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Ann Thorac Surg 1996;61:1153-1157
© 1996 The Society of Thoracic Surgeons
Centre for Cardiopulmonary Surgery Amsterdam, Department of Cardiac Surgery, Academical Hospital Vrije Universiteit, and Central Laboratory of the Netherlands Red Cross Blood Transfusion Service, Amsterdam, the Netherlands
Accepted for publication November 6, 1995.
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Abstract |
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Methods. In a prospective, randomized study involving patients undergoing coronary artery bypass grafting with standard full heparinization, we compared heparin-coated circuits (Duraflo II) (10 patients) with uncoated circuits (10 patients) and assessed the extent of initiation of complement activation by detecting iC3 (C3b-like C3) concentrations, classic pathway activation by C4b/c (C4b, iC4b, C4c) concentrations, terminal pathway activation by soluble C5b-9 concentrations, and C3 activation by C3a (C3a desArg) and C3b/c (C3b, iC3b, C3c) concentrations.
Results. Heparin-coated extracorporeal circuits significantly reduced circulating complement activation product C3b/c and soluble C5b-9 concentrations at the end of cardiopulmonary bypass and after protamine sulfate administration compared with the uncoated circuits, but not iC3, C4b/c, or C3a concentrations.
Conclusions. Heparin-coated extracorporeal circuits reduce complement activation through the alternative complement pathway, probably at the C3 convertase level, and, consequently, the terminal pathway. C3b/c seems to be a more sensitive marker than C3a to assess complement activation during cardiac operations.
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Introduction |
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TopFootnotesAbstractIntroductionMaterial and MethodsResultsCommentAcknowledgmentsReferences |
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Complement activation initiates a systemic inflammatory response during and after cardiac operations [1] and is associated with pathophysiologic events such as postoperative cardiac depression [2], pulmonary capillary leakage [3], and hemolysis [4]. During cardiopulmonary bypass (CPB), the complement system is activated mainly through the alternative pathway. Direct contact between the patient's blood and the artificial surface of the extracorporeal circuit and bubbling air through the blood lead to elevated levels of C3a, C5a, and C5b-9 but not C4a [5–7]. After CPB and the administration of protamine sulfate, however, classic pathway activation is initiated through formation of heparin-protamine complexes and is reflected by increasing C4a levels [8].
Complement activation is carefully controlled by regulatory proteins, but during CPB, the action of these regulatory proteins seems to be inadequate to control the complement system. Laboratory studies [9, 10] revealed that plasmatic as well as immobilized heparin can reduce complement activation. Among its actions, heparin may prevent excessive complement activation through the alternative pathway by binding to C3b at the binding site for factor B, thereby inhibiting the formation of the alternative pathway C3 convertase [11]. Heparin may also potentiate C1-esterase inhibitor activity and reduce classic complement pathway activation [12]. In a laboratory study, Videm and co-workers [13] found that heparin-coated extracorporeal circuits reduced formation of C3 activation products and soluble (S)C5b-9 complexes and thus improved the biocompatibility of the circuits. Although several groups including ours [14–18] have confirmed the finding of this laboratory study in clinical studies, the level at which heparin coating inhibits the complement system in patients undergoing a cardiac operation remains to be clarified.
The present study was undertaken to evaluate whether the combination of passively immobilized heparin-coating and standard heparinization can reduce complement activation in patients undergoing cardiac surgical intervention. If so, at which level in the complement cascade does heparin coating inhibit the formation of complement activation products in vivo and which complement activation product in the circulation is the best representative marker of this reduction? We assessed the extent of initiation of alternative complement pathway activation by detecting iC3 (C3b-like C3) concentrations, classic pathway activation by C4b/c (C4b, iC4b, C4c) concentrations, terminal pathway activation by SC5b-9 concentrations, and C3 activation by C3a (desArg) and C3b/c (C3b, iC3b, C3c) concentrations.
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Material and Methods |
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TopFootnotesAbstractIntroductionMaterial and MethodsResultsCommentAcknowledgmentsReferences |
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Anesthesia and CPB
The extracorporeal circulation circuit (Baxter Healthcare Corp, Irvine, CA) consisted of a soft-shell venous reservoir, roller pump, membrane oxygenator (Univox), arterial filter, cardiotomy reservoir, and polyvinyl tubing. In the heparin-coating group, all parts of the extracorporeal circuit were passively coated with heparin (Duraflo II). The total priming volume of the system was 1,400 mL: 1,250 mL of Ringer's lactate solution, 100 mL of 20% mannitol, 50 mL of 8.4% sodium bicarbonate, and 5,000 IU of heparin sodium. The standard perfusion protocol aimed at a hematocrit of 22% to 23% during CPB.
Cardiopulmonary bypass was performed with standard heparinization of 300 IU/kg, moderate systemic hypothermia (28° to 30°C), nonpulsatile flow (2.2 to 2.4 L•min-1•m-2) and cold (4°C) high-potassium crystalloid cardioplegia. A standard cannulation technique was used with a cannula in the ascending aorta and a cannula in the right atrium (two-stage venous cannula). On the morning of operation, patients received their usual dose of antianginal medication and lorazepam (5 mg).
Anesthesia was induced using intravenous sufentanyl forte (30 µg/kg of body weight), pancuronium bromide (0.1 mg/kg), and midazolam hydrochloride (0.1 mg/kg) and was maintained by supplemental doses of these drugs. Cefuroxime sodium (1,500 mg) was given intravenously as prophylaxis against infection. After endotracheal intubation, patients were ventilated to normocapnia using a mixture of oxygen and air. Radial artery and pulmonary artery catheters were placed for hemodynamic measurements using thermodilution and procurement of blood samples. During CPB, the activated clotting time was longer than 400 seconds. After termination of CPB, heparin was neutralized by administration of an equal dose of protamine (3 mg/kg).
Measurements
Blood samples were taken from the radial artery or from the arterial line of the extracorporeal circuit. The samples were immersed in melting ice immediately after collection and processed within 1 hour. Platelet-poor plasma was prepared by centrifugation at 4°C for 10 minutes at 1,500 g. The samples were stored at -70°C. Blood samples for complement activation were taken in EDTA (ethylenediaminetetraacetic acid)–anticoagulated tubes at the following times: before induction of anesthesia, 10 minutes after the start of CPB, before and after removal of the aortic cross-clamp, at cessation of CPB, and after protamine administration.
Concentrations of iC3 (C3(H2O) or C3b-like C3) and C3a (C3a desArg) were determined as previously described [19, 20]. Briefly, before analysis, native C3 is removed from the sample by precipitation with 11% polyethylene glycol to avoid in vitro generation of C3a, which occurs at room temperature [19]. The recovery of C3a from the plasma sample by this procedure is better than 90% [21]. Concentrations of C3a/C3a desArg in the supernatant were determined with a radioimmunoassay as previously described [20].
Concentrations of C3b/bi/c and C4b/bi/c, further denoted as C3b/c and C4b/c, respectively, were determined as described in 1993 [22]. Briefly, C3b/c was measured using anti-C3-9 as capture antibody, which binds to C3b/c as well as iC3. The detection reagent consisted of biotinylated antibodies to C3c. Finally, streptavidin-conjugated horseradish peroxidase was added, and the reaction was developed with 3,3`,5,5`-tetramethylbenzidine (Merck, Darmstadt, Germany). Levels of C4b/c were measured using anti-C4-1 as capture antibody, which binds to C4b/c as well as to iC4 and its degradation products C4b, iC4b, and C4c.
Soluble C5b-9 (terminal complement complex SC5b-9) concentrations were measured with an enzyme immunoassay (Behringwerke AG Diagnostia, Marburg, Germany) according to the manufacturer's protocol. Soluble C5b-9 concentrations were expressed in nanomoles per liter using an estimated molecular weight of 619 kD during calculation.
Statistical Analysis
Statistical analysis was performed using Statview SE+ Graphics computer software (Abacus Concepts, Inc, Berkeley, CA). Comparisons within groups were assessed with the Wilcoxon signed rank sum test and comparisons between the groups, with the Mann-Whitney U test. Correlation analysis was performed applying the Spearman rank correlation test. In all cases, a two-sided probability of less than 0.05 was considered significant. Data are presented as the median value with the interquartile range in parentheses and are not corrected for hemodilution.
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Results |
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TopFootnotesAbstractIntroductionMaterial and MethodsResultsCommentAcknowledgmentsReferences |
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Equal iC3 (C3b-like C3) concentrations were determined in both groups prior to operation and at cessation of CPB (Fig 1
). In both groups, there was an equal decrease of 19 nmol in iC3 concentrations from before operation (baseline) to after cessation of CPB, which reflected hemodilution.
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. Levels of C3a (desArg) started to increase immediately at the onset of CPB (p < 0.05) and remained elevated during CPB and after protamine administration. The concentrations did not differ between groups, and maximal levels were 23 nmol/L (16 to 34 nmol/L) in the heparin-coating group and 52 nmol/L (29 to 68 nmol/L) in the uncoated group (p = 0.15). Concentrations of C4b/c did not increase in either group during CPB. However, after administration of protamine, a significant, similar increase in C4b/c was observed in both groups (p < 0.05), with no significant difference between groups. Maximal levels of C4b/c were 57 nmol/L (38 to 90 nmol/L) in the heparin-coating group and 90 nmol/L (30 to 118 nmol/L) in the uncoated group (p = 0.53). In both groups, C3b/c concentrations significantly increased at the end of CPB and after protamine administration. Heparin coating significantly reduced C3b/c concentrations at the end of CPB and after protamine administration. Maximal levels of 234 nmol/L (205 to 371 nmol/L) were detected in the heparin-coating group and 579 nmol/L (472 to 1,026 nmol/L) in the uncoated group (p = 0.02).
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Significant correlations were observed between maximal concentrations of C3b/c and C3a (desArg) (r = 0.65; p < 0.01), C3b/c and SC5b-9 (r = 0.76, p < 0.01), C3a (desArg) and C4b/c (r = 0.58; p < 0.05), and C3a (desArg) and SC5b-9 (r = 0.67; p < 0.01). To obtain more information about the mechanism of inhibition of the complement system by heparin-coated circuits and plasma concentrations of complement activation products, we determined the ratios of the maximal levels of these activation products. The ratios between maximal levels of C3b/c and SC5b-9 were 170:1 (range, 153 to 241) for the heparin-coating group and 188:1 (range, 141 to 332) for the uncoated group (p = not significant). The ratios between C3b/c and C3a (desArg) were 9:1 (range, 7.1 to 13) and 13:1 (range, 11 to 21) for the heparin-coating and uncoated groups, respectively (p = not significant).
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Comment |
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TopFootnotesAbstractIntroductionMaterial and MethodsResultsCommentAcknowledgmentsReferences |
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In all studies regarding complement activation during CPB, it has been claimed that the complement system is activated through the alternative pathway because no increase in circulating C4a concentrations is observed during CPB. Only after protamine administration does C4a increase, a finding indicating that the formation of heparin-protamine complexes is responsible for this increase [8]. In this study, we applied an assay for C4b/c (C4 activation products C4b, iC4b, and C4c) to assess classic pathway activation. Our results confirm the initial finding that classic pathway activation is not initiated during CPB until the administration of protamine. However, heparin coating of the circuits does not affect the classic pathway activation and diminish C4b/c concentrations after protamine administration.
Activation of the alternative complement pathway consists of three steps: initiation, recognition of the surface, and amplification. Continuous low-grade initiation occurs by formation of iC3 [23, 24]. Hydrolysis of the internal thioester in native C3 yields iC3, which has C3b-like properties. Before induction of anesthesia, the level of iC3 in the whole group was 88 nmol/L (78 to 97 nmol/L), and it did not increase but rather decreased in parallel with hemodilution for which we did not correct. Heparin-coating had no influence on iC3 formation, which is in agreement with the findings of Nilsson-Ekdahl and colleagues [24], who showed that heparin does not inhibit the formation of this species. To date, enhanced in vivo formation of iC3 has been detected solely in patients undergoing a cardiac operation with use of a bubble oxygenator [23].
The formation of C3b/c was markedly enhanced during CPB but was significantly lower in the group with heparin-coated circuits (see Fig 2
). Several studies have reported the inhibitory effects of heparin on activation of the alternative complement pathway. This inhibition may occur through two specific mechanisms. First, heparin may bind to C3b at the site for factor B, thereby inhibiting the formation of the C3 convertase, C3bBb [11]. Second, both surface-bound and fluid-phase heparin may potentiate factor H activity, thereby enhancing inactivation by C3b by factor I [25]. We assume that the latter mechanism is less likely to explain the observed reduction of C3b/c generation during CPB. Specific measurements of Ba and C3c levels would allow a more definite conclusion regarding the mechanism of inhibition by heparin. We observed a constant ratio between C3b/c and SC5b-9 in both groups: 170:1 for the heparin-coating group and 188:1 for the uncoated group (p = not significant). Further, maximal C3b/c levels strongly correlated with SC5b-9 levels (r = 0.76). These two observations suggest that the inhibitory effect of heparin occurred by inhibition of the formation of the C3 convertase. Otherwise, more C3c would have been formed (which is also detected in the enzyme-linked immunosorbent assay for C3b/c), yielding a less marked difference in total C3b/c levels between groups as well as a different ratio of total C3b/c to SC5b-9 in the heparin-coating group.
In several studies [1–3, 5, 6, 14, 15, 20], C3a (desArg) formation appeared to be a useful marker for complement activation in vivo. In this study, however, no significant differences were observed in circulating C3a (desArg) levels between the groups. These findings do not confirm the supposed effect of heparin coating as was evident from the course of C3b/c levels. However, the observed molar ratios of C3b/c and C3a (desArg) were 9:1 (heparin-coating group) and 13:1 (uncoated group) rather than the expected 1:1. Further, these 9:1 and 13:1 ratios could not be explained by an increased formation of iC3, and therefore it must be concluded that the recovery of C3a (desArg) during CPB was much lower than that of C3b/c. This could possibly be related to the molecular size of C3a (desArg) [20]. The relatively small C3a (desArg) molecule may diffuse more rapidly from the systemic circulation. Alternatively, C3a (desArg) may have been cleared more rapidly from the circulation by binding to cellular receptors. A similar molar ratio of 10:1 between C3b/c and C3a (desArg) has been found in healthy volunteers under nonactivating conditions [19]. We conclude that C3a (desArg), because of its lower plasma concentrations compared with C3b/c, is a less sensitive plasma marker than C3b/c to detect complement activation during CPB.
Unlike other studies [15–17] on heparin-coated extracorporeal circuits during CPB, both groups of patients in this study received equal amounts of systemic heparin. Thus, surface-bound heparin was the only differentiating factor between the two groups. Heparin coating does not affect the initiation of the alternative complement pathway, but it reduces circulating C3b/c concentrations during CPB while no classic pathway activation is detected. We conclude that during CPB, heparin coating inhibits alternative pathway amplification, most likely by reducing C3 convertase formation. The selective inhibition of C3 activation products by heparin coating results in an equal reduction in terminal complement pathway activation.
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Acknowledgments |
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TopFootnotesAbstractIntroductionMaterial and MethodsResultsCommentAcknowledgmentsReferences |
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) and heparin-coated (
) circuits. Data are presented as the median value ± the interquartile range and are not corrected for hemodilution (+ = p < 0.05 compared with baseline; * = p < 0.05 between groups.