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Ann Thorac Surg 2002;73:1897-1904
© 2002 The Society of Thoracic Surgeons
a Department of Cardiovascular Surgery, University Hospital (Inselspital), Berne, Switzerland
Accepted for publication February 15, 2002.
* Address reprint requests to Dr Englberger, Department of Cardiovascular Surgery, Inselspital University Hospital, Freiburgstrasse 3010, Berne, Switzerland
e-mail: lars.englberger{at}insel.ch
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Abstract |
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 Top Abstract IntroductionPatients and methodsResultsCommentReferences |
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Methods. Patients were prospectively randomized into two groups: the control group (C) (n = 14) and the low-dose aprotinin group (A) (n = 15) with (2 x 106 KIU = 280 mg) aprotinin added to the pump prime. Cytokine response (interleukin-6, soluble TNF II receptor), terminal complement production (SC5b-9), and neutrophil activation (lactoferrin) were assessed up to 6 hours postoperatively. Clinical data and hemostatic factors including fibrinopeptide A, thrombin-antithrombin complex, D-dimer, and plasmin/
2-antiplasmin were investigated.
Results. In both study groups, a significant increase of all inflammatory markers was seen (IL-6, sTNF-IIR, SC5b-9, lactoferrin), p less than 0.001. Peak levels of complement production occurred after protamine administration, whereas cytokine increases were more pronounced postoperatively with marked elevation up to 6 hours. The markers did not differ significantly between groups throughout the study period (p > 0.05 at each time of determination). However, after protamine administration reduced fibrinolysis (D-dimer, plasmin/2-antiplasmin) was detected in group A. Measurements for coagulation (fibrinopeptide A, thrombin-antithrombin complex) were not significantly influenced by aprotinin. The total amount of blood loss during the first 24 hours was significantly reduced in group A (p < 0.02).
Conclusions. Low-dose aprotinin added to the pump prime does not inhibit the inflammatory response caused by cardiopulmonary bypass, but improves postoperative hemostasis. A potential effect of high-dose aprotinin on inflammatory markers remains to be elucidated.
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Introduction |
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TopAbstractIntroductionPatients and methodsResultsCommentReferences |
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Aprotinin, a nonspecific serine protease inhibitor is known to improve postoperative hemostasis and may beneficially modify the inflammatory reaction [2]. Many studies confirm that beside the impact of high-dose aprotinin on postoperative blood loss, the efficacy of a single dose (2 x 106 KIU = 280 mg) added to the pump prime [3–5] or other low-dose regimens [6] may be beneficial. There are much less patient study data on the potential of aprotinin to modify the inflammatory response. Wachtfogel and colleagues [7] showed in simulated extracorporeal perfusion that high-dose aprotinin may not only preserve platelet function but also may inhibit complement activation, neutrophil degranulation, and kallikrein production. In a clinical setting, using different dosages of aprotinin, some studies state an anti-inflammatory activity of aprotinin that is characterized by reduced release of tumor necrosis factor-, up-regulation of neutrophil integrin CD11d [8, 9], an enhanced endogenous release of the anti-inflammatory interleukin-10 [10], and lowered levels of interleukin-6 [11]. However, conflicting data also exists [12–14].
Despite the fact that high-dose aprotinin may have a better impact on postoperative blood loss, many surgeons prefer a pump-prime protocol with routine operations. This has been proven to lower drainage volume as well as transfusion requirements [3–5] and it offers greater economic effectiveness compared with high-dose administration [15].
With respect to daily clinical situations and the majority of patients who need routine myocardial revascularization, this study was conducted to evaluate the effects of low-dose aprotinin on inflammatory markers.
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Patients and methods |
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TopAbstractIntroductionPatients and methodsResultsCommentReferences |
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Cardiopulmonary bypass
For CPB, a roller pump (Sarns 9000; 3 mol/L Health Care Group, Ann Arbor, MI) and a cooling system (Sarns TCM II; 3 mol/L Health Care Group) were used. The extracorporeal circuit consisted of a tubing set (Jostra AG, Hirrlingen, Germany) connected to a hard-shell cardiotomy reservoir (Dideco Venocard D 774), a membrane oxygenator (Jostra Quadrox HMO 1000), an arterial line filter (Jostra HBF 40), and a blood cardioplegia system (3 mol/L Conducer 4:1). Identical components were used in all study patients.
The circuits were primed with a mixture of 1000 mL Ringer’s lactate solution and 1000 mL 3.5% gelatin preparation. Only in group A, (2 x 106 KIU = 280 mg) aprotinin was added to the pump prime.
Activated clotting time (ACT) during CPB in both groups was adjusted to 600 seconds. Before aortic cannulation, individual doses of heparin were given through the central venous line to achieve an ACT level approximately 100 seconds below the target. Further prolongation of the ACT could be expected by hemodilution, which occurs when CPB is started. During CPB, ACT was kept around 600 seconds by administration of more heparin as required. The ACT was measured using a kaolin-activated system (automated coagulation timer ACT II; Medtronic HemoTec Inc, Englewood, NJ) before skin incision, after initial heparinization, after first and each further delivery of cardioplegia, after aortic declamping, after protamine administration, and at 2, 4, and 6 hours after the operation. After cessation of CPB, the total amount of heparin given initially, as well as 50% of the additional given dose during CPB, were neutralized by an equivalent dose of protamine sulfate (1 mg per 100 IU heparin).
Extracorporeal circulation was performed with moderate systemic hypothermia (32°C) and nonpulsatile flow at a rate of 2.4 L/min per m2 body surface. If additional volume was required, macromolecular solution (3.5% gelatin preparation) was added to the circuit. Each patient received mannitol during CPB. Transfusion of packed red blood cells was performed at a hematocrit of less than 18%. While the patient was fully heparinized on CPB, a cardiotomy suction devise was used to return pericardial blood. After CPB, unprocessed pump blood retained in the circuits was returned to the patient.
Anesthesia, operation, and postoperative care
After premedication with a benzodiazepine, general anesthesia was induced and maintained with midazolam, fentanyl, and inhalatives (isoflurane or enflurane). Muscle relaxation was achieved with pancuronium bromide. Standard monitoring methods including electrocardiography, radial artery catheter, central venous catheter, two peripheral venous catheters, urinary catheter, and rectal temperature measurement were used in all patients.
All patients were operated on according to a standardized surgical protocol. After midline sternotomy the left internal mammarian artery was harvested in all patients except 1 in group C who had a stenosis of the left subclavian artery. Antegrade cold blood cardioplegia combined with topical cooling was used for myocardial protection. Distal anastomoses were done first; proximal anastomoses after release of the aortic cross-clamp was done by using a side-biting clamp.
Using the venous reservoir of the extracorporeal circuit as a collection box for up to 12 hours after the operation, mediastinal blood shed was retransfused, if nessessary (hemoglobin < 9.5 g/dL; total drainage volume > 250 mL; clinical hypovolemia; ACT < 140 seconds). A red blood cell transfusion was administered when hemoglobin decreased to less than 8.0 g/dL. Transfusion of fresh frozen plasma was indicated to correct suspected deficiency of coagulation factors when drain production was increased (>200 mL/hour). In the intensive care unit the patients were treated according to routine protocol. Intensive care unit personnel were not informed to which study group the patient was randomized.
Laboratory analyses
Arterial blood samples were collected at seven different time points: preoperatively, after induction of anesthesia (base line); 5 minutes after first cardioplegia; 5 minutes after aortic declamping; 10 minutes after protamine administration; and 2, 6, and 24 hours postoperatively. Samples for analysis of the routine hematologic measurements (hematocrit, hemoglobin, erythrocyte count, white blood cell count, platelet count) were given to the central laboratory of the hospital for routine analysis. In all the other samples the corpuscular content was separated from the fluid phase by centrifugation at 2,000 g for 20 minutes at 6°C. Plasma and serum were stored at -80°C before being assayed.
Lactoferrin, released by activated neutrophils, was analyzed from EDTA-plasma using an ELISA (Bioxytech, Oxis International Inc, Portland, OR). SC5b-9, reflecting the final step of complement activation, was analyzed by the ELISA technique (Quidel, San Diego, CA). For cytokine response interleukin 6 (IL-6) and the soluble tumor-necrosis factor II receptor (sTNF-IIR) were measured also using ELISA methods (MQ2 to 39C3, 18882D and 18871D Antibodies, PharMingen, San Diego, CA; Human TNF-RII CytoSets (Biosource Europe SA, Fleurus, Belgium). SC5b-9, IL-6 and sTNF-IIR were determined in serum. To determine factors of coagulation and fibrinolysis, plasma was used treated with CTADPPACK as an anticoagulant [16]. Coagulation factors fibrinopeptide A and thrombin-antithrombin complex were measured by using a radioimmunoassay, (IMCO Corporation Ltd, AB, Stockholm, Sweden) and the ELISA technique (Enzygnost thrombin-antithrombin complex micro; Behring, Marburg, Germany), respectively. D-dimer (Dimertest, Agen Biomedical, Acacia Ridge, Australia) and plasmin/2-antiplasmin complex, (Enzygnost plasmin/2-antiplasmin complex micro, Behring, Marburg, Germany) as reflecting the fibrinolytic status also were measured using the ELISA technique. Measurements were not corrected for hemodilution.
Statistical analysis
Statistical analysis was carried out using standard software (S-Plus 4.5; MathSoft Inc, Seattle, WA). Global testing of laboratory results for groups difference, time effects, and groups interactions was done using the method as outlined by Akritas and Brunner [17], supplemented by the parametrical analogues. The Wilcoxon rank-sums test was applied for direct comparisons between groups at one time point or for distinct patient characteristics. Trends were analyzed with the page test. Holm correction or Bonferroni correction were used if necessary. Laboratory results are shown as median (25th to 75th percentiles), a more precise value for descriptive statistics in small sample sizes. If appropriate, other data are presented as mean ± standard deviation. A p value less than 0.05 was considered statistically significant.
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Results |
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Compared with base line, the median concentration of terminal complement complex (SC5b-9) was elevated at all sampling points. During CPB, median values of SC5b-9 increased markedly in both groups. Peak values were determined after protamine administration (569.5 [416.8 to 652.0] ng/mL in group C and 540.5 [421.8 to 754.0] ng/mL in group A; p = no significance). No significant intergroup differences were determined (Fig 3).
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Lactoferrin levels showed a double peaked time course. A first significant increase during CPB and asecond one was noted 2 hours after operation. After aortic declamping, peak values were measured in both groups (1678 [1236 to 1856] ng/mL in group C vs 1467 [1285 to 2036] ng/mL in group A; p = no significance). Although median levels were lower in the aprotinin group at all sampling points, no significant intergroup differences were detected (Fig 3).
Coagulation and fibrinolysis
As indicator of thrombin formation, the plasma concentration of fibrinopeptide A was elevated perioperatively with peak values after protamine administration (25.1 [21.6 to 39.4] ng/mL in group C vs 31.6 [23.1 to 40.6] ng/mL in group A; p = no significance). After operation, median values returned towards base line, but were still elevated 24 hours postoperatively. No significant intergroup differences were determined (Fig 4).
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Preoperative median levels of D-dimer were within the normal range. After an elevation during CPB and peak values after protamine administration in both groups (362.0 [196.0 to 725.0] ng/mL in group C vs 171.0 [82.8 to 187.0] ng/mL in group A; p < 0.002), D-dimer levels remained markedly elevated in the postoperative period. Higher median values were detected in group C at the end of the operation and up to 24 hours postoperatively (163.0 [104.0 to 195.0] ng/mL in group C vs 63.3 [48.8 to 74.9] ng/mL in group A; p < 0.001) (Fig 4).
Median plasmin/2-antiplasmin complex levels showed an initial decrease during CPB followed byincreasing values postoperatively. Two hours after operation, maximal values were determined with significantly lower levels in group A (1406 [1148 to 1847] µg/L vs 937 [727 to 1125] µg/L; p < 0.05). Twenty-four hours postoperatively, median plasmin/2-antiplasmin complex values dropped beneath base line measurements (Fig 4).
The values of platelets, hemoglobin, and hematocrit were very similar in both the aprotinin and control groups (Table 4).
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Comment |
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In addition, aprotinin may also attenuate the systemic inflammatory reaction initiated by CPB [2]. This complex biochemical response involves activation of kallikrein and complement cascades, activation of neutrophils with degranulation and protease enzyme release, oxygen radical production, and the synthesis of various cytokines. An increasing number of studies demonstrate that elevated concentrations of proinflammatory markers are associated with organ dysfunction and adverse clinical outcome [19]. Furthermore, coagulation and fibrinolytic system become strongly activated during and after CPB. Despite full heparinization during CPB, activation of thrombin is not completely suppressed [20]. Thrombin triggers conversion of plasminogen to plasmin resulting in a hyperfibrinolytic state. In fact, hemostatic and inflammatory cascades are not separated from each other, but there is a strong linkage on different levels [1]. With this physiologic background one may assume direct and indirect effects of aprotinin on the inflammatory response. We tested the hypotheses whether low-dose aprotinin (2 x 106 KIU = 280 mg) added to the pump prime alters the plasmatic and cellular inflammatory response in routine coronary artery bypass grafting.
As expected, the present study confirms the proinflammatory response to CPB with increased levels of IL-6, sTNF-IIR, terminal complement complex (SC5b-9), neutrophil activation (lactoferrin), and elevated white blood cell count. Differences among the inflammatory markers were detected after time. Complement activation occurs rapidly with maximum plasma levels during an operation, whereas the cell-mediated response is reflected by elevated concentrations of cytokines in the postoperative period.
IL-6 is a multifunctional proinflammatory cytokine consistently elevated after a major operation, especially after CPB [21]. Raised plasma levels have been associated with cardiac dysfunction [19, 22]. Endogenous TNF-receptors may attenuate cytotoxicity of TNF-, one of the central mediators of inflammation [23]. Our data show that cytokine response measured in terms of IL-6 and soluble TNF-II receptor are not altered by aprotinin administration. This is consistent with Ashraf and coworkers [12] who could not demonstrate significant effects of low-dose aprotinin on IL-6 levels. Other investigators have demonstrated attenuated release of IL-6 after high-dose aprotinin administration [11] and an equal anti-inflammatory effect of aprotinin compared with methylprednisolone in a clinical setting [8, 11]. These conflicting data may be explained by various doses of aprotinin. In addition, the inhibitor dose of aprotinin for kallikrein, one of the key effectors in the contact phase system, is much higher than for the inhibition of plasmin [24]. Therefore, our results support the use of higher dosages of aprotinin to attenuate the inflammatory response.
Complement activation takes place by blood contact with foreign surfaces primarily through the alternative pathway, leading to formation of the C3 convertase. In addition, after reversal of heparin, heparin-protamine complexes induce activation through the classic pathway. C3b fragments formed by C3 convertase initiate both positive feedback mechanism and formation of C5 convertase. Irrespective of initial pathway (classic or alternative), after generation of C3 convertase, subsequent steps of complement activation are the same, resulting in formation of terminal complement complex. Terminal complement complex consists of an active cytolytic membrane-bound complex (C5b-9) and a biological inactive soluble part (SC5b-9) that circulates. The latter also seems to be a reliable marker to test biocompatibility of artificial surfaces. In the present study, complement activation in the terminal pathway (SC5b-9) showed no significant intergroup differences. This can be noted in accordance with other investigators who also have shown no significant effects of aprotinin in full-dose regimens [13].
However, lactoferrin release has been shown to be unrelated to complement activation [25]. We could not demonstrate significant intergroup differences with respect to this marker of the cellular inflammatory response. It was most likely to find differences with respect to neutrophil activation because in vitro studies [7], animal studies [26], and studies in humans [9, 14] suggest changes in neutrophil response when aprotinin is used.
Nevertheless, focusing changes in coagulation and fibrinolysis during and after CPB, we could demonstrate beneficial effects of aprotinin in the pump prime only regimen. Using individual doses of heparin to reach a target ACT of 600 seconds during CPB, there were no significant differences detected in the coagulation products fibrinopeptide A or thrombin-antithrombin complex. In this study, ACT levels were determined using a kaolin-activated system, which in contrast to celite-activation does not produce prolonged ACT values in the presence of aprotinin [27]. Consecutively comparable amounts of heparin were administered to both groups. In fact, it may be possible that low plasma levels of aprotinin achieved with low-dose protocols may not have anticoagulative properties as stated for high-dose application of aprotinin [18]. Biochemically, our results demonstrate no hypercoagulability with the use of the pump prime dose [3] and do not support concerns about its safety [28], given the fact that proper systemic anticoagulation with heparin was performed. The activation of the clotting system, which could be observed in both study groups, was not followed by hyperfibrinolysis in aprotinin-treated patients. This underlines that improved hemostasis is mainly attributable to the antifibrinolytic action of aprotinin [29], which can be also stated in the low-dose regimen. In accordance with the biochemical findings, we were able to demonstrate clinically reduced blood loss and a decreased need for transfusion of packed red blood cells in patients treated with aprotinin.
According to our data we conclude that low-dose aprotinin added to the pump prime volume does not inhibit the inflammatory response caused by CPB, whereas hemostatic improvement can be observed. The rationale for using low-dose aprotinin in elective coronary artery bypass graft operation is to lower perioperative blood loss. Whether there is a dose-dependent effect of aprotinin on inflammatory markers as described for hemostatic benefits remains to be elucidated.
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