APT070 inhibits complement activation during in vitro cardiopulmonary bypass.

BACKGROUND: The proteins of the complement cascade play an important role in inflammation and the immune response. They have been shown to be activated during cardiopulmonary bypass (CPB), and may be responsible for the inflammatory response to CPB. We looked at the effect of APT070, an anti-complement agent, on human blood during in vitro CPB. MATERIALS AND METHODS: Four hundred millilitres of blood was venesected from healthy human volunteers and heparinised. To the blood was added either APT070 to a concentration of 50 microg/ml (n=5) or vehicle control (n=4). The blood was entered into an in vitro CPB circuit and circulated for 90 min. RESULTS: Our results showed that after 90 min of in vitro bypass APT070 significantly inhibited the activation of compliment as demonstrated by C3a (p=0.03) and sC5b-9 (p=0.01) levels, and reduced neutrophil stimulation as measured by CD11b expression (p=0.04 at 90 min). CONCLUSION: APT070 significantly inhibits complement and neutrophil activation. This result may have considerable implications, especially if it can be shown to decrease the inflammatory sequelae of CPB.     

Complement activation during cardiopulmonary bypass. Comparison of bubble and membrane oxygenators

A prospective randomized trial involving 91 patients undergoing cardiopulmonary bypass compared the effects of bubble oxygenators (with and without methylprednisolone sodium succinate) and membrane oxygenators on complement activation and transpulmonary sequestration of leukocytes. Patients were divided as follows: Group I, 30 patients, bubble oxygenator; Group II, 31 patients, bubble oxygenator and methylprednisolone sodium succinate (30 mg/kg); Group III, 30 patients, membrane oxygenator. In Group I, C3a increased from 323 +/- 171 ng/ml during cardiopulmonary bypass to 1,564 +/- 785 ng/ml at 25 minutes after bypass (p less than 0.0001). A significant decrease in C3a was found in Groups II and III compared to Group I (p less than 0.0001). C5a did not change significantly during cardiopulmonary bypass in any group. Reestablishment of pulmonary circulation at the end of bypass produced significant transpulmonary leukocyte sequestration in Group I; the median cell difference was 1,700/microliter. Transpulmonary sequestration was significantly (p less than 0.0001) less in Group II (median cell difference = 200/microliter) and in Group III (median cell difference = 400/microliter) than in Group I. We conclude that cardiopulmonary bypass with a bubble oxygenator alone initiates significantly (p less than 0.0001) more C3a activation and leukocyte sequestration than when methylprednisolone sodium succinate (30 mg/kg) is given 20 minutes before the start of cardiopulmonary bypass with a bubble oxygenator or when a silicone membrane oxygenator is used.     

Evidence for complement activation by protamine-heparin interaction after cardiopulmonary bypass

Complement activation by the alternate pathway has been implicated in the pathophysiology of cardiopulmonary bypass (CPB), and laboratory studies suggest that the complement cascade may be activated by the protamine-heparin complex. To determine if the administration of protamine to patients receiving heparin activates complement, we studied 100 patients undergoing CPB by assaying levels of C3a and C4a (classic pathway) at regular intervals before and after protamine administration. In group I (90 patients), protamine was given at the usual interval (median 5 minutes) after CPB. In group II (10 patients), protamine was withheld until skin closure (median 45 minutes) after CPB. Results demonstrated that C4a was not activated during CPB in either group. After CPB, the C4a level in group I was 459 ng/dl and increased to 1047 ng/dl 10 minutes after protamine administration (p less than 0.001). In group II, the C4a level was 484 ng/dl at the end of CPB and 354 ng/dl 15 minutes later, which corresponds to the value immediately after protamine administration in group I. The delayed administration of protamine in group II caused a significant increase in C4a at the time of skin closure (1090 ng/dl; p less than 0.001). Corresponding results from C3a analysis before and after protamine administration confirmed the activation of complement cascade. Our study provides the first clinical evidence that the protamine-heparin complex activates complement via the classic (C4a) pathway. The hemodynamic effects of protamine after CPB may be related to complement activation.     

Complement activation in cardiopulmonary bypass, with special reference to anaphylatoxin production in membrane and bubble oxygenators

Complement activation by cardiopulmonary bypass (CPB) was studied in 82 patients divided into membrane (MOG) and bubble oxygenator groups (BOG). The influence of primed homologous to circulating autologous blood volume (H/A) ratio was also evaluated. C4a increased very slowly during CPB in both groups, maintaining slightly higher levels in the BOG than in the MOG, with the exception of a marked initial rise in the BOG with a high H/A ratio (greater than or equal to 20%). Anaphylatoxin C3a levels increased more steeply in the BOG than in the MOG. An obvious rise in anaphylatoxin C5a production was observed in the BOG alone. The influence of high H/A ratio perfusion on complement activation was milder in the MOG than in the BOG. In 20 monkeys (Macaca fascicularis), continuous intraaortic infusion with bubbled autologous blood increased C4a and C3a levels, while autologous blood extracorporeally contacted with nylon increased C3a levels alone. In vitro studies revealed that human immunoglobulin fractions denatured by oxygen bubbling produced C4a, C3a, and C5a in a dose-dependent manner, although human albumin treated identically as human immunoglobulin did not produce these complements. It was thus inferred that (1) during CPB, complement is predominantly activated via the classical pathway in the BOG and via the alternative pathway in the MOG; (2) higher anaphylatoxin levels in the BOG than in the MOG are related to mode and grade of blood trauma; (3) anaphylatoxin level differences in both groups tend to increase with high H/A perfusion; and (4) immunoglobulin-free sera may reduced classical pathway activation.     

Effects of cardiopulmonary bypass on pulmonary leukostasis and complement activation

We measured white blood cell counts and complement component (C3a, C4a, and C5a) and prostacyclin levels, and studied lung biopsy specimens, in 16 patients undergoing cardiopulmonary bypass and compared them with four patients undergoing other pulmonary procedures. Bypass caused no significant elevation in peripheral venous white blood cell counts. Higher counts were present in the right atrium compared with the left atrium. Patients who underwent bypass had elevated complement component and prostacyclin concentrations before operation and these levels increased further during operation. Trapping of polymorphonuclear leukocytes occurred in pulmonary alveolar capillaries and venules after bypass. We conclude that bypass activates complement components primarily of the alternative pathway and leads to increased blood prostacyclin levels. These changes are accompanied by polymorphonuclear leukocyte accumulation in the lungs and may be important in initiation of the adult respiratory distress syndrome in these patients.     

Neutrophil lysosomal enzyme release and complement activation during cardiopulmonary bypass

Complement activation and neutrophil degranulation were concomitantly studied during uncomplicated cardiopulmonary bypass (CPB). Plasma concentrations of complement factor C4, complement split product C3d, the neutrophil lysosomal enzyme elastase complexed with alpha 1-proteinase inhibitor (PI) and fibronectin were measured in 12 patients, C3d and elastase/PI increased significantly during CPB (volume-corrected results). The C3d rise was almost linear, whereas elastase/PI showed exponential increase. Mean elastase/PI and mean C3d concentrations at different times during CPB covaried closely. The study showed that during CPB neutrophil lysosomal enzyme release is intimately related to complement activation, although activation of the two systems may be caused by a common third activator within the extracorporeal circuit.     

Time for new concepts about measurement of complement activation by cardiopulmonary bypass?

Fifty-one patients admitted for routine coronary bypass operations were randomized to cardiopulmonary bypass with a membrane oxygenator (Capiox) or a bubbler (Polystan or William Harvey). Complement activation was measured using enzyme immunoassays for concentrations of C3 activation products and the terminal complement complex. From 5.8 to 8.1 arbitrary units (AU)/mL (medians), the plasma concentrations of C3 activation products increased by 119.9 AU/mL (Capiox), 124.6 AU/mL (Polystan), and 79.5 AU/mL (William Harvey) to a peak at closure of the sternum (not significant when related to baseline concentrations). The increase in C3 activation products and baseline C3 activation were linearly correlated (R2 = 0.30; p less than 0.0001). From 5.5 to 6.1 AU/mL, the plasma terminal complement complex concentrations increased by 45.2 AU/mL (Capiox), 15.4 AU/mL (Polystan), and 17.4 AU/mL (William Harvey) to a peak before termination of cardiopulmonary bypass. Maximal terminal (C5-C9) activation was significantly higher in the membrane oxygenator group (p less than 0.0001) and showed no relationship to C3 activation. Measurement of C3 activation only gives no information about C5-C9 activation. At present, terminal complement complex quantitation is probably the best index of C5-C9 activation during cardiopulmonary bypass.     

Heparin-coated cardiopulmonary bypass equipment. II. Mechanisms for reduced complement activation in vivo

OBJECTIVE: Our objective was to study mechanisms for reduced complement activation by heparin coating of cardiopulmonary bypass equipment in clinical heart surgery. METHODS: Adults undergoing elective coronary artery bypass grafting were randomized to cardiopulmonary bypass with Duraflo II heparin-coated (n = 15) or uncoated (n = 14) sets (Duraflo coating surface; Baxter International, Inc, Deerfield, Ill). Blood samples were analyzed with the use of enzyme immunoassays for C1rs-C1 inhibitor complexes and the activation products Bb, C4bc, C3bc, C5a-desArg, and the terminal complement complex. Data were compared by repeated-measures analysis of variance. RESULTS: C1 was activated during bypass, and increases in C1rs-C1 inhibitor complexes were larger with heparin coating (P =.03). C4bc increased after administration of protamine, without intergroup differences (P =.69). Bb (P =.22) and C5a-desArg (P =.13) tended to increase less with heparin coating. Formation of C3bc (P =.03) and the terminal complement complex (P <.01) was significantly reduced with heparin coating. C5a-desArg increased 2-fold during bypass, whereas the terminal complement complex increased 10- to 20-fold. Maximal terminal complement complex concentrations were significantly correlated to maximal Bb and C3bc (R = 0.6, P <.001), but not to C1rs-C1 inhibitor complexes or C4bc (R < 0.05, P >.8). CONCLUSIONS: C1 activation during bypass was increased by heparin coating, but further classical pathway activation was held in check until administration of protamine. Heparin coating significantly inhibited C3bc and terminal complement complex formation. Terminal complement complex concentrations were related to alternative pathway activation and may be useful for evaluation of differences in bypass circuitry. Increases and intergroup differences in terminal complement complex concentrations were much larger than those in C5a-desArg.     

In vitro evaluation of Capiox FX05 and RX05 oxygenators in neonatal cardiopulmonary bypass circuits with varying venous reservoir and vacuum-assisted venous drainage levels

The purpose of this study was to evaluate the hemodynamic properties and microemboli capture associated with different vacuum-assisted venous drainage (VAVD) vacuum levels and venous reservoir levels in a neonatal cardiopulmonary bypass circuit. Trials were conducted in 2 parallel circuits to compare the performance of Capiox Baby RX05 oxygenator with separate AF02 arterial filter to Capiox FX05 oxygenator with integrated arterial filter. Arterial cannula flow rate to the patient was held at 500 mL/min and temperature maintained at 32°C, while VAVD vacuum levels (0 mm Hg, −15 mm Hg, −30 mm Hg, −45 mm Hg, −60 mm Hg) and venous reservoir levels (50 mL, 200 mL) were evaluated in both oxygenators. Hemodynamic parameters measuring flow, pressure, and total hemodynamic energy were made in real time using a custom-made data acquisition system and Labview software. Nearly 10 cc bolus of air was injected into the venous line and gaseous microemboli detected using an Emboli Detection and Classification Quantifier. Diverted blood flow via the arterial filter’s purge line and mean pressures increased with increasing VAVD levels (P < 0.01). Mean pressures were lower with lower venous reservoir levels and were greater in RX05 groups compared to FX05 (P < 0.01). Microemboli detected at the preoxygenator site increased with higher VAVD vacuum levels and lower venous reservoir levels (P < 0.01). The amount of microemboli captured by the FX05 oxygenator with integrated arterial filter was greater than by the RX05 oxygenator alone, although both oxygenators were able to clear microemboli before reaching the pseudo-patient.     

Effects of methylprednisolone on complement activation and leukocyte counts during cardiopulmonary bypass

Generation of the complement activation products C3dg and terminal complement complex (TCC) and numerical changes in peripheral granulocytes (PMN) and lymphocytes were assessed in patients undergoing aortocoronary bypass surgery with extracorporeal circulation (ECC). Fluid from bronchial lavage performed preoperatively and 4 hours postoperatively was analyzed for granulocyte elastase activity and PMN content. Ten of the 20 patients received methylprednisolone (30 mg/kg b.w.) immediately before ECC. No difference was found between them and the control group regarding C3dg and TCC, and both groups showed similar postoperative decrease of peripheral blood lymphocytes. The postoperative PMN count in peripheral blood was significantly higher in the methylprednisolone group than in the controls from 12 hours onwards. In bronchial lavage fluid the postoperative PMN count was unaltered in the methylprednisolone group, but significantly increased in the controls. No granulocyte elastase activity was found before or after surgery in either group. The results indicated that methylprednisolone does not affect complement activation during cardiopulmonary bypass, but increases the granulocytes in peripheral blood postoperatively.     

两种氧合器对心肺转流炎症反应的影响

目的动态监测室间隔缺损修补术患者在心肺转流(CPB)各时段血清可溶性细胞间粘附分子(sICAM-1)、可溶性E-选择素(sE-selection)及肿瘤坏死因子α(TNF-α)的变化规律,并比较西京-90鼓泡式氧合器和希健-Ⅱ膜式氧合器对其的影响。方法选择择期行室间隔缺损修补术的患者30例,随机均分为鼓泡式氧合器组(B组)和膜式氧合器组(M组)。所有患者分别在麻醉后CPB开始前(T1)、主动脉阻断开放前(T2)、CPB结束时(T3)、术后2h(T4)、6h(T5)、24h(T6)及48h(T7)取静脉血5ml用ELISA法测定sICAM-1、sE-selection及TNF-α的浓度。结果两组患者血清中的TNF-α于T2时开始显著升高,T4时达到峰值(P<0·01)。sICAM-1于T5时开始升高,T6时达峰值。sE-selection于T4时开始升高,T5时达峰值(P<0·01)。M组大部分时点TNF-α、sICAM-1、sE-selection的浓度均低于B组。结论希健-Ⅱ膜式氧合器引起的炎症反应较轻。     

Sequestration of glyceryl trinitrate (nitroglycerin) by cardiopulmonary bypass oxygenators

The effectiveness of glyceryl trinitrate (nitroglycerin) in controlling myocardial ischemia and blood pressure during coronary artery bypass graft surgery is frequently lost during surgery, possibly as a result of drug sequestration by the cardiopulmonary bypass circuit. The objective of this study was to utilize a gas-liquid chromatographic assay to determine the extent of removal of glyceryl trinitrate from the priming fluid by the bubble and membrane oxygenators. The apparatus was maintained at either 25 or 37 degrees C, the two extreme temperatures experienced by the patient during bypass surgery. At apparent steady state, the circulating glyceryl trinitrate concentration was decreased by 20.6%, 46.6%, and 67.3% with the Maxima membrane oxygenator, Cobe membrane oxygenator, and Bentley bubble oxygenator, respectively. The three-layer defoaming filters that are used in the Bentley bubble oxygenator were studied by immersing each of the three filters in fluid containing 60 nM glyceryl trinitrate and monitoring the drug concentration in Plasmalyte. The filters sequestered approximately 90% of the glyceryl trinitrate from the bathing solution of which 31% was recovered with a single methanol wash of the polyurethane filter. These data demonstrate that the different oxygenators used in the cardiopulmonary bypass circuit remove glyceryl trinitrate to varying degrees from the circulating fluid.     

Complement activation with bubble and membrane oxygenators in aortocoronary bypass grafting

Thirty-three patients admitted for coronary bypass grafting were randomized to cardiopulmonary bypass with a bubble oxygenator (Cobe or Polystan) or a membrane oxygenator (SciMed). Plasma concentrations of C3 activation products and the terminal complement complex were measured using enzyme immunoassays. Both variables increased almost linearly after onset of cardiopulmonary bypass, with maximal concentrations at closure of the sternum. From a baseline of 7.5 to 12.0 arbitrary units (AU)/mL (medians), the concentrations of C3 activation products increased by 117.5 AU/mL (Cobe), 120.5 AU/mL (Polystan), and 213.3 AU/mL (SciMed). The increase in the membrane group was significantly higher than in the two bubble oxygenator groups (p less than 0.01). From a baseline of 0.9 to 1.3 AU/mL, the concentrations of terminal complement complex increased by 5.4 AU/mL (Cobe), 6.6 AU/mL (Polystan), and 7.7 AU/mL (SciMed) (differences not significant). The higher C3 activation caused by the membrane oxygenator may be explained by differences in flow profile and surface area in contact with blood. The study cannot confirm the general assumption that membrane oxygenators lead to lower complement activation than do bubble oxygenators.     

Evaluation of Mimesys phosphorylcholine (PC)-coated oxygenators during cardiopulmonary bypass in adults

A new generation of coating extracorporeal circuitry with biocompatible polymers has entered the North American perfusion market. This new biomimetic coating process uses synthetic phosphorylcholine (PC) containing polymers to bond covalently to the surface of the Sorin Monolyth oxygenator, under the brand name of Mimesys. In part one of a three-part investigation, 160 Mimesys-coated oxygenators were randomly evaluated against 36 uncoated oxygenators for blood flow, hemodynamic resistance, and pressure differentials. In part two, retrospective analysis of platelet data collected in this study was compared with platelet data collected from a previous investigation using uncoated Monolyth oxygenators with albumin and crystalloid perfusates. Part three examined the risk-adjusted clinical outcomes of 71 patients treated with Mimesys-coated oxygenators, compared with 71 case-matched patients treated with uncoated oxygenators. There was no difference found in the Mimesys-coated group, when compared to the control group, with regard to pressure differentials or hemodynamic resistance. However, we conclude that platelet protection with PC-coated Monolyth's using crystalloid perfusates, was similar to platelet protection with albumin perfusates, and significantly better than uncoated Monolyths using crystalloid perfusates.     

Washin and washout of isoflurane administered via bubble oxygenators during hypothermic cardiopulmonary bypass

Washin and washout of a volatile anesthetic given through the oxygenator during hypothermic (23.4 +/- 2.1 degrees C) cardiopulmonary bypass were studied in nine patients. The authors administered isoflurane and measured its partial pressure in arterial (Pa) and venous (Pv) blood and the gas exhausted from the oxygenator (PE) at 1, 2, 4, 8, 16, 32, and 48 min during washin. These measurements were repeated during washout, which coincided with rewarming. During washin, PE, Pa, and Pv progressively rose toward inlet gas partial pressure (PI). Equilibration of Pa with PI was 41% after 16 min, 51% after 32 min, and 57% after 48 min of washin. During washout, Pa declined to 24% of its peak after 16 min and to 13% after 32 min. Washin and washout were considerably slower in mixed venous blood. Washin of isoflurane appeared to occur more slowly during cardiopulmonary bypass than during administration via the lungs in normothermic patients, presumably because hypothermia increases tissue capacity, compensating for the effect of hemodilution that otherwise would decrease the blood/gas partition coefficient. During rewarming, washout appeared to occur as rapidly as from the lungs of normothermic patients. This may have resulted from the declining blood/gas partition coefficient (due to rewarming) and relatively limited tissue stores of isoflurane. The relationship between exhaust and arterial partial pressures was reasonably consistent; for clinical purposes, measurement of PE can be used to estimate Pa.     

In vitro and clinical study of complement activation during cardiopulmonary bypass with reference to types of oxygenator, primed autologous blood and patient's factor

Complement activation during cardiopulmonary bypass (CPB) was studied in vitro and in vivo with regard to types of oxygenator, primed autologous blood and patient's factor. In vitro study was performed using human blood in a simple circuit involving an oxygenator, roller pump and connector tubing. In vivo study was carried out in 118 patients and divided into bubble (BO) and membrane oxygenator (MO) groups. The influence of primed homologous to circulating autologous blood volume (H/A) ratio was also examined. In vitro study, C3a and C4a increased steeply in the BO group. On the other hand, in the MO group, C3a and C4a increased up to minute of 60, and afterwards gradually decreased. In clinical study, complement was more activated in the BO group than in the MO group. These results supported that in the BO group, immunoglobulin denatured by blood-gas interface played an important role in complement activation. In membrane oxygenator, blood-material interface was a major cause of complement activation. In order to reduce these complement activation, we introduced fresh concentrated red cells, which was almost free of immunoglobulin, as a primed blood. Application of this method in clinical study, complement activation was reduced and postoperative lung function was improved significantly. These changes were more significant in the BO group. In the high H/A group, differences of anaphylatoxin level between BO and MO group had a tendency to increase. This method is useful to the CPB case of neonate and infant, which was subjected to be primed with a large amount of blood.     

Inflammatory system activation during cardiopulmonary bypass as an indicator of biocompatibility: a randomized comparison of bubble and membrane oxygenators

As the exposure of blood to foreign material during cardiopulmonary bypass (CPB) leads to triggering of inflammatory systems, the inflammatory response was used as an indicator of the biocompatibility of oxygenators. Activation of complement and neutrophil granulocytes during CPB was studied in 96 patients undergoing coronary bypass, with randomized comparisons between four different oxygenators, two of bubble and two of membrane type. Seven patients undergoing thoracotomy without CPB served as controls. During CPB there was significant complement activation, measured as changes in the ratio C3d/C3, with no demonstrable difference between the bubble and membrane oxygenator groups. Such change was not seen in the controls. Neutrophil granulocytes released significant amounts of the granule proteins lactoferrin and myeloperoxidase during CPB, but not during thoracotomy without CPB. The plasma concentrations of lactoferrin and myeloperoxidase were significantly lower in the membrane oxygenator groups, possibly indicating better biocompatibility. The strong inflammatory response with both oxygenator types, however, indicates that presently used CPB devices have unsatisfactory biocompatibility.