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“Low-Dose” Aprotinin Modifies Hemostasis but Not Proinflammatory Cytokine Release
“Low-Dose” Aprotinin Modifies Hemostasis but Not Proinflammatory Cytokine Release Saeed Ashraf, FRCS(CTh), Yi Tian, MCh, Dalia Cowan, BSc, Unnikrishnan Nair, FRCS, Ranjit Chatrath, FFRCA, Nigel R. Saunders, FRCS, Kevin G. Watterson, FRACS, and Paul G. Martin, PhD Cardiothoracic Department, Killingbeck Hospital, Leeds, United Kingdom
Background. Cytokines are implicated in the pathogenesis of the “whole-body inflammatory response” that may complicate the period after cardiopulmonary bypass (CPB). Low-dose aprotinin in the pump during CPB has been shown to improve postoperative hemostasis and platelet preservation. We tested the hypothesis that lowdose aprotinin influences the inflammatory reaction (in terms of cytokine release) after CPB. Methods. In a prospective, randomized study, 38 patients undergoing elective coronary artery bypass grafting were investigated. Nineteen patients received lowdose aprotinin (2 3 106 KIU [280 mg] in the pump), and a control group of 19 did not. Complement activation, cytokine production, leukocyte elastase release, D-dimer level, full blood count, postoperative blood loss, and transfusion requirements were analyzed before, during, and after CPB. Results. Interleukin-1b was not detected in either group, whereas traces of tumor necrosis factor-a were infrequently observed. Plasma elastase, interleukin-6,
interleukin-8, and neutrophil count increased (p < 0.001) during and after CPB compared with the baseline levels, reaching a peak at 2 hours after protamine administration in both groups before returning toward baseline at 24 hours. Proinflammatory cytokine markers did not differ significantly (p > 0.1) between the groups throughout the study period. The C5b-9 level increased (p < 0.001) in both groups perioperatively, reaching its peak 15 minutes after protamine. Twenty-four– hour postoperative blood loss was significantly (p < 0.001) reduced in the aprotinin group in association with markedly reduced D-dimer levels (p < 0.001). Patients in the aprotinin group also received significantly less banked blood postoperatively than the control group (p < 0.01). Conclusions. Low-dose aprotinin fails to modify proinflammatory cytokine release, yet confers hemostatic improvement through reduced fibrinolysis in patients undergoing routine coronary artery bypass grafting. (Ann Thorac Surg 1997;63:68 –73) © 1997 by The Society of Thoracic Surgeons
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tive blood loss in high-risk patients or procedures [13– 16]. Recent studies have shown that aprotinin in “high” doses can attenuate the whole-body inflammatory response by inhibiting kallikrein production completely, and complement and neutrophil activation partially [17, 18]. We conducted the present study to assess the influence of “low-dose” aprotinin (2 3 106 KIU [280 mg], favored by some as a potentially safer and cost-effective [19] alternative to a high-dose regimen) on hemostasis and proinflammatory cytokine levels in patients undergoing routine coronary artery bypass grafting.
ardiopulmonary bypass (CPB) induces hemorrhagic complications and initiates a biochemical and cellular “whole-body inflammatory response,” which may be associated with substantial morbidity and mortality [1–3]. A common mechanism is thought to be responsible, involving the inflammatory mediators of the complement system and cytokines [2– 4]. Cytokines are potent intercellular signaling molecules known to participate in the regulation of cellular growth, function, and differentiation [2– 4]. Interleukin-6 (IL-6) and interleukin-8 (IL-8) levels have been shown to be elevated during and after CPB and are associated with cardiac and pulmonary dysfunction after bypass [5, 6]. Various interventions have been used to reduce the intensity of the systemic inflammatory response secondary to CPB, eg, administration of corticosteroids [7], use of nonsteroidal antiinflammatory drugs [8], use of a leukocyte depletion filter [9], and use of heparin-coated circuits [10 –12]. Aprotinin is a low-molecular-weight peptide inhibitor of trypsin, kallikrein, and plasmin that is commonly used in cardiac operations for its ability to reduce postoperaAccepted for publication July 19, 1996. Address reprint requests to Dr Ashraf, Cardiothoracic Surgery, Killingbeck Hospital, York Road, Leeds, LS14 6UQ United Kingdom.
© 1997 by The Society of Thoracic Surgeons Published by Elsevier Science Inc
Patients and Methods After ethical committee approval and informed consent, 38 patients (31 men, 7 women) undergoing coronary artery bypass grafting were randomized to low-dose aprotinin (n 5 19; 16 men and 3 women) or a “noaprotinin” control group (n 5 19; 15 men and 4 women). Each patient had effort-induced angina pectoris refractory to maximal antianginal therapy and multivessel coronary artery disease (.70% vessel occlusion). Patients entering the study had an ejection fraction greater than 0.40. Exclusion criteria were unstable angina, myocardial infarction within the previous 3 months, reoperation, 0003-4975/97/$17.00 PII S0003-4975(96)00812-0
Ann Thorac Surg 1997;63:68 –73
diabetes mellitus, liver or kidney failure, severe asthma or chronic obstructive airway disease, and oral anticoagulant or immunosuppressive therapy. The techniques of anesthesia and CPB were standardized. After premedication, anesthesia was induced with fentanyl (30 mg/kg, intravenously); muscle relaxation was achieved with pancuronium bromide (0.1 to 0.2 mg/kg, intravenously). Mechanical ventilation was initiated (tidal volume 10 to 15 mL/kg, rate 12 to 15 breaths/min), and anesthesia was supported by inhalation of 1% isoflurane. Operative monitoring was identical in all patients. The extracorporeal circuit comprised a Stockert roller pump (Shiley Inc, Irvine, CA), a Shiley S100 membrane oxygenator, and polyvinylchloride tubes. In the aprotinin group, after a test dose, 2 3 106 KIU (280 mg) of aprotinin (Trasylol; Bayer AG, Leverkusen, Germany) was added to the priming solution of the extracorporeal circuit. The control group received an equivalent prime volume without aprotinin. Patients were given heparin just before the institution of CPB with 300 IU/kg, with additional dosing as necessary to maintain the activated clotting time greater than 480 seconds. Nonpulsatile extracorporeal circulation was initiated at flows of 2.4 to 2.6 L z m22 z min21. Moderate systemic hypothermia (28° to 30°C, nasopharyngeal) was uniformly used. Cardiac arrest was achieved by infusion of 1 L of cold blood cardioplegic solution and topical slush. All distal anastomoses were performed during a single period of crossclamping, and the proximal anastomoses to the aorta were completed during the rewarming period. Extracorporeal circulation was terminated at a nasopharyngeal temperature of 37°C. Heparin was neutralized after the end of CPB with protamine sulfate (1 mg/100 IU heparin).
Laboratory Measurements Venous blood samples (20 mL) were drawn into sodium citrate (0.32% w/v, 1:9 parts blood) immediately after the induction of anesthesia; 5 minutes after the onset and 5 minutes before the end of CPB; and 20 minutes, 2 hours, and 24 hours after protamine sulfate administration. Blood was centrifuged (10 minutes, 3,000 rpm, 4°C) to obtain plasma. Plasma specimens were frozen and stored at 280°C before batch analysis. Enzyme-linked immunosorbent assay techniques were used to measure each of the cytokines (tumor necrosis factor-a [TNF-a], IL-1b, IL-6, and IL-8) (R&D Systems, Minneapolis, MN), Ddimer (Diagnostica Stago Asnieres sur Seine, France), and terminal complement complex C5b-9 (Quidel, San Diego, CA). Plasma neutrophil elastase concentrations were determined using an autoanalyzer technique (E. Merck Diagnostica, Darmstadt, Germany). The limit of sensitivity of each assay was: TNF-a 5 5 pg/mL, IL-1b 5 0.3 pg/mL, IL-6 5 3 pg/mL, IL-8 5 20 pg/mL, C5b-9 5 16 ng/mL, and elastase 5 20 ng/mL. Full blood counts were measured at each sampling point, and no adjustment was made for hemodilution. Blood loss and blood transfusion were recorded until 24 hours after the operation. Packed red blood cells were infused when the hematocrit was less than 30%. Cardiac output was measured by the thermodilution technique
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Table 1. Clinical Characteristics of the 38 Patients Undergoing Coronary Artery Bypass Graftinga Characteristic
Control Group
Aprotinin Group
19 15:4 65 (50 –79) 80 (48 –110) 45 (24 –75) 3 (2–5)
19 16:3 61 (49 –72) 81 (55–124) 45 (33– 68) 4 (2– 4)
No. of patients Sex (male/female) Age (y) CPB time (min) Cross-clamp time (min) No. of distal grafts a
Data are presented as median (range), except as noted.
CPB 5 cardiopulmonary bypass.
through a Swan-Ganz catheter (Baxter Edward) from the mean of three readings. Cardiac index was calculated and reported in milliliters per kilogram. Systemic and pulmonary vascular resistances were calculated by conventional formulas and were not indexed.
Statistical Analysis The Mann-Whitney U test was used to assess the significance of differences between groups, and the Wilcoxon test was used for nonparametric repeated measures. Pearson’s correlation coefficient was used to assess associations (Statistica software). In all cases, less than 0.05 was considered significant.
Results The clinical characteristics of the 38 patients are summarized in Table 1. There were no statistically significant demographic differences observed between the aprotinin and control groups in terms of age, bypass and crossclamp times, or number of grafts. No patient required exploration for postoperative bleeding, and there were no operative deaths or important adverse complications; all patients were discharged from the intensive care unit on the first postoperative day. Twenty-four– hour total blood loss into the chest drain was significantly reduced in the aprotinin group compared with the control group (371 mL [range, 110 to 840 mL] versus 722 mL [range, 345 to 1,140 mL]; p , 0.001). There was also a significant reduction in the amount of bank blood used postoperatively in patients treated with aprotinin compared with controls: 12 versus 25 U, respectively; p , 0.01 (Fig 1).
Tumor Necrosis Factor-a and Interleukin-1b Levels of TNF-a and IL-1b did not change significantly with CPB in either group. Interleukin-1b was not detectable at all in either group, whereas traces of TNF-a were detected in only 5 patients in the control group and 7 patients in the aprotinin group.
D-Dimer Plasma D-dimer levels (Fig 2) were similar in both groups at induction, then increased and continued to increase during and at the end of CPB (p , 0.01), reaching a peak
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Fig 1. Postoperative blood loss (BL) and blood transfusion (BT) expressed in milliliters (median) were significantly (**p , 0.001; *p , 0.01) less in the aprotinin group (open bars) than in the control group (closed bars). Error bars 5 25th to 75th interquartile range.
at 20 minutes after protamine administration in both groups: 2,360 ng/mL (range, 377 to 3,316 ng/mL) in the control group versus 805 ng/mL (range, 322 to 2,092 ng/mL) in the aprotinin group (p , 0.0001). At 24 hours, the level of D-dimer returned to baseline in the aprotinin group but remained significantly elevated (p , 0.01) in the control group. The D-dimer level in the control group correlated significantly with postoperative blood loss: r 5 0.50, p , 0.04 (Fig 3).
Interleukin-6 The baseline level of IL-6 was below the sensitivity threshold of the assay in both groups, but rose progressively during CPB, reaching a peak (p , 0.001) 2 hours after protamine administration (median, 309 pg/mL [range, 97 to 432 pg/mL] in the control group; 260 pg/mL [range, 103 to 522 pg/mL] in the aprotinin group; p 5 not significant). The level returned toward baseline after 24 hours (Fig 4).
Interleukin-8 Changes in plasma concentrations of IL-8 with time are shown in Figure 5. The peak level of IL-8 occurred 2
Fig 2. Perioperative evolution of D-dimer levels in the control group (closed bars) and the aprotinin group (open bars). (CPB 5 cardiopulmonary bypass.)
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Fig 3. Correlation of the circulating D-dimer level (after protamine administration) with 24-hour postoperative blood loss in the control group.
hours after protamine administration; the level fell steadily thereafter toward the preoperative level in both groups after 24 hours. Release of IL-8 in both groups was correlated significantly with plasma elastase in the control group (r 5 0.70; p , 0.001) and in the aprotinin group (r 5 60; p , 0.001), as shown in Figure 6.
C5b-9 Complex The concentration of C5b-9 increased slightly 5 minutes after the initiation of CPB, and by the end of CPB, the concentration was significantly increased (p , 0.01). The maximum level was reached 20 minutes after the administration of protamine: 471 ng/mL (range, 77 to 1,553 ng/mL) in the control group versus 412 ng/mL (range, 44 to 977 ng/mL) in the aprotinin group (p 5 not significant). In both groups, the level of C5b-9 decreased rapidly at 2 hours after protamine, returning to the baseline level at 24 hours (Fig 7).
Plasma Leukocyte Elastase Plasma elastase level was significantly increased in both groups before the end of CPB compared with the postin-
Fig 4. Changes in the circulating concentration of interleukin-6 (IL-6) (median) with time in the control group (closed squares) and the aprotinin group (open triangles). Error bars 5 25th to 75th interquartile range. (CPB 5 cardiopulmonary bypass; NS 5 not significant.)
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Fig 5. Changes in the circulating concentration of interleukin-8 (IL-8) (median) with time in the control group (closed squares) and the aprotinin group (open triangles). Error bars 5 25th to 75th interquartile range. (CPB 5 cardiopulmonary bypass; NS 5 not significant.)
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Fig 7. Changes in the circulating concentration of C5b-9 (median) with time in the control group (closed squares) and the aprotinin group (open triangles). Error bars 5 25th to 75th interquartile range. (CPB 5 cardiopulmonary bypass; NS 5 not significant.)
Comment duction level, and it increased further in both groups to a peak level 2 hours after protamine (median, 126 ng/mL [range, 20 to 340 ng/mL] in the control group versus 190 ng/mL [range, 22 to 448 ng/mL] in the aprotinin group; p , 0.001). The level returned to baseline at 24 hours. There were no statistically significant differences between the groups.
Full Blood Counts Initial neutrophil numbers did not differ significantly between the groups. After initial bypass hemodilution, there was a progressive rise in the neutrophil count that began during rewarming, reaching a similar peak level 2 hours after protamine (median, 11.0 3 109/L [range, 5.2 to 13.8 3 109/L] versus 11.7 3 109/L [range, 6.3 to 19 3 109/L] in the control and aprotinin groups, respectively; p 5 not significant). Levels returned to baseline after 24 hours. Platelet numbers fell in both groups after the institution of bypass and remained low during the operation. Thereafter, the count rose in both groups. There was no significant difference between the groups.
Fig 6. Correlation of circulating interleukin-8 (IL-8) and elastase levels in the control group (closed squares; r 5 0.70, p , 0.001) and the aprotinin group (open triangles; r 5 0.60, p , 0.01).
Several investigators have shown an excellent improvement in hemostasis during and after CPB with low-dose aprotinin in the pump, which was postulated to be due to both inhibition of fibrinolysis and preservation of the platelet adhesive capacity through the platelet glycoprotein 1b receptor. It was suggested that when aprotinin is present in the pump prime during the “first pass” of blood through the circuit, platelet receptors are preserved [14, 15]. In the present study, low-dose aprotinin in the pump significantly reduced 24-hour blood loss and blood transfusions. The need for banked blood was reduced by approximately half in the treated patients. Moreover, in 12 of these 19 patients, no homologous blood was required. These findings are consistent with those of other studies that used the high-dose aprotinin regimen [13, 16, 17]. Fibrinolytic activity, measured as the concentration of plasma D-dimer (a breakdown product of cross-linked fibrin), showed a significantly greater level during and after CPB in the control than in the aprotinin patients, and the D-dimer level in the control group correlated significantly with postoperative blood loss, indicating that increased bleeding was associated with fibrinolysis in the control group. The predominant antifibrinolytic effect of aprotinin on platelet receptor preservation may be supported by the fact that in this and other published studies [13, 16], substantial decreases in platelet counts were similarly observed in both groups. However, platelet count cannot be considered a suitable proxy for platelet function. The present study confirms the proinflammatory response to CPB, with increases in IL-6, IL-8, C5b-9, plasma leukocyte elastase, and absolute neutrophil counts occurring in both groups. The anaphylatoxins C3a and C5a as well as the terminal complement complex C5b-9 have been shown to be elevated during CPB [2, 3, 10], and this rise precedes that of IL-6 [3]. This is consistent with our findings. Studies have shown that terminal complement complex (C5b-9) is a more useful index than C3 and C5 activation for accurate assessment of the inflammatory
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response to CPB [3, 10]. The complement products have been implicated in the increase in neutrophil activation and degranulation and endothelial adhesion. However, in contrast to other authors [13], we found no difference at any measured point between the aprotinin and the control groups in terms of C5b-9 or plasma leukocyte elastase. Although complement activation with the release of elastase has been shown to increase the production of IL-1b and TNF-a [3], reports to date on the plasma IL-1b and TNF-a response to CPB have been conflicting. In this study, no significant increase in IL-1b or TNF-a was demonstrated in either group. This agrees with some [3, 4], but not all previous reports [12, 20], and this disparity in the data may be attributable to the use of highsensitivity assay kits by some groups. Interleukin-6 is a major promoter of the acute-phase response and is synthesized by a variety of cell types, including endothelium and leukocytes [2, 12]. The acutephase response refers to the large and diverse systemic and metabolic changes that occur in response to events such as trauma and infection. This response is characterized by fever, leukocytosis, thrombocytosis, and production of many hormones. It is now evident that the acute-phase response may, in part, be responsible for some of the adverse complications of CPB. We did not find any difference between the groups in terms of IL-6 release, which is consistent with the findings of Seghaye and colleagues [21] in a pediatric cohort, in which lowdose aprotinin therapy proved unremarkable in terms of cytokine release. Interleukin-8 is a potent chemotactic factor that activates neutrophils and induces degranulation, loss of endothelial basement membrane integrity, and ultimately transendothelial neutrophil migration [4, 22]. The positive correlation of IL-8 with plasma elastase release in our study is consistent with that in another study [4], suggesting that elastase is released from damaged neutrophils activated by IL-8. Observations by Finn and associates [4] in pediatric patients undergoing CPB and by Endo and co-workers [23] in septic patients with adult respiratory distress syndrome also indicated that IL-8 was closely involved in the production and release of elastase. Although Wachtfogel and colleagues [18] in vitro and Van Oeveren and associates [13] in vivo have demonstrated an inhibitory effect of aprotinin on neutrophil activation and plasma elastase release and proposed related therapeutic benefits in cardiac operations, we did not observe these effects. Furthermore, no differences were revealed between the aprotinin and control groups with regard to proinflammatory cytokine profiles. This could be due to a dose-dependent phenomenon, as high-dose aprotinin (approximately 4 to 6 3 106 KIU [560 to 840 mg]) was used, in contrast to low-dose aprotinin (2 3 106 KIU [280 mg]) in the present study. Paradoxically, Blauhut and colleagues [24] made observations consistent with our own regarding elastase release, despite using high-dose aprotinin. Ethically, high-dose
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aprotinin use cannot be justified in routine coronary artery bypass grafting because of concerns relating to its impact on graft occlusion and other possible clinical sequelae [19, 25]. Low-dose aprotinin used in the pump is technically simple to administer and confers substantial bloodsaving benefit equivalent to that observed with the highdose protocol, yet at approximately half the cost. The implication of this study for our unit has been to adopt this low-dose regimen in all patients at high risk of postoperative bleeding, in the belief that this measure is likely to be both cost-effective and associated with fewer morbid sequelae than high-dose aprotinin. In summary, low-dose aprotinin fails to blunt the proinflammatory cytokine response to CPB; its major clinically relevant action appears to be at the fibrinolytic level. Further studies are indicated in high-risk cohorts to determine whether high-dose aprotinin use modifies the proinflammatory response and cytokine release.
References 1. Kirklin JK, Westaby S, Blackstone EH, Kirklin JW, Chenoweth DE, Pacifico AD. Complement and damaging effects of cardiopulmonary bypass. J Thorac Cardiovasc Surg 1983; 86:845–57. 2. Butler J, Rocker GM, Westaby S. Inflammatory response to cardiopulmonary bypass. Ann Thorac Surg 1993;55:552–9. 3. Steinberg JB, Kapelanski DP, Olson JD, Weiler JM. Cytokine and complement levels in patients undergoing cardiopulmonary bypass. J Thorac Cardiovasc Surg 1993;106:1008–16. 4. Finn A, Naik S, Nigel K, Levinsky RJ, Strobel S, Elliott M. Interleukin-8 release and neutrophil degranulation after paediatric cardiopulmonary bypass. J Thorac Cardiovasc Surg 1993;105:234– 41. 5. Hennein HA, Ebba H, Rodriguez JR, et al. Relationship of the proinflammatory cytokines to myocardial ischemia and dysfunction after uncomplicated coronary revascularization. J Thorac Cardiovasc Surg 1994;108:626–35. 6. Kalfin RE, Engelman RM, Rousou JA, et al. Induction of interleukin-8 expression during cardiopulmonary bypass. Circulation 1993;88:401– 6. 7. Engelman R, Rousou J, Flack J, Deaton D, Kalfin R, Das D. Influence of steroids on complement and cytokine generation after cardiopulmonary bypass. Ann Thorac Surg 1995; 60:801– 4. 8. Marewitz A, Faist E, Lang S, Endres S, Hultner L, Reichart B. Regulation of acute phase response after cardiopulmonary bypass by immunomodulation. Ann Thorac Surg 1993;55: 389–94. 9. Bando K, Pillai R, Cameron DE, et al. Leukocyte depletion ameliorates free radical mediated lung injury after cardiopulmonary bypass. J Thorac Cardiovasc Surg 1990;99:873–7. 10. Videm V, Svennevig JL, Fosse E, Semb G, Osterud A, Mollnes T. Reduced complement activation with heparincoated oxygenator and tubings in coronary bypass operation. J Thorac Cardiovasc Surg 1992;103:806–13. 11. Gu YJ, van Oeveren W, Akkerman C, Boonstra PW, Huyzen RJ, Wildevuur RH. Heparin-coated circuits reduce the inflammatory response to cardiopulmonary bypass. Ann Thorac Surg 1993;55:917–22. 12. Steinberg BM, Grossi E, Schwartz D, et al. Heparin bonding of bypass circuits reduces cytokine release during cardiopulmonary bypass. Ann Thorac Surg 1995;60:525–9. 13. Van Oeveren W, Jansen NJG, Bidstrup BP, et al. Effects of aprotinin on hemostatic mechanisms during cardiopulmonary bypass. Ann Thorac Surg 1987;44:640–5.
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14. Van Oeveren W, Harder MP, Roozendaal KJ, Eijsman L, Wildevuur CRH. Aprotinin protects platelets against the initial effect of cardiopulmonary bypass. J Thorac Cardiovasc Surg 1990;99:788–97. 15. Lavee J, Raviv Z, Smolinsky A, et al. Platelet protection by low-dose aprotinin in cardiopulmonary bypass: electron microscopic study. Ann Thorac Surg 1993;55:114–9. 16. Bidstrup BP, Harrison J, Royston D, Taylor KM, Treasure T. Aprotinin therapy in cardiac operations: a report on use in 41 cardiac centers in the United Kingdom. Ann Thorac Surg 1993;55:971– 6. 17. Wachtfogel YT, Kucich U, Hack CE, Gluszko P, Colman RW, Edmunds LH. Aprotinin inhibits the contact, neutrophil, and platelet activation system during simulated extracorporeal perfusion. J Thorac Cardiovasc Surg 1993;106:1–10. 18. Wachtfogel YT, Yanina T, Hack E, et al. Selective kallikrein inhibitors alter human neutrophil elastase release during extracorporeal circulation. Am J Physiol 1995;268:H1353–7. 19. Westaby S. Aprotinin in perspective. Ann Thorac Surg 1993; 55:1033– 41. 20. Jansen NJ, van Oeveren W, van den Broek L, et al. Inhibition by dexamethasone of the reperfusion phenomena in cardio-
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pulmonary bypass. J Thorac Cardiovasc Surg 1991;102: 515–25. Seghaye M, Duchateau J, Grabitz RG, et al. Influence of low-dose aprotinin on the inflammatory reaction due to cardiopulmonary bypass in children. Ann Thorac Surg 1996; 61:1205–11. Smith WB, Gamble JR, Clark-Lewis I, Vadas MA. Interleukin-8 induces neutrophil transendothelial migration. Immunology 1991;72:65–72. Endo S, Inda S, Yamashita N, Hoshi S, Yoshida M, Ceska M. Polymorphonuclear leukocyte elastase and plasma interleukin 8 levels in patients with ARDS. J Jpn Assoc Acute Med 1992;3:92– 6. Blauhut B, Gross C, Necek S, Doran JE, Spa¨th P, LunsgaardHansen P. Effects of high-dose aprotinin on blood loss, platelet function, fibrinolysis, complement, and renal function after cardiopulmonary bypass. J Thorac Cardiovasc Surg 1991;101:958– 67. Cosgrove DM III, Heric B, Lytle B, et al. Aprotinin therapy for reoperative myocardial revascularization: a placebocontrolled study. Ann Thorac Surg 1992;54:1031– 8.
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© 1997 by The Society of Thoracic Surgeons Published by Elsevier Science Inc
Ann Thorac Surg 1997;63:73 • 0003-4975/97/$17.00 PII S0003-4975(96)01285-4
Background. Cytokines are implicated in the pathogenesis of the “whole-body inflammatory response” that may complicate the period after cardiopulmonary bypass (CPB). Low-dose aprotinin in the pump during CPB has been shown to improve postoperative hemostasis and platelet preservation. We tested the hypothesis that lowdose aprotinin influences the inflammatory reaction (in terms of cytokine release) after CPB. Methods. In a prospective, randomized study, 38 patients undergoing elective coronary artery bypass grafting were investigated. Nineteen patients received lowdose aprotinin (2 3 106 KIU [280 mg] in the pump), and a control group of 19 did not. Complement activation, cytokine production, leukocyte elastase release, D-dimer level, full blood count, postoperative blood loss, and transfusion requirements were analyzed before, during, and after CPB. Results. Interleukin-1b was not detected in either group, whereas traces of tumor necrosis factor-a were infrequently observed. Plasma elastase, interleukin-6,
interleukin-8, and neutrophil count increased (p < 0.001) during and after CPB compared with the baseline levels, reaching a peak at 2 hours after protamine administration in both groups before returning toward baseline at 24 hours. Proinflammatory cytokine markers did not differ significantly (p > 0.1) between the groups throughout the study period. The C5b-9 level increased (p < 0.001) in both groups perioperatively, reaching its peak 15 minutes after protamine. Twenty-four– hour postoperative blood loss was significantly (p < 0.001) reduced in the aprotinin group in association with markedly reduced D-dimer levels (p < 0.001). Patients in the aprotinin group also received significantly less banked blood postoperatively than the control group (p < 0.01). Conclusions. Low-dose aprotinin fails to modify proinflammatory cytokine release, yet confers hemostatic improvement through reduced fibrinolysis in patients undergoing routine coronary artery bypass grafting. (Ann Thorac Surg 1997;63:68 –73) © 1997 by The Society of Thoracic Surgeons
C
tive blood loss in high-risk patients or procedures [13– 16]. Recent studies have shown that aprotinin in “high” doses can attenuate the whole-body inflammatory response by inhibiting kallikrein production completely, and complement and neutrophil activation partially [17, 18]. We conducted the present study to assess the influence of “low-dose” aprotinin (2 3 106 KIU [280 mg], favored by some as a potentially safer and cost-effective [19] alternative to a high-dose regimen) on hemostasis and proinflammatory cytokine levels in patients undergoing routine coronary artery bypass grafting.
ardiopulmonary bypass (CPB) induces hemorrhagic complications and initiates a biochemical and cellular “whole-body inflammatory response,” which may be associated with substantial morbidity and mortality [1–3]. A common mechanism is thought to be responsible, involving the inflammatory mediators of the complement system and cytokines [2– 4]. Cytokines are potent intercellular signaling molecules known to participate in the regulation of cellular growth, function, and differentiation [2– 4]. Interleukin-6 (IL-6) and interleukin-8 (IL-8) levels have been shown to be elevated during and after CPB and are associated with cardiac and pulmonary dysfunction after bypass [5, 6]. Various interventions have been used to reduce the intensity of the systemic inflammatory response secondary to CPB, eg, administration of corticosteroids [7], use of nonsteroidal antiinflammatory drugs [8], use of a leukocyte depletion filter [9], and use of heparin-coated circuits [10 –12]. Aprotinin is a low-molecular-weight peptide inhibitor of trypsin, kallikrein, and plasmin that is commonly used in cardiac operations for its ability to reduce postoperaAccepted for publication July 19, 1996. Address reprint requests to Dr Ashraf, Cardiothoracic Surgery, Killingbeck Hospital, York Road, Leeds, LS14 6UQ United Kingdom.
© 1997 by The Society of Thoracic Surgeons Published by Elsevier Science Inc
Patients and Methods After ethical committee approval and informed consent, 38 patients (31 men, 7 women) undergoing coronary artery bypass grafting were randomized to low-dose aprotinin (n 5 19; 16 men and 3 women) or a “noaprotinin” control group (n 5 19; 15 men and 4 women). Each patient had effort-induced angina pectoris refractory to maximal antianginal therapy and multivessel coronary artery disease (.70% vessel occlusion). Patients entering the study had an ejection fraction greater than 0.40. Exclusion criteria were unstable angina, myocardial infarction within the previous 3 months, reoperation, 0003-4975/97/$17.00 PII S0003-4975(96)00812-0
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diabetes mellitus, liver or kidney failure, severe asthma or chronic obstructive airway disease, and oral anticoagulant or immunosuppressive therapy. The techniques of anesthesia and CPB were standardized. After premedication, anesthesia was induced with fentanyl (30 mg/kg, intravenously); muscle relaxation was achieved with pancuronium bromide (0.1 to 0.2 mg/kg, intravenously). Mechanical ventilation was initiated (tidal volume 10 to 15 mL/kg, rate 12 to 15 breaths/min), and anesthesia was supported by inhalation of 1% isoflurane. Operative monitoring was identical in all patients. The extracorporeal circuit comprised a Stockert roller pump (Shiley Inc, Irvine, CA), a Shiley S100 membrane oxygenator, and polyvinylchloride tubes. In the aprotinin group, after a test dose, 2 3 106 KIU (280 mg) of aprotinin (Trasylol; Bayer AG, Leverkusen, Germany) was added to the priming solution of the extracorporeal circuit. The control group received an equivalent prime volume without aprotinin. Patients were given heparin just before the institution of CPB with 300 IU/kg, with additional dosing as necessary to maintain the activated clotting time greater than 480 seconds. Nonpulsatile extracorporeal circulation was initiated at flows of 2.4 to 2.6 L z m22 z min21. Moderate systemic hypothermia (28° to 30°C, nasopharyngeal) was uniformly used. Cardiac arrest was achieved by infusion of 1 L of cold blood cardioplegic solution and topical slush. All distal anastomoses were performed during a single period of crossclamping, and the proximal anastomoses to the aorta were completed during the rewarming period. Extracorporeal circulation was terminated at a nasopharyngeal temperature of 37°C. Heparin was neutralized after the end of CPB with protamine sulfate (1 mg/100 IU heparin).
Laboratory Measurements Venous blood samples (20 mL) were drawn into sodium citrate (0.32% w/v, 1:9 parts blood) immediately after the induction of anesthesia; 5 minutes after the onset and 5 minutes before the end of CPB; and 20 minutes, 2 hours, and 24 hours after protamine sulfate administration. Blood was centrifuged (10 minutes, 3,000 rpm, 4°C) to obtain plasma. Plasma specimens were frozen and stored at 280°C before batch analysis. Enzyme-linked immunosorbent assay techniques were used to measure each of the cytokines (tumor necrosis factor-a [TNF-a], IL-1b, IL-6, and IL-8) (R&D Systems, Minneapolis, MN), Ddimer (Diagnostica Stago Asnieres sur Seine, France), and terminal complement complex C5b-9 (Quidel, San Diego, CA). Plasma neutrophil elastase concentrations were determined using an autoanalyzer technique (E. Merck Diagnostica, Darmstadt, Germany). The limit of sensitivity of each assay was: TNF-a 5 5 pg/mL, IL-1b 5 0.3 pg/mL, IL-6 5 3 pg/mL, IL-8 5 20 pg/mL, C5b-9 5 16 ng/mL, and elastase 5 20 ng/mL. Full blood counts were measured at each sampling point, and no adjustment was made for hemodilution. Blood loss and blood transfusion were recorded until 24 hours after the operation. Packed red blood cells were infused when the hematocrit was less than 30%. Cardiac output was measured by the thermodilution technique
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Table 1. Clinical Characteristics of the 38 Patients Undergoing Coronary Artery Bypass Graftinga Characteristic
Control Group
Aprotinin Group
19 15:4 65 (50 –79) 80 (48 –110) 45 (24 –75) 3 (2–5)
19 16:3 61 (49 –72) 81 (55–124) 45 (33– 68) 4 (2– 4)
No. of patients Sex (male/female) Age (y) CPB time (min) Cross-clamp time (min) No. of distal grafts a
Data are presented as median (range), except as noted.
CPB 5 cardiopulmonary bypass.
through a Swan-Ganz catheter (Baxter Edward) from the mean of three readings. Cardiac index was calculated and reported in milliliters per kilogram. Systemic and pulmonary vascular resistances were calculated by conventional formulas and were not indexed.
Statistical Analysis The Mann-Whitney U test was used to assess the significance of differences between groups, and the Wilcoxon test was used for nonparametric repeated measures. Pearson’s correlation coefficient was used to assess associations (Statistica software). In all cases, less than 0.05 was considered significant.
Results The clinical characteristics of the 38 patients are summarized in Table 1. There were no statistically significant demographic differences observed between the aprotinin and control groups in terms of age, bypass and crossclamp times, or number of grafts. No patient required exploration for postoperative bleeding, and there were no operative deaths or important adverse complications; all patients were discharged from the intensive care unit on the first postoperative day. Twenty-four– hour total blood loss into the chest drain was significantly reduced in the aprotinin group compared with the control group (371 mL [range, 110 to 840 mL] versus 722 mL [range, 345 to 1,140 mL]; p , 0.001). There was also a significant reduction in the amount of bank blood used postoperatively in patients treated with aprotinin compared with controls: 12 versus 25 U, respectively; p , 0.01 (Fig 1).
Tumor Necrosis Factor-a and Interleukin-1b Levels of TNF-a and IL-1b did not change significantly with CPB in either group. Interleukin-1b was not detectable at all in either group, whereas traces of TNF-a were detected in only 5 patients in the control group and 7 patients in the aprotinin group.
D-Dimer Plasma D-dimer levels (Fig 2) were similar in both groups at induction, then increased and continued to increase during and at the end of CPB (p , 0.01), reaching a peak
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Fig 1. Postoperative blood loss (BL) and blood transfusion (BT) expressed in milliliters (median) were significantly (**p , 0.001; *p , 0.01) less in the aprotinin group (open bars) than in the control group (closed bars). Error bars 5 25th to 75th interquartile range.
at 20 minutes after protamine administration in both groups: 2,360 ng/mL (range, 377 to 3,316 ng/mL) in the control group versus 805 ng/mL (range, 322 to 2,092 ng/mL) in the aprotinin group (p , 0.0001). At 24 hours, the level of D-dimer returned to baseline in the aprotinin group but remained significantly elevated (p , 0.01) in the control group. The D-dimer level in the control group correlated significantly with postoperative blood loss: r 5 0.50, p , 0.04 (Fig 3).
Interleukin-6 The baseline level of IL-6 was below the sensitivity threshold of the assay in both groups, but rose progressively during CPB, reaching a peak (p , 0.001) 2 hours after protamine administration (median, 309 pg/mL [range, 97 to 432 pg/mL] in the control group; 260 pg/mL [range, 103 to 522 pg/mL] in the aprotinin group; p 5 not significant). The level returned toward baseline after 24 hours (Fig 4).
Interleukin-8 Changes in plasma concentrations of IL-8 with time are shown in Figure 5. The peak level of IL-8 occurred 2
Fig 2. Perioperative evolution of D-dimer levels in the control group (closed bars) and the aprotinin group (open bars). (CPB 5 cardiopulmonary bypass.)
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Fig 3. Correlation of the circulating D-dimer level (after protamine administration) with 24-hour postoperative blood loss in the control group.
hours after protamine administration; the level fell steadily thereafter toward the preoperative level in both groups after 24 hours. Release of IL-8 in both groups was correlated significantly with plasma elastase in the control group (r 5 0.70; p , 0.001) and in the aprotinin group (r 5 60; p , 0.001), as shown in Figure 6.
C5b-9 Complex The concentration of C5b-9 increased slightly 5 minutes after the initiation of CPB, and by the end of CPB, the concentration was significantly increased (p , 0.01). The maximum level was reached 20 minutes after the administration of protamine: 471 ng/mL (range, 77 to 1,553 ng/mL) in the control group versus 412 ng/mL (range, 44 to 977 ng/mL) in the aprotinin group (p 5 not significant). In both groups, the level of C5b-9 decreased rapidly at 2 hours after protamine, returning to the baseline level at 24 hours (Fig 7).
Plasma Leukocyte Elastase Plasma elastase level was significantly increased in both groups before the end of CPB compared with the postin-
Fig 4. Changes in the circulating concentration of interleukin-6 (IL-6) (median) with time in the control group (closed squares) and the aprotinin group (open triangles). Error bars 5 25th to 75th interquartile range. (CPB 5 cardiopulmonary bypass; NS 5 not significant.)
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Fig 5. Changes in the circulating concentration of interleukin-8 (IL-8) (median) with time in the control group (closed squares) and the aprotinin group (open triangles). Error bars 5 25th to 75th interquartile range. (CPB 5 cardiopulmonary bypass; NS 5 not significant.)
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Fig 7. Changes in the circulating concentration of C5b-9 (median) with time in the control group (closed squares) and the aprotinin group (open triangles). Error bars 5 25th to 75th interquartile range. (CPB 5 cardiopulmonary bypass; NS 5 not significant.)
Comment duction level, and it increased further in both groups to a peak level 2 hours after protamine (median, 126 ng/mL [range, 20 to 340 ng/mL] in the control group versus 190 ng/mL [range, 22 to 448 ng/mL] in the aprotinin group; p , 0.001). The level returned to baseline at 24 hours. There were no statistically significant differences between the groups.
Full Blood Counts Initial neutrophil numbers did not differ significantly between the groups. After initial bypass hemodilution, there was a progressive rise in the neutrophil count that began during rewarming, reaching a similar peak level 2 hours after protamine (median, 11.0 3 109/L [range, 5.2 to 13.8 3 109/L] versus 11.7 3 109/L [range, 6.3 to 19 3 109/L] in the control and aprotinin groups, respectively; p 5 not significant). Levels returned to baseline after 24 hours. Platelet numbers fell in both groups after the institution of bypass and remained low during the operation. Thereafter, the count rose in both groups. There was no significant difference between the groups.
Fig 6. Correlation of circulating interleukin-8 (IL-8) and elastase levels in the control group (closed squares; r 5 0.70, p , 0.001) and the aprotinin group (open triangles; r 5 0.60, p , 0.01).
Several investigators have shown an excellent improvement in hemostasis during and after CPB with low-dose aprotinin in the pump, which was postulated to be due to both inhibition of fibrinolysis and preservation of the platelet adhesive capacity through the platelet glycoprotein 1b receptor. It was suggested that when aprotinin is present in the pump prime during the “first pass” of blood through the circuit, platelet receptors are preserved [14, 15]. In the present study, low-dose aprotinin in the pump significantly reduced 24-hour blood loss and blood transfusions. The need for banked blood was reduced by approximately half in the treated patients. Moreover, in 12 of these 19 patients, no homologous blood was required. These findings are consistent with those of other studies that used the high-dose aprotinin regimen [13, 16, 17]. Fibrinolytic activity, measured as the concentration of plasma D-dimer (a breakdown product of cross-linked fibrin), showed a significantly greater level during and after CPB in the control than in the aprotinin patients, and the D-dimer level in the control group correlated significantly with postoperative blood loss, indicating that increased bleeding was associated with fibrinolysis in the control group. The predominant antifibrinolytic effect of aprotinin on platelet receptor preservation may be supported by the fact that in this and other published studies [13, 16], substantial decreases in platelet counts were similarly observed in both groups. However, platelet count cannot be considered a suitable proxy for platelet function. The present study confirms the proinflammatory response to CPB, with increases in IL-6, IL-8, C5b-9, plasma leukocyte elastase, and absolute neutrophil counts occurring in both groups. The anaphylatoxins C3a and C5a as well as the terminal complement complex C5b-9 have been shown to be elevated during CPB [2, 3, 10], and this rise precedes that of IL-6 [3]. This is consistent with our findings. Studies have shown that terminal complement complex (C5b-9) is a more useful index than C3 and C5 activation for accurate assessment of the inflammatory
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response to CPB [3, 10]. The complement products have been implicated in the increase in neutrophil activation and degranulation and endothelial adhesion. However, in contrast to other authors [13], we found no difference at any measured point between the aprotinin and the control groups in terms of C5b-9 or plasma leukocyte elastase. Although complement activation with the release of elastase has been shown to increase the production of IL-1b and TNF-a [3], reports to date on the plasma IL-1b and TNF-a response to CPB have been conflicting. In this study, no significant increase in IL-1b or TNF-a was demonstrated in either group. This agrees with some [3, 4], but not all previous reports [12, 20], and this disparity in the data may be attributable to the use of highsensitivity assay kits by some groups. Interleukin-6 is a major promoter of the acute-phase response and is synthesized by a variety of cell types, including endothelium and leukocytes [2, 12]. The acutephase response refers to the large and diverse systemic and metabolic changes that occur in response to events such as trauma and infection. This response is characterized by fever, leukocytosis, thrombocytosis, and production of many hormones. It is now evident that the acute-phase response may, in part, be responsible for some of the adverse complications of CPB. We did not find any difference between the groups in terms of IL-6 release, which is consistent with the findings of Seghaye and colleagues [21] in a pediatric cohort, in which lowdose aprotinin therapy proved unremarkable in terms of cytokine release. Interleukin-8 is a potent chemotactic factor that activates neutrophils and induces degranulation, loss of endothelial basement membrane integrity, and ultimately transendothelial neutrophil migration [4, 22]. The positive correlation of IL-8 with plasma elastase release in our study is consistent with that in another study [4], suggesting that elastase is released from damaged neutrophils activated by IL-8. Observations by Finn and associates [4] in pediatric patients undergoing CPB and by Endo and co-workers [23] in septic patients with adult respiratory distress syndrome also indicated that IL-8 was closely involved in the production and release of elastase. Although Wachtfogel and colleagues [18] in vitro and Van Oeveren and associates [13] in vivo have demonstrated an inhibitory effect of aprotinin on neutrophil activation and plasma elastase release and proposed related therapeutic benefits in cardiac operations, we did not observe these effects. Furthermore, no differences were revealed between the aprotinin and control groups with regard to proinflammatory cytokine profiles. This could be due to a dose-dependent phenomenon, as high-dose aprotinin (approximately 4 to 6 3 106 KIU [560 to 840 mg]) was used, in contrast to low-dose aprotinin (2 3 106 KIU [280 mg]) in the present study. Paradoxically, Blauhut and colleagues [24] made observations consistent with our own regarding elastase release, despite using high-dose aprotinin. Ethically, high-dose
Ann Thorac Surg 1997;63:68 –73
aprotinin use cannot be justified in routine coronary artery bypass grafting because of concerns relating to its impact on graft occlusion and other possible clinical sequelae [19, 25]. Low-dose aprotinin used in the pump is technically simple to administer and confers substantial bloodsaving benefit equivalent to that observed with the highdose protocol, yet at approximately half the cost. The implication of this study for our unit has been to adopt this low-dose regimen in all patients at high risk of postoperative bleeding, in the belief that this measure is likely to be both cost-effective and associated with fewer morbid sequelae than high-dose aprotinin. In summary, low-dose aprotinin fails to blunt the proinflammatory cytokine response to CPB; its major clinically relevant action appears to be at the fibrinolytic level. Further studies are indicated in high-risk cohorts to determine whether high-dose aprotinin use modifies the proinflammatory response and cytokine release.
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