© 1999 American Heart Association, Inc.
Thrombosis |
Influence of Antithrombin III on Coagulation and Inflammation in Porcine Septic Shock
From Centeon Pharma GmbH, 35002 Marburg (G.D.) and the Medical Clinic, Justus-Liebig-University, 35385 Gießen (B.L.), Germany.
Correspondence to PD Dr Gerhard Dickneite, Centeon Pharma GmbH, Preclinical Pharmacology, PO Box 1230, D-35002 Marburg/Germany, Emil von Behring-Straße 76, 35041 Marburg/Germany. E-mail Dicknei1{at}MSMBWMD.Hoechst.com
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Abstract—The physiological inhibitor of thrombin, antithrombin III (ATIII, Kybernin P) was investigated for its antiinflammatory and anticoagulant effects in a pig model of septic shock. Pigs were infused with a dose of 0.25 µg · kg-1 · h-1 of lipopolysaccharide (LPS) over a period of 3 hours. Animals developed systemic inflammation, disseminated intravascular coagulation (DIC), organ failure and cardiovascular abnormalities, namely pulmonary hypertension and systemic hypotension. Twenty septic pigs were allocated to 2 study groups, treated either with ATIII (n=10) or placebo (n=10). ATIII was administered as a 250-U/kg IV bolus infusion for 30 minutes (-60 to -30 minutes) followed by a single IV bolus of 125 U/kg (t=0) and a second 30-minute infusion of 250 U/kg (120 to 150 minutes). ATIII significantly prevented the development of a DIC; the increase in fibrin monomers (placebo, 11.4±9.1 reciprocal titers, at 6 hours) was completely overcome by ATIII (P<0.05). ATIII significantly prevented the increase in thromboxane (TXB2) levels, which were 809±287 pg/mL in the placebo and 420±174 pg/mL in the verum group after 6 hours (P<0.02). On the other hand, ATIII had no influence on TNF levels. In a lethal study with an increased dose of LPS (0.5 µg · kg-1 · h-1). A significant reduction in mortality was observed in the ATIII group (0 of 7) compared with the placebo group (4 of 6) (P<0.05,
2 test) a significant
reduction of pulmonary hypertension (placebo, 42.0±11.1
mm Hg; ATIII, 23.6±7.5 mm Hg, P<0.05), but no
effect on systemic hypotension, was noted in the ATIII group. It was
thus concluded that modulation of the procoagulatory state by
substitution of ATIII results in a late beneficial antiinflammatory
effect in this model of septic shock.
Key Words: lipopolysaccharide • disseminated intravascular coagulation • pulmonary hypertension • soluble fibrin monomers • thromboxane
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Introduction |
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Sepsis as a severe complication of infection is characterized by systemic inflammation, activation of proteolytic cascades, coagulation abnormalities (DIC) and a gradually developing hypodynamic status with impaired organ perfusion, and finally death in a septic shock. Lung circulation in septic shock is characterized by hypertension and increased pulmonary resistance, resulting in low cardiac output, and, frequently, in lung failure. Mortality in septic shock is usually high, ie, in the range of 40% to 50%.1 If associated with multi-organ dysfunction such as respiratory or renal failure, mortality might exceed 90%.2 The initiating event for the development of sepsis is the activation of macrophages by lipopolysaccharide (LPS) liberated from Gram-negative bacteria via binding to its surface receptor CD14.3 However, sepsis might be induced by other agents, such as Gram-positive bacteria, fungi or viruses. The initial excessive secretion of cytokine mediators such as interleukin 1 (IL-1) and tumor necrosis factor (TNF-
) is followed by the
activation of biological cascades including the coagulation, the
complement, the fibrinolytic and the kallikrein-kinin systems, which
contribute to the maintenance of the inflammatory
reaction. Arachidonic acid metabolites are thought to
play an important role in hemodynamic alterations, thus
thromboxane A2
(TXA2) appears to be associated with
pulmonary arterial hypertension while prostacyclin
(PGI2) might contribute to the lowering of lung
blood pressure. In particular the activation of the coagulation cascade
induces the uncontrolled generation of thrombin from its precursor
molecule prothrombin, and leads to DIC, which is associated with
consumption of coagulation factors and microthrombosis.4
Under the conditions of hemostatic balance the activity of thrombin is
controlled by its physiological
antagonist antithrombin III (ATIII). ATIII is a
single-chain glycoprotein with a Mr
of 58 000 Da,5 which is a progressive
inhibitor of serine proteases. The inhibitor
has a binding site for heparin; its activity is increased dramatically
by heparin through accelerating the binding rate to its target
protease.6 ATIII interacts with several proteases of the
plasma; besides thrombin, it inhibits kallikrein, factors IXa, Xa, XI,
and XIIa.7 8 9 10 ATIII has been shown to be efficacious in several experimental models of sepsis and septic shock, regardless of the species investigated, as shown in baboons,11 dogs,12 13 14 sheep,15 rabbits16 17 rats,18 19 and chicken embryos.20 ATIII in these models proved to be effective after inducing a sepsis or septic shock with different agents including live bacteria (Escherichia coli, Klebsiella pneumoniae), bacterial lipopolysaccharide and lactic acid. In a guinea pig model Kessler et al demonstrated that ATIII could prevent DIC and organ hemorrhage and improve mortality after infection with the Gram-positive bacterium Staphylococcus aureus.21 Clinical efficacy in patients with sepsis or septic shock has been demonstrated by several authors (for a review see Reference 2222 ), the rationale for the therapy being the prevention of DIC.23 A recently published paper on the meta-analysis of ATIII in severe sepsis demonstrated a clear trend towards increased survival.24 However, some authors suggest that ATIII might have antiinflammatory as well as anti-DIC activity.25
In the present study we induced a sepsis by the infusion of Salmonella abortus equi lipopolysaccharide into pigs; the development of systemic inflammation, coagulation activation and hemodynamic changes was followed over time. The influence of ATIII administration on the outcome of the sepsis was investigated.
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Materials and Methods |
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Substances
Salmonella abortus equi lipopolysaccharide (S equi LPS) and Indomethacin were purchased from Sigma (Deisenhofen, Germany). Antithrombin III (Kybernin P) and human serum albumin (Human Albumin 25% Behring) were provided by Centeon Pharma GmbH. Pentobarbital was delivered by Sanofi-Ceva, Ketamine-HCI was from Parke-Davis and Xylazin-HCI from Bayer. Ringer-lactate solution (DAB7) was purchased from Braun-Melsungen.
Endotoxic Pig Model and Treatment Protocol
Male juvenile castrated pigs (German domestic pig, 19 to 32 kg,
3 to 4 months) were purchased from a local supplier. Animals were
housed in conventional stables at an ambient temperature of 18°C to
21°C with straw bedding. They were fed a Deuka V pig chow (Deuka) and
tap water ad libitum.
Animals were subjected to a veterinarian health inspection before use. Animals showing unusual coagulation, hyper- or hypotension, elevated temperature, leukopenia or leucocytosis were withdrawn from the study.
For the maintenance anesthesia, pentobarbital was given as a 7.5-mg/kg IV bolus followed by a 10-mg · kg-1 · h-1 intravenous infusion, performed with a Havard infusion pump 22 (FMI). Pigs were ventilated mechanically via a tracheal tube by a RUS-1302 respirator with a mixture of 600 L/h room air and 150 L/h O2. Respiration rate was 16 breaths/min with 40% inspiration. CO2 in the expiration air was monitored by a Normocap 200 CD2 to 02 (Hoyer).
Test substances were infused via an abdominal vein by a polyethylene catheter (1.4x2.1 mm, Braun–Melsungen, Melsungen), and samples drawn from the vena jugularis externa. A volume substitution of 150 mL/h was performed with Ringer-lactate solution. A polyethylene catheter (0.5x0.9 mm, Braun–Melsungen, Melsungen) was placed into the left femoral artery and connected to a pressure transducer to monitor the systemic arterial pressure throughout the experiment. Mean arterial pressure (MAP) was calculated from the systolic (SAP) and diastolic (DAP) arterial blood pressure according to the following formula: MAP=(2xDAP+SAP)/3. An ECG was recorded permanently.
A 4-lumen Swan-Ganz catheter (5 F thermodilution catheter, Biosensors International) was inserted into a pulmonary artery to measure the pulmonary arterial pressure and, after inflating the ballon, the pulmonary capillary wedge pressure (PCWP).
Two pig studies, with a lethal and a sublethal outcome, were performed.
A sublethal sepsis was induced in a total of 20 pigs by a 3-hour
infusion of 0.25 µg · kg-1 ·
h-1 LPS. The animals were allocated to 2 groups;
the treatment group (n=10) received ATIII according to the following
regimen: 250 U/kg (t= -60 to -30 minutes, IV infusion), 125 U/kg (IV
bolus, t=0) and 250 U/kg (t=120 to 150 minutes, IV infusion). The
placebo group (n=10) received the appropriate amount of protein (given,
that 1 U of ATIII is 
0.2 mg of protein); 50-25-50 mg/kg human serum
albumin (HSA), respectively, was administered according
to the same schedule as described for ATIII. For the lethal endotoxic
pig study, 13 pigs instrumented with a Swan-Ganz catheter into the lung
and a femoral artery catheter were infused with S equi LPS
over 3 hours with a dose of 0.5 µg ·
kg-1 · h-1 LPS.
The animals were treated with ATIII (n=7) or with HSA (n=6) according
to the same schedule as outlined for the sublethal study. Animals
surviving the 6-hour observation period were killed by an overdose of
pentobarbital.
In addition, 1 pig, which was treated neither with LPS nor one of the study medications, served as a baseline control.
Separation of Plasma
All assays besides TXB2,
PGI2 and endotoxin were performed with
citrate-plasma. Blood was taken from the jugular vein and mixed with a
buffered citrate solution (20% vol/vol). Cells were separated from
plasma by centrifugation at 3000g for 15
minutes. For TXB2 and PGI2
detection, indomethacin plasma (10% vol/vol of 2
mmol/L indomethacin/3.8% sodium dihydrogen citrate)
was used. Endotoxin determination required heparin plasma, withdrawn
into pyrogen-free tubes (5-mL Vacutainer, containing 143 USP units of
sodium heparin per tube; Becton Dickinson).
Antithrombin III (ATIII) Plasma Levels
ATIII activity was determined with the Berichrom Antithrombin
III test kit (Behringwerke AG). The assay is based on the inhibition of
thrombin by its natural plasma-derived inhibitor, ATIII,
which detects both porcine and human ATIII. Values are given in % of
standard human plasma (=100% or 1 U/mL).
Thrombin/Antithrombin Complex (TAT)
TAT was determined with the Enzygnost TAT micro ELISA test kit
(Behringwerke AG). The 2 antibodies used were a solid-phase antibody
against human thrombin and a POD-conjugated antibody against ATIII.
This ELISA test, which cross-reacts with pig TAT, has already been
shown to cross-react with rat TAT.26
C1 Esterase Inhibitor (C1-INH) Plasma Levels
C1-INH activity was measured with the C1 Inhibitor
Berichrom test kit (Behringwerke AG).
Platelets, Leukocytes and Erythrocytes
The platelet, leukocyte and erythrocyte counts in whole
blood were determined in a sysmex hematological analyzer
(Digitana).
Fibrinogen
Fibrinogen was determined with the ChromoTimeSystem
(CTS-fibrinogen from Behringwerke AG) in an Uvikon 930 photometer at
405 nm.
Fibrin Monomer Complex (FM)
The soluble fibrin monomer complexes in plasma were determined
with an agglutination test kit (FM-Test No. 565440,
Boehringer). Human erythrocytes (group 0, Rh-negative) were
coated with fibrin monomers from human fibrinogen. In the presence of
fibrin monomers in the plasma under investigation, agglutination will
occur. Plasma was diluted serially (from 1:2 to 1:n) and the highest
dilution giving a positive reaction was defined as the titer. The lower
detection limit of the test was 15 to 20 µg/mL. Values were given as
reciprocal titers.
Coagulation Assays
Activated partial thromboplastin time (aPTT) and
prothrombin time (PT) were determined with Neothromtin or with
Thromborel S (Behringwerke AG) in a Schnitger &
Gross coagulometer.
Coagulation Factor VIII (F VIII)
The principle of the detection of pig factor VIII was to
substitute factor VIII-deficient human plasma (Behringwerke AG) with
the pig samples. The prolonged aPTT in the factor VIII-deficient human
plasma was shortened by admixing defined amounts of standard human
plasma (SHP) to obtain a calibration curve. Pig plasma factor VIII was
expressed as percentage of SHP (defined as 100%).
Tumor Necrosis Factor (TNF)
TNF was determined with the pig TNF- ELISA test kit (Biozol).
The 2 antibodies used were a solid-phase polyclonal antibody against
pig TNF- and a POD-conjugated monoclonal antibody against pig
TNF-.
von Willebrand Factor (vWF)
With an Asserachrom vWF ELISA test kit the vWF concentration in
plasma was determined (Boehringer). The 2 antibodies used were
a solid-phase F(ab)2 anti-human vWF antibody and
an anti-vWF-peroxidase–conjugated antibody.
Detection of Endotoxin Plasma Levels
Endotoxin was measured with the LAL chromogenic QCL
1000 test kit (Bioproducts).
Plasma Levels of Prostacyclin (PGI2)
Prostacyclin was detected in plasma as its stable metabolite,
6-keto PGF1. To avoid secretion of
PGI2 from platelets during blood sampling,
indomethacin plasma was used. 6-keto
PGF1 was tested by a competitive ELISA
(Amersham).
Thromboxane Plasma Levels
As TXA2 is unstable in plasma the stable
metabolite TXB2 was measured with a competition
ELISA (Amersham Life Science). Free TXB2 competes
with POD-labeled TXB2 for the binding to the
solid-phase anti-TXB2.
Clinical Chemistry
Creatinine, urea and GOT (glutamate oxaloacetate
transaminase) were measured in plasma by test strips (Reflotron,
Boehringer).
Statistics
Differences between mortality rates were determined by the
2 test; differences between other
parameters were detected with the Student's
t test.
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Results |
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After infusion of 0.25 µg · kg-1 · h-1 S equi LPS, pigs developed increasing endoxin plasma leveling off at 3 hours, the termination point of infusion (Figure 1
). Endotoxin levels decreased thereafter
to reach baseline levels 1 hour later. The inflammatory reaction was
demonstrated by a rapid increase in TNF- plasma levels, reaching a
plateau between 1 and 2 hours to decrease again after 2 hours, ie,
before the termination of the LPS infusion (Figure 2). The
Table depicts the changes of
parameters during sepsis in pigs (n=10, treated with HSA)
at 6 hours after the start of the LPS infusion. As DIC developed, the
levels of ATIII decreased to 70% of the baseline value; concomitantly
the TAT levels increased about 10-fold. Uncontrolled thrombin
activation led to an increase in soluble fibrin monomers (sFM);
fibrinogen levels decreased slightly. Consumption of coagulation
factors like F VIII resulted in a prolonged aPTT
(nonsignificant) and PT. A decrease of platelets might indicate the
formation of disseminated microthrombi. An increase in circulating vWF
was demonstrated, which was thought to be secreted from the
Weibel-Palade bodies of the disturbed endothelium.
Marked leukocytopenia with a nadir at 2 hours was explained by the
sticking of leukocytes to the activated
endothelium. Cardiovascular changes
were visualized by systemic hypotension and pulmonary
hypertension as well as a slight increase in the pulmonary
wedge pressure. Increased plasma levels of GOT, urea and
creatinine were indicative of hepatic and renal
failure.
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-). Blood was withdrawn at the indicated time points,
heparin-plasma was prepared and tested for endotoxin levels with a LAL
assay. Values are given in European units (EU)/mL as means and standard
deviations. A control animal receiving no LPS (n=1) generated only
baseline values (<0.0125 EU/mL -
-).
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To evaluate the mechanism by which ATIII interferes with sepsis and
septic shock we allocated 20 septic pigs to treatment with ATIII (n=10)
or HSA (n=10). ATIII plasma levels (activity) were up to about 500% to
600% of baseline level at the time when we started the administration
of LPS (Figure 3). After the end of the
third ATIII infusion plasma levels decreased again and the terminal
half-life was calculated at 16 hours. We evaluated the influence of
ATIII on the development of inflammatory cytokine levels. As
shown in Figure 2
, the placebo-treated group and the ATIII group
depicted essentially the same plasma levels of TNF-; in both groups
maximal plasma levels were 700 pg/mL. TXB2
plasma levels increased rapidly in the placebo as well as in the ATIII
group to reach a maximal value of 1100 pg/mL after 30 minutes
(Figure 4). TXB2
levels in the placebo group stayed elevated until termination of the
study, whereas in the ATIII group a significant decrease was obtained
towards the end of the experiment. TXB2 levels at
6 hours were 809±287 pg/mL and 420±174 pg/mL in the placebo and ATIII
groups, respectively (P<0.02, t test).
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We investigated the course of DIC by measuring the formation of soluble
fibrin monomers (sFM). Figure 5
demonstrates a steady increase of this marker fibrinogen
activator in the placebo group. ATIII was able to
totally prevent the increase in sFM (P<0.05, t
test). Marked thrombocytopenia to about 70% of baseline level
developed in the untreated animals (Figure 6). Although ATIII could not prevent
platelet drop, there was a small but significant increase in
platelet numbers when compared with control (P<0.05,
t test). An increased inactivation of thrombin was
demonstrated by the higher formation of TAT complexes in the ATIII
group. Whereas in the placebo control TAT levels were 176.7±96.7
µg/L at the end of the experiment, the ATIII group TAT levels were
526±448.8 µg/L. Both the increase in aPTT and PT were less
pronounced in the ATIII group, although the differences were not
statistically significant (data not shown). None of the 20 animals in
this study died during the observation period.
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In a separate lethality study, with the LPS dose increased to 0.5
µg · kg-1 ·
h-1, we included a total of 13 pigs. Seven
animals were given the same ATIII regimen as in the sublethal study;
HSA was administered to 6 septic pigs. Sixty-six percent (4 of 6) of
the placebo animals were dead at 6 hours post-LPS, whereas none (0 of
7) of the pigs of the ATIII group died during the observation period of
6 hours (P<0.01, 2 test).
Pulmonary artery pressure in the lethal study showed the
typical, 2-peaked increase after the induction of sepsis (Figure 7). In particular, the second increase in
pressure, which had a peak value of 42.0±11.1 mm Hg in the
placebo group at 3 hours, was prevented by ATIII treatment
(23.6±7.5 mm Hg, P<0.05). In contrast, in the
sublethal study the increase of the PAP was less pronounced and was not
different in the ATIII and placebo groups (23.5±6.1 versus 22.3±5.0
at 180 minutes post-LPS).
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No influence of ATIII on systemic arterial blood was detected in the lethal or in the sublethal study (data not shown).
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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The aim of the present study was to provide data on the mechanism of action of ATIII on sepsis and septic shock. We selected the prophylactic ATIII regimen that had been introduced by Taylor et al for their studies in baboons, and which included 3 administrations of ATIII.11 For our studies we used the pig model because the hemodynamic situation in this species resembles the human one. The major data presented here were obtained from a sublethal pig sepsis study, and a smaller part concerning ATIII's effect on mortality rate and pulmonary artery pressure came from a lethal study. In the sublethal study we investigated ATIII's anticoagulant and antiinflammatory mechanism. The scope of these investigations was to provide data for ATIII's mechanism of action. In the lethal study we demonstrated prevention of sepsis-related mortality in animals infused with ATIII in our endotoxic pig model. Whereas mortality was high in the placebo group (66%) and thus in a range observed in patients suffering from severe septic shock, all ATIII-treated animals survived. The results in our pig model are thus in line with the observations of other authors obtained in animal models or in clinical studies.27
However, the precise mechanism by which ATIII exerts its protective
effect in sepsis and septic shock is still under discussion. ATIII is
the principal inhibitor of thrombin and several other
proteases of the coagulation system, thus being responsible for the
hemostatic balance. As sepsis and septic shock are frequently
associated with DIC, resulting in decreased ATIII
levels,28 29 there is a clear rationale for a substitution
therapy with ATIII in the treatment of sepsis and septic shock. The
bulk of evidence in preclinical and clinical studies suggest that ATIII
can modulate excess activation of the coagulation system and prevent
the consumption of clotting factors and the decrease in
platelets,11 14 12 21 30 although a limited impact on
DIC has occasionally been reported.16 During sepsis the
extrinsic pathway of coagulation is activated by the
expression of tissue factor after stimulation of the
endothelium with inflammatory cytokines such as
TNF- or IL-1ß. Our data indicate that ATIII does not interfere
with this early step in the inflammatory response. TNF levels were
essentially the same in the ATIII treatment and placebo groups.
The data demonstrated in our pig model show a modulation of DIC by ATIII as it prevents the increase in fibrin monomers and, in part, the decrease in platelet counts. Moreover, increased TAT levels in the ATIII group indicate that newly generated thrombin was effectively bound to and inhibited by ATIII. As thrombin is inhibited predominantly by ATIII, one can conclude that less free thrombin is present in the ATIII group resulting in reduced levels of fibrin monomers, the primary product of thrombin's action on fibrinogen. It can be concluded that prevention of DIC by ATIII is an important, but not the only contribution of ATIII in overcoming septic shock. Our own previous data in a rat-Klebsiella sepsis model demonstrate only a limited effect of ATIII on DIC parameters18 but a significant prevention of sepsis-related death. On the other hand, the highly potent inhibitor of thrombin, recombinant hirudin, could very efficiently suppress DIC in the same sepsis model. However, no further improvement in mortality was observed with this treatment.31 Thus, the search for an alternative mechanism of action for ATIII appears to be a challenge. Our data are in line with those of Spannagl et al,32 who used a complex of ATIII and heparin in an LPS-pig sepsis model. The authors clearly showed a prevention of FM increase by ATIII/heparin. As heparin accelerates the binding of ATIII to thrombin, this was clearly attributed to ATIII's anti-DIC effect. However, the ATIII/heparin complex failed to show a significant effect on survival. The failure of ATIII/heparin to be protective in that model might be explained by a series of experiments performed by Okajima and coworkers.25 33 This group suggested that heparin impairs the positive effect of ATIII by competing for the glycosaminoglycan binding sites at the endothelium. Direct binding of ATIII to endothelial cells has been demonstrated by Stern et al34 in bovine aortic segments. It has been shown by other authors that ATIII, in vitro35 36 or in vivo,37 stimulates the production of PGI2 from endothelial cells, which might be explained by its binding to the glycosaminoglycan structure. As PGI2 inhibits platelet aggregation and promotes vasodilation in lung arteries, this might explain ATIII's beneficial effects on sepsis. Additional evidence that ATIII binds to endothelial glycosaminoglycans came from studies other than sepsis studies. Pseudorabies viruses bind to endothelial cells via their heparin sulfate receptors. Voigt et al38 demonstrated that ATIII inhibits the binding of the virus to the endothelium. In a recent paper Ostrovsky et al39 describe that the leukocyte-endothelium interaction was inhibited by ATIII in a feline mesenterial ischemia/reperfusion injury.
Our data show that pulmonary hypertension in the pig sepsis model is decreased by ATIII, thus leading to improved lung function. On the other hand, ATIII has no effect on systemic blood pressure. Prevention of pulmonary hypertension by ATIII might be explained by the decrease in plasma TXB2. As thromboxane is secreted by platelets it might be speculated that the decrease in TXB2 is related to the inhibition of thrombin's action on platelets by ATIII.
It must be taken into consideration, however, that this study was a prophylactic one and that these data must be confirmed by a therapeutic ATIII regimen. A series of experiments has begun in pigs with a therapeutic ATIII treatment and a dose regimen adopted from the ongoing phase III sepsis study in humans.
However, an alternative explanation for the prevention of pulmonary hypertension by ATIII has to be considered. Seeger et al40 have shown that fibrin monomers generated by excess thrombin activity might induce vasoconstrictor response in lungs via thromboxane. As ATIII effectively prevented fibrin monomer formation, improvement of lung function might be mediated via this mechanism.
ATIII does not inhibit the early inflammatory event in sepsis, the
production of TNF-, but it reduces the plasma levels of
intermediate or end products of the inflammatory mediator systems.
Thus, it might be concluded that ATIII produces a late antiinflammatory
effect through modulation of the procoagulatory reactions during the
progression of septic shock.
The development of a septic shock is paralleled by the decrease in
ATIII levels, and the mortality rate was shown to be high in patients
with low ATIII levels.41 When ATIII was introduced for the
treatment of sepsis several years ago it was suggested for treatment of
patients with 20 to 40 U/kg ATIII, a dose leading to ATIII levels of
maximally 100%. In recent years evidence accumulated that this dose
might not be sufficient for the treatment of sepsis. In a clinical
study in patients suffering from peritonitis, Jochum et
al42 were able to demonstrate a clinical benefit when
ATIII plasma levels were adjusted to values of 120% to 140%.
Obertacke et al43 reported a shorter stay in the ICU in
polytraumatized patients with ATIII plasma levels brought to
140%. In a placebo-controlled double-blind study conducted by
Fourrier et al,30 ATIII levels were kept at 200% for 
3
days in septic patients. The authors reported a reduction in mortality
of 30%; however, due to the low number of patients, the
difference was not significant. Our own data support the assumption
that substantially higher levels than 100% ATIII are needed. In
accordance with other animal studies ATIII plasma levels should reach a
range of 400% to 500%.11 18 19 The question as to
whether this high concentration really is necessary for a clinical
study in humans cannot be answered conclusively, because one has to
bear in mind that in all animal experimentation a human protease
inhibitor (ATIII) has to interact with an animal protease
(thrombin). It might thus be concluded that in humans lower levels of
ATIII (200%) could be sufficient, as suggested in a recent ATIII
pharmacokinetic study in septic patients.44 Future
clinical trials with supranomal ATIII plasma levels are thus mandatory
to clarify this question.
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Acknowledgments |
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The authors wish to acknowledge the skillful technical assistance of Bärbel Dörr, Franz Kaspereit, and Wilfried Krege, as well as the excellent secretarial work of Petra Zimmermann.
Received June 23, 1998; accepted December 1, 1998.
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References |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
1. Parillo JE, Parker MM, Natanson C, Suffredini AF, Danner RL, Cunnion RE, Ognibene FP. Septic shock in humans. Advances in the understanding of pathogenesis, cardiovascular dysfunction, and therapy. Ann Intern Med. 1990;113:227–242.
2. Centers for Disease Control. Chronic disease reports: Deaths from nine chronic diseases - United States, 1986. Morb Mort Weekly Rep. 1990;39:17–36.
3. Ziegler-Heitbrock HWL, Ulevitch RJ. CD14: Cell surface receptor and differentiation marker. Immunol Today. 1993;14:121–125.[Medline] [Order article via Infotrieve]
4. Bick RL. Disseminated intravascular coagulation: Objective clinical and laboratory diagnosis, treatment, and assessment of therapeutic response. Semin Thromb Hemost. 1996;22:69–88.[Medline] [Order article via Infotrieve]
5. Olds RJ, Lane DA, Mille B, Chowdhury V, Thein SL. Antithrombin: The principle inhibitor of thrombin. Semin Thromb Hemost. 1994;20:353–372.[Medline] [Order article via Infotrieve]
6. Olson ST, Björk I. Regulation of thrombin activity by antithrombin and heparin. Semin Thromb Hemost. 1994;20:373–409.[Medline] [Order article via Infotrieve]
7. Soons H, Tans G, Hemker HC. The heparin-catalysed inhibition of human factor XIa by antithrombin III is dependent on the heparin type. Biochem J. 1998;256:815–820.
8.
Pieters J, Willems G, Henker HC, Lindhout T.
Inhibition of factor IXa by antithrombin
III/heparin during factor X activation. J Biol Chem. 1988;263:15313–15318.
9.
Stead N, Kaplan AP, Rosenberg RD. Inhibition of
activated factor XII by antithrombin-heparin cofactor.
J Biol Chem. 1976;251:6481–6488.
10. Venneröd AM, Laake K, Solberg AK, Strömland S. Inactivation and binding of human plasma kallikrein by antithrombin III and heparin. Thromb Res. 1976;9:457–466.[Medline] [Order article via Infotrieve]
11. Taylor FB Jr, Emerson TE Jr, Jordan R, Chang AK, Blick KE. Antithrombin-III prevents the lethal effects of escherichia coli infusion in baboons. Circ Shock. 1988;26:227–235.[Medline] [Order article via Infotrieve]
12. Hauptman JG, Hassouna HI, Bell TG, Penner JA, Emerson TE. Efficacy of antithrombin III in endotoxin-induced disseminated intravascular coagulation. Circ Shock. 1988;25:111–122.[Medline] [Order article via Infotrieve]
13. Phillips TF, Mammen EF, Selik NR, Starico A, Assarian GS, Kihm JT, Murano G. Experimentelle Verbrauchkoagulopathie - Behandlung mit Antithrombin III. Med Welt. 1984;35:1022–1026.
14. Mammen EF, Miyakawa T, Phillips TF, Assarian GS, Brown JM, Murano G. Human antithrombin concentrates and experimental disseminated intravascular coagulation. Semin Thromb Hemost. 1985;11:373–383.[Medline] [Order article via Infotrieve]
15. Redens TB, Leach WJ, Bogdanoff DA, Emerson TE. Synergistic protection from lung damage by combining antithrombin-III and alpha1-proteinase inhibitor in the E. coli endotoxemic sheep pulmonary dysfunction model. Circ Shock. 1988;26:15–26.[Medline] [Order article via Infotrieve]
16. Triantaphyllopoulos DC. Effects of human antithrombin III on mortality and blood coagulation induced in rabbits by endotoxin. Thromb Haemost. 1984;51:232–235.[Medline] [Order article via Infotrieve]
17. Gómez C, Páramo JA, Colucci M, Rocha E. Effect of heparin and/or antithrombin III on the generation of endotoxin-induced plasminogen activator inhibitor. Thromb Haemost.. 1989;62:694–698.[Medline] [Order article via Infotrieve]
18. Dickneite G, Pâques EP. Reduction of mortality with antithrombin III in septicemic rats: A study of Klebsiella pneumoniae-induced sepsis. Thromb Haemost. 1993;69:98–102.[Medline] [Order article via Infotrieve]
19. Emerson TE, Fournel MA Jr, Leach WJ, Redens TB. Protection against disseminated intravascular coagulation and death by antithrombin-III in the escherichia coli endotoxemic rat. Circ Shock. 1987;21:1–13.[Medline] [Order article via Infotrieve]
20.
Mann LT Jr, Jensenius JCHR, Simonsen M. Antithrombin
III: Protection against death after injection of thromboplastin.
Science. 1969;166:517–518.
21.
Kessler CM, Tang ZC, Jacobs HM, Szymannski LM. The
suprapharmacologic dosing of antithrombin concentrate for
Staphylococcus aureus-induced disseminated intravascular coagulation in
guinea pigs: Substantial reduction in mortality and morbidity.
Blood. 1997;89:4393–4401.
22. Menache D, Grossman BJ, Jackson CM. Antithrombin III: physiology, deficiency, and replacement therapy. Transfusion. 1992;32:580–588.[Medline] [Order article via Infotrieve]
23.
Vinazzer HA. Antithrombin III in shock and disseminated
intravascular coagulation. Clin Appl Thromb Hemostas. 1995;1:62–65.
24. Eisele B, Lamy M, Thijs LG, Keinecke HP, Schuster F, Matthias R, Fourrier F, Heinrichs H, Delvos U. Antithrombin III in patients with severe sepsis. Intensive Care Med. 1998;24:663–672.[Medline] [Order article via Infotrieve]
25.
Uchiba M, Okajima K, Murakami K, Okabe H, Takatsuki K.
Effects of antithrombin III (ATIII) and TRP 49-modified ATIII on plasma
level of 6-keto-PGF 1 in rats. Thromb Res. 1995;80:201–208.[Medline]
[Order article via Infotrieve]
26. Dickneite G, Czech J, Keuper H. Formation of fibrin monomers in experimental disseminated intravascular coagulation and its inhibition by recombinant hirudin. Circ Shock. 1994;42:183–189.[Medline] [Order article via Infotrieve]
27. Dickneite G. Antithrombin III in animal models of sepsis and organ failure. Semin Thromb Hemost. 1998;24:61–69.[Medline] [Order article via Infotrieve]
28. Matthias FR. Hemostasis inbalance with sepsis. Pathogenetic value and therapeutic implications. Intensivmed. 1996;33:1–4.
29. Leithäuser B, Matthias FR. Microcirculation and hemostasis in inflammatory processes. Med Klin. 1997;92:426–431.[Medline] [Order article via Infotrieve]
30.
Fourrier F, Chopin C, Huart JJ, Runge I, Caron C,
Goudemand J. Double-blind, placebo-controlled trial of antithrombin III
concentrates in septic shock with disseminated intravascular
coagulation. Chest. 1993;104:882–888.
31. Dickneite G, Czech J. Combination of antibiotic treatment with the thrombin inhibitor recombinant hirudin for the therapy of experimental Klebsiella pneumoniae sepsis. Thromb Haemost. 1994;71:768–772.[Medline] [Order article via Infotrieve]
32. Spannagl M, Hoffmann H, Siebeck M, Weipert J, Schwarz HP, Schramm W. A purified antithrombin III - heparin complex as a potent inhibitor of thrombin in porcine endotoxin shock. Thromb Res. 1991;61:1–10.[Medline] [Order article via Infotrieve]
33. Okajima K. New therapeutic implications of antithrombin III replacement in disseminated intravascular coagulation and multiple organ failure. Intensivmed. 1996;33:5–11.
34. Stern D, Nawroth P, Marcum J, Handley D, Kisiel W, Rosenberg R, Stern K. Interaction of antithrombin III with bovine aortic segments. J Clin Invest. 1985;75:272–279.
35. Yamauchi T, Umeda F, Inoguchi T, Nawata H. Antithrombin III stimulates prostacyclin production by cultured aortic endothelial cells. Biochem Biophys Res Commun. 1989;163:1403–1411.
36. Horie S, Ishii H, Kazama M. Heparin-like glycosaminoglycan is a receptor for antithrombin III- dependent but not for thrombin-dependent prostacyclin production in human endothelial cells. Thromb Res. 1990;59:895–904.[Medline] [Order article via Infotrieve]
37.
Uchiba M, Okajima K, Murakami K, Okabe H, Takatsuki K.
Attenuation of endotoxin-induced pulmonary vascular injury by
antithrombin III. Am J Physiol. 1996;270:L921–L930.
38. Voigt A, Sawitzky D, Zeichhard H, Habermehl KO. Cellular receptor structures for pseudorabies virus are blocked by antithrombin III. Med Microbiol Immunol (Berl). 1995;184:97–103.
39. Ostrovsky L, Woodman RC, Payne D, Teoh D, Kubes P. Antithrombin III prevents and rapidly reverses leukocyte recruitment in ischemia/reperfusion. Circulation. 1997; 96:2302–2310.
40.
Seeger W, Neuhof H, Hall J, Roka L. Pulmonary
vasoconstrictor response to soluble fibrin in isolated lungs: Possible
role of thromboxane generation. Circ Res. 1988;62:651–659.
41.
Fourrier F, Chopin C, Goudemand J. Septic shock,
multiple organ failure, and disseminated intravascular coagulation.
Compared patterns of antithrombin III, protein C and protein S
deficiencies. Chest. 1992;101:816–823.
42. Jochum M. Influence of high-dose antithrombin concentrate therapy on the release of cellular proteinases, cytokines, and soluble adhesion molecules in acute inflammation. Semin Hematol. 1995;32:14–32.
43. Obertacke U, Kleinschmidt C, Jochum M, Bardenheuer M, Dresing K, Eisold C, Neudeck F. Reduction of shock-related complications and pulmonary microvascular permeability by continuous application of antithrombin III (140% serum activity) for 4 days after trauma - preliminary results of a prospective clinical study. Circ Shock. 1993;(suppl 1):41. Abstract.
44. Thijs LG, Eisele B, Keinecke HO, Knaub S, Heinrichs H. Antithrombin III in patients with severe sepsis - a dosage regimen finding and pharmacokinetic study. Intensive Care Med. 1997;23:58. Abstract.[Medline] [Order article via Infotrieve]
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