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J Thorac Cardiovasc Surg 1995;110:963-970
© 1995 Mosby, Inc.
SURGERY FOR ACQUIRED HEART DISEASE |
Chicago, Ill., Temple, Tex., and Copenhagen, Denmark
From the Departments of Medicine, Surgery, and Anesthesia, Northwestern Memorial Hospital, Chicago, Scott and White Clinic, Temple, Texas, and Novo Nordist A/S, Copenhagen.
Received for publication Dec. 5, 1994. Accepted for publication Feb. 7, 1995. Reprint requests: David Green, MD, PhD, 345 E Superior St, Room 1407, Chicago, IL 60611.
Abstract
Objective: To evaluate the role of recombinant bovine aprotinin in reducing blood loss in coronary artery bypass graft surgery.Design: An open-label, randomized, controlled study evaluating two dosage levels of recombinant aprotinin.Setting: Two acute care hospitals (Northwestern Memorial Hospital, Chicago, Ill., and the Scott & White Memorial Hospital, Temple, Texas).Patients: Patients undergoing primary and reoperation coronary artery bypass grafting were assigned to groups by means of a computer-generated table of random numbers. Treated (n = 48) and control (n= 36) patients did not differ significantly in age, sex, weight, number of grafts, or preoperative hemoglobin level.Interventions: Recombinant aprotinin was given at two dosages. Dosage level 1 consisted of a bolus of 2 mg/kg intravenously immediately after the induction of anesthesia, 1 mg/kg added to each liter of the oxygenator prime, and 0.5 mg kg -1hr -1infused continuously during operation. At dosage level 2, doses were doubled. Intraoperative monitoring of anti–factor Xa activity was performed, and additional doses of heparin were given on the basis of anti–factor Xa results.Main outcome measures:Preoperative and postoperative hemoglobin levels, amounts of autotransfusion device and chest tube drainage blood, and transfusions of allogeneic red blood cells. Adverse clinical events (alterations in renal function, graft thrombosis, myocardial infarction, and death) were recorded.Results:Additional heparin was given to 48% patients in the aprotinin group and to 44% of control patients. Overall red blood cell loss (in milliliters, mean ± standard deviation [SD]) was decreased with aprotinin at dosage level 1 for reoperations (1040 ± 162 vs 1544 ± 198, p < 0.01), and at dosage level 2 for all operations (primary operations, 886 ± 362 vs 1333 ± 618, p = 0.02; reoperations, 1191 ± 560 vs 1815 ± 1116, p = 0.2). Fewer patients in the aprotinin than in the control group had transfusions of donated blood (6/48 vs 12/36, p = 0.02) or reinfusion of chest tube drainage blood (12/48 vs 20/36, p<0.01). Among patients receiving dosage level 1, there were no myocardial infarctions or deaths. At dosage level 2, one patient had profound bradycardia and died on day 12 and two patients had late graft closures. Two control patients had hypotension after bypass necessitating intraaortic balloon pumps, and one of these patients died. Postoperative increases in blood urea nitrogen and creatinine levels were small in both aprotinin and control groups. No hypersensitivity or other allergic reactions occurred.Conclusion:We conclude that, at the dosages given, recombinant bovine aprotinin decreases surgical blood loss and transfusion requirements in patients undergoing coronary artery bypass grafting, but its use requires appropriate monitoring of heparin use during bypass. Whether higher dosages of aprotinin increase the risk of graft thrombosis must be further assessed with a larger patient sample. (J THORAC CARDIOVASC SURG 1995;110:963-70)
Improved surgical techniques and equipment have resulted in major reductions in blood loss during coronary artery bypass grafting (CABG) operations. In December 1993, the U.S. Food and Drug Administration approved aprotinin, a polyvalent proteinase inhibitor derived from bovine tissue. This is a polypeptide of 58 aminoacids that has a molecular weight of about 6500 daltons. In several trials, aprotinin was shown to significantly limit blood loss and the need for blood replacement. 
1-3 Q-wave myocardial infarction, acute vein graft thrombosis, disseminated intravascular coagulation, renal dysfunction, and severe allergic reactions have been reported in conjunction with aprotinin treatment, 3,4 and these reports raise questions about the safety of the drug.
To avoid bovine tissue as the source of production, Novo Nordisk A/S (Copenhagen, Denmark) developed processes to produce recombinant bovine aprotinin (r-Apr) by fermentation using S. cerevisiae as the production organism. The recombinant protein appears to be identical with native aprotinin and is prepared with a purity of about 99%, as determined by high-pressure liquid chromatography. 5 It is supplied in vials of 50 ml containing 1.4 mg/ml of r-Apr with an activity of 10,000 kallikrein inhibitory units (KIU) per milliliter. Studies in rats, cats, and pigs showed no differences between r-Apr and native aprotinin with respect to either pharmacokinetics or pharmacodynamics. 6
We have evaluated the safety and effectiveness of r-Apr in patients undergoing either primary CABG or reoperations. Two dosage levels of the drug were studied; one was equivalent to the currently recommended dosage of native aprotinin and one was half this dosage. The lower dosage was evaluated at Northwestern Memorial Hospital (Chicago, Ill.), and the other dosage was evaluated at both Northwestern and the Scott & White Clinic (Temple, Texas). The effect of r-Apr on blood loss, transfusion requirements, and the results of safety monitoring are presented. The data indicate that r-Apr is a valuable agent for safely decreasing bleeding in patients undergoing CABG.
METHODS
Subjects
Consecutive patients requiring primary CABG or reoperations were recruited for this study. Patients were excluded if they had had a myocardial infarction within the previous 7 days, had an ejection fraction of less than 30%, had intractable congestive heart failure, were older than 79 years, or had received aspirin within 72 hours of operation. In addition, patients were ineligible if serum creatinine level exceeded 2 mg/dl, hematocrit was less than 30%, platelet count was less than 100,000 cells/µl, or the prothrombin time was above the normal reference range.
Study design
This was an open-label, randomized clinical trial conducted in two phases. In phase 1, patients assigned to the r-Apr group received 2 mg/kg (14,300 KIU/kg) as an intravenous bolus given in 20 minutes after the induction of anesthesia, an intravenous infusion of 0.5 mg 
kg-1 hr-1 (3570 KIU kg-1 hr-1 ) until the patient left the operating room, and 1 mg/kg (7143 KIU) added to each liter of lactated Ringer's solution for priming of the membrane oxygenator. In phase 2, each dose was doubled. Studies of dosage level 1 were performed in Chicago (42 patients), and studies of dosage level 2 were conducted both in Chicago (26 patients) and in Temple, Texas (16 patients). Patients were stratified according to center, surgeon (four in Chicago and two in Temple), and type of operation. Within each stratum, patients were assigned to groups by means of a computer-generated table of random numbers in permuted blocks of five for primary operations and two for reoperations. The study was approved by both institutions' Human Subjects Review Boards.
The sample size calculation was based on estimates of blood loss previously determined in patients undergoing CABG at Northwestern Memorial Hospital. For those undergoing primary CABG, the calculations indicated that it would be possible to show a difference greater than or equal to 1.2 SDs with a power of 90% and a significance level of 5% if 30 patients were evaluated at each dosage level. For those undergoing reoperations, 12 patients would be needed at each dosage level to show a difference 1.7 SDs with a power of 90% and a significance level of 5%. For primary operations, random assignment in a 3:2 ratio for the r-Apr versus control groups was calculated to result in a power loss of less than 5%. For reoperations, the ratio of r-Apr to control groups was 1:1. The random assignment was done for each participating surgeon.
Intraoperative monitoring of heparin
All patients received heparin at 300 U/kg immediately before cannulation of the aorta. Because aprotinin prolongs the heparin clotting time, as measured by the Celite-activated clotting time (ACT) test (Manville Service Corp., Denver, Colo.), 7 anticoagulation was monitored during cardiopulmonary bypass by means of a modified amidolytic anti–factor Xa assay. 8 A microcentrifuge (StatSpin Technologies, Norwood, Mass.) and enzyme-linked immunosorbent assay plate reader were mounted on a mobile cart, which was stationed adjacent to the operating theater. Blood samples were obtained at 30-minute intervals until the end of bypass or as clinically indicated. The blood was collected in tubes containing 3.8% sodium citrate and was immediately centrifuged for 2 minutes. The anti–factor Xa activity was measured as follows: plasma was diluted 1:30 in Tris(hydroxymethyl)aminomethane (Tris) buffer, pH 8.4; normal pooled plasma, controls, and standards were similarly diluted. Diluted samples were then placed in the wells of microtiter plates, incubated with antithrombin III (KabiVitrum AB, Stockholm, Sweden), and then factor Xa (Kabi) was added, followed 1 minute later by S-2222 (Kabi). The reaction was stopped at 5 minutes by the addition of COA Stopbuffer (Kabi), pH 3.0. The absorbance of the mixture at 405 nm was recorded with the enzyme-linked immunosorbent assay plate reader, with 620 nm used as reference. The anti–factor Xa levels of the patient samples were determined according to a standard curve constructed from readings of the standards at various heparin concentrations. All samples were run in duplicate.
Additional heparin was given whenever the anti–factor Xa levels fell below 3.0 U/ml according to the following nomogram: anti–factor Xa 2.5 to 2.9 U/ml, 50 U/kg heparin; anti–factor Xa 2 to 2.4 U/ml, 100 U/kg heparin; anti–factor Xa 1.5 to 1.9 U/ml, 200 U/kg heparin; and anti–factor Xa less than 1.5 U/ml, 300 U/kg heparin. The effect of heparin was reversed by giving 1 mg protamine sulfate for every 100 U of heparin given at the time of cannulation.
The Cell-Saver autotransfusion device (Haemonetics Corp., Braintree, Mass.) was used in every operation, and all blood that could be suctioned from the operative field was processed and returned to the patient. This included blood remaining in the pump circuit. In addition, blood from the chest tube drains was also reinfused if the total volume exceeded 150 ml. Units of allogeneic (donor) blood were transfused according to the following criteria: during cardiopulmonary bypass if the hemoglobin level was less than 5 gm/dl; after bypass if all autransfusion device blood had been infused and the hemoglobin level was still less than 7 gm/dl; and on the first and subsequent postoperative days if the hemoglobin level was less than 7 gm/dl.
Laboratory investigations
All patients underwent complete blood cell counts and tests of renal function before operation. Blood was collected at 30-minute intervals during bypass; at 1, 4, and 24 hours after operation; and at postoperative day 5. In these samples hemoglobin, blood urea nitrogen (BUN), and creatinine levels were measured. Samples were taken at 1 and 6 months for determination of antibodies against potential yeast contaminants and r-Apr.
To assess perioperative bleeding, the volume and hematocrit of the chest tube drainage were measured and erythrocytes lost were calculated by multiplying the drainage volume (ml) by the hematocrit. The amounts and types of all transfused fluids were also recorded. Loss of erythrocytes was calculated from the following expression:
Erythrocyte loss = BV x 
Hct
where BV is blood loss weight in kg x 60 ml/kg and Hct is preoperative hematocrit minus day 5 hematocrit. Day 5 was selected because by that time patients were in stable condition and were no longer receiving transfusions. To determine overall erythrocyte loss, the calculated erythrocyte loss and the erythrocyte content (hematocrit times volume) of all transfused blood were summed. This included autotransfused blood (hematocrit 50%) and allogeneic packed erythrocytes (hematocrit 80%). Nonstudy personnel measured the amounts of chest tube drainage, autotransfusion device blood, and allogeneic erythrocytes infused.
Follow-up evaluation
All patients were seen at 1 and 6 months after discharge, and information was recorded regarding chest pain, angina of new onset, myocardial infarction, or other thrombotic events.
Statistical methods
All data were expressed as the mean ± SD. Comparisons between groups were made with a t test for independent samples or the nonparametric Wilcoxon rank-sum test, SAS version 6.08 procedures TTEST and NPAR1WAY, respectively (SAS Institute, Inc., Cary, N.C.). Nonparametric tests were used if the data did not have a normal distribution and equal variance, as determined by a standard F test for equal variances and the Shapiro-Willks test for normality. When categoric measures were compared between patient groups, Pearson's 
2 test was used. A p value of less than 0.05 was considered significant.
RESULTS
Patients
Two hundred consecutive patients were screened, and 116 were excluded for the reasons shown in Table I. Table II shows the baseline characteristics of the study participants. Most patients were male and in their early sixties. There were no significant differences between study groups with respect to weight or number of grafts inserted. As planned, two thirds of the patients undergoing primary operations received r-Apr at each dosage level; half of the patients undergoing reoperations received r-Apr at each dosage level. All of the patients undergoing reoperations had initially undergone CABG and required reoperation for closed grafts, progression of atherosclerotic disease in the native coronary arteries, or both of these conditions. The maximum degree of hypothermia used was 29° C. Bypass times (in hours, mean ± SD) were as follows: dosage level 1 for primary operations, control 2.58 ± 0.9, r-Apr 2.25 ± 0.52; dosage level 1 for reoperations, control 2.79 ± 0.72, r-Apr 2.38 ± 0.37; dosage level 2 for primary operations, control 2.14 ± 0.49, r-Apr 2.22 ± 0.64; dosage level 2 for reoperations, control 3.02 ± 0.74, r-Apr 2.94 ± 0.46 (p > 0.1 for all comparisons by t test). No patients received platelet concentrates, fresh-frozen plasma, desmopressin, or antifibrinolytic agents.
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Blood loss
Table III shows the results of monitoring of blood loss during the perioperative period. Baseline hemoglobin values were similar in all patient groups. Hemoglobin was measured repeatedly after operation; the values on day 5 are representative and do not reveal any significant differences between r-Apr treated or control patients, either at dosage level 1 or 2, or for primary or reoperations. This is because patients were transfused with autotransfusion device blood, chest tube blood, or donated blood as described in the Methods section. However, the amount of autotransfusion device blood (in milliliters of erythrocytes, mean ± SD) given to patients in the r-Apr groups was generally less than that given to control patients: primary operations at dosage level 1, 418 ± 148 versus 441 ± 117 (p = 0.7); primary operations at dosage level 2, 337 ± 245 versus 619 ± 380 (p = 0.02, Wilcoxon rank-sum test); reoperations at dosage level 1, 563 ± 136 versus 539 ± 176 (p = 0.8); reoperations at dosage level 2, 459 ± 386 versus 656 ± 467 (p = 0.8, Wilcoxon rank-sum test). Furthermore, the proportion of patients in the r-Apr groups needing allogeneic blood or chest tube drainage transfusion was significantly lower than in the control group (Table IV).
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Table III). Overall erythrocyte losses (in milliliters, mean ± SD) were as follows: for primary operations, dosage level 1 was 916 ± 363 for r-Apr versus 905 ± 206 for control (p = 0.9) and dosage level 2 was 886 ± 362 versus 1333 ± 618 (p = 0.02); for reoperations, dosage level 1 was 1040 ± 162 versus 1544 ± 198 (p = 0.01) and dosage level 2 was 1191 ± 560 versus 1815 ± 1116 (p = 0.2, Wilcoxon rank-sum test). These differences in blood loss between r-Apr and control groups were mirrored by the measurements of total erythrocytes lost in the chest tube drainage fluid. For primary operations at dosage level 1, erythrocytes lost were 47 ± 35 ml versus 113 ± 55 ml (p < 0.001), and for dosage level 2, they were 60 ± 34 ml versus 152 ± 57 ml (p < 0.001). For reoperations at dosage level 1, erythrocyte losses were 38 ± 16 ml versus 108 ± 19 ml (p < 0.001), and for dosage level 2, they were 33 ± 24 versus 214 ± 151 (p = 0.02).
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Adverse reactions
At dosage level 1 of r-Apr, none of the patients had recurrence of angina, hadnew myocardial infarction, or died. Two patients in the control group, however, had hypotension after bypass and required an intraaortic balloon pump; one of these patients died. At dosage level 2, there were three adverse events. At the Temple site, one patient undergoing primary operation had a late graft closure (day 133); one undergoing reoperation had a myocardial infarction on day 28. The third patient underwent reoperation at the Chicago site; profound bradycardia developed after extubation, and respiratory failure was followed by death on day 12. Creatine kinase measurements and electrocardiography did not reveal evidence of myocardial injury; postmortem examination was not permitted.
The changes in BUN and creatinine levels from baseline to day 5 values were examined for control patients and patients receiving r-Apr (Table V). The changes were small for all groups; there was a statistically significant but clinically unimportant increase in BUN and creatinine levels in patients undergoing primary operations and receiving dosage level 2 of r-Apr. One patient in the r-Apr group who underwent reoperation had an increase in BUN to 70 mg/dl, an increase in creatinine level to 2.6 mg/dl, and a urinary tract infection, which was treated. One week later, the BUN and creatinine values were normal. No hypersensitivity or other allergic reactions were observed.
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The main results of this study are that r-Apr decreased blood loss as well as the need for transfusions in patients undergoing CABG. The conservation of blood noted in this investigation is comparable to that previously reported for aprotinin derived from bovine lungs. 9 Importantly, efficacy was observed at a dosage level of r-Apr that corresponded to only half the dosage currently recommended for bovine lung aprotinin (bolus dose of 280 mg [2 x 10 6 KIU], oxygenator prime of 280 mg [2 x10 6 KIU], and continuous infusion of 70 mg/hr [0.5 x 106 KIU]. Cosgrove and coworkers 3 also reported that efficacy was retained if only half the dosage of bovine lung aprotinin was given. Others have experimented with adding bovine lung aprotinin to the pump prime alone, but efficacy in these studies has been inconsistent. 10-12 It should be noted that the results of others are not directly comparable to those in our studies because other studies did not use r-Apr and aprotinin dose was not based on body weight. The main benefit of the low-dose r-Apr was seen in patients undergoing reoperations; even in patients undergoing primary operations, however, there was a trend toward lessened use of allogeneic blood, a significant decrease in units of blood autotransfused, and a decrease in drainage hemoglobin. Patients undergoing reoperations and receiving standard dosage r-Apr also had less blood loss, but the values between standard dose and control did not reach statistical significance because of the small number of subjects studied and the wide variations in individual blood loss.
Our blood loss data may be biased by the fact that it is extremely difficult to accurately determine intraoperative blood loss. Blood lost to the surgical drapes and sponges cannot be measured or retrieved. In addition, the autotransfusion device blood included blood remaining in the cardiopulmonary bypass circuit at the end of the operation. This volume of blood is variable and depends on patient factors not necessarily associated with blood loss. This variability may have influenced the overall erythrocyte loss calculations. Although the calculated blood losses did not differ between r-Apr and control groups, this was because patients were transfused according to a standard protocol. Importantly, patients in the r-Apr groups received significantly less allogeneic and chest tube drainage erythrocytes. Other factors known to affect perioperative blood loss, including age, sex, surgeon, total cardiopulmonary bypass time, and aspirin use, were controlled for or did not differ between groups.
The mechanisms by which aprotinin decreases bleeding associated with heart operations are still unclear. It has been suggested that aprotinin, by virtue of its ability to inhibit serine proteases, may prevent platelet activation and the degradation of platelet membrane glycoproteins by plasmin as well as averting premature lysis of the hemostatic clot. 13-16 In a companion study, 17 we measured a variety of indexes of platelet function and hemostatic factor activity in subjects treated with r-Apr and in control subjects. The major finding was that expression of platelet GMP-140, a protein located on the inner membrane of the platelet alpha granule, was less increased in treated patients than in control patients. This suggests lesser platelet activation. Other observations included a decline in the expression of platelet surface glycoprotein Ib and an increase in the release of ß-thromboglobulin during bypass, but these were similar in patients treated with r-Apr and control patients. In addition, no differences were noted in platelet numbers or plasma levels of fibrinogen or fibrinopeptide A. It may be that aprotinin acts on the platelet-vessel wall interaction because, as shown by Kestin and colleagues, 18 cardiopulmonary bypass results in poor platelet reactivity to an in vivo wound but normal platelet function in vitro and no intrinsic platelet defects. Future studies should focus on the bleeding time and measurements of thromboxane A2 and other platelet agonists in the shed blood of patients undergoing bypass operations with and without aprotinin.
The principal concern in the use of aprotinin is thrombosis; graft occlusion, myocardial infarction, disseminated intravascular coagulation, and deaths from thrombosis have all been attributed to the drug. 3,4 Some of these thrombotic events, however, may have been caused by inadequate dosing with heparin as a result of interference with the ACT by aprotinin. 19 Aprotinin inhibition of coagulation serine proteases augments the prolongation of the ACT by heparin, making heparin dosage adjustments based on the ACT unreliable. In our study, heparin underdosage was avoided by using a rapid anti–factor Xa assay, which was unaffected by aprotinin. This resulted in patients treated with r-Apr receiving amounts of heparin comparable to dosages given to control subjects. Nevertheless, two patients receiving the higher dosages of r-Apr had graft occlusion (although one occurred 133 days after operation), and a third patient died. Even with adequate heparinization, there may be a thrombotic risk with currently used dosages of aprotinin. Whereas angiographically controlled studies of aprotinin have not shown a greater incidence of graft thrombosis in treated patients than in control subjects, 20 there is a clear need for more studies to evaluate the safety of aprotinin.
Allergic reactions, including anaphylaxis, 21 have been reported in patients treated with bovine lung aprotinin (0.6% in the series reported by Royston 9); no such reactions were observed in this trial at either dosage level of r-Apr. In addition, there were no important changes in renal function attributable to the agent. We conclude that r-Apr effectively diminishes surgical bleeding and the need for transfusions in patients undergoing CABG, but its use requires careful monitoring of intraoperative heparin use.
Footnotes
J THORAC CARDIOVASC SURG 1995; 110:963-70 
References
This article has been cited by other articles:
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  J. R. Brown, N. J.O. Birkmeyer, and G. T. O'Connor Meta-Analysis Comparing the Effectiveness and Adverse Outcomes of Antifibrinolytic Agents in Cardiac Surgery Circulation, June 5, 2007; 115(22): 2801 - 2813. [Abstract] [Full Text] [PDF]
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 T. Miyashita, Y. Hayashi, Y. Ohnishi, and M. Kuro Retrospective analysis of effect of low-dose aprotinin priming on allogeneic blood transfusion in repeated cardiac operations Perfusion, May 1, 1999; 14(3): 189 - 194. [Abstract] [PDF]
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