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Blood, Vol. 93 No. 1 (January 1), 1999:
pp. 157-164
Antithrombin Reduces Ischemia/Reperfusion Injury of Rat Liver by
Increasing the Hepatic Level of Prostacyclin
By
Naoaki Harada,
Kenji Okajima,
Shigeki Kushimoto,
Hirotaka Isobe, and
Keiichi Tanaka
From the Department of Laboratory Medicine, Kumamoto University
School of Medicine, Kumamoto, Japan; and the Department of Emergency
and Critical Care Medicine, School of Medicine, Fukuoka University,
Fukuoka, Japan.
 |
ABSTRACT |
We investigated whether antithrombin (AT) can reduce
ischemia/reperfusion (I/R)-induced injury of rat liver by promoting
prostacyclin release from endothelial cells. Although intravenous
administration of AT (250 U/kg) markedly reduced hepatic injury,
neither dansyl-Glu-Gly-Arg-chloromethyl ketone-treated factor Xa
(DEGR-Xa), a selective inhibitor of thrombin generation, nor
Trp49-modified AT, which lacks affinity for heparin, had
any effect. Hepatic levels of 6-keto-PGF1 , a stable
prostacyclin (PGI2) metabolite, were increased
significantly after I/R of the rat liver. AT significantly increased
the hepatic level of 6-keto-PGF1, whereas neither
DEGR-Xa nor Trp49-modified AT increased it. Hepatic tissue
blood flow was markedly reduced after I/R. Although AT significantly
increased the hepatic tissue blood flow after I/R, neither DEGR-Xa nor
Trp49-modified AT increased the blood flow. Hepatic levels
of cytokine-induced neutrophil chemoattractant (CINC) and
myeloperoxidase (MPO) were significantly increased after hepatic I/R.
The levels of these two indicators were reduced by AT but were
unaffected by either DEGR-Xa or Trp49-modified AT.
Pretreatment of animals with indomethacin (IM) completely inhibited the
protective effects of AT on the I/R-induced hepatic damage and the
leukocyte activation as well as the AT-induced increase in hepatic
6-keto-PGF1 levels after I/R. Iloprost, a stable analog
of PGI2, exhibited effects similar to those of AT and also
significantly inhibited the exacerbation of liver injury, the decrease
in hepatic tissue blood flow, and the increases in hepatic CINC and MPO
levels seen in rats subjected to I/R but pretreated with IM. These
findings suggest that AT may prevent I/R-induced hepatic injury by
increasing the hepatic levels of PGI2 through the
interaction of AT with cell-surface glycosaminoglycans, thus increasing
hepatic tissue blood flow and inhibiting leukocyte activation in
animals subjected to I/R.
© 1999 by The American Society of Hematology.
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INTRODUCTION |
ANTITHROMBIN (AT) inhibits the
coagulation factors generated in the coagulation cascade. The
inhibition of proteases by AT is markedly accelerated by its
interaction with glycosaminoglycans on the endothelial cell
surface.1
Prostacyclin (PGI2) is a well-known cytoprotective agent
that is synthesized in endothelial cells.2 AT promotes the
endothelial release of PGI2 in vitro and in vivo by
interacting with cell surface glycosaminoglycans.3-5 One of
the PGI2 activities is vasodilation.6
PGI2 has also been shown to inhibit leukocyte activation by
inhibiting tumor necrosis factor- (TNF-) production by
monocytes,5 neutrophil activation,7 and
neutrophil adhesion to endothelial cells.8 Because
activated leukocytes release a variety of inflammatory mediators,
including cytokines, neutrophil proteases, and reactive oxygen species,
all of which can damage adjacent endothelial cells, they have been
thought to play a role in tissue injury.9-13
The administration of AT has been found to reduce the mortality rate of
animals challenged with lipopolysaccharide (LPS) or Escherichia
coli.14 AT significantly increased the survival rate of
rabbits exposed to LPS but did not improve coagulopathy.15 Although heparin inhibited coagulopathy in baboons with endotoxin shock, it failed to reduce the mortality rate.16 These
observations suggest that the beneficial effects of AT in sepsis may be
due to both its anticoagulant activity and another unknown activity. Thus, we hypothesize that AT prevents tissue injury by inhibiting leukocyte activation through the promotion of endothelial release of
PGI2. Consistent with this hypothesis, we have previously
demonstrated that AT prevents endotoxin-induced pulmonary vascular
injury by promoting PGI2 release from endothelial cells
independent of its anticoagulant activity.17
Ischemia/reperfusion (I/R) is an important mechanism of tissue injury
in which activated leukocytes are critically involved.18 I/R-induced hepatic injury is an important pathologic process leading
to hepatic damage after circulatory shock or major hepatic surgery.19-21 Because leukocytes are implicated in the
pathology of I/R-induced hepatic injury,22-24 we
hypothesize that AT prevents I/R-induced hepatic injury by promoting
the endothelial release of PGI2.
In the present study, we examined whether AT reduces I/R-induced
hepatic injury in rats by promoting the endothelial release of
PGI2, thus maintaining the hepatic tissue blood flow and
inhibiting leukocyte activation. To examine this hypothesis, we
analyzed the effects of AT, an inactive derivative of factor Xa that
inhibits thrombin generation selectively in vivo, and a chemically
modified AT that lacks affinity for heparin on I/R-induced hepatic
injury in rats.
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MATERIALS AND METHODS |
Reagents.
AT was kindly provided by Green Cross (Osaka, Japan). AT was purified
from heat-treated, pooled human plasma by adsorption on fixed heparin
according to a modified version of the technique of Miller-Anderson et
al.25 The AT concentrate used in the experiments showed a
single band in response to polyacrylamide gel electrophoresis with
sodium dodecyl sulfate. Further characterization of the AT concentrate
demonstrated that its heparin concentration was less than 0.01 U/mL and
that it was free of pathogen. Iloprost was kindly provided by Eizai
Pharmaceutical Co (Tokyo, Japan). Dimethyl(2-hydroxy-5-nitrobenzyl) sulfonium bromide (DHNBSB) and indomethacin (IM) were purchased from
Sigma Chemical Co (St Louis, MO). All other reagents were of analytical
grade.
Animal model of hepatic I/R.
Adult, pathogen-free, male Wistar rats (Nihon SLC, Hamamatsu, Japan),
weighing 220 to 280 g, were used in each experiment. The care and
handling of the animals were in accordance with the National Institute
of Health guidelines. All experimental procedures described below were
approved by the Kumamoto University Animal Care and Use Committee. All
rats were deprived of food, but not water, for 24 hours before each
experiment. The hepatic I/R protocol was performed as described
previously.26,27 After induction of anesthesia in the
animals with ketamine hydrochloride (100 mg/kg, intraperitoneally;
Parke-Davis, Morris Plains, NJ), the liver of each was exposed through
a midline laparotomy. Silk ligatures were placed around the right and
left branches of the portal vein and the hepatic artery. Complete
ischemia of the median and left hepatic lobes was produced by clamping
the left branches of the portal vein and the hepatic artery for 60 minutes. The right hepatic lobe was perfused to prevent intestinal
congestion. During the period of hepatic ischemia, the animal's
abdomen was covered with plastic wrap to prevent dehydration. After the
period of ischemia, the ligatures around the left branches of the
portal vein and hepatic artery were removed. To accurately evaluate
blood flow of the median and left hepatic lobes after ischemia, the
right branches of the portal vein and the hepatic artery were ligated to prevent shunting to the right lobe after reperfusion.26
The wound was closed with 3-0 silk. This procedure directed all portal and hepatic blood flow, except for a small amount of flow to the caudal
hepatic lobe, through the lobes of the liver previously made ischemic.
Sham-operated animals were similarly prepared, except that no ligature
was placed to obstruct the blood flow to the left and median hepatic
lobes. Instead, the blood flow to the right lobe of the liver was
occluded.
Measurement of serum liver enzymes.
Blood samples were taken 12 hours after reperfusion to measure the
level of serum alanine aminotransferase (ALT) and aspartate aminotransferase (AST), as previously described.28 These
blood samples were collected into test tubes from the anesthetized
animals via withdrawal from the abdominal aorta with a 22-gauge needle. ALT and AST levels were measured by standard clinical automated analysis and the results are expressed in international units (IU) per
liter.
Measurement of plasma fibrinogen concentration and serum fibrin and
fibrinogen degradation product (E) [FDP (E)] levels.
The plasma concentration of fibrinogen was defined as the amount of
coagulable protein, as previously described.29 FDP (E) level was determined in the serum samples by the latex agglutination assay, as previously described.30
Histological determination of fibrin deposits in the liver.
After 12 hours of reperfusion, liver specimens were fixed in 10%
buffered formalin and then embedded in paraffin. Sections (4 µm) were
prepared and stained with phosphotungstic acid-hematoxylin, as
previously described.31
Measurement of hepatic tissue blood flow.
Hepatic tissue blood flow was measured by laser-Doppler flowmeter
(ALF21N; Advance, Tokyo, Japan) for 3 hours after reperfusion, as
described previously.32 After anesthesia with ketamine
hydrochloride (100 mg/kg, intraperitoneally), the right jugular veins
of these animals were cannulated with a PE-10 catheter for continuous
infusion of normal saline or test drugs. The Doppler flowmeter probe
was placed on the medial hepatic lobe. Hepatic tissue blood flow was measured from 30 minutes before ischemia until 3 hours after
reperfusion. The results are expressed as the percentage of preischemia
levels.
Determination of hepatic 6-keto-PGF1 levels.
Hepatic 6-keto-PGF1 levels were determined in animals
subjected to I/R before and after 1, 2, 3, and 6 hours of reperfusion according to the methods described previously.33 In brief,
the medial hepatic lobe in which the tissue blood flow had been
measured was weighed and then immediately homogenized in 5 mL of 0.1 mol/L phosphate buffer (pH 7.4) containing 2 mmol/L IM at 5°C. The
homogenate was first centrifuged at 2,000g for 10 minutes to
remove minute amounts of solid tissue debris and the supernatant was
then acidified with 1 mol/L HCl. 6-keto-PGF1 was
extracted from the supernatant using columns packed with ethyl-bonded
silica gel (ethyl C2; Amersham, Buckinghamshire, UK). The columns were
prepared by washing them with 2 mL of methanol, followed by 2 mL of
water. The acidified supernatant was applied to the column, and this
was followed by sequential washes with 5 mL of water, 5 mL of 10%
ethanol, and 5 mL of hexane. The elution of 6-keto-PGF1
was performed with 5 mL of methyl formate, after which the solvent was
evaporated under a stream of nitrogen gas. The concentration of
6-keto-PGF1 was assayed using a specific enzyme
immunoassay kit (Amersham). This enzyme immunoassay was sensitive
enough to detect 10 to 1,280 pg/mL of 6-keto-PGF1. The
results are expressed as ng of 6-keto-PGF1 per gram of
tissue. The cross-reactivities of this assay with PGE2,
PGF2, thromboxane B2, and arachidonic acid
were 2.8%, 1.4%, 0.03%, and 0.01%, respectively, according to the
manufacturer's data sheet. The maximum level of PGE2 in the liver of this animal model was about 4 ng per gram of tissue. The
tissue level of PGE2 was assayed using a specific enzyme
immunoassay kit (Amersham), as described previously.34
Determination of hepatic levels of cytokine-induced neutrophil
chemoattractant (CINC).
Hepatic levels of CINC were determined by a modification of the method
of Clark et al.35 In brief, the medial hepatic lobe in
which the tissue blood flow had been measured was weighed and then
homogenized in 5 mL of 0.1 mol/L phosphate buffer (pH 7.4) containing
0.05% (vol/wt) of sodium azide at 5°C. The homogenate was first
centrifuged at 2,000g for 10 minutes to remove minute amounts
of solid tissue debris. The supernatant was assayed using a Rat
Interleukin-8 (CINC/gro) enzyme-linked immunosorbent assay (ELISA)
system (Amersham). This ELISA was sensitive enough to detect 4.7 to 300 pg/mL of CINC/gro. The results are expressed as picograms of CINC per
gram of tissue.
Determination of hepatic myeloperoxidase (MPO) activity.
After the indicated period of reperfusion, the livers were quickly
removed, and accumulation of leukocytes was assessed by measuring MPO
activity in the liver according to a previously described
method.36 In brief, the livers were weighed and suspended in 6 mL of 50 mmol/L phosphate buffer (pH 6.0) containing 1%
hexadecyltrimethylammonium bromide. The samples were homogenized and
the homogenate was sonicated, freeze-thawed, and then centrifuged
(4,500g for 15 minutes at 4°C). MPO activity in the
supernatant (0.1 mL) was determined after the addition of 0.6 mL of
phosphate buffer (pH 6.0) containing 0.167 mg/mL o-dianisidine
dihydrochloride and 0.0005% hydrogen peroxide. The change in
absorbance at 460 nm over 10 minutes was measured in a
spectrophotometer (DU-54; Beckman, Irvine, CA). One unit of MPO
activity was defined as the amount of enzyme able to reduce 1 µmol of
peroxide per minute. Results are expressed as units of MPO activity per
gram of tissue.
Five separate groups of animals (n = 40 per each group) were used to
assess liver enzyme levels, tissue blood flow, hepatic 6-keto-PGF1 levels, hepatic CINC levels, and hepatic MPO activities. This procedure avoided problems associated with excessive blood loss due to blood sampling or associated with fluid loss due to
blood flow measurement.
Preparation of Trp49-modified AT.
The Trp49 residue of AT was chemically modified by a
version of the method of Karp et al.37 In brief, DHNBSB was
mixed with a continuously stirred solution containing 40 µmol/L AT, 0.1 mol/L Tris-HCl (pH 8.0), and 0.15 mol/L NaCl. The final
concentration of DHNBSB was calculated as 8 mmol/L. After stirring for
15 minutes at 22°C, the insoluble hydroxynitrobenzyl alcohol, which
formed as a hydrolysis product, was removed by centrifugation. The
resulting solution was next subjected to chromatography on a column
(2.6 × 60 cm) of Sephacryl S-200HR that had been equilibrated
with 0.1 mol/L Tris-HCl (pH 8.0) and 0.15 mol/L NaCl and then subjected to chromatography on a column (3 × 6 cm) of heparin-Sepharose CL6B, as previously described.38 The extent of
derivatization of AT was determined spectrophotometrically in 2 mol/L
NaOH at a wave length of 410 nm (molar extinction coefficient, 1.85 × 104 mol/L 1).39 The
progressive antithrombin activity of the Trp49-modified AT
in the absence of heparin was virtually identical to that of intact AT
(data not shown). The study of thrombin inhibition by
Trp49-modified AT was performed as previously
described.39
Preparation of active site-blocked factor Xa.
Factor X was purified from human plasma40 and then
activated with Russell's viper venom, as described
previously.41 The activated factor Xa was then inactivated
by incubating it with a 20-fold molar excess of dancyl
glutamylglycylarginyl chloromethyl ketone (DEGR) for 30 minutes at
25°C, after which the mixture was subjected to extensive dialysis
in a solution containing 20 mmol/L TrisHCl (pH 7.4) and 100 mmol/L
NaCl. Such DEGR-treated factor Xa (DEGR-Xa) selectively inhibits
thrombin generation by competing with intact factor Xa in the formation
of the prothrombinase complex.42 The remaining factor Xa
activity, assayed by using the chromogenic substrate
S-2222,43 was less than 0.01% of the untreated factor Xa.
The DEGR-Xa prepared in this manner prolonged the activated partial
thromboplastin time (APTT) in a concentration-dependent manner (0 to
300 µg/mL; data not shown). APTT was measured using actin FS reagent
(Baxter, Deerfield, IL) according to the manufacturer's instructions.
Administration of AT, Trp49-modified AT, and DEGR-Xa.
We have previously reported that the plasma concentration of
6-keto-PGF1, a stable metabolite of PGI2,
begins to increase 30 minutes after the intravenous (IV) administration
of AT (250 U/kg) in intact rats.44 Thus, AT was
administered 30 minutes before reperfusion in this study.
Trp49-modified AT (250 U/kg) and DEGR-Xa (3 mg/kg) were
also injected IV 30 minutes before reperfusion. At the dosage used in
the present study, Trp49-modified AT and DEGR-Xa have
antithrombin activities comparable to that of 250 U/kg of native
AT.17
IM and iloprost administration.
IM (20 mg/kg) was suspended in bicarbonate-buffered saline and
administered subcutaneously for 30 minutes before ischemia. Control
animals received the same volume of bicarbonate-buffered saline instead
of IM.
Iloprost was dissolved in normal saline and continuously infused (100 ng/kg/min) into the animals via the right jugular vein. For those
groups of animals in which the tissue blood flow was measured, this
infusion began before the onset of hepatic ischemia and continued until
the rats were killed 3 hours after onset of reperfusion. For those
groups of animals in which the liver enzyme levels were measured,
iloprost was infused continuously for 6 hours after the onset of
reperfusion to prevent any respiratory problems associated with
prolonged anesthesia. Control animals received continuous infusion of
the same volume of normal saline instead of iloprost.
Statistical analysis.
Data are expressed as the mean ± SD. The results were compared
using either an analysis of variance followed by Scheffé's post
hoc test or an unpaired t-test. A level of P < .05 was considered statistically significant.
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RESULTS |
Effects of AT, DEGR-Xa, and Trp49-modified AT
on I/R-induced hepatic injury.
Serum levels of ALT and AST were significantly increased after 1 hour
of reperfusion compared with levels in sham-operated animals, peaking
at 12 hours after reperfusion.28 Plasma levels of
fibrinogen and serum levels of FDP (E) were not significantly increased
1, 3, 6, and 12 hours after reperfusion compared with those of
sham-operated animals (data not shown). Fibrin deposition was not
observed histologically in the livers of rats subjected to hepatic I/R
12 hours after reperfusion (data not shown).
Although the IV administration of 250 U/kg of AT significantly
inhibited the increase in serum aminotransferase levels 12 hours after
reperfusion (Fig 1), the lower doses of AT
(50 and 100 U/kg) did not (data not shown). Neither DEGR-Xa (3 mg/kg, IV), a selective inhibitor of thrombin generation, nor
Trp49-modified AT (250 U/kg, IV), which lacks affinity for
heparin, inhibited the increase in serum aminotransferase levels
observed at 12 hours after reperfusion (Fig 1).
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| Fig 1.
Effects of AT and various agents on serum levels of
aminotransferases in rats after hepatic I/R. Animals were subjected to
60 minutes of hepatic ischemia followed by reperfusion as described in
Materials and Methods. Animals were injected IV with AT (250 U/kg) 30 minutes before reperfusion or subcutaneously with IM (20 mg/kg) 30 minutes before ischemia. DEGR-Xa (3 mg/kg) or
Trp49-modified AT (250 U/kg) was injected intravenously 30 minutes before reperfusion. Iloprost was infused continuously starting
just before the onset of ischemia at a rate of 100 ng/kg/min with or
without subcutaneous injection of IM (20 mg/kg). Serum levels of ALT
and AST were determined after 12 hours of reperfusion. Each bar
represents the mean ± SD. *P < .01 versus sham.
 P < .01 versus I/R.  P < .01 versus
AT-treated I/R. ¶P < .01 versus IM-treated I/R.
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Effects of AT, DEGR-Xa, and Trp49-modified AT on hepatic
levels of 6-keto-PGF1.
To determine whether AT promoted the endothelial release of
PGI2 in the liver after I/R by interacting with
glycosaminoglycans on the endothelial cell surface, we examined the
effects of AT and Trp49-modified AT on hepatic levels of
6-keto-PGF1 after I/R. The hepatic levels of
6-keto-PGF1 were significantly increased after
reperfusion, compared with those of the sham-operated animals, and
peaked 1 hour after reperfusion (Fig 2). AT
(250 U/kg) significantly enhanced the I/R-induced increase in hepatic
6-keto-PGF1 levels 1 hour after reperfusion
(Fig 3), whereas neither
Trp49-modified AT (250 U/kg) nor DEGR-Xa (3 mg/kg) had an
effect (Fig 3).
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| Fig 2.
Changes in hepatic 6-keto-PGF1 levels in
rats subjected to hepatic I/R. Animals were subjected to 60 minutes of
hepatic ischemia followed by reperfusion as described in Materials
and Methods. Sham-operated animals were prepared in a similar manner,
except that blood flow to the left and median hepatic lobes was not
obstructed. Mean values of hepatic 6-keto-PGF1 levels
after various periods of hepatic reperfusion are shown. Data represent
the mean ± SD derived from six animal experiments. ( )
Sham-operated animals; ( ) I/R animals. §P < .01 versus
preischemia. *P < .01 versus sham.
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| Fig 3.
Effects of AT, Trp49-modified AT, DEGR-Xa,
and AT plus IM pretreatment on changes in hepatic
6-keto-PGF1 levels in rats subjected to hepatic I/R.
Animals were subjected to 60 minutes of hepatic ischemia followed by
reperfusion as described in Materials and Methods. Animals were either
injected IV with AT (250 U/kg), Trp49-modified AT (250 U/kg), or DEGR-Xa (3 mg/kg) 30 minutes before reperfusion or injected
subcutaneously with IM (20 mg/kg) 30 minutes before ischemia. After 1 hour of reperfusion, hepatic 6-keto-PGF1 levels were
measured by enzyme immunoassay (EIA). Each bar represents
the mean ± SD. *P < .01 versus sham. P < .01 versus I/R. P < .01 versus AT-treated I/R.
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Effects of AT, DEGR-Xa, and Trp49-modified AT on the
changes in hepatic tissue blood flow in rats subjected to hepatic I/R.
During hepatic ischemia, the hepatic tissue blood flow decreased to
approximately 30% of the preischemia level and then increased to 50%
of the preischemia level 3 hours after reperfusion
(Fig 4). Although AT (250 U/kg)
significantly increased the hepatic tissue blood flow after 1 to 3 hours of reperfusion, neither DEGR-Xa nor the
Trp49-modified AT increased the blood flow (Fig 4).
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| Fig 4.
Effects of AT, DEGR-Xa, and Trp49-modified AT
on the changes in hepatic tissue blood flow in rats subjected to
hepatic I/R. Animals were subjected to 60 minutes of hepatic ischemia
followed by reperfusion as described in Materials and Methods. Animals
were injected IV with AT (250 U/kg), DEGR-Xa (3 mg/kg), or
Trp49-modified AT (250 U/g) at 30 minutes before
reperfusion. Hepatic tissue blood flow was continuously measured by a
laser Doppler flowmeter starting 30 minutes before ischemia until 3 hours after reperfusion. Each value represents the mean ± SD derived
from 5 animals. ( ) I/R animals; ( ) AT-treated I/R animals; ()
DEGR-Xa-treated I/R animals; () Trp49-modified
AT-treated animals. P < .01 versus I/R. P < .01 versus AT-treated I/R.
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Effects of AT, DEGR-Xa, and Trp49-modified AT on
I/R-induced changes in hepatic levels of CINC and MPO in rats.
Hepatic levels of CINC, a member of the interleukin-8 (IL-8) family,
were significantly increased after reperfusion, compared with those of
the sham-operated animals, and peaked 2 hours after reperfusion
(Fig 5A). Administration of AT (250 U/kg)
significantly inhibited this increase 2 hours after reperfusion
(Fig 6), whereas the administration of
Trp49-modified AT (250 U/kg) and DEGR-Xa (3 mg/kg) had no
effect. Hepatic MPO activity was increased significantly after
reperfusion, compared with that of the sham-operated animals, and
peaked 6 hours after reperfusion (Fig 5B). Administration of AT (250 U/kg) significantly inhibited this increase 6 hours after reperfusion,
whereas the administration of Trp49-modified AT (250 U/kg)
and DEGR-Xa (3 mg/kg) had no effect (Fig 7).
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| Fig 5.
Changes in hepatic CINC levels (A) and MPO activity (B)
in rats subjected to hepatic I/R. Animals were subjected to 60 minutes
of hepatic ischemia followed by reperfusion as described in Materials
and Methods. Sham-operated animals were prepared in a similar manner,
except that blood flow to the left and median hepatic lobes was not
obstructed. Mean values of the hepatic CINC level and MPO activity
after various periods of hepatic reperfusion are shown. Data represent
the mean ± SD derived from six animal experiments. ()
Sham-operated animals; () I/R animals. §P < .01 versus
preischemia. *P < .01 versus sham.
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| Fig 6.
Effects of AT and various agents on the I/R-induced
increase in hepatic CINC levels 2 hours after reperfusion. Animals were
subjected to 60 minutes of hepatic ischemia followed by reperfusion as
described in Materials and Methods. Mean values of the hepatic CINC
levels after 2 hours of hepatic reperfusion are shown. Data represent
the mean ± SD. P < .01 versus I/R. P < .01 versus AT-treated I/R. ¶P < .01 versus IM-treated
I/R.
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| Fig 7.
Effects of AT and various agents on the I/R-induced
increase in hepatic MPO activity 6 hours after reperfusion. Animals
were subjected to 60 minutes of hepatic ischemia followed by
reperfusion as described in Materials and Methods. Mean values of
hepatic MPO activity after 6 hours of hepatic reperfusion are shown.
Data represent the mean ± SD. P < .01 versus I/R.
P < .01 versus AT-treated I/R. ¶P < .01 versus IM-treated I/R.
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Effect of IM pretreatment on the AT-induced effects in I/R of the rat
liver.
Pretreatment of animals with IM (20 mg/kg, subcutaneously)
significantly reduced hepatic 6-keto-PGF1 levels in rats
subjected to I/R and it completely inhibited the increase in hepatic
6-keto-PGF1 levels seen in response to AT administration
(Fig 3). AT did not inhibit the increase in serum aminotransferase
levels seen 12 hours after reperfusion in animals pretreated with IM
(Fig 1). IM itself significantly enhanced the increase in serum
aminotransferase levels 12 hours after reperfusion (Fig 1). AT did not
increase the hepatic tissue blood flow in animals pretreated with IM
(Fig 8). IM itself significantly reduced
hepatic tissue blood flow (Fig 8). AT did not prevent the I/R-induced
increases in hepatic levels of CINC and MPO in animals pretreated with
IM (Figs 6 and 7). IM itself significantly enhanced the I/R-induced
increases in these indicators for leukocyte activation (Figs 6 and 7).
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| Fig 8.
Effects of IM pretreatment and continuously infused
iloprost on I/R-induced changes in hepatic tissue blood flow in rats
receiving AT. Animals were subjected to 60 minutes of hepatic ischemia
followed by reperfusion as described in Materials and Methods. Animals
were injected IV with AT (250 U/kg) 30 minutes before reperfusion or
subcutaneously with IM (20 mg/kg) 30 minutes before ischemia. Iloprost
was infused continuously starting before the onset of ischemia at a
rate of 100 ng/kg/min with or without subcutaneous injection of IM (20 mg/kg) 30 minutes before ischemia. Hepatic tissue blood flow was
continuously measured by laser Doppler flowmeter starting 30 minutes
before ischemia until 3 hours after reperfusion. Each value represents
the mean ± SD derived from 5 animals. () I/R animals; ()
IM-treated I/R animals; () AT-treated I/R animals; ( ) AT-treated
I/R animals pretreated with IM; ( ) iloprost-treated I/R animals;
() iloprost-treated I/R animals pretreated with IM. P < .01 versus I/R. P < .01 versus AT-treated I/R.
¶P < .01 versus IM-treated I/R.
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Effect of iloprost, a stable analog of PGI2, on hepatic
injury and the other events induced by I/R of the rat
liver.
The continuous IV infusion of iloprost (100 ng/kg/min) significantly
inhibited the increase in serum aminotransferase levels observed 12 hours after reperfusion (Fig 1) and increased the hepatic tissue blood
flow after hepatic I/R (Fig 8). The continuous IV infusion of iloprost
significantly inhibited the I/R-induced increase in hepatic levels of
CINC (Fig 6) and MPO activity (Fig 7) 2 and 6 hours after reperfusion,
respectively. The increases in serum aminotrasferase levels (Fig 1),
hepatic CINC levels (Fig 6), and MPO activity (Fig 7) and the reduction
of hepatic tissue blood flow (Fig 8) in rats subjected to hepatic I/R,
but pretreated with IM, were significantly inhibited by the continuous
IV infusion of iloprost.
| |
DISCUSSION |
In the present study, AT prevented hepatic injury induced by I/R,
whereas Trp49-modified AT did not. Because
Trp49 plays a critical role in AT's interaction with
heparin,39 Trp49-modified AT retains its
progressive antithrombin activity, but lacks affinity for
heparin.17 Because the inhibition of thrombin by AT is
markedly accelerated by its interaction with glycosaminoglycans on the
endothelial cell surface,1 the lack of efficacy of
Trp49-modified AT seen in this model may be due to a
decrease in its inhibitory activity against thrombin. However, this
possibility seems unlikely, because DEGR-Xa, a selective inhibitor of
thrombin generation, did not prevent I/R-induced liver injury in the
present study. DEGR-Xa (3 mg/kg) inhibited endotoxin-induced
coagulation abnormalities to the same extent as AT (250 U/kg).17 These results parallel earlier studies that were
performed on the primate (baboon) in which DEGR-Xa also inhibited
coagulation abnormalities after infusion of LD100 E
coli, without protecting against its lethal effects.45
In addition, we have previously demonstrated that Trp49-modified AT (250 U/kg) alleviates endotoxin-induced
coagulation abnormalities to the same extent as native AT (250 U/kg),17 suggesting that the antithrombin activity of
Trp49-modified AT may be similar to that of AT in respect
to the alleviation of coagulation abnormalities induced by endotoxin.
This observation also suggests that the failure of
Trp49-modified AT to prevent the I/R-induced liver injury
was not due to its decreased anticoagulant activity, but probably due
to a lack of affinity for heparin. These observations suggest that the
preventive effect of AT on I/R-induced hepatic injury may not be a
result of its anticoagulant activity, but may be dependent on its
interaction with glycosaminoglycans on the endothelial cell surface.
AT promotes the release of PGI2 from endothelial cells by
interacting with glycosaminoglycans on the endothelial cell
surface.3-5 In the present study, AT significantly
increased the hepatic level of 6-keto-PGF1 1 hour after
hepatic I/R. Trp49-modified AT does not promote the release
of PGI2 from endothelial cells.44 Neither
Trp49-modified AT nor DEGR-Xa increased the hepatic tissue
level of 6-keto-PGF1 in animals subjected to I/R,
suggesting that AT could increase hepatic production of
PGI2 not through its antithrombin activity, but by
interaction with glycosaminoglycans on the endothelial cell surface.
Because neither Trp49-modified AT nor DEGR-Xa did not
prevent the I/R-induced hepatic injury, we postulate that AT may
prevent I/R-induced hepatic injury by promoting the endothelial release
of PGI2.
In the present study, the critical role of hepatic PGI2 in
the prevention of I/R-induced hepatic injury was illustrated by the
following observations. (1) Iloprost, a stable analog of
PGI2, prevented the hepatic injury induced by hepatic
I/R. (2) Pretreatment of animals with IM, a potent inhibitor of
PGI2 synthesis, significantly exacerbated the I/R-induced
hepatic injury. (3) Iloprost significantly inhibited the IM-induced
exacerbation of hepatic injury. Also, the preventive effects of AT were
not observed in the animals pretreated with IM.
Taken together, these observations further support the hypothesis that
the protective effect of AT against I/R-induced hepatic injury could be
mediated primarily by the actions of PGI2.
Because PGI2 exhibits vasodilator activity, the hepatic
production of PGI2 induced by AT after reperfusion may
increase the hepatic tissue blood flow. Consistent with this hypothesis
is the observation made in the present study that the I/R-induced decrease in hepatic tissue blood flow was markedly inhibited by AT
administration. This inhibitory effect was not observed in animals
pretreated with IM. Iloprost inhibited the I/R-induced decrease in
hepatic tissue blood flow and also prevented the IM-induced decrease in
hepatic tissue blood flow induced by hepatic I/R. These observations
suggest that AT inhibits the I/R-induced decrease in hepatic tissue
blood flow by increasing the hepatic production of PGI2.
These effects contribute to the attenuation of hepatic injury after
I/R.
Activated leukocytes are considered to play a pivotal role in
I/R-induced hepatic injury by releasing various inflammatory mediators
that are capable of damaging endothelial cells.46,47 We
have previously demonstrated the involvement of neutrophil elastase in
an animal model of hepatic I/R.28 Consistent with these
observations are the findings in the present study that hepatic levels
of CINC, a member of the IL-8 family that promotes the accumulation of
neutrophils,46 as well as hepatic MPO activity were
increased after I/R. Because PGI2 inhibits the production of TNF-, which enhances the production of IL-8, a potent activator of neutrophils,48,49 it is possible that AT may also
prevent I/R injury in rats by inhibiting leukocyte activation through the promotion of PGI2 production. This hypothesis is
supported by the observation in the present study that AT inhibited the I/R-induced increase in hepatic levels of CINC and MPO. These effects
of AT were not observed in animals pretreated with IM. Iloprost
inhibited the I/R-induced increase in hepatic levels of CINC and MPO.
It also inhibited the IM-induced enhancement of CINC and MPO levels in
animals subjected to hepatic I/R. These observations suggest that
PGI2 plays an important role in preventing hepatic
I/R-induced activation of leukocytes. Thus, it is likely that AT may
also prevent I/R-induced hepatic injury by inhibiting the hepatic
accumulation of neutrophils through an increase in the hepatic
PGI2 levels. This hypothesis is consistent with our previous report demonstrating that AT prevents in vivo
endotoxin-induced endothelial cell injury by inhibiting the activation
of leukocytes by promoting the endothelial PGI2
release.17 Taken together, these observations strongly
suggest that AT prevents I/R-induced hepatic injury by maintaining
tissue blood flow and by inhibiting leukocyte activation, both of which
can be mediated by the actions of PGI2.
AT appears to prevent I/R-induced hepatic injury independent of its
anticoagulant effect in the present study. Although neither an increase
in the serum levels of FDP nor hepatic fibrin formation was observed in
rats subjected to I/R, these observations cannot completely rule out
thrombin generation in the hepatic microcirculation in this model. In
hepatic injury induced by I/R of whole liver in rats, AT has been shown
to prevent hepatic injury by inhibiting coagulation
abnormalities.50 Thus, it is possible that AT can prevent
I/R-induced hepatic injury by inhibiting thrombin activity as well as
by inhibiting leukocyte activation through the induction of
PGI2 release.
Emerson et al14 has reported that AT prevents hepatic
damage induced by endotoxin in rats without inhibiting
endotoxin-induced coagulation abnormalities. Jochum51 has
demonstrated that an infusion of AT concentrate significantly inhibits
hepatic dysfunction in patients with multiple trauma by inhibiting
leukocyte activation.
We conclude that the beneficial effects of AT on hepatic injury can be
explained partly by its promotion of the release of PGI2 by
endothelial cells independent of its antithrombin activity.
| |
FOOTNOTES |
Submitted February 3, 1998;
accepted August 18, 1998.
The publication costs of this
article were defrayed in part by
page charge payment. This article
must therefore be hereby marked
"advertisement"
in accordance with 18 U.S.C. section
1734 solely to indicate this fact.
Address reprint requests to Kenji Okajima, MD, Department of Laboratory
Medicine, Kumamoto University School of Medicine, Honjo 1-1-1 Kumamoto 860, Japan; e-mail: whynot{at}kaiju.medic.kumamoto-u.ac.jp.
| |
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Abstract
Introduction