Complement activation during cardiopulmonary bypass: Effects of immobilized heparin

Complement Activation During Cardiopulmonary Bypass: Effects of Immobilized Heparin Marcela Pekna, MD, PhD, Leif Hagman, MD, Eric Halden, MD, PhD, Ulf R. Nilsson, MD, PhD, Bo Nilsson, MD, PhD, and Stefan Thelin, MD, PhD Department of Clinical Immunology and Transfusion Medicine, Department of Thoracic and Cardiovascular Surgery, and Department of Anesthesiology, University Hospital, Uppsala, Sweden

The role of complement in biocompatibility reactions and the correlation between complement activation during cardiopulmonary bypass (CPB) and postperfusion syndrome have inspired attempts to improve the biocompatibility of extracorporeal blood oxygenation devices. Here we assessed the effect of immobilized heparin on the generation of C3a and terminal complement complexes during CPB. Thirty patients undergoing aortocoronary bypass were randomized to CPB with either heparin-coated (Duraflo II; Bentley, Irvine, CA) or noncoated control membrane oxygenators (Univox; Bentley). A standard dose of heparin (300 IU/kg) was given to the control group while the heparin dose was reduced to 30% (100 IUlkg) in the heparin-coated group. Significantly lower levels of terminal complement complexes were detected in the heparin-coated group by the end of CPB.

From 28 ± 5 AUlml (heparin-coated group) and 26 ± 3 AU/ml (control group, mean ± standard error of the mean) the terminal complement complex levels increased to 391 ± 35 AU/ml and 602 ± 47 AU/ml, respectively (p < 0.002). This difference was still apparent 180 minutes after CPB. Although there was no difference in C3a levels between the two groups at the end of CPB, C3a levels were significantly lower in the heparin-coated group 30 minutes after CPB (194 ± 18 nglml and 307 ± 18 ng/ml in heparin-coated and control groups, respectively; p < 0.001), We conclude that the heparin-coated surface is more biocompatible with regard to complement activation than is the ordinary unmodified surface in extracorporeal circuits.

C

complement activation have been shown to stimulate reactive oxygen metabolite production by neutrophils [3], to activate human monocytes [4], and to stimulate human platelets (reviewed in [5]). Complement activation during cardiopulmonary bypass (CPB) operation is a well-known phenomenon [6]. Animal studies of CPB using recombinant soluble complement receptor type I, an inhibitor of complement activation, have demonstrated that complement activation participates in lung injury after CPB [7]; based on the positive correlation between complement activation and postperfusion syndrome, a possible role for the complement system in the pathogenesis of this condition has been suggested [8-10]. One attempt to improve the biocompatibility of CPB circuits has been to conjugate heparin to the surface. Heparin is a proteoglycan that exhibits strong anticoagulant properties. It also is known to interfere with multiple steps in both the classic [11-14] and alternative pathways of complement [14-18]. The use of heparin-coated equipment during CPB operations has been reported to reduce complement activation [19, 20]. Moreover, the surface immobilized heparin allows for lower doses of systemic heparin, which could result in reduced blood loss and transfusion requirements [21, 22]. In this study we demonstrate that heparin coating of the whole CPB circuit, including the cardiotomy reservoir with the Duraflo II technique, significantly improves biocompatibility with regard to the complement system.

om p lem en t is a system of plasma and cell membrane proteins that forms a major effector arm of the humoral immune system. It is crucial for initiation of an inflammatory reaction, opsonization and neutralization of pathogens, and clearance of immune complexes. However, in some circumstances the products of complement activation may mediate deleterious effects. Complement can be activated by two different pathways, classic or alternative. Regardless of the activation pathway, activation leads to For editorial comment, see page 285. the formation of an enzymatic complex, a C3-convertase, capable of cleaving the third component (C3). C3 is the key molecule of the whole cascade. Proteolytic activation of C3 generates a small C3a fragment with anaphylatoxic properties and a larger C3b fragment with the capacity to bind to the target surface. The bound C3b mediates adherence of leukocytes and triggers the formation of the terminal complement complex (TCC). This complex has cytolytic functions and can either react with the target surface or be inactivated in the fluid phase [1]. Terminal complement complex inserted into the membrane of nearby cells can also act as a cell-stimulating agent [2]. Nonlethal amounts of TCC that are deposited on cell membranes at sites of Accepted for publication Dec 3, 1993. Address reprint requests to Dr Pekna, Department of Medical Biochemistry, University of Coteborg, Medicinaregatan 9, 5-41390, Giiteborg, Sweden.

© 1994 by The Society of Thoracic Surgeons

(Ann Thorae Surg 1994;58:421-4)

0003-4975/94/$7.00

422

PEKNA ET AL COMPLEMENT ACTIVATION DURING CPB

Material and Methods Patients The study comprised 30 patients undergoing coronary artery operations. The control group (C) consisted of 15 patients operated on with a standard CPB circuit, to be described. They received a normal bolus of heparin (300 IU /kg body weight) with an additional dose if activated clotting time was less than 400 seconds. In the heparin-coated group (HC), also consisting of 15 patients, the heparin-coated circuit (to be described) was used and the heparin bolus was reduced to 100 IU /kg body weight. In this group heparin was added when activated clotting time was less than 200 seconds. In both groups protamine administration for heparin reversal was started within 5 minutes after cessation of CPB. All the patients were selected with regard to the following characteristics: normal coagulation parameters; age less than 75 years; at least two-vessel disease; and no insulin-dependent diabetes mellitus or renal insufficiency. None of the patients were undergoing reoperations. The patients were randomized to treatment and the study was double blind. The protocol of the investigation was approved by the Ethics Committee of the Medical Faculty, Uppsala University, Sweden. Informed consent was obtained from all patients.

Anesthesia All patients were given morphine, 0.125 mg/kg body weight, and scopolamine, 0.005 mg/kg body weight, as premedication. Anesthesia was induced with thiopental, 2 to 4 mg/kg body weight, and fentanyl, 0.005 mg/kg body weight. Pancuronium, 0.1 mg/kg body weight, was administered for muscle relaxation. Ventilation was with 50%/50% 02/N20 before CPB start, then with O 2 in air. Anesthesia was maintained throughout the operation using fentanyl and isoflurane.

Cardiopulmonary Bypass The CPB technique was similar for all patients and used the following equipment: a Stockert heart-lung machine with membrane oxygenator (Univox; Bentley, Irvine, CA); a collapsible soft venous reservoir (Bentley); and a cardiotomy reservoir (BCR 3500; Bentley). In the HC group all parts of the circuit were heparin-coated (Duraflo II). The systems were primed with 2,000 mL of Ringer's acetate. St. Thomas' cardioplegic solution (Plegisol; Abbot, Chicago, IL) was used for myocardial protection. Initial blood flow during CPB was 2.2 L/m 2 body surface area and was decreased by 25% after the body temperature had reached 30°C. During CPB a shunt line was open between the oxygenator and the cardiotomy reservoir to avoid stagnant blood flow in the cardiotomy reservoir. Moreover, the volume in the cardiotomy reservoir was kept as low as possible. The blood in the CPB circuit, including cardiotomy reservoir, was retransfused through a venous cannula within 10 minutes after CPB. Residual blood in the oxygenator and the arterial line (600 to 700 ml.) was collected into a transfusion bag and was retransfused within 30 minutes after CPB.

Ann Thorac Surg 1994;58:421-4

Table 1. Preoperative and Intraoperative Data of HeparinCoated Circuit and Control Group" Characteristic CPB time (min) Cross-clamping time (min) No. of distal anastomoses Heparin (IV/kg body weight) Protamine (mg/kg body weight) Protamine to heparin ratio

HC

C

94:':: 6 48:':: 5 3.8:':: 0.3

88:':: 4 42:':: 3 3.5:':: 0.2

93:':: 5

407:':: 3.59 :':: 0.14b 0.89:':: 0.04

0.84:':: 0.09 0.89:':: 0.10

n-

a Values are presented as mean :+: standard error of the mean. b p < 0.001, comparison of mean values for control and heparin-coated groups.

C = control group; coated circuit.

CPB

=

cardiopulmonary bypass;

HC

=

heparin-

Aliquots of blood were drawn into sterile EDTA Vacutainer tubes (Becton Dickinson, Rutherford, NJ) and centrifuged for plasma, which was frozen at -70°C.

Analysis All the parameters were corrected according to hemodilution as estimated by the value of hematocrit. Enzymelinked immunosorbent assay for the detection of C3a was performed as described previously [23]. Briefly, microtiter plate-fixed MoAb 4SD17.3 against a neoepitope of C3a [24] served as a capture antibody. The C3a/C3adcsArg was detected by biotinylated polyclonal anti-C3a antibody [23], which subsequently reacted with HRP-conjugated streptavidin (Amersharn, Bucks, UK). As standard, zymosanactivated human serum was used. Enzyme-linked immunosorbent assay for the detection of the terminal complement complexes (TCC) was performed by a modification of the assay described by Mollnes [25]. After binding to microtiter plate-fixed antiC9 MoAb MCaE11 specific for a neoantigen expressed on C9 (a gift from Dr T. E. Mollnes, Bodo, Norway), TCC was detected by polyclonal anti-C5 antibodies (Dako A/S, Clostrup, Denmark) followed by HRP-conjugated antirabbit immunoglobulin (Dako A/S). Zymosan-activated serum containing 40,000 arbitrary units (AU)/mL served as standard.

Statistical Analysis Statistical analysis of the data was performed using twotailed Student's t test. The results are presented as mean ± standard error of the mean.

Results Patients There were no significant differences between the two groups with regard to preoperative variables, duration of CPB, aortic cross-clamping time, or number of distal anastomoses. However, the groups differed significantly in heparin (p < 0.001) and protamine doses (p < 0.001) (Table 1). There was no operative mortality, and all patients had uneventful postoperative courses.

C3a Generation Significant C3a generation at the end of CPB was observed in both the HC and C groups (252 ± 23 and 300 ± 24

423

PEKNA ET AL COMPLEMENT ACTIVATION DURING CPB

Ann Thorae Surg 1994;58:421-4

ng/mL, respectively; not significant). However, 30 minutes after cessation of extracorporeal circulation, the C3a levels in the HC group (194 ± 18 ng/mL) were reduced significantly (p < 0.001) relative to the C group (307 ± 18 ng/mL). Significantly lower levels of C3a were detected in the HC group during the follow-up period as many as 180 minutes after CPB (p < 0.001) (Fig 1).

Terminal Complement Complex Generation In both groups there was significant formation of TCC at the end of CPB, but TCC levels were significantly lower in the HC group than in the C group (391 ± 35 and 602 ± 47 AU / mL, respectively; p < 0.002). This difference remained significant during the entire period studied (p < 0.02) despite the continuous decrease in TCC levels after CPB in both groups (Fig 2).

700 600 ,-.

]

500

;;J 400

~

300 U U Eo< 200 100 0

before CPB

end ofCPB

30 min

90 min

180 min

after CPB

Fig 2. Generation of terminal complement complex (TCC) during cardiopulmonary bypass (CPB) in heparin-coated circuit (e) and control groups (0). Values are presented as mean::+: standard error of the mean. (AU = arbitrary uniis.)

Comment In the present study we have confirmed that the biocompatibility of extracorporeal blood oxygenation during CPB operations, as assessed by the activation of complement cascade, may be improved significantly by coating the surface of the circuit by immobilized heparin. In addition, this effect may be enhanced further by the inclusion of a heparin-coated cardiotomy reservoir into the CPB circuit. Although C3a was generated to the same extent in both groups during CPB, there was a significant difference in C3a generation between the two groups after extracorporeal circulation was stopped. Whereas there was a minimal change in C3a levels in the C group 30 minutes after CPB, a decrease was seen in the HC group. Gu and co-workers [20] administered the same dose of heparin in a comparable study to both groups of patients and found a similar, though less pronounced, phenomenon. Furthermore, results from their in vitro experiments with the Duraflo II surface suggested that some components of the classic complement pathway are adsorbed to the heparin-coated surface [20]. This is consistent with findings that immobilized heparin has a very high affinity for the first complement component (Cl ), which leads to activation of complement by the classic pathway, though simultaneously

400

,-.

~

5

300

200

'"

l"l

U 100 O+---~-----,r---~---r--~

before CPB

end ofCPB

30 min 90 min 180 min after CPB

Fig 1. Generation of C3a fragments during cardiopulmonary bypass (CPB) in heparin-coated circuit (e) and control groups (0). Values

are presented as mean::+: standard error of the mean.

the alternative pathway is inhibited strongly [26]. The heparin-protamine complexes, formed due to protamine administration after weaning from CPB, are known to activate the classic pathway [27, 28]. The reduced amount of heparin-protamine complexes in the HC group resulting from the reduction of both heparin and protamine doses could contribute further to the reduced post-CPB C3a generation observed in this group. The results of the TCC analysis agree with previous reports showing that the administration of protamine leads to continued activation of the complement cascade but is not accompanied by any detectable TCC formation [19,29]. At the end of CPB the TCC levels in the HC group were significantly reduced, whereas there was no difference between the groups with regard to C3a generation. These results suggest that the immobilized heparin exerts its inhibitory effect on the complement cascade predominantly at the level of the C5-convertase. However, Videm and co-workers [19] studied a different technique of heparin coating and observed a reduction in both TCC generation and C3 activation markers. Thus, the mode of heparin coating may affect the functions of immobilized heparin with regard to regulation of complement activation. As was suggested earlier [30], binding of TCC to surfaceimmobilized heparin may contribute to the reduction of TCC levels in plasma. We recently have reported that C3a was produced by blood contact with both gas bubbles and the artificial surface of a bubble oxygenator in vitro, whereas TCC was generated only by surface activation [29]. It seems conceivable, therefore, that C3a fragments indicate activation of the complement system by the artificial surface of the heart-lung machine, by the blood-gas interface formed in the oxygenator, and by heparin-protamine complexes. The levels of TCC, on the other hand, reflect predominantly the activation and completion of the complement cascade triggered by the artificial surface. The data presented here support this hypothesis. Thus for evaluation of the impact of CPB on the complement system to be complete, both the initiation of the complement cascade and the extent to which it is completed should be assessed [31].

424

PEKNA ET AL COMPLEMENT ACnVAnON DURING CPB

We conclude that the use of the Duraflo II surface leads to a significant improvement in biocompatibility of CPB with regard to the complement system, which may be strengthened further by use of a heparin-coated cardiotomy reservoir. Moreover, the use of heparin-coated devices permits administration of lower doses of heparin and protamine, which also could contribute to less complement activation after CPB. This study was supported in part by Bentley Laboratories Europe, Uden, The Netherlands. We wish to acknowledge the cooperation of perfusionists Ms Eva Thorne and Ms Elisabeth Ahlvin and the technical assistance of Ms Lena Larsson.

References 1. Muller-Eberhard HJ. Molecular organization and function of the complement system. Annu Rev Biochem 1988;57:321-47. 2. Morgan BP. Complement membrane attack on nucleated cells: resistance, recovery and non-lethal effects. Biochem J 1989;264: 1-14. 3. Hallett MB, Luzio JP, Campbell AK. Stimulation of Ca2+_ dependent chemiluminescence in rat polymorphonuclear leucocytes by polystyrene beads and the non-lytic action of complement. Immunology 1981;44:569-76. 4. Hausch GM, Seitz M, Betz M. Effect of the late complement components C5b-9 on human monocytes: release of prostanoids, oxygen radicals and of a factor inducing cell proliferation. Int Arch Allergy Appl Immunol 1987;82:317-20. 5. Sims PJ, Wiedmer T. The response of human platelets to activated components of the complement system. Immunol Today 1991;12:338-42. 6. Fosse E, Mollnes TE, Ingvaldsen B. Complement activation during major operations with or without cardiopulmonary bypass. J Thorac Cardiovasc Surg 1987;93:860-6. 7. Gillinov AM, DeValeria PA, Wilkenstein JA, et a1. Complement inhibition with soluble complement receptor type 1 in cardiopulmonary bypass. Ann Thorac Surg 1993;55:619-24. 8. Kirklin JK, Westaby S, Blackstone EH, Kirklin JW, Chenoweth DE, Pacifico AD. Complement and the damaging effects of cardiopulmonary bypass. J Thorac Cardiovasc Surg 1983;86: 845-57. 9. Moore FD, Warner KG, Assousa S, Valeri CR, Khuri SF. The effects of complement activation during cardiopulmonary bypass. Ann Surg 1988;208:95-103. 10. Khuri SF, Wolfe JA, [osa M, et a1. Hematologic changes during and after cardiopulmonary bypass and their relationship to the bleeding time and nonsurgical blood loss. J Thorac Cardiovasc Surg 1992;104:94-107. 11. Raepple E, Hill H-U, Loos M. Mode of interaction of different polyanions with the first (C'l, Cl), the second (C2) and the fourth (C4) component of complement-I. Effect on fluid phase Cl and on Cl bound to EA or to EAC4. Immunochemistry 1976;13:251-5. 12. Loos M, Volanakis JE, Stroud RM. Mode of interaction of different polyanions with the first (CI, C'l ), the second (C2) and the fourth (C4) component of complement-II. Effect of polyanions on the binding of C2 to EAC4b. Immunochemistry 1976;13:257-61. 13. Loos M, Volanakis JE, Stroud RM. Mode of interacton of different polyanions with the first (Cl, Cl ), the second (C2) and the fourth (C4) component of complement-III. Inhibition of C4 and C2 binding sitets) on CIs by polyanions. Immunochemistry 1976;13:789-91. 14. Weiler JM, Linhardt RJ. Antithrombin III regulates complement activity in vitro. J Immunol 1991;146:3889-94.

Ann Thorac Surg 1994;58:421-4

15. Weiler JM, Yurt DT, Fearon DT, Austen KF. Modulation of the formation of the amplification convertase of complement, C3b,Bb, by native and commercial heparin. J Exp Med 1978; 147:409-21. 16. Kazatchkine MD, Fearon DT, Metcalfe DD, Rosenberg RD, Austen KF. Structural determinants of the capacity of heparin to inhibit the formation of the human amplification C3 convertase. J Clin Invest 1981;67:223-8. 17. Kazatchkine MD, Fearon DT, Silbert JE, Austen KF. Surfaceassociated heparin inhibits zymosan-induced activation of the human alternative complement pathway by augmenting the regulatory action of the control proteins on particle-bound C3b. J Exp Med 1979;150:1202-15. 18. Meri S, Pangburn MK. Discrimination between activators and nonactivators of the alternative pathway of complement: regulation via a sialic acid/polyanion binding site on factor H. Proc Natl Acad Sci USA 1990;87:3982-6. 19. Videm V, Svennevig JL, Fosse E, Semb G, Osterlund A, Mollnes TE. Reduced complement activation with heparincoated oxygenator and tubings in coronary bypass operations. J Thorac Cardiovasc Surg 1992;103:806-13. 20. Gu YJ, van Oeveren W, Akkerman C, Boonstra PW, Huyzen RJ, Wildevuur CRH. Heparin-coated circuits reduce the inflammatory response to cardiopulmonary bypass. Ann Thorac Surg 1993;55:917-22. 21. Von Segesser LK, Weiss BM, Garcia E, Gallino A, Turina M. Reduced blood loss and transfusion requirements with low systemic heparinization: preliminary clinical results in coronary artery revascularization. Eur J Cardiothorac Surg 1990;4: 639-43. 22. Borowiec J, Thelin S, Bagge L, Hultman J, Hansson H-E. Decreased blood loss after cardiopulmonary bypass using heparin-coated circuit and 50% reduction of heparin dose. Scand J Thorac Cardiovasc Surg 1992;26:177-85. 23. Nilsson-Ekdahl K, Nilsson B, Pekna M, Nilsson UR. Generation of iC3 at the interface between blood and gas. Scand J ImmunoI1992;35:85-91. 24. Nilsson B, Svensson KE, Borwell P, Nilsson UR. Production of mouse monoclonal antibodies that detect distinct neoantigenic epitopes on bound C3b and iC3b but not on the corresponding soluble fragments. Mol Immunol 1987;24:487-94. 25. Mollnes TE, Lea T, Froland SS, Harboe M. Quantification of the terminal complement complex in human plasma by an enzyme-linked immunosorbent assay based on monoclonal antibodies against neoantigen on the complex. Scand J ImmunoI1985;22:197-202. 26. Nilsson UR, Larm 0, Nilsson B, Storm K-E, Elwing H, Nilsson-Ekdahl K. Modification of the complement binding properties of polystyrene: effects of end-point heparin attachment. Scand J Immunol 1993;37:349-54. 27. Kirklin JK, Chenoweth DE, Naftel DC, et al. Effects of protamine administration after cardiopulmonary bypass on complement, blood elements, and the hemodynamic state. Ann Thorac Surg 1986;41:193-9. 28. Cavarocchi NC, Schaff HV, Orszulak TA, Homburger HA, Schnell WA, Pluth JR. Evidence for complement activation by protamine- heparin interaction after cardiopulmonary bypass. Surgery 1985;98:525-31. 29. Pekna M, Nilsson L, Nilsson-Ekdahl K, Nilsson UR, Nilsson B. Evidence for generation of iC3 during cardiopulmonary bypass as the result of blood-gas interaction. Clin Exp Immunol 1993;91:404-9. 30. Videm V, Mollnes TE, Garred P, Svennevig JL. Biocompatibility of extracorporeal circulation. In vitro comparison of heparin-coated and uncoated oxygenator circuits. J Thorac Cardiovase Surg 1991;101:654-60. 31. Videm V, Fosse E, Mollnes TE, Garred P, Svennevig JL. Time for new concepts about measurements of complement activation by cardiopulmonary bypass? Ann Thorac Surg 1992;54: 725-31.