Recombinant Human Antithrombin Research Articles

A randomized phase 3 trial evaluating antithrombin gamma treatment in Japanese patients with early-onset severe preeclampsia (KOUNO-TORI study): Study protocol

IntroductionPreeclampsia is a leading cause of maternal and perinatal morbidity and mortality. Several studies have demonstrated the beneficial effects of antithrombin replacement in patients with preeclampsia. Here, we describe the study protocol of KOUNO-TORI (KW-3357 randOmized, mUlti-center, double-bliNd, placebO-controlled phase 3 sTudy in patients with early Onset pReeclampsIa) to evaluate recombinant human antithrombin gamma (rhAT-gamma) for the treatment of early-onset severe de novo preeclampsia. Material and methodsPatients with early-onset severe de novo preeclampsia who are ≥24 to <32 weeks pregnant at the time of registration and have an antithrombin activity of ≤100% at screening are included. The target population is selected based on a reanalysis of the data of a previous plasma-derived antithrombin phase 3 study. Primary endpoint is the prolongation of pregnancy from the initiation of rhAT-gamma treatment to the pregnancy termination. Secondary endpoints include gestational age in terms of achievement of 32- and 34-weeks'gestation, and gestational age in terms of achievement of 28 weeks' gestation for patients enrolled at <28 weeks' gestation. Maternal, fetal, and neonatal outcomes will be assessed. DiscussionAs we have selected a specifically defined target population based on reanalysis of data of a previous plasma-derived antithrombin phase 3 study, the results of our study are expected to provide efficacy and safety data concerning rhAT-gamma treatment in Japanese patients. This study could help identify an effective novel treatment for such patients with early-onset severe preeclampsia for whom appropriate treatment is unavailable.

Focus on coronavirus disease 2019 associated coagulopathy.

Focus on coronavirus disease 2019 associated coagulopathy.

Prospective, randomized, double-blind, placebo-controlled evaluation of the Pharmacokinetics, Safety and Efficacy of Recombinant Antithrombin Versus Placebo in Preterm Preeclampsia

Despite expectant management, preeclampsia remote from term usually results in preterm delivery. Antithrombin, which displays antiinflammatory and anticoagulant properties, may have a therapeutic role in treating preterm preeclampsia, a disorder characterized by endothelial dysfunction, inflammation, and activation of the coagulation system. This randomized, placebo-controlled clinical trial aimed to evaluate whether intravenous recombinant human antithrombin could prolong gestation and therefore improve maternal and fetal outcomes. We performed a double-blind, placebo-controlled trial at 23 hospitals. Women were eligible if they had a singleton pregnancy, early-onset or superimposed preeclampsia at 23 0/7 to 30 0/7 weeks' gestation, and planned expectant management. In addition to standard therapy, patients were randomized to receive either recombinant human antithrombin 250 mg loading dose followed by a continuous infusion of 2000 mg per 24 hours or an identical saline infusion until delivery. The primary outcome was days gained from randomization until delivery. The secondary outcome was composite neonatal morbidity score. A total of 120 women were randomized. There was no difference in median gestational age at enrollment (27.3 weeks' gestation for the recombinant human antithrombin group [range, 23.1-30.0] and 27.6 weeks' gestation for the placebo group [range, 23.0-30.0]; P=.67). There were no differences in median increase in days gained (5.0 in the recombinant human antithrombin group [range, 0-75] and 6.0 for the placebo group [range, 0-85]; P=.95). There were no differences between groups in composite neonatal morbidity scores or in maternal complications. No safety issues related to recombinant human antithrombin were noted in this study, despite the achievement of supraphysiological antithrombin concentrations. The administration of recombinant human antithrombin in preterm preeclampsia neither prolonged pregnancy nor improved neonatal or maternal outcomes.

Thromboprophylaxis with antithrombin gamma and unfractionated heparin for a pregnant woman complicated by hereditary antithrombin deficiency.

Antithrombin replacement using recombinant human antithrombin (antithrombin gamma) is a well‐tolerated treatment and effective as thromboprophylaxis for a pregnant woman with hereditary antithrombin deficiency.

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

Antithrombin (AT) III physiological levels are decreased during septic shock and supplementation therapy could therefore be beneficial. We hypothesized that the use of recombinant human AT could reduce disseminated intravascular coagulation (DIC) occurrence. We conducted a randomized open label controlled experimental study. Ten female "Large White" pigs were challenged with i.v. infusion of Escherichia coli endotoxin. Two groups of 5 pigs were randomly assigned to receive either recombinant human AT 100 U/kg over 30 min (ATryn group) or 0.9% saline (control group). AT III levels, coagulation, hemostasis, inflammation parameters, hemodynamics, and microcirculatory parameters were measured over a 5-h period. Immediately after euthanasia, kidneys were withdrawn for histology evaluation. Statistical analysis was performed with nonparametric tests and Dunn's test for multiple comparisons. AT III activity was significantly higher in the ATryn group than in the control group from 60% (213% [203-223] vs. 104% [98-115], P = 0.008, respectively) to 300 min (115% [95-124] vs. 79% [67-93], P = 0.03). Recombinant human AT supplementation had no impact on hemodynamics, microcirculatory parameters, and sequential changes of coagulation parameters (platelet count, fibrinogen level, thrombin-AT complexes, and von Willebrand factor). Interleukin 6 and tumor necrosis factor α values were statistically the same for both groups throughout the study. Percentage of thrombosed glomeruli and percentage of thrombosed capillary in glomerulus were not significantly different between both groups. In our model of endotoxic shock, a single low dose of recombinant human AT did not prevent DIC occurrence, severity, inflammatory profile, or hemodynamic alterations.

A Comparison of the Oligosaccharide Structures of Antithrombin Derived from Plasma and Recombinant Using POTELLIGENT® Technology

Human antithrombin (AT) has two isoforms of which the predominant α-form is glycosylated on all four possible glycosylation sites and the lower abundant β-isoform lacks the oligosaccharide on Asn135. The main oligosaccharide structure of human AT consists of biantennary complex-type oligosaccharides lacking a core fucose. Generally, Chinese hamster ovary (CHO) cells produce recombinant human AT (rhAT) with core-fucosylated oligosaccharides. However, rhAT lacking core-fucose oligosaccharides can be produced by POTELLIGENT® technology, which uses FUT8 knockout CHO cells in production. The rhAT has more variable glycan structures, such as tetra-antennary complex type, high-mannose type, and mannose 6-phosphate species as minor components compared to plasma-derived human AT (phAT). In addition, the site-specific glycan profile was different between two ATs. We evaluated the effect of these properties on efficacy and safety based on a comparison of rhAT made by that technology with phAT in terms of their respective oligosaccharide structures, site-specific oligosaccharide profiles, and the ratio of α- and β-forms. Although some structural differences were found between the rhAT and phAT, we concluded that these differences have no significant effect on the efficacy and safety of rhAT.

Isolation of recombinant human antithrombin isoforms by Cellufine Sulfate affinity chromatography

Human antithrombin (hAT) is a major serine protease inhibitor that regulates blood coagulation in human plasma, and it has been applied for the treatment of antithrombin (AT) deficiency and disseminated intravascular coagulation (DIC). In the past, hAT for therapeutic use has been obtained from human plasma; however, hAT can now be sourced from transgenic animals and Chinese hamster ovary (CHO) cells by recombinant technology. The dominant form of hAT in plasma is the α form, which is glycosylated with four oligosaccharides and sialylated at its terminals. However, it would be preferable to remove the poorly sialylated α form of hAT, the minor β form with unoccupied glycosylation sites, and the inactive forms. Cellufine Sulfate, a heparin-mimic affinity resin made of cellulose and modified with sulfate groups, has an affinity for heparin-binding proteins and can be used in cation exchange chromatography. Based on these properties, the α and β forms of hAT could be separated and purified depending on the rate of sialylation. Consequently, Cellufine Sulfate was used to enrich the highly sialylated α form with a considerably higher step yield than that obtained using conventional heparin-immobilized resins. Furthermore, subsequent hydrophobic interaction chromatography could eliminate the inactive forms. These results suggested that Cellufine Sulfate was more effective than heparin-immobilized resins in purifying the highly sialylated α form of hAT for therapeutic applications.

A summary of the Japan septic disseminated intravascular coagulation study.

Over the past few decades, the large, international, randomized controlled trials of anticoagulant therapies for patients with sepsis have not yielded any improvement in mortality rates. However, in Japan, anticoagulant therapies are administered for sepsis patients with disseminated intravascular coagulation (DIC), but not for sepsis patients without DIC. Furthermore, epidemiological data regarding sepsis in Japan are scarce. Therefore, a nationwide multicenter retrospective observational study, the Japan Septic Disseminated Intravascular Coagulation (JSEPTIC DIC) study, was undertaken. The JSEPTIC DIC study enrolled 42 intensive care units and included 3,195 patients with sepsis. The results of the JSEPTIC DIC study indicated the following: (i) anticoagulant therapy may be effective in sepsis‐induced DIC patients at high risk for death, (ii) recombinant human soluble thrombomodulin administration and antithrombin supplementation are associated with survival benefits in patients with sepsis‐induced DIC.

Human Recombinant Antithrombin (ATryn®) Administration Improves Survival and Prevents Intravascular Coagulation after Intraportal Islet Transplantation in a Piglet Model

Human islet transplantation is a viable treatment option for type 1 diabetes mellitus (T1DM). However, pancreatic islet inflammation after transplantation induced by innate immune responses is likely to hinder graft function. This is mediated by incompatibility between islets and the blood interface, known as instant blood-mediated inflammatory reaction (IBMIR). Herein we hypothesized that portal venous administration of islet cells with human recombinant antithrombin (ATryn ® ), a serine protease inhibitor (serpin), which plays a central role in the physiological regulation of coagulation and exerts indirect anti-inflammatory activities, may offset coagulation abnormalities such as disseminated intravascular coagulation (DIC) and IBMIR. The current prospective, randomized experiment was conducted using an established preclinical pig model. Three groups were constituted for digested pancreatic tissue transplantation (0.15 ml/kg): control, NaCl 0.9% (n = 7); gold standard, heparin (25 UI/kg) (n = 7); and human recombinant ATryn® (500 UI/kg) (n = 7). Blood samples were collected over time (T0 to 24 h), and biochemical, coagulation, and inflammatory parameters were evaluated. In both the control and heparin groups, one animal died after a portal thrombosis, while no deaths occurred in the ATryn®-treated group. As expected, islet transplantation was associated with an increase in plasma IL-6 or TNF-α levels in all three groups. However, DIC was only observed in the control group, an effect that was suppressed after ATryn® administration. ATryn® administration increased antithrombin activity by 800%, which remained at 200% for the remaining period of the study, without any hemorrhagic complications. These studies suggest that coadministration of ATryn® and pancreatic islets via intraportal transplantation may be a valuable therapeutic approach for DIC without risk for islets and subjects.

Production, purification and characterization of recombinant human antithrombin III by Saccharomyces cerevisiae

Background: Antithrombin III (ATIII) is a protein that inhibits abnormal blood clots (or coagulation) by breaking down thrombin and factor Xa. ATIII helps to keep a healthy balance between hemorrhage and coagulation. The present work demonstrated the production, purification and characterization of recombinant human antithrombin (rhAT) from yeast Saccharomyces cerevisiae BY4741 was demonstrated. After expression of rhAT by S. cerevisiae , the biomass and rhAT concentration were analyzed through fed-batch fermentation process. Results: In fed-batch fermentation, the biomass (maximum cell dry weight of 11.2 g/L) and rhAT concentration (312 mg/L) of the expressed rhAT were achieved at 84 h of cultivation time. The maximum cell lysis efficiency (99.89%) was found at 8 s sonication pulse and 7 mL lysis buffer volume. The rhAT protein solution was concentrated and partially purified using cross-flow filtration with the recovery yield and purity of 95 and 94%, respectively. The concentrated solution was further purified by the single step ion exchange chromatography with the recovery yield and purity of 55 and ≥ 98%, respectively. The purified rhAT was characterized by various analytical techniques, such as RP-HPLC, FT-IR, CD, SDS-PAGE, western blotting, and Liquid chromatography mass spectrometry (LC-MS) analysis. The biological activity of rhAT was analyzed as heparin cofactor to meet the therapeutic grade applications. Conclusions: The simple, cost-effective and economically viable nature of the process used in the present study for the production of rhAT will be highly beneficial for the healthcare sector. This may also be used to produce other value-added therapeutic recombinant proteins expressed in S. cerevisiae , with greater effectiveness and ease.

Efficient single step chromatographic purification of recombinant human antithrombin (rhAT) from Saccharomyces cerevisiae.

Antithrombin (AT) is a glycoprotein that inactivates the several physiological target enzymes of coagulation system. The effect of purification strategies plays a crucial role in getting maximum recovery of yield, purity and biological activity of recombinant human antithrombin (rhAT). In the present work, the task of purifying rhAT from Saccharomyces cerevisiae BY4741 has been carried out using two different approaches such as cross flow filtration (CFF) system and chromatography methods. In the first approach, the protein was concentrated and partially purified through CFF to achieve maximum recovery yield and purity of 87 and 94 %, respectively. In the second approach, purification involved a single step chromatography with various types of ion exchange and size exclusion resins to analyze the maximum rhAT recovery yield and purity. From the experimental results, it has been observed that the size exclusion chromatography (SEC) technique with Superose 12 matrix was suitable for the purification of rhAT and achieved the maximum recovery yield and purity of 51 and 97 %, respectively. Further, to acquire a high recovery yield and purity of rhAT, the effect of various chromatographic conditions such as mobile phase, mobile phase pH, flow rate, sample volume and sample concentration were also investigated. Under the optimal chromatographic conditions, rhAT was significantly recovered and purified in a single step with maximum recovery yield, purity and biological activity of 67, 99 % and 410 IU/L, respectively. Based on these investigations, it was concluded that SEC with Superose 12 matrix was a more suitable and a potential method for the purification of rhAT.

Optimization of Fermentation Medium for the Production of Recombinant Human Antithrombin III from Saccharomyces cerevisiae through Statistical Experimental Designs

Statistical Optimization of Olanzapine Ternary Solid Dispersions with Pvp K 30 and Peg 20,000 by Response Surface MethodologyAnil Katharia, Ratendra Kumar, Rajiv Sharma, Yogendra Singh, Uday Veer Singh Teotia

Perioperative and peripartum prevention of venous thromboembolism in patients with hereditary antithrombin deficiency using recombinant antithrombin therapy

Recombinant human antithrombin (rhAT; ATryn), isolated from the milk of transgenic goats, provides an alternative to human plasma-derived antithrombin (AT) concentrate for perioperative and peripartum prophylaxis of venous thromboembolism (VTE) in patients with hereditary AT deficiency. Optimized rhAT dosing algorithms and improved plasma AT monitoring protocol were used in an open-label, single-arm, multinational, pivotal safety and efficacy study that was conducted in patients with hereditary AT deficiency in perioperative and peripartum settings. Loading and maintenance doses were calculated on the basis of pretreatment AT activity levels. Specific dosing regimens were used for pregnant and surgical patients; rhAT was to be given for at least 3 days and for 14 days or less. The primary efficacy end point was the incidence of any thromboembolic event during rhAT therapy or within 7 days of rhAT discontinuation. Safety and AT activity levels were secondary end points. Six surgical and 12 pregnant patients were treated for a median of 3.2 days (range 0.9-14 days). With the optimized dosing regimens, a median of 1 dose adjustment (range 0-6 dose adjustments) was needed to maintain AT activity levels within 80-120% of normal. No confirmed VTEs occurred during treatment or in the subsequent 7 days. Overall, rhAT was well tolerated, but some bleeding complications occurred after rhAT discontinuation and anticoagulation reinstitution. No antibodies to rhAT or goat milk proteins were detected. Perioperative and peripartum prophylactic rhAT therapy in patients with hereditary AT deficiency is well tolerated and effective in preventing VTE.

Recombinant human antithrombin prevents xenogenic activation of hemostasis in a model of pig‐to‐human kidney transplantation

Xenogenic activation of hemostasis (XAH) represents a major hurdle for the transplantation of discordant animal organs into humans as it results in thrombotic microangiopathy (TMA). We have previously shown that recombinant human-activated protein C (rhAPC) mitigates XAH and TMA in an ex vivo model of pig-to-human kidney transplantation. However, the use of rhAPC may not be feasible in a perioperative setting due to possible bleeding complications. Here, we explored the effects of another natural inhibitor of coagulation, human recombinant antithrombin (rhAT), in comparison with rhAPC. Unmodified porcine kidneys (n = 25) were perfused ex vivo with porcine blood, human blood, or human blood supplemented with rhAPC or rhAT. Surrogate parameters of organ survival, markers of XAH (D- Dimer, thrombin-antithrombin complex [TAT], fibrinogen, antithrombin activity, plasminogen), endothelial cell and platelet activation (E-selectin, P-selectin), platelet function tests and histological signs of TMA were evaluated. Perfusion was feasible for > 240 min in all experiments with autologous porcine blood, but limited to 126 ± 78 min with human blood due to increased vascular resistance. Addition of rhAT protected from TMA and allowed for perfusion times > 240 min. In addition, there were less signs of XAH with reduced release of P-selectin and overexpression of E-selectin, whereas the progressive loss of platelet function, observed during discordant perfusion, was prevented. The effect of rhAT was dose-dependent with maximum protection obtained at 3 IU/ml. In conclusion, in this ex vivo model of discordant xenotransplantation, rhAT reduced XAH and prevented TMA in doses that appear feasible for use in clinical or preclinical transplantation settings.

Advantages and pitfalls of combining intravenous antithrombin with nebulized heparin and tissue plasminogen activator in acute respiratory distress syndrome

Pulmonary coagulopathy has become an important therapeutic target in adult respiratory distress syndrome (ARDS). We hypothesized that combining intravenous recombinant human antithrombin (rhAT), nebulized heparin, and nebulized tissue plasminogen activator (TPA) more effectively improves pulmonary gas exchange compared with a single rhAT infusion, while maintaining the anti-inflammatory properties of rhAT in ARDS. Therefore, the present prospective, randomized experiment was conducted using an established ovine model. Following burn and smoke inhalation injury (40% of total body surface area, third-degree flame burn, and 4 × 12 breaths of cold cotton smoke), 18 chronically instrumented sheep were randomly assigned to receive intravenous saline plus saline nebulization (control), intravenous rhAT (6 IU/kg/h) started 1 hour after injury plus saline nebulization (AT i.v.) or intravenous rhAT combined with nebulized heparin (10,000 IU every 4 hours, started 2 hours after injury), and nebulized TPA (2 mg every 4 hours, started 4 hours after injury) (triple therapy, n = 6 each). All animals were mechanically ventilated and fluid resuscitated according to standard protocols during the 48-hour study period. Both treatment approaches attenuated ARDS compared with control animals. Notably, triple therapy was associated with an improved PaO2/FiO2 ratio (p = 0.007), attenuated pulmonary obstruction (p = 0.02) and shunting (p = 0.025), as well as reduced ventilatory pressures (p < 0.05 each) versus AT i.v. at 48 hours. However, the anti-inflammatory effects of sole AT i.v., namely, the inhibition of neutrophil activation (neutrophil count in the lymph and pulmonary polymorphonuclear cells, p < 0.05 vs. control each), pulmonary transvascular fluid flux (lymph flow, p = 0.004 vs. control), and systemic vascular leakage (cumulative net fluid balance, p < 0.001 vs. control), were abolished in the triple therapy group. Combining intravenous rhAT with nebulized heparin and nebulized TPA more effectively restores pulmonary gas exchange, but the anti-inflammatory effects of sole rhAT are abolished with the triple therapy. Interferences between the different anticoagulants may represent a potential explanation for these findings.

Pharmacokinetics of Recombinant Human Antithrombin in Delivery and Surgery Patients With Hereditary Antithrombin Deficiency

Population pharmacokinetic (PK) analyses were conducted to refine dosing recommendations for recombinant human anti-thrombin therapy in surgery and delivery patients with hereditary antithrombin deficiency (HD). Single-dose PK data from patients with HD and nonlinear mixed-effects modeling were used to devise a dosing regimen to target antithrombin (AT) activity levels between 80% and 120% of normal. External validation with data from a phase 3 trial confirmed the correctness of a covariate-free model for surgery patients, but dosing adjustment was necessary for delivery patients. After different covariates were tested, the model was updated to incorporate the influential covariate, delivery. Simulations were used to develop a therapeutic drug-monitoring scenario that results in steady state AT activity levels within the target range as quickly as practically feasible. Data from a second clinical trial provided additional external validation and confirmed the accuracy of the dosing model for both groups of patients.

Antithrombin Attenuates Vascular Leakage via Inhibiting Neutrophil Activation in Acute Lung Injury

To test the hypothesis that restoration of antithrombin plasma concentrations attenuates vascular leakage by inhibiting neutrophil activation through syndecan-4 receptor inhibition in an established ovine model of acute lung injury. Randomized controlled laboratory experiment. University animal research facility. Eighteen chronically instrumented sheep. Following combined burn and smoke inhalation injury (40% of total body surface area, third-degree flame burn; 4 × 12 breaths of cold cotton smoke), chronically instrumented sheep were randomly assigned to receive an IV infusion of 6 IU/kg/hr recombinant human antithrombin III or normal saline (n = 6 each) during the 48-hour study period. In addition, six sham animals (not injured, continuous infusion of vehicle) were used to obtain reference values for histological and immunohistochemical analyses. Compared to control animals, recombinant human antithrombin III reduced the number of neutrophils per hour in the pulmonary lymph (p < 0.01 at 24 and 48 hr), alveolar neutrophil infiltration (p = 0.04), and pulmonary myeloperoxidase activity (p = 0.026). Flow cytometric analysis revealed a significant reduction of syndecan-4-positive neutrophils (p = 0.002 vs control at 24 hr). Treatment with recombinant human antithrombin III resulted in a reduction of pulmonary nitrosative stress (p = 0.002), airway obstruction (bronchi: p = 0.001, bronchioli: p = 0.013), parenchymal edema (p = 0.044), and lung bloodless wet-to-dry-weight ratio (p = 0.015). Clinically, recombinant human antithrombin III attenuated the increased pulmonary transvascular fluid flux (12-48 hr: p ≤ 0.001 vs control each) and the deteriorated pulmonary gas exchange (12-48 hr: p < 0.05 vs control each) without increasing the risk of bleeding. The present study provides evidence for the interaction between antithrombin and neutrophils in vivo, its pathophysiological role in vascular leakage, and the therapeutic potential of recombinant human antithrombin III in a large animal model of acute lung injury.

Use of recombinant human antithrombin concentrate in pregnancy

Dear editor We read with great interest James et al’s article “Prevention and treatment of venous thromboembolism in pregnancy in patients with hereditary antithrombin deficiency.” The authors reported a case series of six women with antithrombin (AT) deficiency treated with plasma derived antithrombin concentrate (pdAT; Thrombate III®, Grifols Therapeutics, Clayton, NC, USA).1 In contrast to these cases, we have managed three AT-deficient women over the past two years with low-molecular-weight-heparin during pregnancy and recombinant human antithrombin concentrate (rhAT; ATryn®, GTC Biotherapeutics, Framingham, MA, USA) at delivery. In the Phase III trial leading to approval of rhAT, pregnant women required frequent dose modifications, and an alternate dosing regimen is currently recommended by the manufacturer.2,3 The detailed information provided in these cases may assist future providers in managing pregnant patients with AT deficiency.

Exploring the Role of Antithrombin Replacement for the Treatment of Preeclampsia: A Prospective Randomized Evaluation of the Safety and Efficacy of Recombinant Antithrombin in Very Preterm Preeclampsia (PRESERVE‐1)

Antithrombin (AT) replacement has been described in patients with hereditary AT deficiency undergoing delivery; however, the kinetics of AT replacement in preeclampsia is not adequately understood. Therefore, the Prospective Randomized Evaluation of the Safety and Efficacy of Recombinant Antithrombin in Very Preterm Preeclampsia (PRESERVE-1) study has been proposed. Sixty women aged≥18 years at 24 0/7-28 0/7 weeks' gestation and with hypertension and proteinuria will be enrolled and randomly assigned to receive recombinant human AT or placebo until fetal and/or maternal indications cause cessation of expectant management or until 34 0/7 weeks' gestation. The primary endpoint is the increase in gestational age from randomization to delivery. Safety assessments and laboratory assays will also be performed. PRESERVE-1 study enrollment will begin during the second half of 2013. The PRESERVE-1 study will provide further insight into the pharmacokinetic activity and safety of AT therapy in preeclampsia.

Antithrombin attenuates myocardial dysfunction and reverses systemic fluid accumulation following burn and smoke inhalation injury: a randomized, controlled, experimental study

IntroductionWe hypothesized that maintaining physiological plasma levels of antithrombin attenuates myocardial dysfunction and inflammation as well as vascular leakage associated with burn and smoke inhalation injury. Therefore, the present prospective, randomized experiment was conducted using an established ovine model.MethodsFollowing 40% of total body surface area, third degree flame burn and 4 × 12 breaths of cold cotton smoke, chronically instrumented sheep were randomly assigned to receive an intravenous infusion of 6 IU/kg/h recombinant human antithrombin (rhAT) or normal saline (control group; n = 6 each). In addition, six sheep were designated as sham animals (not injured, continuous infusion of vehicle). During the 48 h study period the animals were awake, mechanically ventilated and fluid resuscitated according to standard formulas.ResultsCompared to the sham group, myocardial contractility was severely impaired in control animals, as suggested by lower stroke volume and left ventricular stroke work indexes. As a compensatory mechanism, heart rate increased, thereby increasing myocardial oxygen consumption. In parallel, myocardial inflammation was induced via nitric oxide production, neutrophil accumulation (myeloperoxidase activity) and activation of the p38-mitogen-activated protein kinase pathway resulting in cytokine release (tumor necrosis factor-alpha, interleukin-6) in control vs. sham animals. rhAT-treatment significantly attenuated these inflammatory changes leading to a myocardial contractility and myocardial oxygen consumption comparable to sham animals. In control animals, systemic fluid accumulation progressively increased over time resulting in a cumulative positive fluid balance of about 4,000 ml at the end of the study period. Contrarily, in rhAT-treated animals there was only an initial fluid accumulation until 24 h that was reversed back to the level of sham animals during the second day.ConclusionsBased on these findings, the supplementation of rhAT may represent a valuable therapeutic approach for cardiovascular dysfunction and inflammation after burn and smoke inhalation injury.