Activation of the Protein C System During Cardiopulmonary Bypass With and Without Aprotinin

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1 Activation of the Protein C System During Cardiopulmonary Bypass With and Without Aprotinin Ron G. H. Speekenbrink, MD, Rogier M. Bertina, PhD, Francisco España, PhD, Charles R. H. Wildevuur, MD, PhD, and Leon Eijsman, MD, PhD Department of Thoracic Surgery, Onze Lieve Vrouwe Gasthuis, Amsterdam, the Netherlands Background. The protein C system is important in the regulation of hemostasis. We studied its behavior during coronary artery bypass grafting procedures with and without aprotinin treatment using assays sensitive for activation of the protein C system. Methods. In a prospective, double-blind, randomized study of 48 patients we investigated the levels of antigen to proteins C and S and of the complexes between activated protein C with its two major plasma inhibitors, protein C inhibitor and 1 -antitrypsin in patients treated with placebo (n 17), low-dose (n 15), and high-dose (n 16) aprotinin during elective coronary artery bypass grafting. Results. The levels of proteins C and S showed a rapid decrease after heparinization, decreased greatly after start of cardiopulmonary bypass, and remained stable during cardiopulmonary bypass. Activated protein C inhibitor complexes were markedly elevated at the start of the procedure. Activated protein C 1 -antitrypsin decreased greatly after the start of cardiopulmonary bypass and remained stable during cardiopulmonary bypass. A significant peak was observed at the intensive care unit. Activated protein C protein C inhibitor levels showed a peak after heparinization in accordance with the accelerating effect of heparin on the complex formation but decreased thereafter. Treatment with aprotinin did not notably alter any of the measured patterns. Conclusions. In this study no evidence was found for increased activation of the protein C system during coronary artery bypass grafting. Administration of aprotinin did not result in different patterns of activation of the protein C system. (Ann Thorac Surg 1998;66: ) 1998 by The Society of Thoracic Surgeons Despite its central role in the regulation of blood coagulation, little is known about the behavior of the protein C system during cardiopulmonary bypass (CPB). The protein C system primarily consists of the plasma factors proteins C and S and the endotheliumbound thrombomodulin. Thrombomodulin present on the endothelium binds circulating thrombin to form a complex that catalyzes the conversion of protein C to activated protein C (APC). Activated protein C, together with its cofactor protein S, inhibits further thrombin generation by inactivating factors Va and VIIIa. In addition to this anticoagulant effect, APC can neutralize the plasminogen activator inhibitors PAI-1 and PAI-3 and enhance fibrinolysis. Congenital deficiencies of protein C, its cofactor protein S, and a resistance of factor Va to inactivation by APC due to a mutation in factor V result in a hypercoagulable state and are associated with an increased risk of venous thrombosis and pulmonary embolism [1]. In previous studies, significant decreases in the levels Accepted for publication June 4, Address reprint requests to Dr Speekenbrink, Department of Thoracic Surgery, Onze Lieve Vrouwe Gasthuis, P.O. Box 95500, 1090 HM Amsterdam, the Netherlands. of antigens of proteins C and S during CPB have been observed [2, 3]. This was interpreted as the result of activation of the protein C system during CPB, although markers for the activation of this system were not measured. However, the observed changes in protein C and S levels can also be explained by other mechanisms like binding of the proteins to the extracorporeal circuit [4]. To be able to distinguish these possibilities we have supplemented our array of assays in this study with recently developed assays that measure the complexes formed by APC with its natural inhibitors protein C inhibitor (PCI) and 1 -antitrypsin ( 1 AT). Because of the relatively long half-lives of these complexes, 40 and 140 minutes, respectively, they are considered to be sensitive markers for APC generation [5]. The protease inhibitor aprotinin is widely used as an adjunct in blood-saving programs [6, 7]. In vitro studies have shown that aprotinin is a competitive inhibitor of APC similar to the natural inhibitors PCI and 1 - antitrypsin [8]. Therefore, aprotinin might contribute to the inactivation of APC in vivo. In this double-blind placebo-controlled study we investigated the behavior of the protein C system during coronary artery bypass grafting with and without the use of aprotinin by The Society of Thoracic Surgeons /98/$19.00 Published by Elsevier Science Inc PII S (98)

2 Ann Thorac Surg SPEEKENBRINK ET AL 1998;66: PROTEIN C DURING CPB 1999 Patients and Methods The study was performed at the Onze Lieve Vrouwe Gasthuis in Amsterdam, The Netherlands, and the study protocol was approved by the hospitals medical ethical committee. Written informed consent was obtained from all patients. Forty-eight patients scheduled for elective primary coronary bypass grafting were randomly selected. These patients were part of a larger group of patients in which the clinical effects of low- and high-dose aprotinin were studied. The results of the latter study have been published previously [9]. Anesthesia consisted of premedication with lorazepam, induction with sufentanyl, pancuronium bromide, and etomidate or propofol, and was maintained with a continuous infusion of sufentanyl. Routinely, an infusion of dopamine and nitroglycerin was started at the termination of CPB. The extracorporeal circuit contained either a William- Harvey HF 5400 (Bard, Tustin, CA) or an Avecor Ultrox I (Avecor, Plymouth, MN) oxygenator primed with 2,000 ml of Ringer s lactate, 200 ml of 20% human albumin, 100 ml of 20% mannitol, 50 ml of 8% sodium bicarbonate, 50 mg of heparin, and 2 g of cefamandole. Before cannulation, bovine heparin (3 mg/kg) was administered. Additional doses of 50 mg were given after every hour of extracorporeal circulation irrespective of the cecite-activated clotting time, because aprotinin interferes with this measurement. Heparin was neutralized with protamine sulfate in a 1:1 ratio with the initial heparin dose. Additional dosage of protamine was guided by repeat activated clotting times after the residual volume in the extracorporeal circuit had been returned to the patient. Flow ranges were between 2.0 and 2.4 L m 2 min 1 during moderate hypothermia (28 to 32 C). Cardioplegia was achieved with ice-cold crystalloid cardioplegia infused in the ascending aorta after clamping. The study medication was provided by Bayer Leverkusen A.G., Germany, in coded bottles. The code was kept by the manufacturer. All patients received 12 bottles containing 50 ml. Four bottles marked pump were added to the pump prime, four bottles marked infus were administered as a loading dose in 30 minutes after induction of anesthesia, and four bottles marked infus were given as a maintenance dose at a rate of one bottle per hour. The bottles contained either placebo (placebo group) or 500,000 KIU (70 mg) aprotinin (high-dose group). In the low-dose group only the pump bottles contained 500,000 KIU (70 mg) aprotinin and the infus bottles contained placebo. Blood Sampling and Assays Blood samples were drawn from a central venous line or the extracorporeal circuit after induction of anesthesia, after administration of heparin, 5 minutes and 30 minutes after onset of extracorporeal circulation, before and after release of the aortic cross-clamp, after administration of protamine sulfate, and 2 and 4 hours after arrival at the intensive care unit. The samples were collected in tubes containing sodium citrate (final concentration, 1.05 mmol/l). For the measurement of APC inhibitor complexes blood was collected in 5-mL tubes containing 0.2 ml of a medium composed of g of ethylenediaminetetraacetic acid, g of benzamidine, and 0.25 g of soybean trypsin inhibitor per 100 ml. The samples were centrifuged at 3,000 rpm for 10 minutes at 4 C, and the plasma obtained was stored in aliquots at 20 C. Protein C antigen was measured by enzyme-linked immunosorbent assay using a monoclonal antibody (C12) as coating and rabbit antiprotein C immunoglobulin G, conjugated to horseradish peroxidase as the second antibody. Protein S antigen was measured by polyclonal enzyme-linked immunosorbent assay as previously described [10]. Activated protein C PCI complex and APC- 1 -antitrypsin complex concentrations were measured with sandwich enzyme-linked immunosorbent assay techniques according to previously described methods [11, 12]. In all results the ratio between the initial hematocrit and the hematocrit at the sampling point was used to correct data for hemodilution. Statistics Graphs depict mean values standard error. Categorical data were analyzed with the Kruskal-Wallis statistic. Continuous data were analyzed with analysis of variance. The Bonferroni-Holm procedure was used to correct for the multiple comparison artifacts. For laboratory assays differences between groups were analyzed with analysis of variance for repeated measures. All analyses were performed with SPSS software (SPSS Inc, Chicago, IL). Results Patient Data A total of 48 patients entered the study (17 patients in the placebo group, 15 in the 2 million KIU [280 mg] group and 16 in the 6 million KIU [840 mg] group). The demographic data and baseline coagulation studies of the groups are shown in Table 1. The differences between the groups were not significant. The perioperative course in the patients was uneventful. No major complications occurred. Protein C Antigen The concentrations of protein C in the plasma samples were measured in an enzyme-linked immunosorbent assay using a coating antibody (C12) directed at the active site of protein C. This antibody does not recognize APC inhibitor complexes. The results are shown in Figure 1. The course of protein C antigen levels was essentially the same in the three groups, with a rapid decrease after heparinization and start of CPB. This decrease was significant in the placebo and high-dose group (p and p 0.006, respectively). In the placebo group the levels seemed to increase slowly during and after CPB to reach almost the preoperative level, but in the aprotinin-

3 2000 SPEEKENBRINK ET AL Ann Thorac Surg PROTEIN C DURING CPB 1998;66: Table 1. Patient Data Variables Placebo Low-Dose High-Dose Number Age (y) Sex (% male) BSA (m 2 ) Preoperative ASA 12/17 10/15 12/16 Until preoperative day Distal anastomoses LIMA used (%) Perfusion time (min) Cross-clamp time (min) APTT (s) PTT (s) Bleeding time (s) Chest tube drainage (ml) hours postoperative Transfusion (U) Packed cells Fresh frozen plasma Data are expressed as the mean the standard deviation. No significant differences between the groups. APTT activated partial thromboplastin time; ASA acetylsalicylic acid; BSA body surface area; LIMA left internal mammary artery; PTT prothrombin time. treated patients the levels remained unchanged during this period. However, analysis of variance for repeated measures did not show a significant difference in the course of the protein C levels between the groups. Total Protein S The course of the levels of protein S (Fig 2) resembled the course seen for protein C; a sharp decrease after heparinization and start of CPB, which was significant in the Fig 2. Concentrations of protein S antigen corrected for hemodilution. The levels changed significantly in all groups (p ), but did not differ between groups. Within group differences and abbreviations are marked as in Fig 1. placebo and high-dose group (p ) and almost no change during and after CPB. With analysis of variance for repeated measures a significant difference in the course of protein S levels between the groups could not be demonstrated. Activated Protein C Protein C Inhibitor The levels of APC PCI were far above normal ( ng/ml) at the first sampling point. The levels of the complexes increased significantly (p 0.006) in all groups after heparinization (Fig 3), which is not surprising as heparin promotes APC PCI complex formation. The decrease after initiation of CPB was significant in the placebo and low-dose groups, but not in the high-dose group. In the high-dose group a sharp decrease was seen after neutralization of heparin with protamine sulfate (p 0.001). The levels continued to decrease in all groups to reach low values 4 hours after arrival in the intensive care unit. Although the levels in the high-dose aprotinin group are consistently higher during CPB, analysis of Fig 1. Concentrations of protein C antigen corrected for hemodilution. The bar represents cardiopulmonary bypass (CPB) and the triangle indicates the release of the aortic cross-clamp. (Hep administration of heparin; Prot administration of protamine sulfate.) The levels changed significantly in all groups (p ), but did not differ between the groups. Significant within group changes are marked by an asterisk for the placebo group; for the low-dose group, and # for the high-dose group. Fig 3. Concentrations of activated protein C protein C inhibitor corrected for hemodilution. The levels changed significantly in all groups (p ) but did not differ between groups. Within group differences and abbreviations are marked as in Fig 1.

4 Ann Thorac Surg SPEEKENBRINK ET AL 1998;66: PROTEIN C DURING CPB 2001 Fig 4. Concentrations of activated protein C 1 -antitrypsin corrected for hemodilution. The levels changed significantly in all groups (p ) but did not differ between groups. Within group differences and abbreviations are marked as in Fig 1. variance for repeated measures did not demonstrate a significant difference between the groups. Activated Protein C 1 Antitrypsin Activated protein C 1 AT levels were far above normal (7 2 ng/ml) at the first sampling point (Fig 4) but decreased sharply after the start of CPB (p in the aprotinin-treated groups) and remained stable during the rest of CPB. At the intensive care unit an increase followed by a significant decrease (p 0.005) was seen in all groups. Analysis of variance for repeated measures did not show a significant difference between the groups. Comment This study was performed to elucidate the pattern of protein C activation during CPB with and without the use of aprotinin. Interestingly, we measured elevated levels of APC PCI (normal, ng/ml) and APC 1 AT (normal, 7 2 ng/ml) before CPB at the first sampling point (ie, after induction of anesthesia). The levels of APC 1 AT are in the range of those found in such procoagulant states as preeclampsia or hemodialysis [13]. Because we did not measure these complexes before the procedure we cannot differentiate whether the observations are typical for this patient group with atherosclerotic complications or result from the anesthetic procedures. Further studies to elucidate this finding are warranted. At the onset of CPB we found sharp decreases of protein C and S antigen levels, which persisted through the remainder of the procedure. However, the levels did remain within normal limits (75% to 125%). These findings are in accordance with the results of Tanaka and colleagues [3], but differ from those of Knöbl and associates [2]. The latter found no decrease at the onset of CPB, and only a small but significant decrease of protein C antigen after release of the aortic cross-clamp. Furthermore, free protein S was increased during the entire CPB procedure, possibly resulting from release by activated platelets [14]. On the basis of these data both investigators and their colleagues concluded that the protein C system was activated during CPB. Although all these reported decreases of protein C antigen appear to be small, about 20%, similar reductions in physiologic circumstances are associated with massive activation and result in high levels of APC. However, our results with the new APC inhibitor complex assays show the highest concentration before the operation, but they decrease rapidly at the onset of CPB independent of hemodilution. Clearly these results are not compatible with the massive production of APC that would be expected when the decrease of protein C would be the consequence of activation of the system. This might indicate that the decrease of proteins C and S is not attributable to activation but could be explained by binding of these proteins to the extracorporeal circuit. The generation of thrombin and the activation of the protein C system are closely coupled through the formation of thrombin thrombomodulin complexes on the endothelium. Therefore, the patterns for thrombin generation and activation of the protein C system would be expected to be very similar. In previous studies we and other investigators have found a typical pattern with increased thrombin generation at the onset of CPB and after release of the aortic cross-clamp [9, 15]. Our present data, however, show no indication for increased activation of protein C surrounding these events. An explanation for this discrepancy could be that the thrombin measured after the initiation of cardiopulmonary bypass and cross-clamp release was generated outside the blood vessels where there is no coupling with protein C activation. Tabuchi and colleagues [16] have shown that pericardial blood contains high concentrations of thrombin antithrombin complexes and low heparin levels. Recirculation of this cardiotomy suction blood resulted in increased levels of thrombin antithrombin complexes in the systemic blood. Because cardiotomy suction is used mostly after the start of bypass and release of the aortic cross-clamp, this mechanism could very well be responsible for the observed discrepancy between thrombin generation and protein C activation. In vitro studies have shown that aprotinin is an inhibitor of APC with a mechanism of action similar to that of PCI and 1 AT [8]. This inhibitory effect of aprotinin has been implicated in the pathogenesis of perioperative thromboembolic complications [17, 18]. Westaby and colleagues [19] specifically warned of the combination of deep hypothermic circulatory arrest and the use of aprotinin as their retrospective series demonstrated an increased incidence of diffuse intravascular coagulation in aprotinin-treated patients. The combination of low flow or circulatory arrest combined with inhibition of plasmin and the protein C system by aprotinin was thought to be responsible for this effect. Other clinical studies [20 22] have both confirmed and rejected the findings of Westaby and colleagues [19]. Although the affinity of aprotinin for APC is not as strong as that of PCI and 1 AT, in vitro studies have demonstrated that the affinity of aprotinin for APC is increased by heparin to approach

5 2002 SPEEKENBRINK ET AL Ann Thorac Surg PROTEIN C DURING CPB 1998;66: that of kallikrein [9]. Moreover, high concentrations of aprotinin are used during CPB. On the other hand, numerous other proteases present during CPB (eg, plasmin), compete for binding sites on aprotinin. Nevertheless, aprotinin treatment might still result in additional inhibition of APC. This inhibition would result in a shift of APC from PCI and 1 AT to aprotinin and therefore, lower concentrations of APC PCI and APC 1 AT would be expected in the aprotinin-treated patients. Because the high-dose regimen uses not only three times the amount of aprotinin as the low-dose regimen, but also it is administered during the entire procedure, these changes would be most notable in the high-dose group. Our present results are contrary to these predicted findings. We found very similar patterns of APC 1 AT complexes with no significant difference between the groups. The patterns for APC PCI complexes were practically identical in the placebo and low-dose groups. There appears to be a different, albeit not significant, pattern in the high-dose group with persistently higher levels of APC PCI during CPB in contrast to the expected lower levels. The course and especially the abrupt decrease after protamine administration suggest a role for heparin. Heparin is known to enhance APC PCI complex formation and this effect is responsible for the peak we observed in all groups after heparinization. There was, however, no significant difference between the groups in the initial and maintenance doses of heparin used during the procedure (data not shown). Alternatively, the higher levels of APC PCI in the high-dose group could be the result of a heparin-sparing effect of aprotinin resulting in APC PCI complex formation. This, however, is unlikely because of the identical patterns we found in the lowdose aprotinin and placebo groups. It should be emphasized that we have used an indirect way to demonstrate the absence of an inhibitory effect of aprotinin on APC in vivo. Definitive evidence can only be provided by an assay specific for APC aprotinin complexes. Unfortunately this assay is not available. In conclusion, the results from this study do not provide evidence for activation of the protein C system during CPB. Moreover, our results do not show an influence of aprotinin administration on the activation of the protein C system during CPB. We are indebted to J. J. P. Nauta, MSc, for his statistical advice. References 1. Clouse LH, Comp PC. The regulation of hemostasis: the protein C system. N Engl J Med 1986;314: Knöbl PN, Zilla M, Muller MM, et al. The protein C system in patients undergoing cardiopulmonary bypass. J Thorac Cardiovasc Surg 1987:94: Tanaka K, Wada K, Morimoto T, et al. The role of the protein C thrombomodulin system in physiologic anticoagulation during cardiopulmonary bypass. 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