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Eur J Cardiothorac Surg 2007;32:340-345. doi:10.1016/j.ejcts.2007.02.039
Copyright © 2007, European Association for Cardio-Thoracic Surgery. Published by Elsevier B.V. All rights reserved
a Department of Cardiac Surgery, University of Heidelberg, Heidelberg, Germany
b Department of Cardiovascular Surgery, Semmelweis University, Budapest, Hungary
c Bayer HealthCare, Wuppertal, Germany
d Department of Cardiovascular Surgery, University of Freiburg, Germany
Received 14 November 2006; received in revised form 15 February 2007; accepted 28 February 2007.
* Corresponding author. Address: Laboratory of Cardiac Surgery, Department of Cardiac Surgery, University of Heidelberg, INF 326. OG 2, 69120 Heidelberg, Germany. Tel.: +49 6221 566246; fax: +49 6221 564571. (Email: dzsi{at}hotmail.com).
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Abstract |
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 Top Abstract 1. Introduction2. Materials and methods3. Results4. DiscussionReferences |
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Key Words: Aprotinin • Cardiopulmonary bypass • Blood loss • Coronary endothelial function • Vasorelaxation
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1. Introduction |
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TopAbstract1. Introduction2. Materials and methods3. Results4. DiscussionReferences |
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Currently, only bovine-derived aprotinin is available for the clinical setting. As aprotinin is in widespread clinical use, the possibility of an allergic reaction must be considered whenever this drug is used. The anaphylactic potential of aprotinin has been a major concern, the overall risk of anaphylactoid reactions to aprotinin is estimated to be 0.5% and in reexposed patients is higher, approximately 2.8% [7]. Allergic or severe anaphylactic reactions have been reported [8], but mainly with reexposure. Because it is a bovine protein, the possibility of an infectious disease is also raised.
The vascular effect of aprotinin is only partially clarified. Many authors showed that aprotinin causes graft occlusion, especially vein graft occlusion [9,10]. However, no clinical association could be identified between aprotinin use and graft occlusion in another study [11]. Experimental studies resulted in conflicting data. Khan et al. [12] have described favourable effect of aprotinin on endothelial function in experimental model. There have been also some experimental observations that aprotinin impaired endothelium-dependent vasorelaxation [13,14].
Currently, a new recombinant technology has been developed for production of aprotinin and thereby reducing the risk of allergic reaction and transmission of animal diseases. The primary aim of the present study was to investigate the efficacy of recombinant aprotinin on blood loss in comparison to bovine aprotinin in a canine model of extracorporeal circulation. As the vascular effects of aprotinin remains unclear, the secondary aim of our experiment was to examine the effect of recombinant aprotinin on endothelium-dependent and -independent vasorelaxation of coronary arteries.
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2. Materials and methods |
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TopAbstract1. Introduction2. Materials and methods3. Results4. DiscussionReferences |
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,9-epoxy-methanoprostaglandin F2), acetylcholine, adenosine and sodium nitroprusside were from Sigma–Aldrich, Germany.
2.2 Animals
Twenty-four dogs (foxhounds) weighing 14.0–26.4 kg (19.5 ± 3.6 kg) were used in these experiments. All animals received human care in compliance with the ‘Principles of Laboratory Animal Care’ formulated by the National Society for Medical Research and the ‘Guide for the Care and Use of Laboratory Animals’ prepared by the Institute of Laboratory Animal Resources and published by the National Institutes of Health (NIH Publication No. 86-23, revised 1996). The experiments were approved by the Ethical Committee of the Land Baden-Württemberg for Animal Experimentation.
2.3 Surgical preparation and general management
The dogs were premedicated with propionylpromazine and anaesthetized with a bolus of pentobarbital (15 mg/kg initial bolus and then 0.5 mg/kg/h i.v.), paralyzed with pancuronium bromide (0.1 mg/kg as a bolus and then 0.2 mg/kg/h i.v.) and endotracheally intubated. The dogs were ventilated with a mixture of room air and O2 (FiO2
= 60%) at a frequency of 12–15/min and a tidal volume starting at 15 ml/kg/min. The settings were adjusted by maintaining arterial partial carbon dioxide pressure levels between 35 and 40 mmHg. The femoral artery and vein were cannulated for recording mean arterial pressure (MAP) and taking blood samples for the analysis of blood gases, electrolytes and pH, and parameters of blood coagulation. Basic intravenous volume substitution was carried out with Ringer's solution at a rate of 1 ml/min/kg. If necessary, the rate of volume substitution was modified according to the continuously controlled input–output balance in order to maintain cardiac output at baseline levels. According to the values of potassium, bicarbonate and base excess, substitution included administration of potassium chloride and sodium bicarbonate (8.4%). Neither catecholamines nor other hormonal or pressor substances were administered. Rectal temperature and standard peripheral electrocardiogram were monitored continuously.
After left anterolateral thoracotomy in the fourth intercostal space, pericardiotomy and isolation of the great vessels, a perivascular ultrasonic flow probe was attached to the ascendent aorta. Aortic pressure was monitored with 5F Millar catheter tip manometer (Millar Instruments Inc., Houston, TX, USA).
2.4 Cardiopulmonary bypass (CPB)
After systemic anticoagulation with sodium heparin (300 U/kg), the left subclavian artery was cannulated for arterial perfusion. The venous cannula was placed in the right atrium. The extracorporeal circuit consisted of a heat exchanger, a venous reservoir, a roller pump and a membrane oxygenator primed with Ringer lactate solution (1000 ml) supplemented with heparin (150 U/kg) and 20 ml sodium bicarbonate (8.4%). Normothermic CPB was performed for 90 min. CPB were performed without aortic cross-clamping and cardioplegia. After weaning from CPB, heparin was antagonized by protamin i.v. over 10 min and the animals were monitored for 2 h. Thereafter, coronary arteries were excised for further investigation.
2.5 Experimental groups
Twenty-four dogs were divided into three experimental groups in a blinded fashion: control animals (n
= 8) received placebo, dogs of aprotinin treatment groups received bovine (n
= 8) or recombinant aprotinin (n
= 8). The treatment scheme was applied according to Hammersmith (for a body weight 30 kg) starting with a bolus i.v. (0.85 million Kallikrein Inactivator Unit (KIU)) just before CPB was initiated, followed by a pump prime (0.85 million KIU) and an infusion during bypass at a rate of 0.2 million KIU/h for 90 min (0.3 million KIU). The applied dose of aprotinin was adjusted to the actual body weight of the animals. Accordingly, the animals received 28,333 KIU/kg as an initial bolus and 28,333 KIU/kg into the pump prime. During CPB, 10,000 KIU/kg aprotinin was infused over 90 min. The doses of bovine and recombinant aprotinin were the same.
2.6 Measurements of blood loss, biochemical and haemodynamic parameters
The primary endpoint of the study was the total blood loss after weaning from cardiopulmonary bypass during the first 2 h after application of protamin. Secondary endpoints were parameters of coagulation (activated clotting time (ACT), normalized prothrombin time (Quick), activated partial thromboplastin time (aPTT)). Blood loss was measured by gauze bandages during reperfusion at different time points (in 105, 120, 160, 220 min after initiation of CPB). Bandages were placed into the operating area (pericardial sack and surrounding tissues). Weight of gauze bandages was measured before and after cleaning the operating area. We calculated blood loss from difference of weight of gauze bandages. ACT, Quick and aPTT were monitored regularly during and after the 90-min CPB. ACT (celite method), Quick and aPTT were assessed by routine clinical assays. Haemodynamic parameters, included heart rate (HR) and arterial pressure were monitored continuously.
2.7 Vascular function
In addition, endothelium-dependent and -independent vasorelaxation were investigated in isolated coronary arterial rings of the dogs. After the end of the experiments, the coronary arteries were excised and the left anterior descendent (LAD) coronary arteries were isolated and placed in cold (+4 °C) Krebs–Henseleit solution (118 mM NaCl, 4.7 mM KCl, 1.2 mM KH2PO4, 1.2 mM MgSO4, 1.77 mM CaCl2, 25 mM NaHCO3, 11.4 mM glucose; pH = 7.4). The coronary arteries were prepared and cleaned from periadventitial fat and surrounding connective tissue and cut transversely into 4-mm width rings using an operation microscope.
Isolated coronary aortic rings were mounted on stainless steel hooks in individual organ baths (Radnoti Glass Technology, Monrovia, CA, USA), containing 25 ml of Krebs–Henseleit solution at 37 °C and aerated with 95% O2 and 5% CO2. Special attention was paid during the preparation to avoid damaging the endothelium.
Isometric contractions were recorded using isometric force transducers (Radnoti Glass Technology, Monrovia, CA, USA), digitized, stored and displayed with the IOX Software System (EMKA Technologies, Paris, France).
The coronary aortic rings were placed under a resting tension of 3.5 g and equilibrated for 60 min. During this period, tension was periodically adjusted to the desired level and the Krebs–Henseleit solution was changed every 30 min. Potassium chloride (KCl) was used in these experiments to prepare vessels for stable contractions and reproducible dose–response curves to other vasoactive agents. Coronary rings were contracted twice with KCl (80 mM) and rinsed after each contraction until resting tension was again obtained. Thromboxane A2-receptor agonist U46619 (5 x 10–7 M) was used to precontract the rings until a stable plateau was reached, and relaxation responses were examined by adding cumulative concentrations of endothelium-dependent dilator acetylcholine (ACh, 10–9–10–4 M) and bradykinin (BK, 10–10–10–4 M), as well as the endothelium-independent dilator sodium nitroprusside (SNP, 10–10–10–5 M) and adenosine (ADO, 10–6–10–3 M). Contractile responses are expressed as grams of tension, relaxation is expressed as percent of contraction induced by U46619.
2.8 Statistics
All values were expressed as mean ± standard deviation (SD) or standard error of the mean (SEM, in case of vascular function). Paired t-test was used to compare two means within groups. Individual means between the groups were compared by one-way analysis of variance followed by an unpaired t-test with Bonferroni's correction for multiple comparisons and the post-hoc Scheffe's test. A probability value less than 0.05 was considered statistically significant. In the figures, only the significances between the groups were indicated. Significant changes over the time within each group were indicated in the text.
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3. Results |
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TopAbstract1. Introduction2. Materials and methods3. Results4. DiscussionReferences |
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In contrast, application of aprotinin did not affect the endothelium- and receptor-independent, cGMP-mediated vasorelaxation to sodium nitroprusside (SNP) (Fig. 4C).
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4. Discussion |
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TopAbstract1. Introduction2. Materials and methods3. Results4. DiscussionReferences |
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High-dose aprotinin reduces postoperative blood loss and transfusion requirements by 40–50% in the first-time [1,3] and in reoperative patients and in patients who have been pretreated by aspirin [4]. This study demonstrates for the first time that recombinant aprotinin significantly decreases postoperative total blood loss after CPB in a canine model. The effects of bovine and recombinant aprotinin were equivalent. A recent clinical study showed that aprotinin significantly reduced total blood loss by over 50% in comparison with placebo [1]. Our experiments resulted in similar data, postoperative total blood loss was significantly reduced (approximately by 50%) in the recombinant aprotinin-treated group and in the bovine-derived aprotinin group in comparison with control (p < 0.05) (Fig. 2).
Measurement of activated clotting time (ACT) is a standard monitoring procedure for guiding heparin-induced anticoagulation. Although the optimum ACT value for CPB has not been established, values between 400 and 480 s are commonly maintained. Initial investigations demonstrated that celite ACT values are prolonged by aprotinin [15]. The increase in ACT is artifactual because of an in vitro interaction between the celite activator, heparin and aprotinin. In contrast, aprotinin does not affect kaolin ACT [16]. We used a celite-based ACT in our experiment, we continuously measured ACT values every 15 min and maintained more than 500 s during CPB. In the present study, there was only a tendency towards prolonged ACT values without reaching the level of significance (Fig. 3C). As expected, aPTT and ACT increased significantly in all groups after heparinisation and remained elevated during CPB. But there was no significant difference between all three groups regarding Quick, aPTT and ACT (Fig. 3) at any time points.
To the best of our knowledge, this is the first study investigating effects of aprotinin on endothelial function of epicardial coronary arteries in a clinically relevant mammalian model of cardiopulmonary bypass. Previous investigations on the endothelial effects of aprotinin in other models resulted in conflicting results. Ülker et al. [13] found impaired endothelium-dependent vasorelaxation to ACh and to the calcium ionophore A23187 in rat thoracic aortic rings and inhibition of bradykinin-induced endothelium-dependent coronary vasodilatation in coronary arteries of rat Langendorff hearts in the presence of aprotinin [14]. Contrasting these findings, Khan et al. [12] report aprotinin-induced improvement of both endothelium-dependent and -independent vasorelaxation of coronary microvessels after regional ischaemia and cardioplegic arrest in a porcine model.
Our present results demonstrate no significant influence of bovine or recombinant aprotinin on endothelial relaxant responsiveness of canine coronary arterial rings (Fig. 4A and B). These data are in line with the results of Fischer and Steinhoff. Using substance P for endothelium-dependent vasorelaxation, they found no significant effect of aprotinin at clinical dosage levels on endothelial function in porcine coronaries [17]. Similar to our results, endothelium-dependent relaxation to ACh was unaffected by aprotinin treatment on human saphenous vein rings (explanted for coronary bypass) in a previous study [18].
To test the endothelium-dependent vasorelaxation of canine coronary arterial rings, we applied the universally used acetylcholine that stimulates the endothelial production of nitric oxide by binding to muscarinic receptors on endothelial cells. Endothelium-dependent relaxant bradykinin acts in a similar manner by binding to endothelial B2 receptors. Bradykinin is a member of the kallikrein–kinin system that is strongly influenced by aprotinin. The serine protease kallikrein induces redistribution and activation of B2 bradykinin receptors independent of BK-release [19]. Acting as a serine protease inhibitor, aprotinin inhibits kallikrein, thereby blocking B2 bradykinin receptor activation and redistribution. These mechanisms of effects of aprotinin might explain the impairment of bradykinin-induced vasodilatation in rat coronary arteries in the presence of aprotinin, as reported by Ülker et al. [14]. Furthermore, one may hypothesize that these observations might be the reason for our current findings showing a tendency towards reduced bradykinin-induced vasorelaxation, however, without reaching statistical significance (Fig. 4B).
There are only sporadic studies investigating the effect of aprotinin on endothelium-independent dilatory function of vascular smooth muscles. Most of them report no influence of aprotinin on endothelium-independent vasorelaxation to sodium nitroprusside (SNP) [13,14], a nitric oxide donor substance, which acts in a receptor-independent, cGMP-mediated manner by directly activating the guanylyl cyclase enzyme in vascular smooth muscle cells. Our present data completely correspond with these results, the SNP-induced vasorelaxation of coronary arterial rings was unaffected by bovine and recombinant aprotinin (Fig. 4C).
Adenosine (ADO), the fourth vasorelaxant tested in this study, leads to dilatation of vessels by complex and not fully understood mechanisms and its vascular effects involve different pathways depending on species and vessel type. Binding to A2 adenosine receptors on smooth muscle cells, adenosine activates the cAMP-mediated pathway of relaxation [20]. Other studies report partial involvement of the endothelium in the ADO-mediated vasorelaxation [21,22]. The significantly increased vasorelaxation of coronary arterial rings to ADO, but not to SNP in the aprotinin treatment groups indicates that application of aprotinin improved the receptor-dependent cAMP-mediated, but not the receptor-independent cGMP-mediated dilatory function of vascular smooth muscle. We hypothesize that the enhanced ADO-mediated vasorelaxation may reflect aprotinin-induced alterations in adenosine receptor density, sensitivity and/or receptor/effector coupling, however further investigations are needed to elucidate the exact underlying mechanisms.
In conclusion, we documented that the effectiveness of recombinant aprotinin on blood loss and coagulation parameters were equivalent with bovine-derived aprotinin. Neither recombinant aprotinin nor bovine aprotinin impaired the endothelium-dependent vasodilatative function of coronary arteries in our clinically relevant canine model of cardiopulmonary bypass. As recombinant aprotinin probably reduces risk of hypersensitivity reaction and transmission of animal diseases, it should be utilized clinically, however, further clinical investigations are needed.
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Acknowledgments |
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Footnotes |
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1 These authors contributed equally to this work. 
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References |
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TopAbstract1. Introduction2. Materials and methods3. Results4. DiscussionReferences |
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