatol 2011; 40:269-278.
27. Li C, Fung LS, Chung S, et al.: Monoclonal anti-prothrombinase
(3D4.3) prevents mortality from murine hepatitis virus (MHV3) infection. J Exp Med 1992; 176:689-697.
References
2. 3. 4. 5. 6. 7. 8. 9. 10. 11. 12. 13. 14. 15. Halonen KI, Pettilä V, Leppäniemi AK, et al.: Multiple organ
dysfunctions associated with severe acute pancreatitis. Crit
Care Med 2002; 30(6):1274-1279.
Foitzik T, Eibl G, Hotz B, et al.: Persistent multiple organ microcirculatory disorders in severe acute pancreatitis: experimental
findings and clinical implications. Dig Dis Sci 2002; 47:130-138.
Kakafika A, Papadopoulos V, Mimidis K, Mikhailidis DP:
Coagulation, platelets, and acute pancreatitis. Pancreas 2007;
34:15-20.
Dobosz M, Mionskowska L, Hac S, et al.: Heparin improves organ microcirculatory disturbances in caerulein-induced acute
pancreatitis in rats. World J Gastroenterol 2004; 10(17):25532556.
Dobosz M, Hac S, Mionskowska L, et al.: Organ microcirculatory disturbances in experimental acute pancreatitis. A role of
nitric oxide. Physiol Res 2005; 54:363-368.
Forgács B, Eibl G, Wudel E, et al.: RES function and liver microcirculation in the early stage of acute experimental pancreatitis.
Hepato-gastroenterol 2003; 50(51):861-866.
Panek J, Zasada J, Poźniczek M: Microcirculatory disturbance
in the course of acute pancreatitis. Przegl Lek 2007; 64(6):435437.
Folch-Puy E: Importance of the liver in systemic complications
associated with acute pancreatitis: the role of Kupffer cells. J
Pathol 2007; 211(4):383-388.
Zhou ZG, Chen YD: Influencing factors of pancreatic microcirculatory impairment in acute pancreatitis. World J Gastroenterol 2002; 8(3):406-412.
Satoshi H, Hideo I, Chihiro S, et al.: Antithrombin III prevents
cerulein-induced acute pancreatitis in rats. Pancreas 2009;
38:746-751.
Levy GA, Liu M, Ding J, et al.: Molecular and functional analysis of the human prothrombinase gene (HFGL2) and its role in
viral hepatitis. AM J Pathol 2000; 156:1217-1225.
Chan CW, Chan MW, 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 2002;
168:5170-5177.
Marsden PA, Ning Q, Fung LS, et al.: The Fgl2/fibroleukin prothrombinase contributes to immunologically mediated thrombosis in experimental and human viral hepatitis. J Clin Invest
2003; 112:58-66.
Mendicino M, Liu M, Ghanekar A, et al.: Targeted deletion
of Fgl-2/fibroleukin in the donor modulates immunologic response and acute vascular rejection in cardiac xenografts. Circulation 2005; 112:248-256.
Kackstedt MK, Zenclussen AC, Hertwig K, et al.: Th1 cytokines and the prothrombinase fgl2 in stress-triggered and inflammatory abortion. Am J Reprod Immunol 2003; 49:210-220.
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