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American Journal of Respiratory and Critical Care Medicine Vol 167. pp. 1200-1209, (2003)
© 2003 American Thoracic Society

Blockade of Tissue Factor

Treatment for Organ Injury in Established Sepsis

Martha Sue Carraway, Karen E. Welty-Wolf, Debra L. Miller, Thomas L. Ortel, Steven Idell, Andrew J. Ghio, Lars C. Petersen and Claude A. Piantadosi

Department of Medicine, Duke University Medical Center, Durham, North Carolina; Department of Medicine, University of Texas, Tyler, Texas; and Novo-Nordisk, Copenhagen, Denmark

Correspondence and requests for reprints should be addressed to Martha Sue Carraway, M.D., Division of Pulmonary and Critical Care Medicine, Box 3315, Duke University Medical Center, Durham, NC 27710. E-mail: carra001{at}me.duke.edu


   ABSTRACT
TOP
 ABSTRACT
METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Blockade of tissue factor before lethal sepsis prevents acute lung injury and renal failure in baboons, indicating that activation of coagulation by tissue factor is an early event in the pathogenesis of acute lung injury and organ dysfunction. We hypothesized that blockade of tissue factor would also attenuate these injuries in established sepsis by prevention of further fibrin deposition and inflammation. Twelve male baboons received heat-killed Escherichia coli intravenously followed 12 hours later by live E. coli infusion. Six animals were treated 2 hours after the live bacteria with site-inactivated Factor VIIa, a competitive tissue factor inhibitor, and six animals were vehicle-treated sepsis control subjects. Animals were ventilated and monitored for 48 hours. Physiologic and hematologic parameters were measured every 6 hours, and pathologic evaluation was performed after 48 hours. Animals treated with site inactivated Factor VIIa had less severe lung injury, with preserved gas exchange, better lung compliance and histology scores, and decreased lung wet/dry weight. In treated animals, urine output was higher, metabolic acidosis was attenuated, and renal tubular architecture was protected. Coagulopathy was attenuated, and plasma interleukin-6, interleukin-8, and soluble tumor necrosis factor receptor-1 levels were significantly lower in the treated animals. These results show that blockade of coagulation attenuates acute lung and renal injury in established Gram-negative sepsis accompanied by antiinflammatory effects of therapy.

Key Words: systemic inflammatory response • acute respiratory distress syndrome • multiple organ dysfunction syndrome • coagulation

Leading causes of morbidity and mortality in sepsis are acute respiratory distress syndrome and multiple system organ dysfunction (1). Although the pathogenesis of septic acute respiratory distress syndrome is not precisely understood, inflammation, elaboration of cytokines, and activation of coagulation are important mechanisms of injury to the lungs and other organs (2–4). As a part of the inflammatory response, coagulation is activated by increased expression of tissue factor (TF), which initiates the cascade by binding activated Factor VII (FVIIa), forming the TF-FVIIa complex. Assembly of this complex activates Factor X to Xa, which cleaves prothrombin to thrombin (5). Thrombin cleaves fibrinogen to fibrin, which is deposited in the tissues. Activation of coagulation also promotes pulmonary and systemic inflammation and is increasingly recognized as an important pathogenic factor in sepsis-associated organ injury (6–9).

TF expression increases in the lung, kidney, and other organs in sepsis and localizes to endothelial, epithelial, and inflammatory cells (10). TF activity contributes to the procoagulant environment in both the intravascular and extravascular compartments (4). Lipopolysaccharide and selected cytokines, including tumor necrosis factor-{alpha} (TNF-{alpha}), increase TF expression in inflammatory and endothelial cells, which could activate intravascular coagulation (7, 10, 11). It is postulated that enhanced vascular TF expression initiates coagulation and thereby promotes microvascular thrombosis that contributes to organ injury during sepsis (6, 10). In addition, TF expression by parenchymal and recruited lung cells can activate coagulation at extravascular sites in the lung (10, 12). Extravascular coagulation is demonstrated by increased procoagulant and decreased fibrinolytic activity in bronchoalveolar lavage (BAL) fluid from patients with acute respiratory distress syndrome and primates with acute lung injury (13, 14). Activation of extravascular coagulation and transendothelial leakage of plasma proteins promote alveolar fibrin deposition, a key feature of acute lung injury in sepsis (15). In conjunction with nonhydrostatic pulmonary edema, inflammatory cell infiltration, and surfactant dysfunction, alveolar fibrin deposition contributes to disordered gas exchange. Lung fibrin deposition has been implicated in the pathogenesis of acute lung injury in experimental animals (13) and has been proposed to be integral to acute respiratory distress syndrome in humans (4, 14). In addition, intravascular fibrin has been identified in other organs in baboons after Escherichia coli infusion, particularly in glomerular vessels, where it contributes to renal dysfunction (10, 16).

In addition to their procoagulant activity, the TF-VIIa complex and downstream coagulation proteins amplify multiple steps of the inflammatory response and may thereby contribute to tissue injury during sepsis (17, 18). During sepsis in nonhuman primates, preventing initiation of coagulation by TF blockade reduces mortality (19–21). Improved survival was associated with selective reduction in the cytokines interleukin (IL)-6 and IL-8. In humans with sepsis, mortality is decreased by treatment with recombinant activated protein C (22), which interferes with thrombin generation and may have antiinflammatory effects. We have previously shown that pretreatment of septic baboons with active site-inactivated FVIIa (FVIIai) and TF pathway inhibitor attenuates acute injury of the lung and other organs in septic multiple organ failure (16). FVIIai competitively inhibits binding of FVIIa to TF and suppresses both TF-VIIa signaling and coagulant activities. This study demonstrates that this method of TF blockade provides similar effects when used after the onset of sepsis at the time of antibiotic administration. The data demonstrate that attenuation of sepsis-initiated lung and kidney injury is associated with decreased elaboration of specific proinflammatory cytokines.


   METHODS
TOP
 ABSTRACT
 METHODS
RESULTS
 DISCUSSION
 REFERENCES
 
Animal Model
Adult male baboons (Papio cyanocephalus) weighing 14 to 20 kg were obtained from the Southwest Foundation for Biomedical Research (San Antonio, TX) and were used for the studies. They were handled in accordance with American Association for the Accreditation of Laboratory Animal Care guidelines using a protocol approved by the Duke University Institutional Review Board and Institutional Animal Care and Use Committee. As reported, animals were sedated, intubated, and mechanically ventilated. Arterial, venous, and pulmonary arterial catheters were placed (16, 23).

Experimental Protocol
Twelve baboons were used for the study, six animals in each group. We compared septic baboons (sepsis control) with a group of septic animals that received FVIIai (Novo Nordisk, Copenhagen, Denmark). This protein binds TF competitively and blocks initiation of extrinsic coagulation. The protocol for initiation of sepsis was identical in the two groups (16). The baboons received heat-killed E. coli ( 1 x 109 cfu/kg) intravenously at the beginning of the experiment (t = 0 hours). Twelve hours later (t = 12 hours), live E. coli (1 x 1010 cfu/kg) was administered intravenously over 1 hour. Two hours after the start of the live E. coli infusion (t = 14 hours), all 12 animals were treated with gentamicin sulfate (3 mg/kg intravenously) and ceftazadime (1 g intravenously). In the FVIIai-treated group, 1-mg/kg intravenous bolus of FVIIai was given over 5 minutes at the time of the antibiotic administration, 2 hours after the start of live E. coli infusion (t = 14). The drug bolus was followed by continuous infusion of 50 µg/kg/hour until the experiment ended at 48 hours.

Baboons were maintained on the protocol for 36 hours after infusion of live E. coli (t = 48 hours) unless they met criteria for early termination of the experiment. These criteria included hypotension refractory to intravenous fluids and dopamine, refractory metabolic acidosis (pH < 7.1), or hypoxemia requiring 40% inspired oxygen. Temperature, heart rate, arterial blood pressure, pulmonary artery pressures, ventilator parameters, and fluid balance were recorded every hour. Every 6 hours, pulmonary artery wedge pressure, cardiac output, and arterial and mixed venous blood gases were obtained. Animals were killed at the end of the experiment after induction of deep anesthesia and infusion of a saturated potassium chloride solution. An autopsy was performed immediately to harvest and prepare tissues for processing.

Tissue Collection and Preparation
After death, tissue was collected as reported (16). Briefly, the left lung was removed for BAL and to freeze tissue. The right lung was inflation fixed in situ and removed en bloc. Tissue from the heart, liver, kidney, small bowel, and left lung were snap frozen for later quantitation of myeloperoxidase (MPO) and for protein and RNA determination. Lung and small bowel were drained of blood, weighed immediately, then dried in a vacuum oven at 60°C for a minimum of 72 hours for determination of wet:dry (W:D) weight ratio.

Histologic Scoring for Lung Injury
Histologic analysis of lung injury was performed using a semiquantitative scoring system. Eight randomly selected sites from each inflation-fixed right lung were embedded in paraffin, and sections were cut for hematoxylin and eosin staining. Five slides from each animal were analyzed: two upper lobes, one middle lobe, and two lower lobes. Three sites on each slide were visualized, scanned, digitized, and analyzed by an observer unaware of the exposure conditions (Nikon Optiphot-2 light microscope, EPSON Expression 800 scanner). Four findings (alveolar fibrin/edema, alveolar hemorrhage, septal thickening, and intra-alveolar inflammatory cells) were scored on each slide for (1) severity and (2) extent of injury. For grading extent of injury, each component was assigned a score of 0 (absent), 1 (less than 25% involved), 2 (25–50%), or 3 (more than 50), and for severity, each component was graded at the most involved site as 0 (absent), 1, 2, and 3. A mean score for each component for each animal was derived and expressed as the product of extent and severity. These values were used to compare the two groups.

Biochemical Measurements
MPO activity and protein content of tissue homogenate were performed as reported (23). MPO activity is expressed as {Delta} absorbance/minute/g wet weight tissue. Protein and lactate dehydrogenase (LDH) in BAL fluid were also measured as previously reported.

Measurements on Whole Blood, Plasma, and Serum
Blood samples were drawn at baseline (t = 0) and at 12, 14, 18, 24, 36, and 48 hours. Complete blood counts, blood urea nitrogen and creatinine, prothrombin time (PT), activated partial thromboplastin time (aPTT), antithrombin III activity, fibrinogen, thrombin–antithrombin (TAT) complexes were measured on these blood samples. FVIIai levels were measured in plasma and BAL, and serum samples were also assayed for IL-1ß, IL-6, IL-8, and TNF receptor-1 (TNFR-1) using ELISA.

Data Analysis
Selected data are shown as mean ± SE. Physiologic measurements and serial hematologic, arterial blood gas, and cytokine measurements were compared using two-factor analysis of variance (StatView Software; SAS Institute, Inc., Cary, NC). Analysis was based on the difference between the two groups in the change from baseline of the measured variable after sepsis. Because sepsis was initiated at t = 12 hours, the analyses were performed on data expressed as change from t = 12. Post hoc analysis was performed with Fisher's test. Biochemical assays on BAL fluid and tissues from the end of the experiments were compared using unpaired t-test. Data are expressed as mean ± SEM, and p < 0.05 was considered significant. Trends are noted for p < 0.1.


   RESULTS
TOP
 ABSTRACT
 METHODS
 RESULTS
DISCUSSION
 REFERENCES
 
Initiation of sepsis with live E. coli 12 hours after a low dose of heat-killed bacteria results in coagulopathy, proinflammatory cytokine release, and organ failure, which is not related to hemodynamic shock (23). The pathogenesis involves initiation of coagulation because blockade of TF activation before sepsis alleviates organ failure. In this study, intervention with blockade of TF was performed in an established model of sepsis characterized by organ failure, coagulopathy, inflammation, and cytokine expression. Although mortality was not an endpoint, all septic baboons treated with FVIIai survived for the full experimental period, whereas two of six sepsis control animals died before the 48-hour endpoint. Both animals died with refractory metabolic acidosis, renal failure, and hypoxemia.

Hemodynamic Responses to Sepsis
Hemodynamic measurements showed the hyperdynamic responses typical of septic control baboons exposed to the two-dose regimen of E. coli (23). After the initial infusion of heat-killed E. coli, tachycardia and transient hypotension occurred. Cardiac output also increased in response to heat-killed bacteria, and this response persisted after live bacteria were administered. In Table 1 , these data are expressed in measurement units per kilogram of body weight. The two groups were compared statistically by analyzing the changes in measurements after live E. coli infusion at t = 12 hours. There were no significant differences in the response of mean arterial pressure, cardiac output, or heart rate after live E. coli between the groups. Despite similar cardiac output and hemoglobin levels between groups, oxygen delivery was significantly higher in FVIIai-treated animals (p < 0.01), partly reflecting better preservation of arterial oxygen saturation in the treated septic animals (Table 1). Another significant physiologic difference was that the depression of systemic oxygen consumption (VO2/kg) in sepsis control animals was not present in FVIIai-treated animals. By 36 hours in sepsis control baboons, VO2/kg was significantly reduced from 12-hour measurement at 6.3 ± 0.5, whereas in FVIIai-treated animals, VO2/kg was 7.5 ± 0.3, similar to baseline (p < 0.01).


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  		TABLE 1. Hemodynamic measurements in untreated septic baboons and septic baboons treated with fviiai

 
Complete blood counts measured every 6 hours (see Table E1 in the online supplement) showed no significant differences in hematocrit or platelet counts between the groups. Hematocrit fell similarly in both groups over the course of the experiment, indicating that coagulation blockade did not increase bleeding. In both groups, blood leukocyte counts dropped significantly at 14 hours (after administration of live bacteria) and then subsequently increased. In untreated animals, the leukocyte counts increased more rapidly than in animals who received FVIIai and were significantly higher by 48 hours (p < 0.01). Platelet counts dropped significantly after infusion of live E. coli in both groups and continued to decrease over the remaining 36 hours.

FVIIai Attenuated Acute Lung Injury in Sepsis
Lung inflammation and injury was compared by physiologic, pathologic, and biochemical measurements. Physiologic variables, including pulmonary system compliance, alveolar to arterial oxygen gradient (A:a gradient), mean pulmonary artery pressure, and pulmonary vascular resistance (PVR), are plotted in Figure 1 as change in the measurement relative to the value before infusion of live E. coli at t = 12 hours. In untreated animals, lung injury led to an increase in A:a ratio and a decrease in lung compliance over 36 hours after live E. coli infusion. In animals treated with FVIIai after E. coli, lung injury by these measurements was significantly attenuated (p < 0.01 for both). The A:a gradient progressively increased during sepsis and was increased by 22.4 ± 1.7 by t = 48 hours in septic control animals, whereas the change in A:a ratio was lower at all times measured in septic baboons treated with FVIIai (14.5 ± 3.2 at 48 hours, p = 0.0004 vs. sepsis control). Pulmonary system compliance decreased by 7.5 ± 1.6 ml/cm H2O by 48 hours in untreated baboons and decreased by 5.9 ± 1.7 in the treated group (p = 0.02). In sepsis control animals, mean pulmonary artery pressure and PVR peaked 12 hours after live bacteria (t = 24 hours) and remained elevated at 48 hours. In animals receiving FVIIai, mean pulmonary artery pressure and PVR did not increase acutely but slowly increased over the next 36 hours. The trend toward a statistical difference between the two groups (p = 0.07 for PVR and 0.08 for mean pulmonary artery pressure) predominately reflects the differences at 18 and 24 hours.



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  		Figure 1. Changes in A:a oxygen gradient, pulmonary system compliance (Cs), mean pulmonary artery (PA) pressure, and PVR in untreated septic control baboons (solid circles) and septic baboons treated with FVIIai (open circles). In untreated septic animals, lung injury was demonstrated by increased A:a gradient, decreased system compliance, and increased mean pulmonary artery and PVR. In animals treated with FVIIai, less change in A:a gradient and system compliance indicated that injury was significantly attenuated (*p <= 0.05 versus sepsis control subjects). Data are shown as mean change from time of live E. coli infusion at t = 12 hours, + SEM. Treatment with FVIIai was at t = 14 hours. Solid circles, sepsis control (n = 6). Open circles, sepsis + FVIIai (n = 6).

 
After the experiment, the extent of lung injury was further assessed by determining lung W:D weight ratio and measuring BAL fluid protein and LDH. W:D weight ratios indicate that blocking TF in sepsis diminished lung edema significantly (Figure 2) . In septic control animals, lung W:D weight ratio was 6.9 ± 0.8, indicating significant lung edema. In contrast, septic animals treated with FVIIai had a significantly lower W:D ratio (4.8 ± 0.13, p = 0.03), similar to values from normal baboons (23). Protein and LDH in BAL fluid indicated a trend toward lower BAL protein and LDH in the FVIIai-treated baboons (p <= 0.1) (Figure 2).



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  		Figure 2. Lung W:D ratios and BAL protein and LDH 36 hours after sepsis was initiated with live E. coli in untreated septic control baboons and septic baboons treated with FVIIai. (A) Lung W:D ratios were significantly lower in FVIIai-treated septic baboons. (B) BAL fluid LDH and protein values showed a trend toward significant difference between the two groups. Dark gray bars, BAL protein. Light gray bars, BAL LDH (*p <= 0.05 vs. sepsis control subject subjects).

 
By pathologic assessment, lung injury was attenuated by treatment with FVIIai. Typical gross pathology and light microscopy photomicrographs are shown in Figure 3 . Figure 3A shows the gross appearance of a lung from an untreated septic animal with diffuse consolidation and hemorrhage. In Figure 3B, the lung from a septic baboon treated with FVIIai shows minimal changes. Figures 3C–3E show typical light microscopy sections of lungs stained with hematoxylin and eosin from a normal baboon, an untreated septic baboon, and a baboon treated with FVIIai after the onset of sepsis. In the lungs of untreated septic baboons 36 hours after live E. coli (Figure 3D), the alveolar–septal membrane was thickened; alveolar edema was present, and there were increased numbers of inflammatory cells in the interstitium and alveolar spaces. In contrast, lung sections from animals treated with FVIIai showed less alveolar and interstitial inflammatory cells and in many areas appeared uninjured (Figure 3E). Results of semiquantitative analysis of lung injury are shown in Figure 3F. Lung injury in the untreated sepsis animals was characterized primarily by deposition of intra-alveolar fibrin/edema and by septal thickening. FVIIai-treated animals had significantly less alveolar fibrin and septal thickening than untreated septic animals. Mean scores for alveolar fibrin were 1.57 and 0.42 (p < 0.03) and for septal thickening were 5.72 versus 3.69 (p < 0.02) for untreated sepsis and sepsis + FVIIai, respectively.



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  		Figure 3. Gross pathology, light microscopy, and immunohistochemistry for TF in lungs from normal control baboon, untreated septic, and FVIIai-treated septic baboons 36 hours after the onset of sepsis. (A) A representative photograph of a lung from an untreated septic baboon shows lung injury characterized by diffuse edema and consolidation with patchy hemorrhage. (B) This representative lung of a septic animal treated with FVIIai was relatively normal in appearance. (C) Hematoxylin and eosin staining of lung from a normal baboon has thin alveolar septae and minimal inflammation. (D) Hematoxylin and eosin staining in lung from an untreated septic baboon shows thickening of alveolar–septal membrane, alveolar edema, and increased numbers of inflammatory cells. (E) Representative hematoxylin and eosin of lung from a septic animal treated with FVIIai shows decreased inflammatory cells and interstitial thickening compared with sepsis control subjects. (F) Histologic lung injury scores. In untreated sepsis, alveolar fibrin/edema and septal thickening were significantly greater than in FVIIai-treated animals (intra-alveolar fibrin, p < 0.03, and for septal thickening, p < 0.02).

 
Septic lung injury in the baboon is also characterized by increased lung TF expression (16). Figures 4A–4C show representative immunohistochemical stains for TF after 36 hours in a normal control, untreated septic, and septic baboon treated with FVIIai. In normal baboon lung, TF is expressed primarily in alveolar macrophages. During sepsis in untreated animals, TF expression was detected in intra-alveolar mononuclear inflammatory cells and diffusely along the alveolar epithelium. In the lungs of baboons treated with FVIIai, staining for TF in the alveolar region was patchy, consistent with the overall effect of decreased injury.



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  		Figure 4. Immunohistochemistry for TF in lungs from normal, untreated septic, and septic baboon treated with FVIIai. (A) Normal control lung has TF staining that is primarily in alveolar macrophages. (B) In untreated septic animals after 36 hours, TF expression occurs in the intra-alveolar inflammatory cells and along the alveolar region. (C) In septic baboons treated with FVIIai, lung staining for TF was present in occasional inflammatory cells.

 
FVIIai Attenuated Renal Injury and Metabolic Acidosis
Over 36 hours after live E. coli infusion, untreated baboons developed progressive renal dysfunction manifested by progressive oliguria, rising serum creatinine, and development of metabolic acidosis (Figure 5) . Blockade of TF attenuated renal injury in sepsis, as shown by significant improvements in these functional parameters. Urine output (ml/kg body weight) was significantly higher in septic animals treated with FVIIai than in septic control baboons (Figure 5A). Figure 5B shows that septic control baboons developed progressive metabolic acidosis characterized by decreased serum [HCO3-] levels over 36 hours. In contrast, serum [HCO3-] remained similar to control values in animals treated with FVIIai. Serum creatinine increased significantly by t = 48 hours in septic control baboons, which was largely prevented by FVIIai treatment. In both groups of baboons, intravenous fluid support was directed to maintain pulmonary artery occlusion pressure of 8–12 mm Hg, and fluid balance (total fluid intake - total output) was not statistically different (Figure 5B).



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  		Figure 5. Renal injury and metabolic acidosis in sepsis were attenuated with FVIIai. (A) Urine output (ml/kg body weight) was measured every 6 hours and was significantly higher in septic animals treated with FVIIai than in untreated septic control baboons. (B) Fluid balance (total fluid intake - total output) was similar in the two groups. (C) Septic control baboons developed progressive decrease in serum [HCO3-] levels over 36 hours, whereas [HCO3-] remained similar to initial values in septic animals treated with FVIIai. (Solid circles) Sepsis control (n = 6). (Open circles) Sepsis + FVIIai (n = 6). (D) Serum creatinine increased significantly by t = 48 hours in septic control baboons but not in septic animals treated with siVIIa. White bars, Sepsis control. Gray bars, Sepsis + FVIIai (*p <= 0.05 compared with sepsis control; {dagger}p <= 0.05 compared with t = 0 hours).

 
Figures 6A–6C show light microscopy photomicrographs of the kidney from a septic untreated and FVIIai-treated baboon. In Figure 6A, a kidney from a control animal shows normal-appearing glomeruli and tubules. In the untreated septic animal (Figure 6B), renal tubules were filled with amorphous sediment. The tubular epithelial cells were edematous, and the glomeruli contained numerous inflammatory cells. Many of the small vessels contained fibrin clots. These changes were greatly abrogated in the treated animals (Figure 6C).



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  		Figure 6. Light microscopy photomicrographs of the kidney from a normal, untreated septic and FVIIai-treated septic baboon. (A) A kidney from a normal control shows typical glomeruli and tubules. (B) In the untreated septic animal, renal tubules were filled with amorphous sediment, the tubular epithelial cells were edematous, and the glomeruli contained inflammatory cells. Many of the small vessels contained fibrin clots. (C) Kidney from FVIIai treated baboon. Tubular architecture was preserved and glomeruli lacked inflammatory changes.

 
FVIIai Attenuated Sepsis-induced Coagulopathy
To ascertain the effect of blocking TF on sepsis-induced coagulopathy, PT, PTT, fibrinogen, platelet counts, antithrombin III, and TAT levels were measured at 0, 12, 14, 18, 24, 36, and 48 hours. PT, PTT, platelet counts, and FVIIai levels are reported in Table E1. Fibrinogen and TAT complex levels are shown in Figure E1 (online supplement). Plasma concentrations of FVIIai measured by ELISA showed peak drug levels in the treated animals of 3,300 ± 1,460 ng/ml at 14 hours, shortly after the bolus, and FVIIai levels remained elevated until the end of the experiment at t = 48 hours. FVIIai was detectable in the BAL fluid, where levels were 17–33% of the plasma levels, indicating the drug penetrated into the lung compartment.

After heat-killed E. coli are infused at t = 0, the baboon develops a slightly prolonged aPTT and elevated fibrinogen at t = 12 hours as part of the acute phase response. After live bacteria, the animals develop severe coagulopathy with progressive increases in PT and aPTT and a decrease in fibrinogen and platelet count. In septic animals treated with FVIIai, PT increased and platelet counts decreased similar to sepsis control subjects. Because TF inhibition prolongs PT, this measure cannot be used to monitor efficacy of this treatment for sepsis-associated coagulopathy. Figure E1 and Table E1 demonstrate that blockade of TF reversed several important parameters of septic coagulopathy, including aPTT prolongation, fibrinogen depletion, and TAT complex formation. After the onset of sepsis, aPTT increased progressively (Table E1). In septic animals treated with FVIIai, this prolongation of aPTT was attenuated significantly (p < 0.01). Plasma fibrinogen levels decreased significantly over 6 hours and remained depressed in the sepsis control animals (Figure E1). In septic animals treated with FVIIai, fibrinogen levels decreased initially at t =14 hours, but after FVIIai was administered, they did not decrease further (p < 0.01 vs. sepsis control). Additional evidence that blockade of TF in sepsis attenuates dysregulated coagulation is shown by the effect of FVIIai treatment on TAT complex formation in septic animals. In the untreated septic group, TAT complexes increased significantly after onset of sepsis at 14 hours and remained elevated until the end of the experiment. In septic baboons treated with FVIIai, TAT complex levels initially increased after sepsis was initiated but subsequently decreased to control values after FVIIai treatment at 14 hours.

FVIIai Attenuated Organ Inflammation and Selectively Decreased Inflammatory Cytokine Production
In conjunction with attenuated septic organ injury in baboons treated with FVIIai, TF inhibition also significantly diminished accumulation of neutrophils in specific tissues and attenuated elaboration of selected proinflammatory cytokines. Total organ neutrophil content was estimated by measurement of MPO activity (Figure 7) . Lungs of untreated septic animals had a significantly higher neutrophil content than those from FVIIai-treated animals (36.8 ± 4.1 U/min/mg vs. 20.1 ± 1.7 U/min/mg, p <= 0.01). Similarly, kidneys from untreated animals had significantly higher MPO content than those of treated baboons (3.6 ± 1.1 U/min/mg vs. 0.4 ± 0 0.1 U/min/mg, p <= 0.01). This effect was organ specific, and tissue MPO values in the liver, small bowel, and heart were similar between the two groups.



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  		Figure 7. MPO content of the lung (A) and kidney (B) in sepsis were reduced by blockade of extrinsic coagulation with FVIIai. (A) Lungs of untreated animals had a significantly higher MPO levels than those of FVIIai-treated animals. (B) In untreated septic animals, kidney MPO values were significantly higher than those of FVIIai-treated baboons (*p = 0.01 compared with sepsis control).

 
Plasma cytokines were measured to determine the extent of the systemic inflammatory response. The data demonstrated attenuated levels of plasma IL-6 and IL-8 in sepsis when TF was blocked (all p < 0.01). As seen in Figure 8 , the predominant effect of FVIIai was to suppress the sustained elevation of these cytokines in the later stage of the experiment. After administration of live E. coli, IL-6 and IL-8 peaked 1 hour later at 14 hours in both groups. Blockade of coagulation with FVIIai at the time of peak cytokine expression (t = 14 hours) subsequently decreased plasma levels of IL-6 and IL-8 to control values within 24 hours (at t = 36 hours), whereas these cytokines remained significantly elevated in untreated baboons. Similarly, soluble TNF receptor-1 levels increased significantly in untreated baboons over 36 hours after live E. coli infusion, whereas inhibition of TF prevented sustained elevation of this cytokine (p < 0.01). The difference between the two groups (p < 0.0001) suggests that blocking TF attenuates TNF-{alpha} expression in sepsis. In contrast, blockade of TF did not diminish elaboration of the early response cytokine IL-1ß during sepsis.



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  		Figure 8. Inflammatory cytokine elaboration was diminished in sepsis with FVIIai. (A) IL-6 levels increased 1 hour (t = 14 hours) after sepsis was initiated at t = 12 hours in both groups. IL-6 remained elevated in sepsis control subjects but returned toward initial values in septic animals treated with FVIIai. (B) Similarly, IL-8 peaked early in sepsis in both groups, remained significantly elevated in the sepsis control baboons, and returned to initial values in FVIIai-treated animals. (C) IL-1ß levels were transiently elevated early in sepsis in both groups. (D) Soluble TNF receptor-1 levels increased progressively in septic baboons over 36 hours after live E. coli infusion. In FVIIai-treated animals, sustained elevation of this cytokine was attenuated. Solid circles, sepsis control group (n = 6). Open circles, sepsis + FVIIai group (n = 6) (*p < 0.05 compared with sepsis control).

 
The same cytokines were measured in BAL fluid collected at the end of the experiments. Similar to plasma values, the IL-6 levels in BAL fluid were significantly higher in the untreated septic baboons compared with FVIIai-treated animals (277.4 ± 21 vs. 82.1 + 44 pg/ml, p < 0.05). There were trends toward significantly lower IL-8, IL-1, and TNFr-1 levels in the FVIIai-treated animals. These include the following values in pg/ml for untreated sepsis versus sepsis with FVIIai: (IL-8: 1,487 ± 791 vs. 338.8 + 190, p = 0.09; IL-1: 6.3 ± 0.8 vs. 3.2 ± 0.9, p = 0.07; and TNF-r1: 298 ± 102 vs. 108.9 ± 31, p = 0.06).


   DISCUSSION
TOP
 ABSTRACT
 METHODS
 RESULTS
 DISCUSSION
REFERENCES
 
It has been established that blockade of initiation of the procoagulant response before sepsis decreases mortality in nonhuman primates. Effective strategies to block initiation of extrinsic coagulation have included use of monoclonal antibodies to TF, the natural TF pathway inhibitor, and inactive analogs of FVIIa. In a recent study in baboons, it was demonstrated that blockade of the TF-VIIa complex with FVIIai at the onset of sepsis attenuated sepsis-induced multiple organ injury and dramatically protected the lungs and kidneys (16).

This study reports for the first time that inhibiting initiation of coagulation at the time of antibiotic treatment, after the response to sepsis has been established, attenuates subsequent lung and renal injury. This protection occurs in conjunction with less elaboration of inflammatory cytokines. These important findings support current hypotheses that TF blockade could be a potentially important therapeutic strategy in established sepsis. The results argue that early institution of this type of therapy in sepsis could prevent multiple organ failure, which is an important cause of mortality. Furthermore, the data provide new insight into the importance of coagulation in perpetuating inflammation and organ dysfunction in sepsis. These results also imply that the initial inflammatory response to Gram-negative sepsis is appropriate and not deleterious to organ function and suggest that a sustained inflammatory response is a more important cause of organ injury. Although this study does not answer the important issue of whether established organ dysfunction can be reversed by blockade of TF, it suggests this as a reasonable avenue of investigation.

A recent clinical trial in human sepsis showed that therapy with activated protein C decreased absolute mortality by 6%, confirming experimental work in animals that attenuation of the coagulation response is an effective treatment strategy (19–21). The mechanisms responsible for the mortality benefit in that clinical study and in previous animal experiments have not been elucidated. Proposed mechanisms include prevention of intravascular thrombosis and attenuation of the inflammatory response. These data suggest that a common mechanism of critical organ failure in sepsis involves dysregulated coagulation.

One widely proposed mechanism whereby anticoagulant agents might attenuate the complications of sepsis is by abrogating the coagulopathy of disseminated intravascular coagulation and preventing intravascular thromboses. That microvascular obstruction contributes to organ injury in sepsis is often implied by pathological findings (24), yet the data are not totally compelling because of the low frequency of fibrin clot, particularly in the lung. Although sepsis-induced coagulopathy was modified in this study after FVIIai, some key elements were not affected by the treatment. The progressive decreases in platelet count in septic control and treated baboons suggest ongoing consumptive coagulopathy in both groups. Differences were found in fibrinogen depletion, TAT formation, and aPTT values between the groups. Because coagulopathy was not completely prevented, this implies other salutary mechanisms of TF blockade such as prevention of extravascular fibrin deposition and attenuation of inflammation by lesser cytokine responses and neutrophil migration.

The lung histology scores support the notion that extravascular fibrin deposition is an important mechanism or marker of lung injury. Although such data are limited by the low resolution of light microscopy, they do illustrate that a major effect of TF blockade was in attenuating alveolar fibrin accumulation and septal thickening. Qualitatively, septal thickening in this injury appears to be primarily caused by increased capillary cellularity, particularly with inflammatory cells. It is very interesting to note that TF blockade had specific protective effects on certain aspects of septic lung injury. These observations raise important questions about relative contributions to lung injury of the TF-FVIIa complex, downstream coagulation proteins, and fibrin.

An important therapeutic concept in support of anti-TF strategies to target organ failure involves the known cross-talk between coagulation and inflammation through the TF-VIIa complex, Xa, thrombin, and fibrin (7–9, 25). TF-VIIa has intracellular calcium-dependent signaling functions (26) and perhaps other intracellular signaling effects via phosphorylation of mitogen activated protein kinases (27, 28). These effects enhance cytokine production (29), inflammation, and may alter endothelial permeability. Factor Xa and thrombin also have potent proinflammatory effects, mediated in part by nuclear factor-{kappa}B activation (25, 30, 31). Fibrin promotes an acute cellular inflammatory response, contributes to surfactant dysfunction, and provides a scaffold for collagen deposition in the lung (32).

Potential antiinflammatory effects of TF blockade are highlighted in this study by attenuated expression of certain proinflammatory cytokines and decreased neutrophil content in two organs. The protection from septic lung and kidney injury was associated with attenuated IL-6 and TNFr levels in serum and BAL fluid and decreased MPO content of lung and kidney in septic baboons treated with FVIIai. This result indicates that organ protection achieved by blockade of TF was associated with decreased neutrophil content in these tissues and suggests important effects of TF blockade on neutrophils, for example, adherence, migration, and/or survival. An intriguing aspect of this finding is the apparent organ-specific effect of TF blockade on neutrophil content because overall adherence–migration were not affected in liver, heart, or intestines by FVIIai. It is thus tempting to speculate that TF-VIIa complex regulates cytokine or other proinflammatory pathways integrally involved in neutrophil segregation in specific tissues.

Interestingly, certain cytokines such as IL-6 are consistently lower after TF is blocked in nonhuman primates in sepsis. IL-6 levels are also lower in septic patients treated with activated protein C (22). However, it is not clear whether attenuated cytokine expression contributes to organ protection or merely indicates suppression of more fundamental inflammatory responses, such as nuclear factor-{kappa}B activation. Multiple interactions between coagulation and inflammation allow for many mechanisms whereby blocking TF-VIIa complex could influence organ injury and thus mortality in sepsis. For example, TF-VIIa blockade would also inhibit downstream activation of FX and generation of thrombin and fibrin, all of which have potential proinflammatory actions that could be important. This may be an advantage compared with downstream coagulation-based strategies that could allow TF effects to proceed. Blockade of coagulation affects endpoints differently depending on which aspects of the cascade are modified. TF expression results in further elaboration of TF in a "feed-forward loop" consistent with the notion that persistent TF expression contributes to the pro-inflammatory milieu. This work also shows that TF blockade decreases TF expression during sepsis in multiple lung cell types.

The use of therapeutic coagulation blockade in sepsis raises concerns about the potential risk of bleeding, particularly because clinically significant bleeding can occur in septic patients. Although increased bleeding has not been observed after experimental treatment with FVIIai, this study was small. FVIIai inhibits factor Xa generation on TF-bearing monocytes without effect on other hemostatic reactions, including factor Xa or thrombin generation on activated platelets (33). In addition, in the baboon, FVIIai abrogated inflammation apparently without interfering with normal hemostasis. This impression is supported by laboratory observations that the threshold for coagulation function of TF differs from its other functions (34). Thus, less activation of TF-FVIIa may be required for coagulation than for amplification of inflammation. This principle has important implications for the use of TF blockade in human patients for whom bleeding is a concern.

In summary, this study demonstrates that organ dysfunction in sepsis can be abrogated by blockade of extrinsic coagulation in established sepsis. Organ protection is pronounced in lung and kidneys and is associated with attenuated elaboration of selected proinflammatory cytokines and fewer inflammatory cells. These data suggest that progressive organ dysfunction in sepsis involves continued activity of TF or downstream proteases that promote fibrin deposition and perpetuate injurious proinflammatory pathways during sepsis.


   Acknowledgments
 
The authors thank John Patterson, Craig D. Marshall, P. Owen Doar, Eric Alford, Albert Boso, Jennifer Mele, and Lynn Tatro for technical support.


   FOOTNOTES
 
This article has an online supplement, which is accessible from this issue's table of contents online at www.atsjournals.org

Received in original form April 5, 2002; accepted in final form November 17, 2002


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 RESULTS
 DISCUSSION
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