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ABSTRACT |
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ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
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Sepsis-induced tissue factor (TF) expression activates coagulation in the lung and leads to a procoagulant environment, which results in fibrin deposition and potentiates inflammation. We hypothesized that preventing initiation of coagulation at TF-Factor VIIa (FVIIa) complex would block fibrin deposition and control inflammation in sepsis, thereby limiting acute lung injury (ALI) and other organ damage in baboons. A model of ALI was used in which adult baboons were primed with killed Escherichia coli (1 × 109 CFU/kg), and bacteremic sepsis was induced 12 h later by infusion of live E. coli at 1 × 1010 CFU/kg. Animals in the treatment group were given a competitive inhibitor of TF, site-inactivated FVIIa (FVIIai), intravenously at the time of the infusion of live bacteria and monitored physiologically for another 36 h. FVIIai dramatically protected gas exchange and lung compliance, prevented lung edema and pulmonary hypertension, and preserved renal function relative to vehicle (all p < 0.05). Treatment attenuated sepsis-induced fibrinogen depletion (p < 0.01) and decreased systemic proinflammatory cytokine responses, for example, interleukin 6 (p < 0.01). The protective effects of TF blockade in sepsis-induced ALI were confirmed by using tissue factor pathway inhibitor. The results show that TF-FVIIa complex contributes to organ injury in septic primates in part through selective stimulation of proinflammatory cytokine release and fibrin deposition.
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INTRODUCTION |
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ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
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Keywords: adult respiratory distress syndrome; multiple organ failure; Papio; septicemia; thromboplastin
Patients with gram-negative sepsis have a high incidence of acute respiratory distress syndrome (ARDS) and multiple organ failure (MOF). The lungs of these patients characteristically show fibrin deposition in alveolar and interstitial compartments (1, 2), although evidence that this procoagulant state contributes to the pathogenesis of ARDS in sepsis is circumstantial. Strategies designed to treat sepsis by preventing disseminated intravascular coagulation (DIC) decrease mortality in humans (3) and nonhuman primates with shock (4), but these studies have been limited by significant residual mortality, lack of organ-specific analyses, and failure of the animal models to reproduce an acute lung injury (ALI) that resembles ARDS (7). Because ARDS causes significant morbidity and mortality in septic patients, we used a nonhuman primate model of ARDS and MOF (7) to investigate the contribution of tissue factor (TF)-initiated coagulation and fibrin deposition to lung and systemic organ damage in sepsis.
Coagulation is activated in sepsis in humans in parallel with inflammation and is also a feature of the inflammatory response to sepsis in other primates. It is activated by systemic and local injury to the pulmonary and other systems and is part of the innate response to bacterial infection in soft tissues. It has not been known, however, whether coagulation proteins have a critical role in regulating tissue inflammation in vivo. Extrinsic coagulation is rapidly activated when bacteria enter the circulation, and a procoagulant environment develops in the vascular space. This state is dependent on TF and is associated with increases in inflammatory cytokines that mediate the effects of endotoxin (11). Similar procoagulant environments are found in the lungs of animals after endotoxin infusion or during experimental ALI (17, 18) and in bronchoalveolar lavage (BAL) fluid of patients with ARDS (19). As in the systemic circulation, procoagulant activity in the lung appears to be related to TF expression, suggesting that extravascular inflammation also activates the extrinsic pathway (19). Despite the association between procoagulant activity and lung injury, specific etiologic roles for TF and other coagulation factors have not been defined in the injury responses of the lung. Like TF, activated Factors VII (FVIIa) and X (FXa), thrombin, and fibrin have specific effects on cell signaling that could alter vascular permeability, inflammatory cell migration, and surfactant dysfunction in the lung (20). The exact contribution of this complex cross-talk between coagulation and inflammation in the responses to sepsis is not known.
We hypothesized that blocking initiation of coagulation during gram-negative sepsis would prevent ALI and other organ damage by attenuating the coagulation-related inflammatory response. The hypothesis was tested in an established baboon model of Escherichia coli sepsis, where hyperdynamic cardiovascular and systemic inflammatory responses are preactivated by a priming infusion of killed bacteria. After a second, lethal dose of bacteria, the animals develop pulmonary and renal failure similar to humans with ARDS (7). We blocked initiation of coagulation at the TF-FVIIa complex after administration of the priming dose of bacteria by using a site-inactivated FVIIa (FVIIai), which competitively inhibits FVIIa because of its 5-fold higher affinity for TF relative to native FVIIa (24). The following study shows that coagulation blockade by FVIIai decreases systemic inflammation and fibrinogen depletion and prevents injury to the lung and kidneys after E. coli sepsis.
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METHODS |
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ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES
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Animal Preparation
Adult male baboons (Papio cyanocephalus) weighing 14 to 20 kg were quarantined for a minimum of 4 wk, and determined to be tuberculosis free before use (7). Animals were handled in accordance with American Association for Accreditation of Laboratory Animal Care (AAALAC) guidelines, and the experimental protocol was approved by the Duke University (Durham, NC) Institutional Animal Care and Use Committee. They were divided randomly into treatment and sepsis control groups (n = 6 each). Treated animals received active site-inactivated FVIIa (FVIIai; Novo Nordisk, Copenhagen, Denmark), 1 mg/kg intravenously at time = 12 h, immediately before the infusion of live bacteria, followed by 50 µg/kg per h, intravenously. Untreated animals received an intravenous infusion of vehicle only. The drug is derived from human recombinant FVIIa, the active site of which has been blocked by incorporation of a small peptide (D-Phe-L-Phe-L-Arg chloromethyl ketone), and the dose was selected on the basis of safety studies of human patients. The modification blocks proteolytic activity and enhances TF affinity 5-fold (24). To confirm findings with an independent inhibitor of TF, two additional baboons were treated according to the same protocol with tissue factor pathway inhibitor (TFPI; gift of A. Creasey, Chiron, Emeryville, CA) at the same dose.
After an overnight fast each animal was sedated with intramuscular ketamine (20-25 mg/kg) and intubated. Heavy sedation was maintained with ketamine (3-10 mg/kg per h) and diazepam (0.4-0.8 mg/kg every 2 h). Animals were ventilated with a volume-cycled ventilator and paralyzed intermittently with pancuronium (4 mg intravenously) before respiratory measurements. The fraction of inspired oxygen (FIO2) was 0.21, the tidal volume was 12 ml/kg, the positive end-expiratory pressure was 2.5 cm H2O, and respiratory rate adjusted to maintain an arterial PCO2 of 40 mm Hg. An indwelling arterial line and a pulmonary artery catheter were placed via femoral cutdown for hemodynamic monitoring. Detailed descriptions of the model have been published (7).
All animals received heat-killed E. coli (~ 109 CFU/kg) as a 60-min
infusion at t = 0 h, 12 h before live E. coli. Sepsis was induced at t = 12 h by infusing live E. coli at 1010 CFU/kg in a volume of 50 ml over
60 min. Gentamicin (3 mg/kg, intravenous) and ceftazidime (1 g, intravenous) were administered 60 min after completion of the live E. coli
infusion. This results in a circulating colony count of ~ 1.5 × 103, 1 h
after completion of the infusion, just before antibiotic administration.
Fluids were given as needed to maintain pulmonary capillary wedge
pressure (Ppc,we) at 8-12 mm Hg and to support blood pressure. Dopamine was used for hypotension when mean arterial pressure fell below 65 mm Hg despite fluids. After 48 h (36 h after the live bacteria infusion) animals were deeply anesthetized and killed by KCl injection.
Predefined termination criteria included refractory hypotension (
less than 60 mm Hg), hypoxemia (need for FIO2 greater than 40%), or
refractory metabolic acidosis (pH < 7.10 with normal PaCO2).
Hemodynamic Monitoring
Physiologic parameters including heart rate (HR), temperature, arterial blood pressure, pulmonary artery pressure, ventilator parameters,
and fluid intake were recorded every hour. Measurements were obtained every 6 h of cardiac output (
) by thermodilution, central
venous pressure (Pcv), Ppc,we, arterial and mixed venous blood gases,
oxygen saturation, oxygen content, and hemoglobin (Hb) as reported
(7). Urinary catheter output was measured every 6 h and fluid balance was calculated as total intravenous fluid intake minus urine output.
Preparation of Escherichia coli
Escherichia coli (American Type Culture Collection, Rockville, MD; serotype 086a:K61) was prepared as described (7) and adjusted to give a final dose of 1 × 1010 CFU/kg for each baboon (100% lethal dose, LD100). Heat-killed E. coli were prepared by heating tubes of bacteria in a water bath at 65° C for at least 30 min. The number of organisms and efficacy of heat killing were confirmed by colony counting, using pour plates.
Measurements on Whole Blood, Plasma, and Serum
Blood samples were drawn at 0, 12, 13, 18, 24, 36, and 48 h. Complete
blood counts were performed on whole blood (Sysmex-1000 hemocytometer; Sysmex, Long Grove, IL). Plasma (from citrated blood) and
serum were separated and stored at 
80° C. Fibrinogen was measured
with an ST4 mechanical coagulation analyzer (Diagnostica Stago, Parsippany, NJ). Prothrombin time (PT) and activated partial thromboplastin time (aPTT) were measured in duplicate, and antithrombin III
(ATIII) activity was measured on an MDA coagulation analyzer (Organon Teknika, Durham, NC) with a chromogenic assay and expressed as a percentage of the kit standard. An enzyme-linked immunosorbent assay (ELISA) was used to measure plasma thrombin-
antithrombin (TAT) complexes (Dade Behring, Deerfield, IL) and
FVIIai levels in plasma and BAL (Novo Nordisk). Serum samples
were assayed for interleukin 1
(IL-1), IL-6, IL-8, and tumor necrosis factor receptor 1 (TNFR-1), using ELISA kits (R&D Systems,
Minneapolis, MN). Blood urea nitrogen (BUN) and creatinine were
measured by standard clinical techniques.
Tissue Collection and Preparation
After the experiments the thorax was opened, the left mainstem bronchus was ligated, and the left lung was removed. BAL was performed
on the left upper lobe with 240 ml of 0.9% saline. Samples of lung tissue from the left lower lobe were manually inflated and immersed in
4% paraformaldehyde for light microscopy and immunohistochemistry. Four samples were taken at random from the remainder of the
left lung for wet/dry weight determination, taking care to avoid large vascular and bronchial structures. Additional samples from lung, kidney, liver, small bowel, heart, and adrenal were flash frozen in liquid
nitrogen and stored at 80° C for Western blotting and biochemical
studies. The entire right lung was inflation fixed for 15 min at 30 cm
H2O fixative pressure with 2% glutaraldehyde in 0.85 M sodium cacodylate buffer (pH 7.4). Additional tissue from kidney, liver, small
bowel, heart, and adrenal was fixed by immersion in 4% paraformaldehyde. Four samples of small bowel were selected randomly for wet/
dry weight determination.
Biochemical Measurements
Myeloperoxidase (MPO) activity and protein concentration of lung homogenates and protein and lactate dehydrogenase (LDH) concentrations in BAL fluid were measured as described (8, 10). MPO activity was expressed as a change in absorbance per minute per gram (wet weight) tissue. LDH values were expressed as units of activity per liter (U/L).
Western Blotting
Lung samples were homogenized in cold lysis buffer (150 mM NaCl,
50 mM Tris [pH 7.6], 1% sodium dodecyl sulfate [SDS], 3% Nonidet
P-40, 5 mM EDTA, 1 mM MgCl2, 2 mM 1,3-dichloroisocoumarin, 2 mM
1,10-phenanthroline, and 0.4 mM E-64) and centrifuged at 15,000 × g
for 10 min. The supernatants were mixed with Laemmli buffer and frozen at 80° C. Electrophoresis was done under reducing conditions, using 12% polyacrylamide gels. Lanes were loaded with equivalent amounts
of protein and electrophoresis was performed on a Hoefer (San Francisco, CA) minigel system. After transfer, blots were probed for TF expression with anti-TF monclonal antibody (MAb) (mouse anti-human;
American Diagnostica, Greenwich, CT) and horseradish peroxidase
(HRP)-conjugated secondary Ab (goat anti-mouse; Transduction Laboratories, Lexington, KY). Signals were developed by enhanced chemiluminescence (ECL) detection and blots were densitized by using commercially available software (Quantity One; Bio-Rad, Hercules, CA).
Histology and Immunohistochemistry
Paraformaldehyde-fixed tissues were embedded in paraffin, sectioned
and stained with hematoxylin and eosin (H&E), and examined by
light microscopy. Immunolocalization for fibrin was performed with
an MAb (anti-human fibrinogen chain; American Diagnostica) on
paraffin sections of lung, kidney, adrenal, and small bowel. This Ab
reacts strongly with fibrin and weakly with fibrinogen. Sections (5 µm)
were deparaffinized in xylene, rehydrated in graded alcohol, and
washed before incubating overnight at 4° C with anti-fibrin Ab. Sections were then washed and incubated with biotinylated secondary Ab
and the signal was developed with peroxidase-conjugated avidin and
diaminobenzidine. Negative controls were processed as described
above, except that primary incubations were done with nonimmune
mouse serum (Jackson Laboratories, Bar Harbor, ME).
Statistics
Data were entered into a computer spreadsheet and analyzed by commercially available software (StatView, Calabasas, CA). Physiologic data and data from serial blood draws were analyzed by two-factor analysis of variance (ANOVA). Biochemical data from BAL and tissues obtained at the end of the experiments were analyzed by unpaired t tests. Means ± SEM and p values are provided in the Figures 1-3 and 5-7 and in Table 1; p < 0.05 was considered significant and trends are noted for p < 0.10.
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p < 0.05 versus sepsis controls).
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p = 0.07 and *p < 0.01). There
was no difference in BAL
protein between the two
groups. Data are shown
as means ± SEM and
were analyzed by t test.
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RESULTS |
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DISCUSSION
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Both coagulation and inflammation were activated by dead bacteria before infusion of a lethal dose of live E. coli. Just before administration of live E. coli, the animals had a mild coagulopathy with increases in TAT complexes and aPTT, decreased platelets, and increased fibrinogen consistent with an acute-phase response. The inflammatory mediators IL-6, IL-8, and TNFR-1 were increased 2- to 10-fold. Infusion of live bacteria in these animals caused extensive lung injury, renal insufficiency, and damage to other vital organs including liver, bowel, and adrenals. Intravenous administration of FVIIai as a constant infusion effectively blocked further activation of coagulation and inflammation, prevented organ injury, and diminished both intra- and extravascular fibrin deposition. The effect on tissue deposition of fibrin was most prominent in the lung and kidney, where FVIIai-treated animals showed remarkable improvements in gas exchange and renal function compared with vehicle-treated septic controls. Untreated sepsis control animals had strong upregulation of TF in the lungs that was prevented by FVIIai (p < 0.05; Figure 1). Drug levels were measured in plasma and BAL fluid, and showed penetration of FVIIai into the alveolar compartment, where levels in BAL fluid were 194.2 ± 34.7 ng/mg protein at the end of the experiments. Plasma levels are shown in Table 1. Analysis of the pulmonary and renal protection by FVIIai treatment in these animals is provided below.
Acute Lung Injury in Sepsis
FVIIai treatment prevented sepsis-induced hypoxemia, pulmonary hypertension, and loss of pulmonary system compliance. These physiologic data are shown in Figure 2, plotted as
change from t = 12 h to show the drug effect. Historical data
from earlier untreated animals (n = 11) and two septic animals
treated with TFPI are shown as broken lines on the graphs for
comparison only (data not included in statistical analyses). Alveolar-arterial oxygen gradient (DA-aO2) increased in both
groups after infusion of killed bacteria and progressively worsened in the sepsis control group after the onset of live bacterial sepsis at t = 12 h. One animal in the sepsis control group
required supplemental oxygen. Treatment with FVIIai prevented deterioration in gas exchange during sepsis (p < 0.0001),
and the final DA-aO2 actually improved in those animals compared with 12 h. Sepsis-induced increases in mean pulmonary
artery pressure (
) and pulmonary vascular resistance · kg
(RL · kg) were attenuated by FVIIai (p < 0.001 and p < 0.02 versus untreated sepsis controls). FVIIai also prevented the
loss of pulmonary system compliance seen in sepsis control animals (p < 0.001). Dead space increased similarly (calculated at
approximately 40% to 45% of tidal volume) and both groups required a 30% to 35% increase in minute ventilation (
E) during
the experiment (Table 1). The PaCO2 was controlled at 40 mm
Hg in both groups (p = NS for both E and PaCO2).
At postmortem, the lungs of animals treated with FVIIai appeared normal, similar to lungs from uninjured ventilated animals. In contrast, the lungs from sepsis control animals were dense and hemorrhagic. Quantitative measures of lung wet/dry weight, neutrophil (PMN) accumulation, and lavage LDH were all improved in the treated group (Figure 3). Lung wet/dry weights were 5.81 ± 0.19 in septic controls compared with 5.05 ± 0.09 in FVIIai-treated animals (p < 0.01, normal reference range is 4.6-5.0). BAL fluid LDH decreased almost 60% (p < 0.01) and lung MPO activity was decreased by more than 40% (p = 0.07). BAL protein was not significantly different between the two groups.
Lung histology showed marked protection in septic animals treated with FVIIai. Representative sections of the lungs stained with anti-fibrin antibody are shown in Figure 4. The lungs of sepsis control animals had thickened alveolar septae, patchy alveolar edema and hemorrhage, and intra-alveolar inflammatory cell infiltration with macrophages and PMNs. Anti-fibrin staining showed extensive diffuse fibrin deposition along the septae, on intra-alveolar inflammatory cells, and in alveolar fluid. Some small vessels in the lungs contained fibrin clots. Lungs of treated animals had normal alveolar septal architecture, minimal alveolar PMN infiltration, and no alveolar edema. In these animals, septal staining for fibrin was heterogeneous and less extensive than in sepsis controls. In the treated animals, fibrin staining was frequently limited to areas immediately surrounding small vessels; however, intravascular fibrin clots were not apparent. Alveolar macrophages and intravascular monocytes stained focally.
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Renal and Other Ogan Damage in Sepsis
FVIIai also prevented renal failure in sepsis (Figure 5). Serum
creatinine doubled in the sepsis control group but remained normal in the treatment group (p = 0.059). In untreated animals, there was a corresponding decrease in urine output after
infusion of live E. coli. In contrast, urine output was maintained or increased in the treatment group (p < 0.0001). This
was not due to differences in resuscitation because fluid balance (Figure 5) and systemic hemodynamics (Table 1) were
similar in the two groups. Blood pH and serum [HCO3
] were
lower in untreated animals (p < 0.001 and p < 0.01, respectively; Figure 5).
Kidneys from untreated animals were swollen and hemorrhagic at postmortem but appeared normal in FVIIai-treated animals. H&E-stained sections of the kidneys of untreated animals had patchy areas of acute tubular necrosis (ATN) and loss of glomeruli. The kidneys of treated animals, except for a few small foci of ATN, showed normal renal architecture. Immunostaining showed fibrin deposition in glomeruli of sepsis control animals with obliteration of capillary architecture (Figure 4E). Tubular epithelium also stained, and some tubules contained amorphous material that was also positive for fibrin. Vessels occluded by fibrin clot were readily identified. In the treated animals, glomerular fibrin deposition was absent and minimal tubular epithelial staining was seen in only a few animals (Figure 4F).
The appearance of the adrenals, liver, and small bowel was also normal in the FVIIai-treated animals. In contrast, the adrenals from untreated animals were swollen and hemorrhagic and small bowel was grossly edematous. Small bowel wet/dry weights were higher in untreated animals, but high variability in the bowel injury did not permit a significant difference to be achieved between the groups (6.36 ± 0.51 in treated versus 8.30 ± 1.13 in untreated animals, p = 0.15). In contrast to the decreased fibrin staining in the lungs and kidneys, focal fibrin deposition was seen in adrenals and small bowel in both treated and untreated septic animals. Despite this, adrenal cortical congestion and hemorrhage and small bowel hemorrhage and edema were diminished in septic animals treated with FVIIai. There was no significant effect of FVIIai on PMN content in organs other than the lung. MPO activity in kidney, liver, and small bowel was variable in control animals and differences were not significant between the two groups.
Sepsis-induced Coagulopathy
Intravascular activation of coagulation was decreased in septic animals treated with FVIIai compared with controls (Figure 6). Initial values for coagulation parameters were within the normal range for this species. Drug treatment prevented plasma fibrinogen depletion as expected with therapeutic blockade of coagulation (p < 0.0001). TAT complexes increased after live E. coli in sepsis controls, peaking at 13-18 h, and then declined as ATIII activity levels decreased. The increase in TAT complexes was attenuated in treated animals (p < 0.0001); however, the decrease in ATIII activity was not significantly different. Although TAT levels decreased late in the experiment in untreated septic animals, coagulation was ongoing in those baboons. The aPTT increased progressively in both groups but was higher in untreated animals (p < 0.01). PT was higher in the treatment group because of the drug effect on the assay, between 53 and 67 s for the duration of drug infusion (p < 0.0001). In the untreated group PT increased progressively from 17.8 ± 0.4 s at 12 h (before live E. coli was infused) to 25.5 ± 3.6 s at the end of the experiment.
Both groups of animals developed neutropenia, thrombocytopenia, and anemia after infusion of live E. coli (see Table 1). White blood cells reached a nadir of approximately 1,500 (× 103/µl) in both groups 1 h after the infusion (t = 13 h) and progressively increased to near baseline levels by the end of the experiment (9,400 ± 1,800 in treated animals versus 13,000 ± 3,900 in untreated animals [× 103/µl], p = 0.08). All animals were thrombocytopenic by 12 h after the infusion of live E. coli (t = 24 h) and mean platelet counts were 30,000 or less in both groups at the end of the experiment. Hb decreased similarly in both groups without evidence of significant hemorrhage in either (Table 1).
Proinflammatory Cytokine Levels
Elevations of inflammatory cytokines were attenuated by
treatment with FVIIai (Figure 7). Circulating levels of IL-1,
IL-6, IL-8, and TNFR-1 rose sharply after infusion of live E. coli in both treated and untreated animals. Peak IL-6 levels
were not different between the two groups, but IL-6 declined
more rapidly in FVIIai-treated animals (p < 0.001) and returned to levels found in naive animals. Likewise, IL-8 and
TNFR-1 levels were attenuated compared with controls (p < 0.01 and p < 0.001, respectively). There was no difference in
IL-1 levels between the two groups.
Systemic Hemodynamic Parameters
Hemodynamic measurements, including HR, Ppc,we, /kg,
and systemic vascular resistance · kg (Rsv · kg), were not altered by treatment with FVIIai (Table 1). Hypotension responded to intravenous fluids in both groups; one animal in
the treatment group required low-dose dopamine briefly after
live bacteria were infused. Ten of the 12 animals survived until
the scheduled termination point of the protocol. One sepsis
control animal died at 30 h (18 h after live bacteria infusion)
from ALI, with refractory hypoxemia and respiratory acidosis,
and one animal in the FVIIai treatment group died 3 h before
the end of the study from a complication of endotracheal intubation. Two animals in each group developed self-limited hematuria during the experiment and one animal in the FVIIai
treatment group had a clot in the bronchus intermedius at
postmortem. Most animals in the two groups had some blood-tinged secretions associated with suctioning at some point in
the study. No severe or life-threatening bleeding complications occurred in either group.
Pulmonary and Renal Injury after TFPI Infusion
To confirm the effects of TF blockade on ALI in E. coli sepsis, two baboons were treated with TFPI according to the same experimental protocol. Activation of coagulation was blocked in sepsis after TFPI infusion with similar improvements in plasma fibrinogen levels. Terminal fibrinogen levels (t = 48 h) in those animals were 75% and 95% of 12-h values. Like FVIIai, TFPI did not alter systemic hemodynamic parameters. Gas exchange and pulmonary mechanics were protected in both animals (see Figure 2). Histopathology and fibrin immunostaining of lung tissue after TFPI showed decreased inflammatory cell infiltrates, decreased septal thickening, and decreased fibrin deposition in the lung. As in the FVIIai-treated group, renal architecture was normal and fibrin staining in the kidneys was absent after TFPI.
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ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
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This is the first study to show specific improvements in end organ function after blocking initiation of coagulation by TF in gram-negative sepsis. The findings establish an etiologic role for TF in sepsis-induced respiratory and renal failure and show that blockade of TF effectively preserves both pulmonary and renal function in baboons with multiple organ failure. Previous animal studies using a variety of strategies to block coagulation in sepsis-induced DIC have reported improved survival after either TF blockade or anticoagulation, but assessment of treatment effect on end organ injury has been complicated by the presence of severe septic shock, lack of continuous physiologic monitoring and standardized volume resuscitation, and variable survival. This has hampered acquisition of physiologic correlates of survival and made it difficult to compare responses to treatment in different tissues (4, 25). In our approach, priming preactivates inflammation and causes mild, self-limited alterations in pulmonary gas exchange, mechanics, and hemodynamics similar to experimental endotoxemia in humans (7, 28). However, subsequent overwhelming gram-negative sepsis results in progressive lung and renal injury, persistent elevation of inflammatory cytokines, and coagulopathy. The immune response in these animals is complex, and certain therapies, for example, MAb to leukocyte adhesion molecules, significantly worsen outcome in primed animals (8, 10). In contrast, blockade of the TF- FVIIa complex attenuates coagulopathy and fibrin deposition and prevents lung and renal injury after lethal E. coli infusion.
In the past, a primary goal of coagulation blockade in sepsis has been to inhibit fibrin deposition in the vascular compartment. Here we have demonstrated that extravascular fibrin coagulation contributes to organ injury and is also amenable to intervention. Fibrin deposition provides the critical matrix for cell migration and collagen formation in tissue repair but may also stimulate inflammation (2, 29). In the lungs, parenchymal accumulation of fibrin may contribute to inflammatory cell migration, surfactant dysfunction, and profibrotic processes (23, 30). Although gas exchange and lung wet/ dry weights were greatly improved in our study, residual fibrin was detected in the alveolar region and around small vessels in the lungs of FVIIai-treated animals. This suggests that the strong protective effects of TF blockade were not entirely due to the absence of fibrin and that key repair processes involving coagulation might remain intact during treatment with FVIIai.
FVIIai did prevent intralumenal fibrin clots in the lungs and kidneys after 36 h of sepsis, which may have contributed to tissue protection. Intravascular fibrin deposition contributes to organ failure as a direct result of obstructive thrombus in small nutrient vessels (33) and via enhancement of endothelial-leukocyte interactions (34). Although intravascular fibrin is likely to be important in some tissues and in certain clinical settings, for example, when overwhelming shock and tissue hypoperfusion occur, extravascular TF expression by epithelial cells and tissue macrophages also initiates procoagulant, proinflammatory events (35). Both resident and infiltrating macrophages, as well as fixed cell populations, have been implicated as sources of TF in inflammatory lung and in renal disease (36, 37), suggesting coagulation is regulated differently in extravascular parenchyma.
TF is a Group II cytokine receptor that may regulate immune
functions either directly or through generation of FXa, thrombin, and fibrin, all of which exhibit cross-talk with inflammation (38-
40). Each component has independent effects on the inflammatory response, and blocking initiation at TF has the advantage of
curtailing inflammatory interactions at subsequent steps in the
pathway. In particular, TF-activated mitogen-activated protein
kinase (MAPK) cytokine regulation is relevant to the development of ALI. IL-6, for example, has been associated with persistent inflammation and poor outcomes in ARDS (41). In vitro,
FVIIai inhibits MAPK activation, demonstrating that catalytically active FVIIa is required for TF signaling via these pathways
(39). Ligation of TF by FVIIa induces a number of immunoregulatory genes, including IL-1, IL-8, and other chemokines, coagulation and growth factors, and collagenases (40). In our septic
baboons, FVIIai decreased the plasma levels of IL-6, IL-8, and
TNFR-1. This effect could stem from decreased TF signaling or
decreased downstream production of FXa and thrombin, which
also induce proinflammatory cytokines (42, 43). IL-6 and IL-8
further increase TF expression (44), and TF blockade with FVIIai
notably decreased sepsis-induced TF expression in the lung. Regulation of other important mediators of acute lung injury, for example, vascular endothelial growth factor (VEGF), may require
the proteolytic activity of FVIIa and/or the cytoplasmic tail of TF
(20, 45). Finally, some data suggest that when TF is highly expressed it functions as a cofactor to present FVIIa to other transmembrane proteins that initiate signaling events (29). If such interactions are important when TF is highly overexpressed as in
sepsis, direct targeting of FVIIa would have an advantage over
other interventions that inhibit TF.
In earlier studies of animals with fulminant sepsis, three experimental agents, TFPI (4), anti-TF MAb (5), and DEGR- FVIIa (26), have been targeted at TF-initiated coagulation. These agents improved survival; however, natural inhibitors of proteases distal to the TF-FVIIa complex, including activated protein C (APC) and antithrombin III (ATIII), have also shown survival efficacy (6, 25, 27). Because these strategies have all been tested in previously unchallenged animals that develop rapidly progressive shock, it is possible that coagulant activity contributes to mortality in shock downstream from the TF-FVIIa complex. Like the anti-TF agents, their impact on ALI and MOF has not been studied, although a human trial showed a survival benefit in patients with severe sepsis who were treated with APC. The majority of those patients had shock that required vasopressor therapy at study entry. The effects of APC responsible for improved outcome in those patients are not clear (3). APC inhibits thrombin generation by inactivating Factors Va and VIIIa, but like other coagulation system proteins, APC may have independent effects on inflammation.
One potential mechanism for improved survival in these studies was a decrease in systemic cytokine expression, particularly IL-6 and IL-8 (4, 25). Critical effects for these mediators have been difficult to localize and do not consistently link coagulation and cytokines with survival. ATIII, which inhibits coagulation at FXa and thrombin, decreased mortality, coagulopathy, and IL-6 production in baboons (25); however, these results were not duplicated in human trials (46). By comparison, DEGR-FVIIa attenuated coagulopathy and IL-6 production but had a variable effect on survival that did not correlate with cytokine levels (26). Also, inactivated FXa attenuated coagulopathy but did not improve survival in acute septic shock (47). In humans TF blockade with TFPI did not affect IL-6 levels after low-dose endotoxin infusion, although it did prevent activation of coagulation (48). Together these studies imply different thresholds for inflammatory and clotting functions of coagulation proteases in primates, especially as the inflammatory challenge progresses.
In our study, FVIIai abrogated lung inflammation without altogether blocking coagulation. This novel observation, if applicable to human sepsis, may offer promise to patients in whom bleeding is a concern. Although FVIIai effectively binds TF, it incompletely blocks coagulation in vitro (49). Thus greater activation of TF-FVIIa may be required for inflammation than for coagulation (50), and at the dose of FVIIai used in this study no serious bleeding was seen. The effect of the drug on coagulation can be reversed with human recombinant FVIIa if bleeding does occur, however. Thrombocytopenia persisted in treated animals, which is another reason to closely monitor for bleeding in clinical studies.
In summary, specific blockade of coagulation at the TF- FVIIa complex prevents lung and renal injury during E. coli sepsis in nonhuman primates. Other tissues were protected to varying degrees, suggesting TF contributes differently among organs to injury in sepsis. We tested this strategy in the presence of ongoing inflammation because persistent cytokine expression may have critical implications for outcome in critically ill humans with ARDS. Previous strategies for treating septic shock based on other aspects of coagulation have had varying clinical success (3, 46). This likely reflects both the heterogeneous injury of sepsis and complex interactions among coagulation proteases with respect to inflammation. Our data do suggest that intervening proximally in the coagulation cascade will have a positive impact on pulmonary and renal injury in sepsis.
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Footnotes |
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Correspondence and requests for reprints should be addressed to Karen Welty-Wolf, M.D., Department of Medicine, Box 3518, Duke University Medical Center, Durham, NC 27710. E-mail: welty001{at}mc.duke.edu
(Received in original form May 8, 2001 and accepted in revised form August 10, 2001).
Acknowledgments: Thanks to Abla Creasey of Chiron (Emeryville, CA) for providing the TFPI used in this study, and to Craig Marshall, John Patterson, Jackie Carter, and Jennifer Mele for expert technical assistance.
Supported by grant P01 HL 31992-18 from the National Institutes of Health, and by Novo Nordisk.
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References |
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TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
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