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Blood, Vol. 93 No. 1 (January 1), 1999:
pp. 157-164
By
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.
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
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- 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.
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 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.
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 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 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.
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.
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).
Effects of AT, DEGR-Xa, and Trp49-modified AT on hepatic
levels of 6-keto-PGF1
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).
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).
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
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.
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.
Submitted February 3, 1998;
accepted August 18, 1998.
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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Top
Abstract
Introduction
, 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
(TNF-
1).
P < .01 versus I/R. 
P < .01 versus
AT-treated I/R. ¶P < .01 versus IM-treated I/R.
)
Sham-operated animals; (
) I/R animals. §P < .01 versus
preischemia. *P < .01 versus sham.
) I/R animals; (
) AT-treated I/R animals; (
) AT-treated
I/R animals pretreated with IM; (
) iloprost-treated I/R animals;
(