Impact of Graded Hypothermia on Coagulation and Fibrinolysis

Journal of Surgical Research 167, 125–130 (2011) doi:10.1016/j.jss.2009.07.037

Impact of Graded Hypothermia on Coagulation and Fibrinolysis Chryssoula Staikou, M.D., D.E.S.A.,*,1 Anteia Paraskeva, M.D., D.E.S.A.,* Elias Drakos, M.D.,† Ioanna Anastassopoulou, M.Sc.,‡ Eleni Papaioannou, M.D.,§ Ismini Donta, D.V.M.,k and Michael Kontos, M.D.{ *1st Department of Anesthesiology, Medical School, University of Athens, Aretaieio Hospital, Athens, Greece; †Laboratory of Pathology, Laiko General Hospital, Athens, Greece; ‡Department of Hematology, Laiko General Hospital, Athens, Greece; §Department of Anesthesiology, Laiko General Hospital, Athens, Greece; kExperimental and Surgical Animal Research Laboratory of Medical School, University of Athens; and {1st Department of Surgery, Medical School, University of Athens, Laiko General Hospital, Athens, Greece Submitted for publication March 26, 2009

Background. Hypothermia has a detrimental effect on hemostatic mechanism. The purpose of this experimental study was to investigate the effect of graded hypothermia on markers of the anticoagulant system (antithrombin III and protein C) and fibrinolytic system (plasminogen, a2-antiplasmin), and on vascular wall and other tissue specimens. Materials and methods. Ten New Zealand rabbits were subjected to mild and then moderate core hypothermia of 32 C for 60 min. Blood samples were obtained at normothermic (T1), mild (T2), and moderate (T3) hypothermic conditions. Chromogenic assay methods were used to determine quantitatively (%) the activity of antithrombin III, protein C, plasminogen, and a2-antiplasmin. Hypothermic values were compared with the normothermic values. Tissue and vessel wall specimens were examined under light microscope. Results. Reduction of activity (%) from normothermia (T1) to mild (T2) and moderate (T3) hypothermia was found for antithrombin III (103.40 ± 12.54, 87.40 ± 13.50, and 82.70 ± 20.78, respectively, with statistically significant difference between T1–T3: P[0.03), for protein C (70.1 ± 7.51, 56.30 ± 8.34, and 53.1 ± 7.34, with statistically significant difference between T1–T2 and T1–T3: P[0.015 for both comparisons) and a2-antiplasmin (97 ± 9.63, 80.60 ± 11.73, and 83.70 ± 13.94, with statistically significant difference between T1–T2: P[0.006). Plasminogen activity was increased (14.50 ± 0.52, 16.30 ± 1.63, and 17.30 ± 2.45, with statistically significant difference between T1–T2 and T1–T3: P[0.033 for both 1 To whom correspondence and reprint requests should be addressed at: Department of Anesthesiology, Aretaieio Hospital Medical School, University of Athens, 76 Vassilissis Sophias Avenue, 11528, Athens, Greece. E-mail: [email protected].

comparisons). Histologic examination revealed no significant lesions on tissue and vessel wall specimens. Conclusions. The results of our study suggest that even though the hypothermia period was relatively short, the processes of coagulation and fibrinolysis were altered with simultaneous changes. Ó 2011 Elsevier Inc. All rights reserved.

Key Words: hypothermia; antithrombin-III; protein C; plasminogen; a2-antiplasmin.

INTRODUCTION

Hypothermia has been associated with defects in coagulation, contributing to the morbidity and mortality of surgical and trauma patients by promoting further hemorrhage [1, 2]. The exposure of trauma victims to the cold temperature of environment, emergency, and operating room, along with bleeding and transfusions, considerably increase the risk of complications, such as the triad of hypothermia, acidosis, and coagulopathy, which may be even fatal [3]. Similar complications are induced by prolonged surgical procedures where the low operating room temperature, the heat loss from visceral exposure, along with the multiple transfusions of cold blood products may lead to hypothermia with hemorrhagic diathesis [1, 3]. Moreover, hypothermia-induced coagulation defects are usually underestimated; in fact, they are usually concealed since standard clotting studies are routinely performed at 37 C. The result is a rather late detection of the coagulopathy, when diffuse oozing appears in the surgical field [2, 4, 5]. The impact of different degrees of hypothermia on coagulation has been investigated. Deep hypothermia of less than 30 C has been found to cause platelet

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dysfunction due to platelet sequestration in the liver and spleen and decreased platelet aggregation, prolongation of clotting times and increased fibrinolytic activity [6, 7]. It has been reported that hypothermia and rapid re-warming may lead to lethal disseminated intravascular coagulation (DIC) [8]. Moderate hypothermia (>30 C) in humans produces a decrease of prothrombin time and platelet count, while the clot strength remains unaltered [9]. Local cooling of 30 C has been found to increase bleeding time [10]. In animal studies, moderate hypothermia (32 –34 C) has been related with reversible platelet dysfunction, prolonged bleeding, prothrombin, and partial prothrombin times, and also reduced thromboxane B2 due to impaired platelet ability to produce it [11, 12]. Hypothermia also impairs blood circulation by increasing blood viscosity and reducing the velocity of capillary blood flow. Tissue lesions have been found in hypothermic victims: gastric and brain hemorrhage, ischemic and hemorrhagic macro- and micro-infarcts in brain and viscera, hypoxic-type lesions and fatty changes in vital organs, pancreatic necrotic and hemorrhagic lesions, as well as vessel wall necrotic areas and thrombus formation in arterioles and small veins [8, 13, 14]. The hemostatic mechanism consists of platelets and enzymes taking part in a cascade of reactions, which are regulated by positive and negative feedback mechanisms. This cascade results in a balance between coagulation (production of thrombin and fibrin) and fibrinolysis (fibrin degradation). If this balance is distorted, the consequences may be either excessive hemorrhage and/or a tendency towards thromboembolism. Hypothermia may affect this balance, since it inhibits the series of enzymatic reactions of the coagulation cascade and impairs platelet reactivity [9]. Most studies have investigated the impact of deep hypothermia on coagulation [6–8]. On the other hand, studies on moderate hypothermia are either in vitro experiments or clinical studies on major trauma victims and are mainly focusing on platelet and clotting times changes [1, 2, 9–12, 15, 16]. To our knowledge, there are limited data for the in vivo impact of mild and moderate hypothermia on the markers of anticoagulant and fibrinolytic systems: antithrombin III (AT-III), protein C (PC), plasminogen (PLG), and a2-antiplasmin (a2-APL). The aim of this study was to investigate the isolated effects of graded hypothermia on AT-III, PC, PLG, a2APL, and also on vascular wall and other tissue specimens of rabbits without trauma or surgical intervention. MATERIALS AND METHODS Ten male New Zealand white rabbits (age 3–4 months, weight 3.35– 4.1 kg) were used in this study, which was conducted at the

Experimental and Surgical Animal Research Laboratory of Medical School of the University of Athens. The protocol was approved by the Committee on the Care of Experimental Animals, and the rabbits were housed and cared in conformance with the Animal Care Committee guidelines (National law 160/91-12). Premedication, consisting of ketamine HCl (Ketaset; Ceva, Paris, France) 25 mg/kg and xylazine HCl (Rompun; Bayer, Leverkusen, Germany) 5 mg/kg, was administered intramuscularly to the rabbits to facilitate their shaving and positioning on the table in the operating theatre (room temperature of 22–24 C). An esophageal thermistor inserted at the heart level and connected to a monitor Graseby-Rigel 429 was used for continuous core temperature monitoring, since it has been shown that there is a good correlation between lower esophageal and blood temperature [2]. Electrocardiograph (Graseby-Rigel 429), pulse oxymetry (Kontron Instruments Pulse Oximeter 7840, Watford, UK) and blood pressure monitoring (Dinamap-Criticon Vital Signs Monitor 1840, Tampa, FL) were applied to all animals which were receiving N/S 0.9% (10 mL/kg) through a 21 G ear vein catheter. After surgical exposure of the carotid artery, a 20 G catheter was placed into the vessel for blood sample withdrawal. First blood samples (baseline) were obtained at this point at normothermic conditions (time point T1). The carotid artery catheter was kept open during the experiment with tiny amounts of N/S 0.9%. Heparin was strictly excluded from the protocol. Anesthesia was induced with propofol 10 mg/kg IV (Propofol 1%; Fresenius, Upsala, Sweden) and a tracheotomy (tracheal tube of internal diameter (ID) 3–3.5 mm) was performed for continuous mechanical ventilation (Harvard Rodent Ventilator, model 683, South Natick, MA) with FiO2 0.4 in air, tidal volume 9 mL/kg, and respiratory rate 22/min with adjustments targeting to normocarbia. General anesthesia was maintained with propofol (infusion rate 30 mg/kg/h) and incremental doses of fentanyl 30 mg/kg (Fentanyl-Janssen, Beerce, Belgium). Second blood samples were obtained 60 min after the first sample at mild hypothermic conditions (time point T2). After that, cooling was achieved by applying icepacks directly to the animal skin, in order to subject the animals to moderate hypothermia. After 30–40 min, the target of esophageal temperature of 32 C was achieved and the icepacks were withdrawn. Under the guidance of the esophageal thermistor, the animals were kept for 60 min at a steady core temperature (32 C), exposed to a room temperature of 22–24 C, and being under general anesthesia. If needed, icepacks were used again for short periods of time for the animals to maintain a core temperature of 32 C. At that time point (T3), blood samples were withdrawn and tissue specimens were obtained surgically, and the animal was sacrificed. The duration of the whole procedure (from T1 to T3) was about 160 min. Blood samples for AT-III, PC, PLG and a2-APL measurements were obtained at T1, T2 and T3 and the first 2 mL were discarded. Arterial blood gasses (1 mL of blood in a syringe with 50 IU of heparin) were also measured for proper adjustment of ventilator settings in order to avoid hypercarbia and hypoxemia. Blood samples (1.8 mL) were collected in 0.109 M trisodium citrate tubes (9:1 vol/vol), centrifuged immediately for 15 min at 2500 g, and plasma was stored at –80 C until AT-III, PC, PLG, and a2-APL assay during the following 30 d by the use of the synthetic chromogenic substrate method. This method was used for the quantitative determination of AT-III, PC, PLG, and a2-APL activity. For measurement of AT-III activity level in plasma, a known excess of thrombin was used in the presence of heparin, (kit STA-Stachrom ATIII, Asnieres-Sur-Seine, France), for PC a special activator was added (kit STA-Stachrom Protein C), for PLG a known excess of streptokinase was added (kit STA-Stachrom Plasminogen), and for a2-APL a known excess of plasmin was added (kit STA-Stachrom Antiplasmin). Tissue specimens obtained from the animals were cut appropriately, immersed in 10% formalin solution for 48 h, and routinely processed and embedded in paraffin. Paraffin-embedded sections were then stained with hematoxylin-eosin stain and evaluated by light microscopy. The Statistical Package for Social Sciences (SPSS, ver.16.0) (SPSS Inc., Chicago, IL) was used for analysis. AT-III, a2-APL, mean arterial

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STAIKOU ET AL.: GRADED HYPOTHERMIA INDUCED COAGULOPATHY pressure (MAP), and partial carbon dioxide tension in arterial blood (PaCO2) followed normal distribution, and ANOVA repeated measures was used for within time analysis and paired Student’s test for paired comparisons. PC, PLG, Heart rate (HR), hemoglobin saturation with oxygen in arterial blood (SatO2), pH, partial oxygen tension in arterial blood (PaO2), and base excess (BE) did not follow a normal distribution, and Friedman test was used for within-time analysis, while Wilcoxon signed rank test was used for paired comparisons. Values are expressed as mean 6 SD. A statistical level of 0.05 (P < 0.05) was considered statistically significant. Bonferroni correction was used for pairwise comparisons.

acidosis was manifested at the end of the experiment with animal arterial pH: 7.18 6 0.156 and BE: –8.93 6 5.06. Blood pressure and heart rate were gradually reduced during the procedure, but the animals remained clinically stable, without signs of circulatory collapse. Inotropes or vasoactive drugs were not used in any case. Markers of Coagulation and Fibrinolysis

RESULTS

The rabbits were subjected to mild hypothermia (time period from normothermia to mild hypothermia blood samples at T2: 60 min), external cooling and then steady core moderate hypothermia (32 C) for 60 min (blood samples and specimens at T3). We performed a quantitative determination of the activity AT-III, PC, PLG, and a2-APL, comparing their values in blood samples obtained from rabbits under normothermia, mild, and moderate hypothermia; esophageal temperatures: 39.31 6 0.31 C, 36.43 6 0.63 C, and 31.9 6 0.37 C, respectively. Hemodynamic and Blood Gas Values

HR, MAP, SatO2, pH, PaO2, and BE differed significantly within the three time points of measurement (X2 ¼ 16.200 and P ¼ 0.0005, F ¼ 17.800 and P ¼ 0.0005, X2 ¼ 16.632 and P ¼ 0.0005, F ¼ 12.239 and P ¼ 0.001, X2 ¼ 15.200 and P ¼ 0.001, and X2 ¼ 14.600 and P ¼ 0.001, respectively). PaCO2 did not differ at any time point (F ¼ 1.362, P ¼ 0.281). Paired comparisons for variables HR, MAP, SatO2, pH, PaO2, and BE are shown in Table 1. Oxygenation and normocarbia were maintained throughout the procedure, but a significant metabolic

AT-III, PC, PLG, and a2-APL differed significantly within the three time points of measurement (F ¼ 5.458 and P ¼ 0.017, X2 ¼ 15.800 and P ¼ 0.0005, X2 ¼ 11.529 and P ¼ 0.003, and F ¼ 7.258 and P ¼ 0.02, respectively). Paired comparisons for variables AT-III, PC, PLG, and a2-APL are shown in Table 2. Histologic Findings

The tissue specimens obtained from organs and vessels of the animals at the end of the experiment were examined under the light microscope. No significant lesions related to hypothermia were observed in tissue specimens obtained from the heart, kidney, liver, spleen, pancreas, intestine, esophagus, and lung. Vessel wall endothelium from aorta, mesenteric artery, and inferior vena cava was found intact without lesions or thrombi. No skin damage from the use of icepacks was macroscopically observed. DISCUSSION

In this study, we evaluated the effect of graded moderate hypothermia on specific markers of the anticoagulant and fibrinolytic systems (AT-III, PC, PLG, a2-APL) and on tissue specimens obtained from organs

TABLE 1 Heart rate (HR), Mean Arterial Pressure (MAP), Partial Oxygen Tension in Arterial Blood (PaO2), Partial Carbon Dioxide Tension in Arterial Blood (PaCO2), Base Excess (BE), and Hemoglobin Saturation with Oxygen in Arterial Blood (SatO2) at Three Different Time Points Representing Normothermia (T1), Mild (T2), and Moderate (T3) Hypothermia Parameters

T1

T2

T3

HR (bt/min) MAP (mm Hg) SatO2 (%) pH PaCO2 (mm Hg) PaO2 (mm Hg) BE

189.10 6 17.90y* 73.30 6 12.18y* 93.34 6 1.93y* 7.34 6 0.085y* 43.78 6 6.125 83.3 6 5.94y* 0.46 6 7.5y*

168 6 22.15yz 58.60 6 11.82yz 99.53 6 0.35yz 7.36 6 0.105yz 46.46 6 5.34 167.10 6 19.39yz 2.36 6 5.75yz

124.40 6 6.48z* 49.60 6 6.05z* 99.48 6 0.36z* 7.18 6 0.156z* 42 6 7.09 158.10 6 18z* -8.93 6 5.06z*

Values are mean 6 SD. P value <0.05 was statistically significant. * T1 versus T3. y T1 versus T2. z T2 versus T3.

P y

0.021*0.015z0.021 0.009*0.0015z0.24 y 0.015*0.015z0.816 y 0.555*0.015z0.036  y 0.0015*0.0015z0.555 y 0.417*0.024z0.015 y

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TABLE 2 Antithrombin III (AT-III), Protein C (PC), Plasminogen (PLG), and a2-Antiplasmin (a2-APL), at Three Different Time Points Representing Normothermia (T1), Mild (T2), and Moderate (T3) Hypothermia Parameters

T1

T2

T3

AT-III (%) PC (%) PLG (%) a2-APL (%)

103.40 6 12.54y* 70.10 6 7.51y* 14.5 6 0.527y* 97 6 9.63y*

87.40 6 13.50yz 56.30 6 8.34yz 16.30 6 1.63yz 80.60 6 11.73yz

82.70 6 20.78z* 53.10 6 7.34z* 17.30 6 2.45z* 83.70 6 13.94z*

P y

0.06*0.03z1.626 0.015*0.015z0.342 y 0.033*0.033z1.08 y 0.006*0.072z1.644 y

Values are mean 6 SD. P value <0.05 was statistically significant. * T1 versus T3. y T1 versus T2. z T2 versus T3.

and vessel wall. Our results showed that progressive hypothermia significantly reduces the activity of PC, and increases the activity of PLG. We also found that mild hypothermia reduces significantly the activity of a2-APL, and that moderate hypothermia reduces significantly the activity of AT-III. Histologic examination of tissue and vessel wall specimens revealed no lesions related to hypothermia. Rabbits were selected as the study animals because of the similarities between rabbit and human hemostatic system [17, 18]. We evaluated the effect of mild and moderate hypothermia on specific factors of coagulation and fibrinolysis, since it has been suggested that at temperatures above 33 C, coagulopathy results primarily from platelet dysfunction, while at temperatures below 33 C, defects in both enzyme activity and platelet function contribute to the disorder [16]. Kettner et al. also found that graded hypothermia to 32 C in humans resulted to a slowing of both enzymatic reactions and the speed of interaction between platelets and coagulation cascade according to TEG measurements [9]. Moreover, it has been demonstrated that hypothermia mainly affects the kinetics of clotting factors, while it has a minimal effect on their concentrations [15]. AT-III and PC are glycoproteins, natural inhibitors of coagulation, synthesized in the liver [19]. AT-III exerts a powerful inhibitory action on thrombin, especially in the presence of heparin. It also inhibits factor Xa, and to a lesser extent other factors too. Our finding that AT-III activity was significantly reduced at moderate hypothermia is in agreement with a relatively recent in vitro study, which showed that the rate of inhibition of thrombin by AT-III was reduced by 5% at 33 C and by 64% at 23 C compared with normothermia (37 C) [16]. On the contrary, Yoshihara et al. found that in dogs, AT-III activity was not affected by deep hypothermia 20 C, while Boldt et al. found that the thrombin/antithrombin III complex was increased in patients undergoing cardiopulmonary bypass at 27 C, indicating a tendency for bleeding diathesis [7, 20]. It is possible that the use of different animals or the different

settings of the studies may explain the different findings of the above studies. PC is vitamin-K dependent and its transformation from pro-enzyme to the active form is thrombin-dependent and potentiated by thrombomodulin. The activated PC proteolytically degrades factor Va and inactivates factor VIIIa. PC also neutralizes the inhibitor of tissue plasminogen activator and facilitates fibrinolysis [21]. Our finding that the activity of PC was significantly reduced at both mild and moderate hypothermia is consistent with the findings of Boldt et al. that in patients undergoing hypothermic (T < 30 C), cardiopulmonary bypass PC was significantly decreased from 88% to 60% [20]. This hypothermiainduced reduction of activity of the two important coagulation inhibitors AT-III and PC could possibly lead to an increased thrombotic tendency. PLG is a precursor inactive b2-globulin synthesized in the liver and is converted to the active plasmin under the influence of tissue or plasma activators. Plasmin degrades fibrin and fibrinogen, leading to the production of D-dimers and fibrin/fibrinogen degradation products (FDPs) [22]. a2-APL is the specific inhibitor of plasmin and the main regulator of fibrinolysis by limiting any possible excessive action of plasmin. a2-APL deficiency may lead to excessive fibrinolysis and subsequent hemorrhagic diathesis [22]. According to our results, a2-APL activity is reduced significantly at mild hypothermia but shows a tendency to increase at moderate hypothermia, while the activity of PLG is gradually increased significantly while the temperature drops. Since a2-APL activity shows only a temporary significant reduction, it seems that a possible tendency towards increased fibrinolysis at moderate hypothermia could be mainly due to changes in PLG activity. To our knowledge, there are no reports about fibrinolysis at mild and moderate hypothermia in order to compare our findings with other studies. At deep hypothermia of 20 C a marked increase in fibrinolytic activity was observed in dogs. It was attributed to plasminogen activator release from the vascular wall stimulated by

STAIKOU ET AL.: GRADED HYPOTHERMIA INDUCED COAGULOPATHY

intrinsic catecholamines [7]. On the contrary, studies in multiple trauma victims showed no significant relation between hypothermia and enhanced fibrinolysis [23, 24]. These different findings may be related to many other factors, apart from hypothermia, that coexist in trauma, such as extensive tissue and vital organ damage, hemorrhagic shock, and acidosis. It is interesting to note that trauma patients may present with some degree of hypercoagulability, which is attributed to massive release of tissue thromboplastin from extensively damaged soft-tissue [23, 25]. Tissue thromboplastin may be related to hypothermia induced lethal DIC. Mobilization of tissue thromboplastin from damaged hypothermic tissue into the circulation in severe hypothermia or during aggressive rapid rewarming may cause DIC, while circulatory collapse, which often accompanies severe, lethal hypothermia, may also play a role [8, 26]. According to our results singnificant metabolic acidosis appeared at the end of the experiment. This finding is consistent with other reports [2, 15, 27]. Ao et al. have found a significant base deficit along with hypokalemia during prolonged canine hypothermia [15]. The hypothermia induced acidosis is possibly due to a decreased cardiac output, peripheral tissue hypoperfusion, and renal dysfunction with retention of acid end metabolic products [2, 27]. It has been suggested that acidosis in hypothermia is associated with alterations in the plasma ionic balance, and increased weak acid buffers due to increases in albumin and inorganic phosphate [27]. At cellular level, the effect of hypothermia on the membrane ion pump for exchanging Kþ with Hþ has also been proposed as the mechanism causing the metabolic acidosis [15]. In massively transfused trauma patients, Ferrara et al. did not identify platelet dysfunction over a pH range 7.18 to 7.04, which was the pH of the survivors and nonsurvivors respectively [1]. In our study, we found that PC, PLG, and a2-APL were significantly changed quite early at T2, when pH was still normal. In our study, histologic examination did not reveal tissue lesions, such as infarcts, or pancreatic damage, or fatty changes in the specimens obtained from the animals’ viscera. Reports of post-mortem and histologic examinations in hypothermic victims have shown hemorrhagic, ischemic, and hypoxic type tissue lesions. Hemorrhage in body cavities and stomach, multiple ischemic and hemorrhagic infarcts in visceral and brain, pancreatic lesions with focal areas of hemorrhage and necrosis, micro-infarcts in the heart muscle and fatty changes in the heart, liver and kidney, as well as gastric erosions were induced by hypothermia [8, 13, 14]. These lesions are attributed to the hypothermia-induced reduction of cardiac output and impairment of blood rheology, and, consequently circulation; blood viscosity

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increases, and deformability of cells is reduced, impending capillary passage, and thus compromising tissue perfusion [2, 13]. The degree of hemoconcentration and hypothermic circulatory collapse are probably dependent on the degree and duration of hypothermia. Histologic examination in cases of severe hypothermia-related deaths have also revealed vessel wall lesions, such as partial necrosis of arteriole wall with thrombus formation at necrotic sites and multiple thrombi in small veins [13, 14]. According to a study of Lindenblatt et al., systemic moderate hypothermia accelerates microvascular thrombus formation in arterioles and venules in mice [28]. They suggest that this thrombotic tendency may be mediated by increased activation of the fibrinogen receptor GPIIb-IIIa on platelets and subsequent fibrinogen binding. In our study, the examination under light microscope revealed no vascular damage related to hypothermia; vessel wall endothelium was intact, and vascular thrombus formation was not identified. In our experiment, the induced hypothermia was moderate, relatively short-term, and the animals remained hemodynamically stable and well oxygenated throughout the procedure, so no tissue lesions or vessel wall endothelium changes attributed to shock and hypoxia were found. The hypothermia-induced changes in vascular endothelium have attracted the interest of many investigators. It has been found that in cardiopulmonary bypass thrombomodulin plasma level, which is considered to be a marker of endothelial cell damage, was increased more in hypothermia than in normothermia [20]. Moreover, Kazanskaya et al. have found under electron microscopy that temperature <30 C without perfusion produces adaptive changes in myocardiac capillary endothelial cells [29]. We induced hypothermia in step-wise fashion, normothermia followed by mild and then by moderate hypothermia, because we consider that this model may reflect quite accurately the clinical conditions in real life. Nevertheless, it should be noted that probably in this hypothermic model the effects of moderate hypothermia on coagulation are also a function of the preceding hypothermic phase. This first phase of mild hypothermia is the impact of induction of general anesthesia and exposure of animals to the low room temperature. Anesthetic drugs, especially propofol, affect body heat balance by producing peripheral vasodilatation, which leads to an internal redistribution of heat from core to periphery [30]. Yet, no relation was found between propofol and coagulation disorders when it is administered in ICU for long term sedation [31]. The fact that we induced short term hypothermia (160 min) could be considered as a limitation of our study, and we can make no conclusions about the effects of hypothermia of longer duration. Ao et al. have found

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that long term (72 h) moderate hypothermia in animals is related to a decreased platelet aggregation and a prolonged coagulation time in TEG [15]. It would also be interesting if we obtained blood samples at various different temperatures to provide a ‘‘dose-response.’’ The aim of our study was to investigate the isolated effects of hypothermia on coagulation in rabbits without trauma or surgical intervention, so we considered that repeated blood sampling could possibly lead to significant blood loss or dilution of clotting factors due to resuscitation, thus affecting our results. We conclude that even mild and short-term moderate hypothermia impairs the hemostatic system, leading to an increased thrombotic and fibrinolytic tendency, which appears quite early in the course of temperature reduction. Vessel wall or tissue lesions were not found under the present study design. More research and clinical studies are needed to investigate the relation of graded hypothermia with coagulation and fibrinolysis, and to clarify its clinical significance in humans.

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