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HEMOSTASIS, THROMBOSIS, AND VASCULAR BIOLOGY
From the Department of Laboratory Medicine, Kumamoto
University School of Medicine, Japan.
Antithrombin (AT) prevents Escherichia coli-induced
hypotension in animal models of sepsis, and it further reduces the
mortality of patients with septic shock. In the present study, we
examined whether AT may prevent the endotoxin (ET)-induced hypotension by promoting the endothelial release of prostacyclin (PGI2)
in rats. Intravenous administration of AT (250 U/kg) prevented both hypotension and the increases in plasma levels of
NO2 Antithrombin (AT) is an important serine protease
inhibitor of the coagulation system.1 Inhibition by AT of
thrombin and the other serine proteases generated from the coagulation
cascade is markedly accelerated by its interaction with the endothelial surface glycosaminoglycans (GAGs).1 Patients with
congenital AT deficiency and those with the variant AT that lacks
affinity for GAGs develop thrombosis, showing the importance of the
interaction of AT with the endothelial cell surface GAGs for regulation
of the coagulation cascade.2,3
AT has been shown to promote endothelial release of prostacyclin
(PGI2) by interacting with the endothelial surface GAGs in vitro4,5 and in vivo.6 PGI2
potently inhibits platelet aggregation7 and induces
vasodilation,8 thereby maintaining proper organ
microcirculation. In addition, PGI2 inhibits the endotoxin
(ET)-induced monocytic production of tumor necrosis factor- Excessive production of NO by the inducible form of NOS (iNOS) plays
critical roles in the pathophysiology of septic shock.12 Septic shock, associated with infection with Gram-negative and Gram-positive bacteria and with fungus, is characterized by
hypotension, organ dysfunction, and disseminated intravascular
coagulation, leading to multiple organ failure.13 The
mechanism of septic shock is now considered to be the marked reduction
of vascular reactivity to vasoconstrictors.14 The
hyporeactivity has been shown to be attributable to the action of
excessively produced NO by iNOS expressed within the
vasculature.15 NO activates soluble guanylyl cyclase,
thereby increasing the cytoplasmic concentration of cyclic guanosine
monophosphate followed by the reduction of the intracellular calcium
concentration.16 The cyclic guanosine monophosphate-independent mechanism for the vasodilation has also been
postulated. For example, peroxynitrite, an oxidant produced by the
reaction of NO and superoxide, has been suggested to activate membrane
potassium channels, leading to vasodilation.17-19
Furthermore, myocardial depression induced by NO might also contribute
to hypotension induced by ET.20
AT significantly reduced the lethal effects of Escherichia
coli infusion in baboons, and the therapeutic effects could not be
explained by its anticoagulant activities.21,22 In the
study using the baboon model of sepsis, AT significantly inhibited the decrease in the mean systemic arterial pressure.21 We
previously reported that AT reduced both ET-induced pulmonary vascular
injury23 and ischemia/reperfusion-induced liver injury by
inhibiting leukocyte activation in rats.24 Neither
DEGR-F.Xa, an inactive derivative of F.Xa that selectively inhibits
thrombin generation, nor Trp49-modified AT, which is
incapable of promoting the endothelial release of PGI2 due
to the lack of the affinity for heparin, reduced these organ
injuries.23,24 These observations strongly suggested that
the therapeutic effects induced by AT could not be mediated by its
anticoagulant effects but could be mediated by the increase in the
endothelial production of PGI2.23,24
Taken together, these observations suggest the possibility that AT
prevents ET-induced hypotension by inhibiting induction of iNOS and
that such effects can be mediated by PGI2, which
inhibits the monocytic production of TNF- In the present study, we examined this possibility using a rat model of
septic shock. Because the lung is one of the main organs expressing
large amounts of iNOS in response to ET,15 we investigated
whether AT inhibits the induction of iNOS by inhibiting TNF- Materials
Preparation of active site-blocked factor Xa
Preparation of Trp49-modified AT The Trp49 residue of AT was chemically modified by a version of the method of Karp et al.28 In brief, DHNBSB was mixed with a continuously stirred solution containing 40 µM AT, 0.1 M Tris-HCl (pH 8.0), and 0.15 M NaCl. The final concentration of DHNBSB was calculated as 8 mM. After 15 minutes at 22°C, the insoluble hydroxynitrobenzyl alcohol, which formed as a hydrolysis product, was removed by centrifugation. The solution was subjected to chromatography on a column (2.6 × 60 cm) of Sephacryl S-200HR (Pharmacia, Uppsala, Sweden) that had been equilibrated with 0.1 M Tris-HCl (pH 8.0) and 0.15 M NaCl and then subjected to chromatography on a column (3 × 6 cm) of heparin-Sepharose CL6B (Pharmacia), as previously described.3 The AT that was present in the void volume was collected and concentrated by filtration with an Amicon YM-10 membrane (Amicon, Danvers, MA). The concentration of AT in the sample was determined immunologically as previously described.29 The extent of derivatization of AT was determined spectrophotometrically in 2 M NaOH at 410 nm (molar extinction coefficient 1.85 × 104 M 1).28 Although the heparin cofactor
activity of Trp49-modified AT was markedly decreased, the
progressive AT activity measured in the absence of heparin was
virtually identical to that of native AT (data not shown). The study of
thrombin inhibition was performed as previously
described.29 Because Trp49-modified AT
prevented the coagulation abnormalities induced by ET in rats to the
same extent as native AT,23 the anticoagulant activity of
Trp49-modified AT could be comparable to that of native AT
in vivo.
ET shock in rats and measurement of MAP The study protocol was approved by the Kumamoto University School of Medicine Animal Care and Use Committee, and the care and handling of the animals were in accordance with the guidelines of the National Institutes of Health. Specific pathogen-free male Wistar rats weighing 220 to 280 g were obtained from Kyudo (Kumamoto, Japan). Animals were anesthetized with pentobarbital sodium (50 mg/kg, intraperitoneally). Mean arterial pressure (MAP) equals the diastolic pressure plus one third of the pulse pressure, which is the difference between the systolic and diastolic pressure. The right femoral artery was cannulated and connected to a pressure transducer for the measurement of MAP. The right jugular vein was cannulated for administration of reagents. Upon completion of the surgical procedure, MAP was allowed to stabilize for 15 minutes. After recording the baseline MAP, animals were treated with vehicle alone (saline) or reagents such as AT (250 U/kg). Thirty minutes after administration of vehicle or reagents, animals intravenously received ET (5 mg/kg) as a slow injection over 5 minutes. MAP was continuously monitored for 180 minutes after ET administration.Measurement of plasma levels of
NO2 and NO3
are the primary oxidized products of NO reacting with water, and
therefore total concentration of
NO2/NO3 in plasma
was used as an indicator of NO production in vivo.30 Blood
was collected from the abdominal aorta into tubes containing 3.8%
sodium citrate. The blood samples were placed on ice and centrifuged at
1000g for 20 minutes to prepare plasma. Total concentration of NO2/NO3 was
measured using a Griess reaction kit (Roche Diagnostics, Mannheim,
Germany). Briefly, plasma protein was removed by adding 5%
ZnSO4 and NaOH (0.3 M) solution. After centrifugation
(2300g for 10 minutes), the supernatant was incubated with
nitrate reductase, NADPH, and flavin adenine dinucleotide for 30 minutes at room temperature to reduce NO3 to
NO2. After incubation, the samples were
placed in 96-well plates and incubated with 1% sulfanilic acid and
0.2% naphthylethylenediamine dihydrochloride/20%
H3PO4 for 5 minutes at room temperature. The absorbance of the mixture at 550 nm was determined using a microplate reader. NO2 concentration was calculated by
comparison with the absorbance of a standard solution of
KNO2.
Measurement of lung level of NOS activity A total of 180 minutes after ET administration, rats were anesthetized by pentobarbital sodium and the lung vasculature was perfused via the right cardiac ventricle with 10 mL cold 0.9% NaCl. The lungs were removed and frozen in liquid nitrogen. These lung samples were homogenized on ice in HEPES buffer (pH 7.5, 30 mM). The homogenate was sonicated and centrifuged at 12 500g for 15 minutes at 4°C. Conversion of [3H]-L-arginine to [3H]- L-citrulline was measured in the supernatant as described by Szabó et al.15 Briefly, tissue homogenate was incubated in the presence of [3H]- L-arginine (0.5 mCi/µM [18.5 MBq/µM]), NADPH (1 mM), calmodulin (30 nM), tetrahydrobiopterin (50 µM), flavin mononucleotide (20 µM), flavin adenine dinucleotide (20 µM), L-valine (60 mM), and CaCl2 (2 mM) for 20 minutes at 37°C in HEPES buffer (pH 7.5, 30 mM). Reactions were stopped by dilution with HEPES buffer (pH 5.5, 100 mM) containing ethyleneglycotetraacetic acid (2 mM) and ethylenediaminetetraacetic acid (2 mM). Reaction mixtures were applied to Dowex 50 W (sodium form) columns (Bio-Rad Laboratories, Hercules, CA), and the radioactivity of eluted [3H]-L-citrulline was measured using a scintillation counter (TRI-CARB 2300TR, Packard, Meriden, CT). Reaction mixtures were prepared without calcium and with ethyleneglycotetraacetic acid (5 mM) to determine the level of calcium-independent iNOS activity. Protein concentration of lung homogenate was measured spectrophotometrically by the Lowry method with bovine serum albumin as a standard.Measurement of lung levels of TNF-  in the supernatant were determined using an enzyme-linked
immunosorbent assay kit for rat TNF- (Genzyme, Cambridge, MA).
Isolation of RNA and Northern blotting analysis Total RNA from rat lungs was prepared by the acid-guanidinium-phenol-chloroform extraction procedure.31 After electrophoresis in formaldehyde-containing agarose gels, RNAs were transferred onto nylon membranes. Hybridization was performed using digoxigenin-labeled rat iNOS antisense RNA32 or rat TNF- antisense RNA as the probe. The antisense RNA was synthesized
using complementary DNA (cDNA) cloned by reverse
transcription-polymerase chain reaction and subcloned into pcDNAII as
the template and a digoxigenin-RNA labeling kit (Roche Diagnostics).
Chemiluminescence signals derived from hybridized probe were detected
on X-ray film using a digoxigenin luminescence detection kit (Roche
Diagnostics) and quantified by densitometry.
Data analysis Data are presented as means ± SD. The results were compared using either analysis of variance followed by Scheffé post hoc test or an unpaired t test. A level of P < .05 was accepted as statistically significant.
Effect of AT, DEGR-F.Xa, or Trp49-modified AT on MAP
and plasma levels of
NO2 /NO3 were
significantly increased 90 minutes after ET administration compared
with those of control animals, reaching the maximum at 180 minutes
(Figure 1B).
AT showed a significant inhibitory effect on the ET-induced decrease in
MAP, while neither DEGR-F.Xa nor Trp49-modified AT showed
any effect (Figure 2A). Although
intravenous administration of AT 60 minutes after the ET challenge did
not inhibit the ET-induced hypotension (data not shown), AT
administered 30 minutes after ET administration significantly prevented
the hypotension (Figure 2B). Although AT (250 U/kg)
significantly inhibited increases in plasma levels of
NO2
Effect of AT, DEGR-F.Xa, or Trp49-modified AT on increase in iNOS activity in the lungs of rats administered ET Lung iNOS activity was significantly increased 90 minutes after ET administration and reached its maximum at 180 minutes (Figure 4). AT (250 U/kg) significantly inhibited increase in lung iNOS activity 180 minutes after ET administration, while neither DEGR-F.Xa (3 mg/kg) nor Trp49-modified AT (250 U/kg) showed any effects (Figure 5).
Effect of AT on the expression of iNOS mRNA in the lungs of rats administered ET Expression of iNOS mRNA in the lungs was increased 60 minutes after ET administration and reached its maximum at 180 minutes (Figure 6). The expression of iNOS mRNA in the lungs 180 minutes after ET administration was significantly inhibited by administration of AT (250 U/kg) (Figure 7).
Effect of AT, DEGR-F.Xa, or Trp49-modified AT on the
tissue levels of TNF- began to increase 60 minutes
after ET administration, peaking at 90 minutes (Figure
8). Intravenous administration of 50 or
100 U/kg AT did not inhibit the increases in the tissue levels of
TNF- 90 minutes after ET administration (data not shown). However,
AT at the dosage of 250 U/kg significantly inhibited these increases
(Figure 9). Neither DEGR-F.Xa (3 mg/kg) nor Trp49-modified AT (250 U/kg) showed any effect on
increases in the lung tissue levels of TNF- (Figure 9).
Effect of AT on the expression of TNF- mRNA began to increase 30 minutes after ET administration, peaking at 60 minutes, and gradually
decreased to prelevels (Figure 10). The
expression of lung tissue TNF- mRNA 60 minutes after ET
administration was significantly inhibited by AT (250 U/kg) (Figure 11).
Effect of anti-rat TNF- /NO3 seen 180 minutes after ET administration were inhibited in animals treated with
antirat TNF- Ab (Figures 12A and 3).
The increase in lung tissue level of iNOS activity was significantly
inhibited in animals treated with anti-rat TNF- Ab 180 minutes after
ET administration (Figure 5).
Effect of indomethacin pretreatment on AT-induced effects Neither the inhibitory effect of AT on the ET-induced decrease in MAP nor that on the ET-induced increases in the plasma levels of NO2/NO3 were
observed in animals pretreated with indomethacin (5 mg/kg) (Figures 12B
and 3). When administered to animals pretreated with indomethacin (5 mg/kg), AT (250 U/kg) did not inhibit increase in the lung tissue level
of iNOS activity 180 minutes after ET administration (Figure 5).
Increases in the lung tissue levels of TNF- 90 minutes after ET
administration were not inhibited by AT in animals pretreated with
indomethacin (Figure 9).
Effect of iloprost, a stable derivative of PGI2, on
changes in MAP, plasma levels of
NO2 /NO3 and lung
tissue level of iNOS activity 180 minutes after ET administration (Figures 3 and 5). Iloprost significantly inhibited the ET-induced increases in the lung tissue levels of TNF- 90 minutes after ET
administration (Figure 9).
In the present study, AT significantly inhibited both the
increases in plasma levels of
NO2 Because endothelial NOS (eNOS) was shown to be induced in rat brain astrocytes by ET administration,35 eNOS could be induced in the lungs of rats given ET, contributing to the development of hypotension by producing NO. We measured eNOS activity in lung samples of rats 180 minutes after ET administration according to the method described by Szabó et al.15 However, there was no significant increase in the lung eNOS activity 180 minutes after ET administration (data not shown). Thus, NO implicated in the development of ET-induced hypotension in this rat model could be derived from iNOS. AT also inhibited the ET-induced increases in both tissue levels of
TNF- Because neither AT nor iloprost completely inhibited the hypotension
and production of TNF- DEGR-F.Xa, a selective inhibitor of thrombin generation, did not show any effects induced by AT in this animal model of septic shock. DEGR-F.Xa (3 mg/kg) inhibited ET-induced coagulation abnormalities to similar extents as AT (250 U/kg).23 Thus, the effects of AT seen in the present animal model might not be mediated by the anticoagulant effects of AT. AT has been shown to promote the endothelial release of
PGI2 by interacting with heparinlike GAGs on the
endothelial cell surface in vitro4,5 and in
vivo.6 The effects induced by AT in this rat model of
septic shock appeared to be mediated by PGI2, which may
have been released from the endothelial cells. This conclusion was
supported by the following observations. (1) Trp49-modified AT (250 U/kg) is a chemically modified AT
that shows AT activity similar to that of unmodified AT but has no
ability to promote the endothelial release of PGI2 due to a
lack of affinity for heparin.6 This compound did not show
any effects such as those induced by AT. (2) Indomethacin pretreatment
abrogated the effects induced by AT. (3) Iloprost, a stable derivative
of PGI2, showed effects similar to those of AT.
PGI2 inhibits TNF- Consistent with the observations in the present study, Taylor et al21 also demonstrated that AT prevents hypotension in baboons challenged with E coli independent of its anticoagulant effect. Because administration of AT after, in addition to its administration before, significantly inhibited the ET-induced hypotension in rats, AT might be effective for the treatment of patients with septic shock. Consistent with this hypothesis are the findings of a previous study showing that AT significantly reduced the mortality of patients with septic shock.46 Because TNF- Although AT has been shown to reduce organ injury by increasing the endothelial production of PGI2 in vivo, the precise mechanism(s) by which AT promotes the endothelial release of PGI2 have not been well understood. In this process, interaction of AT with the endothelial heparinlike GAGs is critical for the promotion of the endothelial release of PGI2.5,6 Further studies are necessary to clarify the mechanism(s).
We thank Drs Masataka Mori and Akitoshi Nagasaki for providing the
iNOS cDNA probe and Drs Yasuo Yamaguchi and Kazutoshi Okabe for
providing the TNF-
Submitted June 11, 2001; accepted October 12, 2001.
The publication costs of this article were defrayed in part by page charge payment. Therefore, and solely to indicate this fact, this article is hereby marked "advertisement" in accordance with 18 U.S.C. section 1734.
Reprints: Kenji Okajima, Department of Laboratory Medicine, Kumamoto University School of Medicine, Honjo 1-1-1, Kumamoto, 860-0811, Japan; e-mail: whynot{at}kaiju.medic.kumamoto-u.ac.jp.
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 P. Molor-Erdene, K. Okajima, H. Isobe, M. Uchiba, N. Harada, and H. Okabe Urinary trypsin inhibitor reduces LPS-induced hypotension by suppressing tumor necrosis factor-{alpha} production through inhibition of Egr-1 expression Am J Physiol Heart Circ Physiol, March 1, 2005; 288(3): H1265 - H1271. [Abstract] [Full Text] [PDF]
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Top
Abstract
Introduction
P < .05 versus
ET plus saline. 
B
activation.