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Prepublished online as a Blood First Edition Paper on December 12, 2002; DOI 10.1182/blood-2002-08-2406.
HEMOSTASIS, THROMBOSIS, AND VASCULAR BIOLOGY
From the Intensive Care Unit, Oita Medical University,
Japan; Department of Laboratory Medicine, Kumamoto
University School of Medicine, Japan; Department of
Pharmacology, Fukuoka University School of Medicine,
Japan; and Department of Maternal-Pediatric Nursing, Oita
Medical University, Japan.
Antithrombin (AT) supplementation in patients with severe sepsis
has been shown to improve organ failures in which activated leukocytes
are critically involved. However, the precise mechanism(s) for the
therapeutic effects of AT is not well understood. We examined in rats
whether AT reduces ischemia/reperfusion (I/R)-induced renal injury by
inhibiting leukocyte activation. AT markedly reduced the I/R-induced
renal dysfunction and histologic changes, whereas neither dansyl
glutamylglycylarginyl chloromethyl ketone-treated factor Xa
(DEGR-F.Xa), a selective inhibitor of thrombin generation, nor
Trp49-modified AT, which lacks affinity for heparin, had any effect.
Renal tissue levels of 6-keto-PGF1 Antithrombin (AT) is an important inhibitor of
serine proteases, which are generated within the coagulation
cascade.1 Interaction of AT with heparin-like
glycosaminoglycans (GAGs) on endothelial cells is important for the
acceleration of thrombin inhibition by AT.1 Thrombotic
episodes have been frequently observed in patients with congenital AT
deficiency and in those with variant AT that lacks affinity for
heparin,2,3 suggesting that the interaction of AT with the
endothelial cell surface heparin-like GAGs is important for regulation
of the coagulation cascade by AT. AT has been shown to reduce the
mortality of baboons challenged with Escherichia coli not
only by inhibiting coagulation abnormalities but also by attenuating
inflammatory responses in which activated leukocytes are critically
involved.4 Furthermore, AT supplementation limited the
inflammatory responses, thereby alleviating the respiratory failure,
liver dysfunction, and renal failure in patients with severe
sepsis.5,6 However, the anti-inflammatory mechanism of AT
is still not well understood. We previously reported that AT promoted
the endothelial release of prostacyclin (PGI2) in vivo by
interacting with heparin-like GAGs on the cell surface.7 PGI2 inhibits tumor necrosis factor- In the present study, we investigated the anti-inflammatory
mechanism(s) of AT using a rat model of ischemia/reperfusion
(I/R)-induced renal injury in which activated leukocytes are
critically involved.16 Our results suggested that
anti-inflammatory effects of AT were not mediated by its direct action
but by mainly by the action of PGI2 released from the
endothelial cells.
Materials
Determination of the content of latent and cleaved form of AT
in the AT concentrate
Preparation of DEGR-treated factor Xa DEGR-treated factor Xa (DEGR-F.Xa), a selective inhibitor of thrombin generation,19 was prepared as described previously. Briefly, factor Xa was purified from human plasma and activated with Russell viper venom.20,21 Activated factor X was inactivated with a 20-fold molar excess of DEGR for 30 minutes at 25°C. The mixture was then subjected to extensive dialysis against a solution containing 20 mM tris(hydroxymethyl)aminomethane(Tris)-hydrochloric acid (pH 7.4) and 100 mM sodium chloride. DEGR-F.Xa competes with intact factor Xa for prothrombinase complex formation.22 DEGR-F.Xa generated as described above showed no clotting activity and a prolonged activated partial thromboplastin time in a concentration-dependent manner (0 to 300 µg/mL).Preparation of Trp49-modified AT The Trp49 residue of AT was chemically modified by a version of the method of Karp et al.23 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 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 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 as previously described.7 The extent of derivatization of AT was determined spectrophotometrically in 2 M NaOH at a wavelength of 410 nm (molar extinction coefficient, 1.85 × 104 M 1).7 The progressive thrombin
inhibitory 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.7
Animal model of renal I/R The care and handling of the animals used in the present study was in accordance with the guidelines of the National Institutes of Health. All experimental procedures were approved by Kumamoto University and Oita Medical University Animal Care and Use Committee. Adult pathogen-free male Wistar rats (Nihon SLC, Hamamatsu, Japan) weighing 180 to 220 g were housed in a temperature-controlled (22°C) room with alternating 12-hour light/dark cycles and were given water but no food for 24 hours before the experiments. During surgery, core temperature was monitored using a rectal probe and maintained with a heating pad and a heating lamp at 35.5°C to 37°C. The renal I/R protocol was performed as described previously,24 with some modifications. In brief, rats were anesthetized with an intraperitoneal injection of pentobarbital sodium 50 mg/kg (Abbott Laboratories, North Chicago, IL), followed by 20 mg/kg every hour for 4 hours and supplemented with an intraperitoneal injection of buprenorphine 2 mg/kg every 12 hours for 2 days. To perform nephrectomy, a midline incision was made in each rat and the left kidney was mobilized to allow the left renal vessels to be ligated. To cause ischemia, the right pedicle was clamped with a noncrushing microvascular clamp for 45 minutes. Ischemia was confirmed visually by blanching of the kidney. During the period of renal ischemia, the rats were covered with plastic wrap to prevent evaporation. After 45 minutes of ischemia, the clamp was removed and the wound was closed with a 3-0 silk suture. The animals were then returned to their cages and allowed free access to food and water. Sham-operated animals underwent the same operation but without clamping. At specified time points (before ischemia and 3, 6, 12, 24, and 48 hours after reperfusion, except for measurement of renal tissue level of 6-keto-PGF1 ), the
rats were anesthetized by intraperitoneal administration of
pentobarbital (50 mg/kg) and killed. Blood samples were taken from the
abdominal aorta. Blood was collected in tubes and centrifuged at
2000g for 10 minutes. Renal dysfunction was evaluated by
measuring serum levels of blood urea nitrogen (BUN) and creatinine by
standard urease assays and picric acid reactions.25
Coagulation abnormality was evaluated by serum levels of fibrin
degradation products (FDP(E)) measured with a latex agglutination assay
as described previously.25 The kidneys were harvested for
examination of histologic changes, renal tissue level of
6-keto-PGF1, renal vascular permeability, assay of renal
MPO activity, and renal tissue content of cytokines.
Experimental design We initially examined the effects of various doses of AT (50, 100, and 250 U/kg intravenously) on renal dysfunction in rats with I/R injury. To elucidate the therapeutic mechanisms of AT, the rats were subjected to the following 9 experimental groups after left nephrectomy: sham-operated, vehicle-treated, AT-treated, DEGR-F.Xa-treated, Trp49-modified AT-treated, IM-treated, AT pretreated with IM-treated, iloprost-treated, and iloprost pretreated with IM-treated.Administration of AT, DEGR-F.Xa, and Trp49-modified AT We previously reported that the plasma concentration of 6-keto-PGF1, a stable metabolite of PGI2,
begins to increase 30 minutes after the intravenous administration of
AT (250 U/kg) in intact rats.26 Therefore, AT was
administered 30 minutes prior to reperfusion in the present study.
DEGR-F.Xa (3 mg/kg) and Trp49-modified AT (250 U/kg) were also
administered intravenously 30 minutes before reperfusion. At the dosage
used in the present study, DEGR-F.Xa and Trp49-modified AT have
anticoagulant activities comparable to that of 250 U/kg native
AT.7
IM and iloprost administration IM (20 mg/kg) was suspended in bicarbonate-buffered saline and administered subcutaneously 30 minutes prior to ischemia. Control animals received the same volume of bicarbonate-buffered saline instead of IM. Iloprost was dissolved in saline and continuously infused (100 ng/kg/min) via the right jugular vein for 3 hours after the onset of renal reperfusion. In other treated groups animals received continuous infusion of the same volume of saline instead of iloprost.Histopathologic studies of the kidneys The kidneys were removed 24 hours after reperfusion, fixed in 10% formalin, embedded in paraffin, cut into 5-µm-thick sections, and stained with hematoxylin and eosin. Samples were analyzed by a pathologist who was blinded to the experimental groups. Tubular necrosis, vascular congestion, and neutrophil accumulation in the outer medulla were evaluated in 20 random fields as described by Kelly et al27 and Chiao et al,28 with some modifications. Tubular necrosis was evaluated by determining the percentage of tubules in the outer medulla in which epithelial necrosis or necrotic debris was observed. Vascular congestion was assessed by counting the erythrocytes in the outer medulla. Neutrophil accumulation was quantified as numbers of neutrophils in the outer medulla. The cells were counted using an eyepiece graticule at a magnification of × 400.Measurement of renal 6-keto-PGF1 (a metabolite of
PGI2) levels were determined in animals subjected to I/R
before and 1, 3, 6, 12, 24, and 48 hours after reperfusion according to
the methods described previously,29 with some
modifications. In brief, the kidneys were weighed and then immediately
homogenized in 5 mL of 0.1 M phosphate buffer (pH 7.4) containing 2 mM
IM at 5°C. The homogenate was first centrifuged at 2000g
for 10 minutes to remove minute amounts of solid tissue debris, and the
supernatant was then acidified with 1 M HCl. The
6-keto-PGF1 was extracted from the supernatant using
columns packed with ethyl-bonded silica gel (ethyl C2; Amersham, Buckinghamshire, United Kingdom). The columns were prepared by washing
them with 2 mL methanol followed by 2 mL water. The acidified supernatant was applied to the column, and this was followed by sequential washes with 5 mL water, 5 mL of 10% ethanol, and 5 mL
hexane. The elution of 6-keto-PGF1 was performed with 5 mL methylformate, 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 1280 pg/mL of
6-keto-PGF1. The results are expressed as nanograms of
6-keto-PGF1 per gram of tissue. The cross-reactivities of this assay with prostaglandin E2 (PGE2),
prostaglandin F2, 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.30
Measurement of renal tissue blood flow Renal tissue blood flow was evaluated by measuring renal cortical blood flow using a laser-Doppler flow meter (ALF21N; Advance, Tokyo, Japan) as described previously.25 The Doppler probe was directed toward the cortex. Renal cortical blood flow was measured from 30 minutes before ischemia until 3 hours after reperfusion. Electrical signals from the probe were digitized and recorded in real time using MacLab software (ADInstruments, Castle Hill, Australia).24 Results are expressed as percentages of the preischemic levels.Evaluation of renal vascular permeability Renal vascular permeability was assessed by measuring extravasated dye as described.25 In brief, Evans blue dye (25 µg/kg) was injected intravenously 10 minutes before the rats were killed by exsanguination from the abdominal aorta. The kidneys were removed, weighed, and placed in 5 mL dimethylformamide (Wako, Osaka, Japan) for 7 days. After centrifugation (2000g for 10 minutes), the concentration of Evans blue dye extracted in the dimethylformamide was measured by a spectrophotometer (DU-54; Beckman, Irvine, CA) at a wavelength of 610 nm and compared with results obtained with standards. Evans blue concentrations are expressed in micrograms per gram of tissue.Assay of renal MPO activity and tissue cytokine content Accumulation of neutrophils in the kidney was evaluated by measuring renal MPO activity as described previously.25,31 The rats were killed at various times points after reperfusion. The kidneys were removed, weighed, and homogenized (Physcotron; Niti-on, Tokyo, Japan) in 10% (wt/vol) 0.05 M phosphate buffer (pH 6.0) containing 0.5% hexadecyltrimethylammonium bromide and sonicated for 20 seconds. After centrifugation (4500g for 20 minutes at 4°C), 0.1 mL of the supernatant was added to 0.55 mL of 0.1 M phosphate buffer (pH 6.0) containing 1.25 mg/mL o-dianisidine and 0.05% hydrogen peroxide. After 5 minutes, changes in absorbance at 460 nm were measured using a spectrophotometer. The activity of purified known human neutrophil MPO was used as the standard. Results are expressed as units of MPO activity per gram of tissue.Renal levels of TNF- Statistical analysis Data are expressed as means ± SDs. Differences between groups were examined for statistical significance using an unpaired t test for single comparison and analysis of variance followed by the Scheffé post hoc test for multiple comparison. A P value below .05 denoted the presence of a statistically significant difference.
Effects of AT, DEGR-F.Xa, and Trp49-modified AT on I/R-induced renal injury Serum levels of BUN and creatinine were significantly increased and reached a peak level at 24 hours after renal I/R in control animals (Figure 1). We determined the effects of 3 different dosages of AT on renal I/R-induced increases in serum levels of BUN and creatinine. AT, at a dosage of 250 U/kg, significantly inhibited these increases at 24 hours after reperfusion (Figure 2), whereas lower doses of AT (50 and 100 U/kg) had no effect (data not shown). Thus, all subsequent experiments were performed at the dosage of 250 U/kg of AT. Neither DEGR-F.Xa nor Trp49-modified AT had any effect on the renal injury (Figure 2).
Histologic examinations of kidneys were performed at 24 hours after
reperfusion (Figure 3). Microscopic
assessment of the outer medulla of the kidney after I/R revealed severe
tubular necrosis with cast formation, vascular congestion, and
neutrophil accumulation in the vasa recta of the outer medulla (Figure
3A). Fibrin deposition was not observed in kidneys from any group. Intravenous administration of AT resulted in a reduction of these histologic changes (Figure 3B), whereas neither DEGR-F.Xa nor Trp49-modified AT had any effects (data not shown). We also analyzed the histologic changes quantitatively using a scoring system for tubular necrosis, vascular congestion, and neutrophil accumulation. These changes were significantly fewer in rats treated with AT than in
control animals and those given DEGR-F.Xa or Trp49-modified AT (Table
1).
Effects of AT, DEGR-F.Xa, and Trp49-modified AT on serum levels of FDP(E) in animals subjected to renal I/R Serum levels of FDP(E) were increased after renal I/R, peaking at 24 hours after reperfusion in control animals (Figure 4A). Increases in serum levels of FDP(E) at 24 hours after reperfusion were significantly inhibited by administration of AT, DEGR-F.Xa, and Trp49-modified AT (Figure 4B).
Effects of AT, DEGR-F.Xa, and Trp49-modified AT on renal levels
of 6-keto-PGF1 after reperfusion. Renal tissue levels of
6-keto-PGF1 were increased after renal I/R in control
animals, peaking at 1 hour after reperfusion (Figure
5A). These levels were significantly higher than those of sham-operated animals (Figure 5A). AT
significantly enhanced I/R-induced increases in renal tissue levels of
6-keto-PGF1 1 hour after reperfusion (Figure 5B),
whereas neither DEGR-F.Xa nor Trp49-modified AT had any effect on these
changes (Figure 5B).
Effects of AT, DEGR-F.Xa, and Trp49-modified AT on renal cortical blood flow Renal cortical blood flow decreased to approximately 40% of the preischemic level at 3 hours after reperfusion (Figure 6). AT significantly inhibited the I/R-induced reduction in renal cortical blood flow. Neither DEGR-F.Xa nor Trp49-modified AT had any effect on the renal cortical blood flow (Figure 6).
Effects of AT, DEGR-F.Xa, and Trp49-modified AT on renal vascular permeability Renal vascular permeability, as assessed by Evans blue dye leakage, increased significantly after reperfusion, reaching a maximum level at 6 hours after injury (Figure 7A). AT significantly inhibited the increases in renal vascular permeability 6 hours after reperfusion, whereas neither DEGR-F.Xa nor Trp49-modified AT inhibited these changes (Figure 7B).
Effects of AT, DEGR-F.Xa, and Trp49-modified AT on renal tissue
levels of TNF-  and CINC were increased after
renal I/R, reaching peak levels at 3 hours after reperfusion and gradually decreasing thereafter (Figure
8A-B). Accumulation of neutrophils in
renal tissue was assessed by determination of renal MPO activity. Renal
tissue levels of MPO were increased after renal I/R, reaching a maximum
at 6 hours after reperfusion (Figure 8C). These variables reflecting
leukocyte activation in animals subjected to renal I/R were
significantly higher than those of sham-operated animals (Figure 9).
Increases in renal tissue levels of TNF-, CINC, and MPO were
significantly inhibited by AT (Figure 9).
Neither DEGR-F.Xa nor Trp49-modified AT had any effect on these changes
(Figure 9).
Effects of IM pretreatment on AT-induced effects in renal I/R Pretreatment of animals with IM significantly prevented increases in renal tissue levels of 6-keto-PGF1 in control animals and those in animals given AT at 1 hour after reperfusion (Figure 5B).
AT did not inhibit I/R-induced increases in serum levels of BUN and
creatinine at 24 hours after reperfusion in animals pretreated with IM.
IM itself also significantly exacerbated these increases at 24 hours
after reperfusion compared with controls (Figure 2B). Pretreatment
with IM reversed AT-induced effects on histologic findings in animals
subjected to renal I/R (Table 1). AT significantly inhibited
I/R-induced increase in serum levels of FDP(E) in animals pretreated
with IM (Figure 4B). IM itself significantly reduced renal cortical
blood flow and increased vascular permeability (Figures 7B and
10). Effects of AT on I/R-induced changes in both renal cortical blood
flow and renal vascular permeability were reversed by pretreatment with
IM (Figures 7B and 10). AT did not
reduce the I/R-induced increases in renal tissue levels of TNF-,
CINC, and MPO at 24 hours after reperfusion in animals pretreated with
IM (Figure 9). IM itself significantly enhanced the I/R-induced
increases in renal tissue levels of TNF-, CINC, and MPO (Figure
9).
Effects of iloprost on the I/R-induced renal changes Iloprost, a stable derivative of PGI2, produced effects similar to those induced by AT. Iloprost significantly reduced renal dysfunction (Figure 2), inhibiting both reduction of renal cortical blood flow (Figure 10) and increase in renal vascular permeability (Figure 7B) in animals subjected to renal I/R. Increases in renal tissue levels of TNF-, CINC, and MPO were significantly
inhibited by iloprost (Figure 9). Furthermore, iloprost reversed the
IM-induced exacerbation of renal changes in animals subjected to renal
I/R (Figures 2,7B,9,10). Iloprost inhibited renal I/R-induced increases in serum levels of FDP(E) and those in animals pretreated with IM
(Figure 4B).
In the present study, we demonstrated that AT reduced renal dysfunction and histologic changes of the kidney in rats subjected to renal I/R independently of its anticoagulant activity, because DEGR-F.Xa did not reduce I/R-induced renal injury despite its anticoagulant activity comparable to that of AT. Activated leukocytes are critically involved in the development of the
I/R-induced renal injury.25 This notion was confirmed in
the present study by the findings that renal tissue levels of TNF- AT inhibited I/R-induced reduction of renal cortical blood flow and increase in renal vascular permeability as shown in the present study. Endothelial cell injury induced by inflammatory mediators released from activated neutrophils such as neutrophil elastase and oxygen-free radicals are critically involved in the renal I/R-induced increase in vascular permeability, leading to the reduction of renal cortical blood flow.25 Our preliminary experiments demonstrating that ONO-5046, a specific inhibitor of neutrophil elastase,33 inhibited the reduction of renal cortical blood flow by inhibiting increase in renal vascular permeability. These observations strongly suggested that AT might inhibit I/R-induced decrease in renal cortical blood flow by inhibiting neutrophil activation. Renal I/R-induced histologic changes included tubular necrosis, vascular congestion, and neutrophil accumulation in the outer medulla as shown in the present study. Because the main site of inflammation where neutrophils are activated is the postcapillary venules feeding proximal tubules in the kidney,34 activated neutrophil-induced endothelial cell injury might lead to ischemia of the proximal tubules in the outer medulla. Consequently, plug formation in the lumen of proximal tubules might lead to renal dysfunction after the renal I/R.35 Thus, AT might improve the I/R-induced renal dysfunction by reducing the tubular necrosis induced by activated neutrophils in the present study. It is well known that neutrophils are activated by TNF- Recently, results of in vitro experiments raised the possibility that
AT might inhibit leukocyte activation directly. Dunzendorfers et
al13 reported that AT directly inhibited neutrophil
chemotaxis by binding to the cell surface-heparan sulfate
glycosaminoglycans. Souter et al14 demonstrated that
AT directly inhibited interleukin-6 production by monocytes
stimulated with lipopolysaccharide. Furthermore, Oelschläger et
al15 demonstrated that AT directly inhibited TNF- Indomethacin inhibits activities of cyclooxygenases, thereby inhibiting production of some eicosanoids other than PGI2. Therefore, some other eicosanoids might be involved in both the IM-induced aggravation of I/R-induced renal injury and therapeutic mechanisms of AT in the present study. Because PGE2 as well as PGI2 have also been shown to inhibit leukocyte activation and both prostaglandins are synthesized from PGH2 in endothelial cells,9,37 it is likely that PGE2 might be involved in therapeutic effects of AT in the present study. Consistent with this hypothesis are the results of our preliminary experiments showing that AT reduced the I/R-induced hepatic injury by promoting the I/R-induced increases in hepatic tissue levels of PGE2 in rats. This possibility should further be examined using this rat model of I/R-induced renal injury. Singbartl et al38 demonstrated that platelets as well as neutrophils could also be critically involved in the development of I/R-induced renal injury by promoting neutrophil infiltration to the outer medulla of kidney, the most vulnerable region of the kidney after I/R. Because PGI2 potently inhibits platelet activation,37 AT might reduce the I/R-induced renal injury by inhibiting activation of platelets as well as neutrophils in the present study. The molecular mechanism by which AT increases the endothelial
production of PGI2 remains to be elucidated. We
demonstrated that AT did not increase the production of
PGI2 directly in cultured endothelial cells,39
suggesting that some factors other than endothelial cells might be
involved in the AT-induced endothelial production of PGI2
in vivo. We recently demonstrated that activation of
capsaicin-sensitive sensory neurons led to an increase in the endothelial production of PGI2 in rats subjected to hepatic
I/R.40 We also reported briefly that AT reduced
I/R-induced liver injury by increasing the hepatic tissue levels of
6-keto-PGF1
Submitted August 8, 2002; accepted December 5, 2002.
Prepublished online as Blood First Edition Paper, December 12, 2002; DOI 10.1182/blood- 2002-08-2406.
Supported in part by the departmental funds of Kumamoto University School of Medicine.
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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8.
D'Acquisto F, Sautebin L, Iuvone T, Di Rosa M, Carnuccio R.
Prostaglandins prevent inducible nitric oxide synthase protein expression by inhibiting nuclear factor- 9. Oh-ishi S, Utsunomiya I, Yamamoto T, Komuro Y, Hara Y. Effects of prostaglandins and cyclic AMP on cytokine production in rat leukocytes. Eur J Pharmacol. 1996;300:255-259[CrossRef][Medline] [Order article via Infotrieve].
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Ollivier V, Parry GC, Cobb RR, de Prost D, Mackman N.
Elevated cyclic AMP inhibits NF- 11. Uchiba M, Okajima K, Murakami K. Effects of various doses of antithrombin III on endotoxin-induced endothelial cell injury and coagulation abnormalities in rats. Thromb Res. 1998;89:233-241[CrossRef][Medline] [Order article via Infotrieve]. 12. Isobe H, Okajima K, Uchiba M, Harada N, Okabe H. Antithrombin prevents endotoxin-induced hypotension | |||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
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Abstract
Introduction
B (NF-
80°C until use. Results are expressed as picograms of cytokine per
gram of tissue.
P < .01 versus AT-treated group;
¶P < .01 versus IM-treated group.
