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Single Low Dose of Human Recombinant Antithrombin (ATryn) has no Impact on Endotoxin-Induced Disseminated Intravascular Coagulation: An Experimental Randomized Open Label Controlled Study

Duburcq, Thibault∗,†; Durand, Arthur; Tournoys, Antoine; Gnemmi, Viviane; Bonner, Caroline; Gmyr, Valery; Hubert, Thomas; Pattou, François∗,§; Jourdain, Mercedes∗,†

Author Information
doi: 10.1097/SHK.0000000000001274

Abstract

BACKGROUND

Sepsis is a multiple organ dysfunction syndrome, characterized by physiological, pathological, and biochemical abnormalities induced by infection (1). Indeed, sepsis is a major public health concern responsible for considerable morbidity and mortality worldwide, if not promptly diagnosed and treated. Reducing the mortality from sepsis remains one of the most significant unmet medical needs, whereby failure of host defense homeostasis leads to systemic symptoms and organ failure. In this cascade of events, the inhibition of coagulation activation and platelet function prevails, resulting in primary and secondary hemostasis or fibrinolysis alterations. Excessive and uncontrolled systemic activation of hemostasis reactions lead disseminated intravascular coagulation (DIC) (2). Numerous studies imply that DIC is associated with microvascular thrombosis, multiple organ failure development (3), and mortality in patients with serious infections (4).

Antithrombin (AT) is a natural glycoprotein and functions as a potent anticoagulant with anti-inflammatory properties, mainly localized in the vascular compartment (5). Recent studies have shown that an early decrease in AT is observed after endotoxin or bacterial infusion (6). In humans with septic shock, AT concentrations are dramatically reduced below the normal range from 40% to 60% (7). The presence of thrombin–antithrombin complex (TAT) with clotting factors and degradation by elastase indicates ongoing thrombin formation and the consumption of AT. In patients with septic shock, a low AT III level is a strong predictor of death and multiple organ failure development (8), thus emphasizing the urgent need for supplemental AT therapeutic substitution.

There are 2 means of producing AT for supplemental therapy. First, human pooled AT (hpAT) can be obtained by purification of combined units of human plasma. Animal studies of hpAT supplementation in septic DIC have shown concordant results. High dose (> 250 U/kg) of hpAT concentrations prevent death and DIC occurrence (9, 10). However, clinical trials using hpAT in humans showed conflicting results. In heterogeneous septic shock patients, mortality remained the same, and hpAT supplementation induced morbidity from bleeding complications, particularly in patients who were given heparin (11, 12). hpAT seemed more promising in the most patients with severe DIC and after heparin cessation (13, 14). However, indication, timing, and the concentration level of AT supplementation remain in question (15). The second source of AT III comes from recent developments in genetic engineering that enabled the production of human recombinant AT (rhAT) in milk of genetically modified goats (16). In experimental studies, rhAT was shown to possess similar anti-inflammatory properties to natural plasmatic AT (17, 18), which may be superior to hpAT (19). These studies used models of Pseudomonas aeruginosa-induced acute lung injury (20, 21) and burn and smoke inhalation injury (22). Only 1 animal study measured the effects of rhAT on sepsis-induced DIC in a pretreatment manner with high dose of rhAT (23). Similarly, the clinical efficacy of rhAT was only evaluated in healthy male volunteers submitted to an endotoxin infusion immediately after receiving a high dose of rhAT (24). Endotoxemia represents a reproducible model of acute inflammation, leading to DIC occurrence, but the physiopathological mechanism with human sepsis may differ. Finally, these promising results with high dose and/or pretreatment protocol are not compatible with clinical practice.

Thus, we hypothesized that the subsequent infusion of a lower dose of rhAT could reduce DIC occurrence and severity in a model of pigs submitted to an endotoxin challenge. In parallel, we studied inflammation profile, hemodynamics, and microcirculatory parameters.

MATERIALS AND METHODS

We performed a randomized, controlled, experimental study. The experimental protocol (CEEA n°132012) received the approval of the Nord-Pas-de-Calais Animal Ethics Committee (Comité d’Ethique en Expérimentation Animale Nord-Pas-de-Calais; C2EA–75) and the French Ministry of Education and Research. Care and handling of the animals were in accordance with the experimental animal use guidelines of the French Ministry of Agriculture and Food.

Animal preparation

All animal procedures were performed in the Department of Experimental Research of the Lille University, Lille, France. Animals were premedicated with intramuscular injection of ketamine (Kétalar, Virbac, France; 2.5 mg/kg of body weight) and xylazine (Sédaxylan, CEVA Santé Animale, France; 2.5 mg/kg of body weight). Isoflurane (Aerrane, Baxter, France) was used for the intubation process, and maintenance of anesthesia was performed with a continuous infusion of midazolam (Hypnovel, Roche, France; 1 mg/kg body weight/h) for the whole experiment. All animals were mechanically ventilated (Osiris 2, Taema, France) with a tidal volume of 8 mL/kg, a positive end-expiratory pressure set at 4 cmH2O to limit cardiovascular effects, FiO2 0.6 to prevent fatal hypoxemia during the study, and respiratory rate 20 to 24 breaths/min only adjusted to maintain normocapnia (40–45 mmHg) at baseline. We chose to maintain ventilation similarly in all animals during the experiment. No recruitment maneuvers were done. Muscle relaxation was obtained by a continuous i.v. infusion of cisatracurium besylate (Nimbex, Hospira, France; 2 mg/kg body weight/h). Analgesia was achieved by a s.c. injection of buprenorphine (Vetergesic, Sogeval, France; 0.1 mg/kg body weight). After dissection of neck vessels, catheters were inserted in the pulmonary artery via the right external jugular vein (Swan-Ganz; Baxter 110 H 7.5F; Baxter Edwards Critical Care, Irvine, CA) and in the right carotid artery for continuous blood pressure monitoring and blood sampling.

Study design

Ten female “Large White” pigs (2 months old) were used in this study. The study was carried out as depicted in Figure 1. During the preparation period, animals received 25 mL/kg 0.9% NaCl, to prevent hypovolemia. Hemodynamic measurements were taken over a 5-h period: at baseline (T) after the stabilization period (T0) and at 30 (T30), 60 (T60), 120 (T120), 210 (T210), and 300 (T300) min. All animals were administered 5 μg/kg/min E. coli lipopolysaccharide (LPS) (serotype 055:B5; Sigma Chemical Co., St. Louis, MO). The endotoxin was diluted in 50 mL of 0.9% NaCl and infused over a 30-min period (from T0 to T30) intravenously. Two groups of 5 animals were studied: control group (NC group) using 0.9% NaCl and ATryn group using rhAT (single low dose of 100 U/kg). We used a low dose of rhAT to avoid hemorrhagic risk and to allow clinical application, if appropriate. Based on the previous work in the same model (6), we chose the same single low dose of 100 U/kg. ATryn (a gift from rEVO Biologics, Framingham, MA) powder (7 U/mg) was dissolved just before administration in solution distilled water and infused over a 30-min period (from T30 to T60) intravenously. In the 2 groups, animals received 5 mL/kg/h (from T60 to T300) of 0.9% NaCl. The only resuscitation endpoint was mean arterial pressure (MAP). If MAP felt below 65 mmHg, 2.5 mL/kg infusion of NaCl 0.9% was given as rescue therapy every 15 min. Bolus infusions were performed to maintain MAP above 65 mmHg as recommended by Sepsis Surviving Campaign (25). At the end of the study period, all animals were sacrificed with T61 administration (T61, 0.3 mL/kg of body weight; Intervet International GmbH, Köln, Germany).

F1
Fig. 1:
Study design.

Macrocirculatory and oxygenation parameters

Invasive arterial and pulmonary catheters are routine monitoring in our model. These parameters are necessary in our resuscitation protocol (invasive MAP). Pulmonary pressure is also used to assed the severity of LPS-induced shock, as the increase of pulmonary systolic pressure during LPS infusion is correlated with endotoxic shock severity.

Heart rate, systemic, and pulmonary arterial pressures were continuously monitored (90308 PC Express Portable Monitor, Spacelabs, Snoqualmie, WA) as well as cardiac output and mixed venous oxygen saturation (Vigilance monitor; Baxter Edwards, Irvine, CA). Systemic arterial and venous blood samples from carotid and pulmonary arteries were obtained simultaneously. Arterial and venous blood gas tensions and lactate levels were measured in an acid–base analyzer (ABL-800, Radiometer, Copenhagen, Denmark). Using a standard formula, we computed global oxygen delivery (DO2), global oxygen consumption (VO2), oxygen extraction ratio (OER), cardiac index (CI) (L/min/m2), systolic index (mL/bpm/m2), systemic vascular resistance (SVR), and pulmonary vascular resistance (PVR; dynes/s/cm5). Body surface area was calculated by Kelley's formula (26).

Microcirculatory parameters

Microcirculatory assessment is a well-established technique in our institution. As microcirculatory dysfunction is closely related to coagulation and inflammation pathways (27), we consequently selected those parameters to basal monitoring in our model.

Skin microvascular blood flow was measured continuously using a laser Doppler flowmeter probe and device (Periflux PF407; Perimed, Järfälla, Sweden). The fiber optic probe was applied on the left hind paw of the animals. Skin blood flow was measured at rest and during reactive hyperemia. Reactive hyperemia was further analyzed (Perisoft 2.5 software, Perimed, Järfälla, Sweden) according to its duration and initial reactive hyperemia uphill slope. Basal tissue oxygen saturation (StO2) was evaluated using a near-infrared spectrometer (InSpectra 850 model, Hutchinson Technology, Hutchinson, MN), with a probe attached on pectoral skin area. A vascular occlusion test was not possible because the probe was too large and could not be applied on a paw. Images of the sublingual microcirculation were obtained with sidestream dark field (SDF) videomicroscopy (Microscan; Microvision Medical, Amsterdam, The Netherlands). Assessment of microcirculatory parameters of convective oxygen transport (microvascular flow index, MFI) was made in parallel, in an offline evaluation, blinded from group treatment.

Biological methods and histological analysis

In the absence of specific porcine DIC definition, and although human DIC definition is debated, we chose to refer to the 2001 International Society on Thrombosis and Haemostasis definition (28). Nevertheless, because of different basal values between human and pig model, and the discussion about the cutoff values of coagulation tests, kinetic of classic parameters (platelets, fibrinogen, prothrombin time, and fibrin monomer) was used to define endotoxin pig DIC. No cutoff values could be used to exactly define DIC in our model. Finally, DIC occurrence was confirmed by histological analysis and the presence of microvasculature thrombosis.

Biologic measurements were taken at T0, T60, T120, T210, and T300. Leukocyte, hemoglobin, and platelet counts were obtained on ethylene diamine triacetic acid anticoagulated blood. For coagulation assays, blood (4 parts) was collected in tubes containing 3.8% sodium citrate (1 part). Prothrombin time and fibrinogen levels were rapidly measured by standard procedures. Immunoassay methods were used to determine quantitative TAT (Enzygnost TAT micro, Siemens, Munich, Germany). Fibrin monomer (Liatest FM Stago, Asnières-sur-Seine, France) was performed by immunoturbidimetric assay. Quantitative AT activity was determined by chromogenic assay (Berichrom AT; Siemens, Munich, Germany). Considering rhAT anti-inflammatory effects could be superior than hpAT, inflammatory parameters were studies to act as a substantial effect of rhAT on LPS-induced pro-inflammatory response. Tumor necrosis factor-α (TNFα) and interleukin 6 (IL-6) were detected by ELISA assays with porcine anti-TNFα antibodies (Quantikine Porcine TNFα, R&D Systems, Minneapolis, MN) and anti-IL-6 antibodies (Quantikine Porcine IL-6, R&D Systems,). The quantitative determination of von Willebrand factor (vWF) antigen (Ag) was measured by turbidimetric assay (vWF Ag Reagent, Siemens, The Nederland) (n = 70%–100%).

At the end of the experiment and immediately after euthanasia, kidneys were withdrawn for histological analysis. Kidney samples were fixed with acidified formal alcohol (AFA) and/or frozen by liquid nitrogen and stored at −80°C. The samples fixed in AFA were embedded in paraffin and sectioned (3–4 μm width). After deparaffinization and rehydration, sections were stained with Masson's trichrome, Periodic Acid Schiff, and Hematoxylin Eosin Safran and evaluated in light microscopy. Frozen tissue cryosections of 5 μm were cut on a cryostat, and incubated 30 min with an antifibrinogen antibody directly conjugated with fluorescein for a direct immunofluorescence using polyclonal rabbit antibody, which recognized fibrinogen (ref. F0111, Dako SA, Trappes, France). We established 2 semiquantitative scores for histologic abnormalities defined as: Score (%) = (number of glomeruli damaged)/(number of glomeruli examined) and (number of capillary sections thrombosed)/(number of capillary sections examined) per damaged glomeruli. At least 50 glomeruli were observed in each sample. The pathologist was blinded to the groups examined.

Data analysis

Statistical analysis was performed with GraphPad Prism 6 software (San Diego, CA). As the distribution was not normal (Shapiro–Wilk test), quantitative data were expressed using median and interquartile range. For multiple intergroup testing, we used Kruskal–Wallis test with Dunn's multiple comparisons test and Mann–Whitney U test. Intragroup comparisons were realized by Friedman test with Dunn's multiple comparisons test and Wilcoxon matched-pairs signed rank test. The 2-tailed significance level was set at P < 0.05.

RESULTS

Median weight was similar in ATryn group 23 (22.5–24.5) kg and control group 22.5 (18.25–23.75) kg (P = 0.33). No animals died within the 5-hour experiments. No macroscopic hemorrhagic complications were observed in both groups.

Effects of supplementation on AT

First we verified that the dosage regimen of AT supplementation was sufficient to achieve circulating supranormal AT plasma levels (Fig. 2). In the ATryn group, the AT activity increased from 106% (100–116) to 213% (203–223) (P < 0.001) just after infusion and then regularly declined to reach 115% (94–124) at the end of the study period (P = 0.62 compared to baseline). Conversely, in the control group, AT III activity progressively decreased until the end of the study period from 105% (98–113) to 79% (67–93) (P = 0.12).

F2
Fig. 2:
Changes in antithrombin level.

Macrocirculatory and oxygenation parameters

No changes in the studied variables were observed during stabilization period. In all animals, endotoxin infusion resulted in a progressive decrease in CI, SvO2, and DO2 from 0 to 300 min. MAP and SPAP increased just after the end of endotoxin administration and then decreased at 60 min. From 120 to 300 min, we observed a progressive rise in SVR, SPAP, PVR, OER, and lactate levels. These results were consistent with the constitution of a severe endotoxin-induced hypodynamic shock as is usually described in the pig model when a bolus infusion of endotoxin is administered. Compared to control group, the animals receiving ATryn presented the same hypokinetic state, and no significant differences were seen in the different studied variables (Fig. 3 and Supplemental Figure 1, https://links.lww.com/SHK/A823). The 2 groups received the same amount of vascular filling during the experiment (500 mL [450–1125 mL] in control group and 450 mL [450–800 mL] in ATryn group; P = 0.41). Three (2 in control group and 1 in ATryn group) animals required 0.9% NaCl boluses to maintain MAP ≥ 65 mmHg according to study protocol. Inspired oxygen fraction ratio (PaO2/FiO2) decreased significantly in the 2 groups (P < 0.05 at T300 compared to baseline). This decrease was nonsignificantly less pronounced in ATryn group. No significant difference on acid–base status was observed between groups. Nevertheless, due to a less increase in partial pressure of carbon dioxide, ATryn group developed a nonsignificant less severe acidosis (pH 7.36 [7.28–7.44] at T300) compared to control group (pH 7.21 [7.17–7.30]; P = 0.05) (Supplemental Figure 2, https://links.lww.com/SHK/A824).

F3
Fig. 3:
Changes in heart rate, mean arterial pressure, cardiac index, systolic pulmonary arterial pressure, mixed venous oxygen saturation, and lactate level.

Microcirculatory parameters

Skin microvascular blood flow (rest and peak flow), StO2, and sublingual MFI score decreased significantly in both groups (P < 0.05 compared to baseline, respectively) without any significant difference between groups (Supplemental Figure 3, https://links.lww.com/SHK/A825).

Coagulation assays

Significant sequential changes characteristic of an early activation of coagulation were observed in all animals (Fig. 4). Similarly, in the 2 groups white blood cell counts decreased immediately after endotoxin infusion (−78% in control group and −66% in ATryn group at 60 min), with the nadir at 210 min. Because of the endotoxin-induced capillary leak, we observed a progressive hemoconcentration in all animals without any significant difference between groups. The prothrombin time decreased similarly in both groups without significant difference. Circulating platelets progressively declined during the whole experiment in all animals (454 g/L [371–502 g/L] at T0 vs. 170 g/L [112–223 g/L]; P < 0.0001). In the same way, we observed a significant decline in circulating fibrinogen in all animals (2 g/L [1.8–2.2 g/L] at T0 vs. 1 g/L [0.7–1.6 g/L] at T300; P = 0.0014). Fibrin monomer increased similarly from 60 to 120 min in both groups (104 μg/mL [52.5–150 μg/mL] in control group and 94 μg/mL [66–150 μg/mL] in ATryn group; P > 0.99). Unfortunately, we could not interpret the results at 210 and 300 min in the 2 groups because some values were over 150 μg/mL, the upper limit of measurement, despite dilutions. TAT concentration started to increase at 60 min to achieve a maximum level at 210 min in control group (P < 0.001 compared to baseline) and 300 min in ATryn group (P < 0.01 compared to baseline). vWF increased in all animals and peaked at 120 min. Overall and compared with the control group, ATryn administration failed to induce any significant differences in levels of leukocytes, platelets, fibrinogen, TAT, and vWF.

F4
Fig. 4:
Changes in leucocyte, hemoglobin, platelet count, fibrinogen, prothrombin time, fibrin monomer, TAT and vWF.

Cytokine levels

Changes in IL-6 and TNFα are illustrated in Figure 5. We observed a same evolution of TNFα levels in the 2 groups without any significant differences. TNFα increased rapidly, peaked at 60 min (129700 pg/mL [92409–139212 pg/mL] in control group vs. 168055 pg/mL [124384–186971 pg/mL] in ATryn group; P = 0.15), and subsequently decreased until the end of the experiment without returning to baseline levels. In control group, IL-6 increased significantly from 60 to 300 min (P < 0.01) whereas, in ATryn group, IL-6 increased significantly from 60 to 210 min (P < 0.001) and then decreased at 300 min (9033 pg/mL [6277–16011 pg/mL] vs. 19270 pg/mL [9544–39229 pg/mL] in control group; P = 0.07).

F5
Fig. 5:
Changes in IL-6 and TNFα.

Histological results

Kidneys appeared macroscopically enlarged and swollen in both groups. Glomeruli showed signs of edema uniformly, and fibrin thrombi were mainly observed in glomerular capillaries. Percentage of thrombosed glomeruli and percentage of thrombosed capillary in glomerulus were not significantly different between groups (Table 1).

T1
Table 1:
Histological scores

Histological scores: Percentage of thrombosed glomeruli (%) = (number of glomeruli damaged)/(number of glomeruli examined) × 100. Percentage of capillary sections thrombosed (%) = (number of capillary sections thrombosed)/(number of capillary sections examined) per damaged glomeruli × 100. Results are expressed as median with interquartile ranges. Mann–Whitney U test were used for intergroup comparisons.

DISCUSSION

In this study, we report that AT supplementation with a single low dose of rhAT (ATryn) did not restore hemostasis impairment of DIC course nor hemodynamics or inflammation alterations. Pigs were chosen as a clinically relevant species, resembling humans in coagulation reactions (29). In our experiment, administration of endotoxin resulted in an activation of coagulation pathways and in a severe and progressive decrease of AT III activity. The coagulation activation was reflected by the increase in TAT complexes, vWF release, and the decrease in prothrombin time, platelets, and fibrinogen concentrations, characterizing septic DIC process. Inflammation process seems also to be involved with a stimulation of pro-inflammatory cytokines (IL-6, TNFα). All these elements lead to organ failure as shown by CI impairment, lactate increase, and microcirculatory dysfunction.

AT III is a vitamin K-independent glycoprotein, which inhibits coagulation process by neutralization of thrombin and other serine proteases by forming a 1:1 stoichiometric TAT complex. In our model, rhAT with a single dose of 100 U/kg bolus did not prevent DIC occurrence or hemostasis impairment course. While AT III activity remains over 100% only in ATryn group, the decrease of prothrombin time, platelet count, and fibrinogen remains the same in both groups. Our results were concordant to Fourrier et al.(6), using the same experimental model. The authors did not find any improvement in the coagulation and fibrinolysis parameters with hpAT (100 U/kg) alone or with hpAT and protein C combined therapy (6). According to our observations, a single low dose of rhAT does not seem to represent a more efficient drug than hpAT in the prevention or correction of endotoxin-induced DIC. By contrast, Dickneite and Kroez (30) demonstrated promising results of higher doses (250 U/kg) and continuous infusion of hpAT in porcine endotoxic shock, but hemostasis alteration was poorly monitored. In fact, the effect of the higher dose of hpAT on coagulation alteration was better studied in endotoxic rats. In these models, obtaining an AT III activity over 100% resulted in a less significant decrease of platelets or fibrinogen (31). Minnema et al.(23) also showed the modest effect of a higher dose (>250 U/kg) of rhAT on hemostasis impairment in a lethally challenged with E. coli baboon model. Timing of AT III administration could be an explanation for these apparently discordant results, indeed Minnema et al.(23) used in their model a pretreatment with rhAT.

Along with its anticoagulant activity, AT exerts anti-inflammatory effects, implicating several mechanisms. Inhibition of pro-inflammatory cytokines (IL-6, TNFα) production can be due to binding of AT to endothelial cell surfaces, or inhibition of thrombin and factor Xa formation (32). Induction of prostacyclin synthesis or binding to syndecan-4 receptor also inhibits leucocyte activation, adherence, and chemotaxis (33). First, indirect evaluation of inflammation can be assessed, in our model, by a slightly better PaO2/FiO2 ratio in ATryn group, assuming a lower destruction of alveolar–capillary barrier. Concordant experimental results had showed that hpAT supplementation could decrease pulmonary inflammation, with fewer neutrophil accumulation, alveolar septal thickening, and neutrophil extracellular trap formation, resulting in a better efficiency of lung function (10). In an ovine model of pneumonia and smoke inhalation Murakami et al.(20) reported similar interesting results. Posttreatment with similar dose regimen (100 U/kg of continuous infusion over 24 h) of rhAT was effective in treating acute lung injury. Nevière et al.(33) observations suggest that rhAT reduces endothelium–leukocyte interactions in a high-dose regimen (500 U/kg) by interacting with local prostacyclin production. In contrast to Nevière et al.(33) and Hoffmann et al.(9) we did not find a reversal of microcirculatory dysfunction in the AT group. Microcirculatory flow alteration evaluated with sublingual SDF imaging analysis or indirect parameters (StO2, P(v,a)CO2) was not significantly different in the 2 groups. However, both parameters did not evaluate endothelium–leucocyte interaction, which could not be assessed from our results. Second, direct evaluation of inflammation profile was modestly modified by rhAT supplementation. We observed a nonstatistically significant reduction of IL-6 in ATryn group at T300 (P = 0.07), whereas TNFα increase was the same in both groups. This observation could be highlighted with previous experimental data using AT higher dose. A modulation of cytokine profile with a decrease of pro-inflammatory cytokines burst was observed with a preconditioning strategy of high dose rhAT supplementation in a baboon septic model (23). Postconditioning with rhAT, to increase AT levels to 200% and 500%, also result in a blockade of IL-6 formation, inhibiting peak of IL-6 release in a human endotoxemia (24). AT supplementation with supranormal activities are mandatory to exert anti-inflammatory activities as a dose-dependent manner (34). Thus, most of recent experimental data are claimed for higher dose or continuous infusion to induce anti-inflammatory properties. Furthermore, while the anti-inflammatory activity of AT seems to be independent from its anticoagulant function, recent data demonstrate that the effects of AT for the adequate mediation of anticoagulation crucially depend on its anti-inflammatory mode of action (35). As a consequence, in our study, a single low dose of 100 U/kg rhAT without continuous infusion may have been insufficient to exert anti-inflammatory effects and prevent DIC occurrence. Nevertheless, most probing observations results of supratherapeutic doses of AT. Such resulting AT activity could not be obtained in human sepsis without a major risk of bleeding adverse effects.

Our model presented several limits. First, in the control group, the AT levels did not drop to the 40% to 60% of normal value as observed in human septic shock. This lack of severity in AT III deficit may have reduced the impact of rhAT supplementation. Second, endotoxic model is not a model of hyperdynamic septic shock. Our model is a hypodynamic shock with a pronounced pulmonary vascular response and an increased right ventricular afterload. The peritonitis-induced septic shock model would probably be more appropriate considering the pathophysiological mechanisms involved in human septic DIC. However, endotoxin reproduces some of the main alterations of inflammatory and pro-coagulant state of sepsis, e.g., DIC occurrence, pro-inflammatory burst, macrocirculatory, and microcirculatory dysfunction. Third, we used the i.v. route for sepsis induction while patients are often infected by natural route. The temporal evolution of the aggression was imposed when the pathological process of patients following an individual natural progression. The endotoxin challenge is responsible for a more explosive pro-inflammatory response than in a septic shock. These limits, without questioning the validity of our pathophysiological model, could impair the comparability with DIC observed in human septic shock. Finally, our study was underpowered because a single low dose of rhAT did not reduce DIC severity. The power achieved with the main coagulation parameters was around 50%. For a conventional statistical power of 80%, an unethical sample of 13 to 26 animals per group would have been necessary.

CONCLUSION

In our model of endotoxic shock, a single low dose of 100 U/kg rhAT did not improve DIC occurrence, hemostasis impairment, inflammatory profile, nor hemodynamic alterations. Further investigations are warranted to assess the potential experimental benefits of higher dose or continuous administration of rhAT in this model.

Acknowledgments

The authors acknowledge Dr Yann Echelard, rEVO Biologics Inc., Framingham, Massachusetts, USA, for providing ATryn.

We thank all the Department staff of Experimental Research of the Lille University, Lille, France for the technical support.

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Keywords:

Antithrombin; disseminated intravascular coagulation; hemodynamics; inflammation; microcirculation; septic shock; AT; Antithrombin; hpAT; human pooled antithrombin; rhAT; human recombinant antithrobin; DIC; Disseminated Intravascular Coagulation; MAP; Mean Arterial Pressure; TAT; Thrombin-Anti-Thrombin complex; vWF; von Willebrand Factor; TNFα; Tumor Necrosis Factor alpha; IL-6; Interleukin 6; E.coli; Escherichia coli; LPS; Lipopolysaccharide

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