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Blood, Vol. 95 No. 4 (February 15), 2000:
pp. 1117-1123
FOCUS ON HEMATOLOGY
Recombinant human antithrombin III improves survival and
attenuates inflammatory responses in baboons lethally challenged
with Escherichia coli
M. C. Minnema,
A. C. K. Chang,
P. M. Jansen,
Y. T. P. Lubbers,
B. M. Pratt,
B. G. Whittaker,
F. B. Taylor,
C. E. Hack, and
B. Friedman
From the Sanquin Blood Supply Foundation, Amsterdam,
The Netherlands; the Laboratory for Experimental and Clinical
Immunology, University of Amsterdam, Amsterdam, The
Netherlands; Oklahoma Medical Research Foundation, Oklahoma
City, OK; and Genzyme Corporation, Framingham, MA.
 |
Abstract |
Plasma-derived antithrombin III (ATIII) prevents the lethal effects
of Escherichia coli infusion in baboons, but the
mechanisms behind this effect are not clear. In the
present study, we evaluated the effects of recombinant human
ATIII (rhATIII) on the clinical course and the inflammatory
cytokine and coagulation responses in baboons challenged with lethal
dose of E coli. Animals in the treatment group (n = 5)
received high doses of rhATIII starting 1 hour before an E coli
challenge. Those in the control group were administered saline.
Survival was significantly improved in the treatment group
(P = .002). Both groups had similar hemodynamic responses
to E coli challenge but different coagulation and inflammatory responses. The rhATIII group had an accelerated increase of
thrombin-ATIII complexes and significantly less fibrinogen
consumption compared to controls. In addition, the rhATIII group had
much less severe thrombotic pathology on autopsy and virtually no
fibrinolytic response to E coli challenge. Furthermore, the
rhATIII group had a significantly attenuated inflammatory response as
evidenced by marked reduction of the release of various cytokines. We
conclude that the early administration of high doses of rhATIII
improves the outcome in baboons lethally challenged with E
coli, probably due to the combined anticoagulation and
anti-inflammatory effects of this therapy.
(Blood. 2000;95:1117-1123)
© 2000 by The American Society of Hematology.
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Introduction |
Antithrombin III (ATIII) plays a central role in
regulating hemostasis. When bound to glycosaminoglycans, it is an
important inhibitor of several serine proteases, including factors Xa,
IXa, XIa, and thrombin,1 that are involved in blood
coagulation. Because of this central inhibitory role in the hemostatic
system, ATIII is believed to play an important role in the clinical
syndrome of diffuse intravascular coagulation (DIC), which may occur in patients with sepsis. Consistent herewith are observations that plasma
levels of ATIII are decreased in patients with DIC, and this is most
likely due to increased consumption and decreased synthesis.2
In baboons challenged with a lethal dose of Escherichia coli,
infusion of plasma-derived human ATIII has been shown to promote survival and protect against DIC.3 It was hypothesized that this protection was due to inhibition of thrombin and the
thrombin-mediated coagulation response. A later study showed that
active site blocked Xa (DEGR Xa) could not prevent
shock, organ damage, and mortality in this model despite an efficient
blockade of the coagulation response.4 Hence, the
protective effect of ATIII infusion on lethality after
LD100 E coli in the baboon model
cannot solely result from its effect on fibrin formation, but it may be
due to other effects such as modulation of the inflammatory reaction.
Infusion of LD100 E coli in the baboon model has
been shown to induce tumor necrosis factor- (TNF-), interleukin-6
(IL-6), IL-8, and other cytokines that appear to regulate inflammatory as well as hemostatic responses to E coli
challenge.5
In the present study of a lethal E coli baboon
model, we investigated the effects of recombinant human ATIII, which
was produced in the milk of transgenic goats, on markers of
inflammation, coagulation, and fibrinolysis.
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Materials and methods |
Test article
Recombinant human ATIII (rhATIII) was produced in the milk of
transgenic goats, purified to >99%, and provided as a sterile lyophilized preparation. When reconstituted in water for injection, the
350 units/mL solution contained: sodium chloride, 0.138 mol/L; sodium
citrate, 0.01 mol/L; and glycine, 0.133 mol/L, pH 7.4 (Genzyme Transgenic, Framingham, MA). The specific
activity of the preparations used was 7 units/mg. Structurally, the
rhATIII used in these experiments was identical to plasma-derived human
ATIII except for differences in glycosylation. Plasma-derived ATIII has
disialylated, biantennary complex carbohydrate
structures at all 4 N-linked glycosylation sites, with very little
fucosylation. In contrast, rhATIII contains oligomannose structures at
1 of the sites (Asn 155) and less highly sialylated, more highly
fucosylated complex carbohydrate structures at the other 3 sites.
rhATIII also contains 2 sugars not present in pATIII
(N-acetyl-galactosamine instead of galactose and N-glycolyl-neuraminic acid instead of N-acetyl-neuraminic acid).6
Experimental design
Experiments were performed on 10 juvenile baboons
(Papio Anubis/Cynocephalus), 1 at a time. They were
fasted overnight and given water ad libitum. The E coli (type
B) preparation was performed as described.7 Each animal was
sedated with ketamine hydrochloride (14 mg/kg, intramuscularly) on the
morning of the study and anesthetized with sodium pentobarbital (2 mg/kg) via a percutaneous catheter positioned in the cephalic vein. The
femoral artery and both femoral veins were cannulated aseptically and
used for measuring aortic pressure, obtaining blood samples, infusing
organisms and rhATIII, and administrating fluids and anesthetics, as
reported elsewhere.7 All baboons were challenged with a
2-hour infusion of a lethal dose of E coli (about
4 × 1010 organisms/kg) at t = 0 hour. Gentamycin
was given as a 60-minute infusion (9 mg/kg, intravenously) 2 hours
after the start of the experiment, followed by 30-minute infusions (4.5 mg/kg) at 6 and 9 hours. Gentamycin (4.5 mg/kg) was further given
intramuscularly at 12 hours and twice daily for 3 days.
The treatment group, consisting of 5 animals, received the following 3 administrations of rhATIII (approximate doses): (1) a 30-minute
infusion of 470 units/kg starting at t = -1 hour (1 hour before E
coli challenge), (2) a bolus of 230 units/kg administered at
t = 0 hours, and (3) a 30-minute infusion of 460 units/kg starting at
t = 3 hours.
The control group of animals, which consisted of a pool of controls,
was treated during a 6-month period preceding the rhATIII studies
described above. They received a continuous 9-hour infusion of
saline.8 All animals were maintained under anesthesia and monitored for 10 hours. They were observed continuously for an additional 36 hours and daily for a maximum of 7 days. Blood samples were collected at given time points for hematology, clinical chemistry, and ATIII determinations. Furthermore, additional samples were collected on final concentrations of 10 mmol/L ethylene diamine tetra-acetic acid (EDTA) and 0.1 mg/mL soy bean trypsin inhibitor before t = 0 and at 1, 2, 3, 4, 6, 8, and 10 hours after the start of
the E coli infusion. The samples, which were used to determine cytokines, neutrophil degradation products, and coagulation and fibrinolytic parameters, were stored frozen until all samples were
available for assay.
Baboons surviving for 7 days were considered permanent survivors and
were subsequently killed with sodium pentobarbital. Necropsy was
performed on all animals. All animal studies were approved by the
Institutional Review Board for Animal Studies.
Assays
Plasma levels of ATIII activity were measured by assessing
ATIII-mediated thrombin inhibition in the presence of heparin. The
protocol is a 2-stage colorimetric end point assay in a microplate format, and residual thrombin activity is measured using the
amidolytic substrate S2238.9 Plasma concentrations of
TNF, IL-6, IL-8, and IL-10 were measured by ELISA as previously
described10-12 and expressed as ng/mL or, in the case of
IL-10, pg/mL.
Levels of tissue-type plasminogen activator (tPA), plasminogen
activator inhibitor type 1 (PAI-1), and thrombin-ATIII (TAT) complexes
were determined by ELISA as described previously.13-15 Values were expressed as ng/mL. Plasmin-2-antiplasmin (PAP)
complexes were measured by radioimmunoassay as described.15
PAP complex levels were expressed as a percentage of the level
present in normal baboon plasma (NBP), in which a maximum amount of
complexes was generated by incubation with an equal volume of urokinase (UK, 50 µg/mL) in the presence of 0.2 mol/L methylamine (final concentration).15 This standard is further referred to as
NBP-MA-UK.
Statistical analysis
Results are expressed as mean ± SEM (standard error of
the mean). Comparisons between groups during the course of the
observation period were performed using the analysis of variance
program (ANOVA) or, in case of abnormally distributed parameters, the
Mann-Whitney U test. Statistical significance in survival was analyzed
by log rank test. A 2-sided value of P < .05 was considered
to indicate a significant difference.
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Results |
Establishment of sepsis
Table 1 shows the weight, sex, actual
E coli dose administered, the number of organisms circulating
at 2 hours, and the survival times of the animals in the control and
treatment groups. Although the dose of E coli administered was
significantly lower in the treatment group
(6.2 ± 0.5 × 1010 CFU/kg) than in the control group (8.7 ± 0.45 × 1010 CFU/kg),
P < .05, the amounts infused were all within the
LD100 range (4-8 × 1010 CFU/kg)
established over a 10-year period. After 2 hours, the E coli
concentration was 5-fold lower in the treatment group
(0.9 ± 0.6 × 107 CFU/kg) compared with the
control group (4.8 ± 2.6 × 107 CFU/kg), P < 0.05.
Plasma ATIII concentration-time data
Figure 1 depicts ATIII activity
in plasma of animals in the treatment group. Increases in ATIII
activity were noted following each administration of rhATIII. The data
indicate that baboon No. 2 did not receive the third rhATIII dose, and
we have not been able to determine how this occurred. For the other
animals, plasma ATIII activity was maintained at or above 10 units/kg
of body weight for 10 hours after initiation of E coli
challenge, and maximum plasma concentrations of ATIII activity
(measured at t = 4 hours) averaged 18 units/mL. After the final
infusion, ATIII activity disappeared from the circulation with an
apparent half-life of approximately 6 hours.
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| Fig 1.
Course of ATIII activity levels in septic baboons treated
with rhATIII.
Recombinant human ATIII was infused 1 hour before E coli
infusion, at t = 0, and 3 hours after infusion in concentrations
of 66.7, 33.3, and 66.7 mg/kg, respectively (indicated by arrows).
ATIII activity levels were measured as previously described.
Baboon No. 1 ( ), No. 2 ( ), No. 3 ( ), No. 4 ( ), and No. 5 ( ).
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Overall outcome
Administration of rhATIII promoted survival against a lethal
infusion of E coli (Table 1, Figure
2). The mean survival of controls was 19.4 hours. In this group, all animals died of multiple organ failure and
DIC. These controls behaved consistently and comparably with other
control groups in studies done by the Oklahoma Medical Research
Group.3,4,7 In contrast, the mean survival rate of the
treated animals was significantly longer: 3 animals survived >168
hours to scheduled euthanasia; 1 animal died 57 hours after E
coli administration as a result of capillary leakage in the lungs,
consistent with acute respiratory distress syndrome (ARDS), but no
evidence of DIC; and 1 animal died 28 hours after E coli
administration as a result of multiple organ failure and moderate DIC.
Notably, the latter animal (baboon No. 2) had not received the final
administration of rhATIII at t = 3 hours after the onset of infection
(Figure 1). As a result, the plasma ATIII activity levels in this
animal at time points beyond 3 hours were half those measured in the 3 animals that survived until scheduled sacrifice. Plasma ATIII activity
levels in the animal that only survived 57 hours postinfection (baboon
No. 4) were consistently lower following E coli challenge than
the levels in animals that survived until study termination.
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| Fig 2.
Survival in control and rhATIII-treated baboons.
The Kaplan-Meier curve of 5 control baboons () and 5 rhATIII-treated
baboons () after lethal challenge with E coli. Animals
surviving for 168 hours (7 days) were considered permanent survivors.
Survival was significantly higher in the rhATIII-treated animals, as
measured by log rank test (P = .002).
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Physiological and hematological responses
The effect of rhATIII infusion on E coli-induced
physiological and hematological response patterns is shown in Table
2. In all baboons, the infusion of E
coli produced a severe hypotensive shock, which was not affected by
rhATIII infusion. A decline in MSAP was observed at
t = 2 hours and maintained throughout the 10 hour-observation period.
Heart rates were similarly elevated in either group, but temperature
was significantly higher in the treatment group than in the control
group (P < .05). Administration of the first infusion of
rhATIII resulted in a significant rise in white blood cell (WBC)
counts, from 5.7 ± 0.8 (103/µL) at t =  1 to
13.2 ± 3 (103/µL) at t = 0 (P < .05).
Saline-infused controls had WBC counts of 5.9 ± 0.7
(103/µL) and 5.8 ± 1.7 (103/µL) at
these respective time points. After E coli challenge, WBC
counts decreased equally in both treatment groups.
Changes in hematocrit were not different between the groups, but the
fall in fibrinogen concentration varied. Fibrinogen levels in the
treatment group were 29±7.4% of baseline at 6 hours and 41.4 ± 13.9% of baseline at 10 hours (P < .05).
These figures are significantly higher than those in the control group
at these time points, 4.2 ± 3.2% and 2 ± 0.8%,
respectively (Figure 3).
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| Fig 3.
Fibrinogen and TAT complexes in control and treatment
group.
Mean ± SEM plasma levels of fibrinogen (upper panel) and
TAT complexes (lower panel) in control () and rhATIII-treated ()
baboons. After infusion of E coli, fibrinogen concentration
(%) was significantly higher in the treatment group at 6 and 10 hours,
whereas TAT complexes were significantly higher in the treatment group
at 2 and 3 hours. *P < .05; **P < .01.
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No differences between the 2 treatment groups were observed in
biochemical markers related to organ damage, such as lactate hydrogenase, creatinine, blood urea nitrogen, and
alanine aminotransferase, either before the experiment
or after 10 hours (results not shown).
Cytokine responses
The administration of E coli was associated with
changes in TNF, IL-10, IL-6, and IL-8 plasma concentrations that
differed in magnitude and/or duration between the control and rhATIII
treatment groups. In both groups of animals, there was a transient rise in plasma TNF concentrations, which peaked 2 hours after the onset of
E coli challenge and returned to baseline by the 6-hour time point (Figure 4, upper panel). However, the
concentration-time data for these groups were not identical. Plasma TNF
concentrations rose more rapidly and were greater in magnitude in the
treatment group (58.6 ± 16.1 ng/mL) than in the controls
(16.3 ± 2 ng/mL) at t = 2 hours (P < .05).
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| Fig 4.
TNF and IL-10 levels in control and treatment groups.
Mean ± SEM plasma levels of TNF (upper panel) and IL-10 (lower
panel) were measured upon lethal E coli infusion. The TNF
response was significantly higher (P < .05) in the
treatment group () compared with the control group (). After 3 hours, levels of IL-10 were lower in the treatment group compared with
the control group. *P < .05; and **P < .01.
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Increases in plasma IL-10 were noted in both groups in
response to E coli challenge (Figure 4, lower panel), with peak
values occurring 2 to 3 hours after the onset of sepsis:
1776 ± 148 pg/mL at 2 hours for the treatment group and
3566 ± 772 pg/mL at 3 hours for the control group. Plasma IL-10
concentrations were significantly higher for the control group compared
with the treatment group (P < .01). Plasma IL-6 (Figure
5, upper panel) values started to increase
2 hours after the onset of sepsis and continued to increase
in both groups up until the 6-hour time point. At this juncture, IL-6 continued to rise in the control group (peak level of
1174 ± 236 ng/mL at 10 hours) but not in the treatment
group (peak level of 220 ± 79 ng/mL at 4 hours), and
significant differences in IL-6 concentrations were noted
from this time point forward. Increased IL-8 and IL-6 plasma
concentrations were first noted 2 hours after the onset of sepsis
(Figure 5, lower panel). In both groups of animals, peak values of IL-8
were reached approximately 4 hours after the onset of sepsis: treatment
group (103 ± 36 ng/mL) and control group (308 ± 62 ng/mL),
P < .01.
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| Fig 5.
IL-6 and IL-8 levels in control and treatment groups.
Mean ± SEM plasma levels of IL-6 (upper panel) and IL-8 (lower
panel) in control () and rhATIII- treated () baboons. The
differences between groups were significant after 4 hours.
*P < .05; ** P < .01.
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Clotting and fibrinolytic responses
Increase in TAT complexes in plasma were noted 2 hours
after induction of sepsis for both groups of animals. There were
significantly more TAT complexes in the treatment group at 2 hours
(1699 ± 410 ng/mL) and 3 hours (2952 ± 48 ng/mL) than in
the control group at the same time points (223 ± 35 ng/mL
and 964 ± 142 ng/mL, respectively), P < .01 (Figure
3). Thereafter, TAT complexes were comparable in both groups and
decreased over time.
As shown in Figure 6, rhATIII
infusions almost completely blocked the tPA response after the E
coli challenge. In the treatment group, plasma concentrations
reached plateau levels from 1 hour on, with the highest concentration
being 27.2 ± 8.1 ng/mL. In contrast, tPA levels in the control
group continuously increased, and they reached the highest
concentration at 8 hours, 88.8 ± 15.2 ng/mL
(P < .01).
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| Fig 6.
Fibrinolytic response in control and treatment groups.
Mean ± SEM plasma levels of tPA (upper panel), PAI-1 (middle
panel), and PAP complexes (lower panel) in control () and
rhATIII-treated () baboons. PAP complexes are expressed as a
percentage of NBP-MA-UK. tPA and PAP complex levels were significantly
lower in the treatment group compared with controls, whereas PAI-1
levels were significantly higher in the treatment group compared with
controls at 2 and 3 hours after the E coli infusion.
*P < .05; **P < .01.
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The appearance of PAI-1 in plasma was also different between the 2 study groups (Figure 6). PAI-1 levels in the ATIII group increased
sharply after 2 hours, with peak concentrations of 6021 ± 1328
ng/mL at 4 hours, while the PAI-1 levels in the control group increased
more slowly and reached the highest levels of 5180 ± 877 ng/mL at
8 hours (P < .01).
The most striking difference between the groups was observed in PAP
complexes (Figure 6). The course in PAP complexes in the control
baboons was as expected in this sepsis model, while in the rhATIII
treatment group, the generation of PAP complexes was virtually
abrogated (P < .01). Plasma concentrations were
10.3 ± 3.5% at 2 hours in the control group, whereas the highest
values observed in the treatment group were 2.1 ± 0.4%.
| |
Discussion |
In the present study we show that rhATIII, just as
plasma-derived ATIII, affords significant protection against the lethal effects of E coli in the baboon model. Supraphysiological
levels of ATIII were achieved by preinfusion of rhATIII and maintained by repeated infusions. Maintenance of these levels was possibly necessary to afford protection against the lethal effects of E coli, as baboon No. 2, who did not receive the third rhATIII
infusion, had the shortest survival time, although survival time was
still longer than any in the control group. High doses of rhATIII
attenuated the coagulation response to E coli challenge and,
perhaps as importantly, attenuated the inflammatory response that
sepsis induces. Both activities may be necessary to protect against lethality.
The dose of E coli administered to the rhATIII
treatment group was slightly less than that administered to controls.
However, this dose was above the LD100 and therefore
expected to induce 100% lethality in baboons. The difference in
circulating E coli organisms after 2 hours postinduction can
reflect either differences in amounts inoculated or an effect of
rhATIII on E coli clearance. As was shown in a previous
study,3 plasma-derived ATIII also enhanced the clearance of
the E coli organisms from the circulation in this baboon model,
although this effect was less than that observed for rhATIII in the
present study. Enhanced clearance of E coli from the
circulation was also observed with a monoclonal antibody against factor
XII in this same model of E coli-induced lethality in
baboons.13
Unexpectedly, a rise in WBC count was observed following the
first infusion of rhATIII. This suggests that either an inflammatory reaction was induced by the rhATIII preparation or there was a demarginating effect on the marginal pool of neutrophils. A follow-up study in noninfected baboons, which evaluated the effect of rhATIII dose on WBC and neutrophil counts, indicated that the administration vehicle alone, containing sodium chloride, sodium citrate, and glycine,
pH 7.4, was all that was required to induce increases in these
parameters (data not shown). After the E coli infusion, the WBC
count decreased equally in both groups, and no significant differences
in clinical, biochemical, or hematological markers were observed. The
only measurable difference was in body temperature. Because animals
were kept warm with a heating pad during anesthesia, the biological
significance of this finding is not known.
Administration of rhATIII had a significant impact on the coagulatory
response to E coli challenge. TAT complexes formed more rapidly
in the treatment group and reached significantly higher concentrations
at earlier time points. The more rapid inhibition of thrombin at early
time points may have had an impact on fibrinogen consumption in the
rhATIII treatment group. In this group, fibrinogen consumption was
significantly attenuated relative to controls, and levels did not drop
below 40% of baseline. In controls, fibrinogen levels plummeted to
<10% starting values by the 6-hour time point. The protective effect
of rhATIII on fibrinogen consumption was similar to that noted for
plasma-derived ATIII and consistent with the ability of rhATIII to
prevent DIC.3
Treatment with rhATIII in lethal septic baboons had a clear inhibitory
effect on fibrinolysis. In particular, its effects on the course of tPA
was striking: After an immediate rise, as is normally observed in this
model, tPA levels in the treatment group decreased but
remained slightly elevated during the observation time. In accordance
with the reduced tPA levels, PAP complexes were not formed, while PAI-1
peak levels were mainly unaffected. The particular course of tPA
appearance in plasma has not been observed by other interventions, such
as administration of C1 inhibitor, in the same E
coli model.8 In general, TNF is thought to mediate the
fibrinolytic response after E coli challenge, although this
consideration is based on observations in low-grade endotoxemia models.5 Since TNF levels were higher in the rhATIII group than in the control group, this cannot explain the observed lack in tPA
response. The main source of tPA is probably the endothelium. Stimuli
for its release are venous occlusion as well as vasoactive substances
like bradykinin and vasopressin.16 However, these mechanisms for tPA release do not provide an explanation for the effect
of rhATIII observed in the septic baboons. As it appears that a small
fraction of ATIII is bound to glycosaminoglycans associated with the
endothelial surface,1 we postulate that ATIII bound to the
endothelial surface via heparin-like substances blocks tPA release.
In addition to attenuating the coagulation and
fibrinolytic response, rhATIII directly or indirectly influenced the
cytokine responses. The concentration of TNF was higher in the
treatment group, whereas the IL-10, IL-6, and IL-8 concentrations were
lower (Figures 4 and 5).
IL-10 is considered an anti-inflammatory cytokine with autoregulatory
effects on pro-inflammatory cytokines such as TNF, IL-6 and
IL-8.17 Thus, the enhanced release of TNF in the treatment group may have reflected the reduced release of IL-10 in this group. It
is tempting to speculate that the diminished IL-10 response in the
treatment group was due to the reduced number of circulating E
coli organisms at 2 hours, since in a previous study, the IL-10 response was lower in a sublethal dose compared to a lethal dose of
E coli in a baboon model.18 Treatment with rhATIII
markedly attenuated the release of the more distal cytokines IL-6 and
IL-8. Four hours after the E coli infusion
increased, IL-6 and IL-8 levels were abrogated, and concentrations
nearly returned to baseline values at 10 hours. This is the first study
reporting such anti-inflammatory effects of ATIII. The mechanism behind
this effect is poorly understood. An earlier study had shown that the
release of IL-6 in the baboon model is mediated by TNF.19
In the present study we observed increased levels of TNF without
increased IL-6. Thus, TNF did not appear to exert its expected
biological effect on IL-6 up-regulation.
The interaction of ATIII with endothelium may be the key to the ability
of ATIII to promote survival. In a sublethal porcine model of
endotoxin-induced DIC, administration of a purified ATIII heparin
complex prevented development of DIC but failed to significantly influence survival.20 In contrast, administrations of ATIII alone in several animal sepsis models did show a positive effect on
survival.3,21,22 Because rhATIII binds strongly to heparin, it is also likely to bind strongly to the endothelium via cell surface
heparin sulfate proteoglycans.6 Although the mechanism of
action has not been deciphered, it has been shown that ATIII promotes
endothelial release of prostacyclin, which may be the key to modulating
the inflammatory response.23
In conclusion, we propose that the early administration of high doses
of rhATIII, through the combined anticoagulation and anti-inflammatory
effects of this therapy, improves the outcome in baboons lethally
challenged with E coli. The direct binding of ATIII to
endothelium may be key to this dual activity.
| |
Acknowledgments |
The authors would like to thank G. Peer and D. Carey for their
excellent technical assistance with the E coli baboon model.
| |
Footnotes |
Submitted March 5, 1999; accepted September 9, 1999.
Reprints: M. C. Minnema, CLB, Sanquin, PO Box 9190, 1006 AD,
Amsterdam, The Netherlands; email: muntar{at}db.nl.
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.
| |
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Abstract
Introduction