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Ann Thorac Surg 1995;60:307-310
© 1995 The Society of Thoracic Surgeons

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Original Articles: Cardiovascular

Does Aprotinin Increase the Myocardial Damage in the Setting of Ischemia and Preconditioning?

Division of Cardiothoracic Surgery, Department of Surgery, Deaconess Hospital, Harvard Medical School, Boston, Massachusetts

	Abstract

Top Footnotes Abstract Introduction Material and Methods Results Comment Acknowledgments References

 
Background. Aprotinin reduces postoperative bleeding in cardiac operations, but its association with perioperative myocardial infarction remains controversial. Ischemic preconditioning is a novel method of myocardial protection.

Methods. To answer whether aprotinin increases postischemic myocardial damage and also to characterize the effect of aprotinin on ischemic preconditioning, four groups of sheep were fully heparinized to keep activated clotting time readings greater than 750 seconds and subjected to 60 minutes of normothermic regional ischemia (diagonal artery occlusion) with 3 hours of reperfusion. Group I was the control with no treatment, group II received aprotinin (1 million KIU load followed by 250,000 KIU/h), group III underwent ischemic preconditioning (three 5-minute intervals of ischemia and reperfusion) before prolonged 1-hour ischemia, and group IV underwent similar ischemic preconditioning and received aprotinin. Area at risk was delineated by monastryl blue pigment, and infarction size by tetrazolium staining.

Results. The ratios of weight of area at risk to left ventricular weight and left ventricular weight to body weight were constant between groups. Infarction size to area at risk ratio data demonstrated that aprotinin increases infarction size by 60% (infarction size to area at risk ratio from 52% ± 10% to 84% ± 10% for I versus II; p < 0.001). Aprotinin also attenuates the protective effect of ischemic preconditioning (infarction size to area at risk ratio from 25% ± 4% to 41% ± 6%; p < 0.001).

Conclusions. In the setting of ischemia, aprotinin increases myocardial damage. If, however, the heart is provided with protective preconditioning, then the deleterious effect of aprotinin may be neutralized. From these data we suggest that aprotinin should not be used routinely in cardiac operations unless extensive blood loss is anticipated, such as in redo open heart operations.

	Introduction

Top Footnotes Abstract Introduction Material and Methods Results Comment Acknowledgments References

 
See also page 310.

To reduce postoperative bleeding in cardiac surgical patients, aprotinin is used extensively in Europe [1–3], but its safety has been questioned in the United States, where a higher prevalence of perioperative myocardial infarction is reported [4]. However, the risks associated with routine blood transfusions, as well as the potential limitations of the banked blood supply, have generated great interest in methods to limit patient exposure to homologous blood transfusions. The indication for the routine use of aprotinin in cardiac surgical procedures remains debatable as renal, hepatic, neural, and myocardial organ system damage is associated with its use.

Ischemic preconditioning (IPC) is of increasing interest as an intraoperative myocardial management strategy because of its remarkable potency and consistent efficacy [5]. In the present investigation, we sought to determine whether aprotinin had any effect on the myocardial damage produced by regional normothermic ischemia and whether this effect could be influenced by IPC.

	Material and Methods

Top Footnotes Abstract Introduction Material and Methods Results Comment Acknowledgments References

 
Animals received humane 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 National Institutes of Health (NIH publication 85-23, revised 1985).

Surgical Preparation
Twenty-four sheep (Dorset or Suffolk of either sex weighing 35 to 45 kg) were sedated with ketamine hydrochloride (20 mg/kg intramuscularly) and then anesthetized with sodium pentobarbital (25 mg/kg intravenously). A tracheostomy was performed through a midline cervical incision and ventilation begun with a volume-cycled ventilator (Harvard Apparatus, Natick, MA). The right internal jugular vein was cannulated for intravenous access and the left common central artery for arterial blood sampling and intraarterial pressure monitoring. Through an anterior bilateral thoracotomy, the pericardial sac was exposed and opened to form a pericardial cradle. Heparin sodium (Elkins-Sinn, Inc, NJ) was given (300 IU/kg intravenously) after thoracotomy, and the same dose was repeated hourly to the end of the experiment. The activated clotting time was measured repeatedly throughout the procedure using celite activating reagent and an 801 Hemochron machine (International Technidyne Corporation, Edison, NJ). The activated clotting time was measured before heparin, after heparin bolus, after aprotinin bolus, during regional ischemia, and during reperfusion. The aim was to keep the activated clotting time greater than 750 seconds to meet the recommended level in the literature [1, 6]. Heart rate (80 to 120 beats/min), mean aortic pressure (60 to 100 mm Hg), and oxygen tension (>100 mm Hg) were constant throughout the study.

Experimental Protocol
The 24 sheep were assigned randomly to the four study groups as follows. Group I was the control as neither aprotinin nor IPC was provided. To complete the model, regional ischemia was performed by snaring the second or third diagonal coronary artery at normothermia for 60 minutes followed by release of the snare and reperfusion for 180 minutes. Group II received aprotinin (Miles Inc, West Haven, CT) both during and after ischemia (1,000,000 KIU load followed by 250,000 KIU/h infusion) but no IPC. Group III underwent IPC (three 5-minute intervals of ischemia and reperfusion) without aprotinin. Group IV received aprotinin after the IPC but before the prolonged normothermic ischemic insult.

Data Collection
Area at risk (AR) was delineated by monastryl blue pigment injection into the aorta after ligation of the involved artery at the end of the procedure. Infarction size (IS) was obtained by tetrazolium staining and computerized planimetry [7]. The ratios of AR to left ventricular weight (LV) and LV to total body weight were calculated. Infarction size to AR ratios were derived from the planimetery data. All data were normalized as a percentage.

Statistical Analysis
A two-way analysis of variance with respect to the effects of IPC and aprotinin was performed. Subgroup analyses were performed by post hoc Tukey test. A p value less than 0.05 was considered significant. All data are presented as mean ± standard deviation.

	Results

Top Footnotes Abstract Introduction Material and Methods Results Comment Acknowledgments References

 
The AR/LV and LV/total body weight ratios were equivalent in all groups (Table 1). In the control group the mean IS/AR was 52% ± 10%. Aprotinin increased the infarct size in group II (84% ± 10%; p < 0.001) (Table 2, Fig 1). With ischemic preconditioning (group III), the mean IS/AR was reduced to 25% ± 4%, and this protective effect was abolished when aprotinin was combined with IPC (group IV), as the mean IS/AR was 41% ± 6% (p < 0.008) (see Table 2, Fig 1).

View larger version (29K): [in this window] [in a new window]   Fig 1. . Normalized infarction size to area at risk ratios in the respective study groups. Aprotinin (AP) augmented ischemic damage, whereas ischemic preconditioning (IPC) attenuated the response. Data are shown as mean ± standard deviation. (Significance: *I versus II, p < 0.001; **I versus III; p < 0.001; #II versus IV, p < 0.001; ##III versus IV, p < 0.008; I versus IV is not significant.)

	Comment

Top Footnotes Abstract Introduction Material and Methods Results Comment Acknowledgments References

 
These data provide new evidence that aprotinin administration may be deleterious in the setting of acute myocardial ischemia. One hour of regional coronary arterial occlusion at normothermia produced a myocardial infarction that was approximately 50% the size of the region at risk in this ovine model. These control group data (group I) are consistent with many other models of regional normothermic ischemia. By providing a preconditioning stimulus, namely, the three brief episodes of repeated normothermic ischemia, the myocardial infarction response to a prolonged normothermic ischemic insult was attenuated to approximately one quarter of the AR (50% reduction in infarct size, group III) as has been previously described [8]. Adding aprotinin by continuous infusion during the ischemic interval without preconditioning resulted in a marked increase in the infarct size (group II). It should be noted that this occurred in the presence of maximal anticoagulation, thereby minimizing the potential for small-vessel or microvascular thrombosis as an etiologic mechanism. When aprotinin administration was preceded by a preconditioning stimulus (group IV), the infarction size was limited back to the control level (group I). Although the preconditioning stimulus nullified the deleterious effects of aprotinin, the resultant infarction was twice as large as that observed with preconditioning alone (group III). These data suggest, then, that aprotinin administration in the setting of repeated regional ischemia (ischemic preconditioning) negates the beneficial effects of the IPC and is, therefore, deleterious.

As noted above, all animals were maintained in a maximally anticoagulated state. Although not proved by pathologic examination, one may suggest that these animals were not subject to microvascular thrombosis as a potential etiologic mechanism of the deleterious effects of aprotinin. One may conclude, then, that a direct negative cardiac effect may be operative. Whether this direct effect occurs on the coronary vascular endothelium or on the myocyte remains to be elucidated. However, consideration of the mechanism of action of aprotinin and IPC may yield a plausible schema.

Aprotinin acts on blood constituents by preventing fibrinolysis, preserving platelets, and inhibiting the kallikrein-mediated amplification of contact coagulation [1–3]. It inhibits activated protein kinase-C [3, 9, 10], and by inhibiting plasma and tissue kallikrein reduces the prostacyclin/thromboxane ratio [11] and nitric oxide release [12]. These effects of aprotinin on the endothelium may lead to microvascular vasoconstriction or occlusion, which could explain our findings of a higher myocardial infarction size in the group given aprotinin without IPC (group II) compared with the control group (p < 0.001).

Although the precise mechanism of IPC is not well defined, the presently favored theory is that adenosine A₁ or ₁-adrenergic receptor stimulation by transient ischemia may activate protein kinase-C [13], which in turn increases the activity of 5`-nucleotidase and increases the production of adenosine [14, 15]. Adenosine plays a multifactorial role in the protection of the ischemic myocardium [16]. Pretreatment with either of the nonselective adenosine antagonists 8-sulphophenyl-theophylline or PD 115,199 abolished the protective effect of IPC [17]. Adenosine A₁ receptor activation preserves the intracellular adenosine triphosphate and, coupled to GI proteins, attenuates ß-adrenoceptor–mediated increases in myocardial contractility and Ca²⁺ influx into the myocytes [5, 17–21]. Ischemic preconditioning protects the coronary arteriolar endothelium and enhances postischemic coronary blood flow, which is mediated by adenosine A₂ receptors [19, 20, 22–24].

Bradykinin is thought to be a primary mediator of the protective antiarrhythmic effect of IPC because it enhances the endothelial release of nitric oxide and prostacyclin [12, 25, 26]. Wall and associates [26] showed that 5 minutes of intraatrial bradykinin infusion followed by 10 minutes of recovery similarly reduces the infarct size relative to the area at risk. The protective effectiveness was blocked in both groups by the bradykinin blocker HOE 140 [26].

The data of the present study indicate a complex interaction between the IPC response, mediated by adenosine A₁ or A₂ receptor stimulation or ₁-adrenergic receptor stimulation, and the direct effects of aprotinin on those same receptors, as well as the bradykinin kallikrein system. Future studies using adenosine receptor blockade (8-sulphophenyl-theophylline) or with -antagonists are clearly warranted. Whether the stress response mediated by extracorporeal circulation mediates the findings of normothermic ischemia and aprotinin administration on infarction size, likewise, requires further work. However, the data of the present study suggest that aprotinin increases the myocardial infarction size in the setting of acute regional ischemia. Similarly, it abolishes the myocardial protector effect of IPC. From these data we propose that aprotinin should not be used routinely in cardiac operations, particularly for acute myocardial ischemia (eg, myocardial revascularization). A ``selective'' use may be adopted to include redo operations, coronary revascularization after failed thrombolytic therapy, religious contraindication of blood transfusion, and with postcardiotomy ventricular assist devices.

	Acknowledgments

Top Footnotes Abstract Introduction Material and Methods Results Comment Acknowledgments References

 
This work was supported in part by National Institutes of Health grants HL29077 and R29 HL48751, and by the Prince Sultan Cardiac Center, Riyadh, Saudi Arabia. We express our appreciation to Debra A. Sheldon for manuscript preparation.

	Footnotes

Top Footnotes Abstract Introduction Material and Methods Results Comment Acknowledgments References

 
Presented at the Thirty-first Annual Meeting of The Society of Thoracic Surgeons, Palm Springs, CA, Jan 30–Feb 1, 1995.

Address reprint requests to Dr Krukenkamp, Deaconess Hospital, 110 Francis St, Suite 2-C, Boston, MA 02215.

	References

Top Footnotes Abstract Introduction Material and Methods Results Comment Acknowledgments References

 

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This Article Abstract Alert me when this article is cited Alert me if a correction is posted Citation Map Services Email this article to a friend Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Add to Personal Folders Download to citation manager Author home page(s): Irvin B. Krukenkamp Paul G. Burns Sidney Levitsky Permission Requests Citing Articles Citing Articles via HighWire Citing Articles via Google Scholar Google Scholar Articles by Bukhari, E. A. Articles by Levitsky, S. Search for Related Content PubMed PubMed Citation Articles by Bukhari, E. A. Articles by Levitsky, S. Related Collections Related Article