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(Circulation. 1999;100:553-558.)
© 1999 American Heart Association, Inc.

Basic Science Reports

Role of C3 Cleavage in Monocyte Activation During Extracorporeal Circulation

Christine S. Rinder, MD; Henry M. Rinder, MD; Kirk Johnson, PhD; Michael Smith, PhD; David L. Lee, MD; Jayne Tracey, BS; Glenda Polack, DVM; Paul Higgins, PhD; C. Grace Yeh, PhD; Brian R. Smith MD

From the Department of Laboratory Medicine (C.S.R., H.M.R., J.T., B.R.S.) and the Department of Anesthesiology, Yale University School of Medicine (C.S.R., D.L.L.), New Haven, Conn; Chiron Corp, Emeryville, Calif (K.J., G.P.); Quinnipiac College, Hamden, Conn (M.S.); and Cytomed, Inc, Cambridge, Mass (P.H., G.Y).

Correspondence to Christine S. Rinder, MD, Department of Laboratory Medicine, Fitkin 617, Yale University School of Medicine, 333 Cedar St, PO Box 208035, New Haven, CT 06520-8035. E-mail christine.rinder{at}yale.edu

	Abstract

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Background—We previously demonstrated that inhibiting formation of terminal complement components (C5a and C5b-9) prevents platelet and neutrophil (PMN) but not monocyte activation during simulated extracorporeal circulation (SECC). This study examined whether earlier complement inhibition during SECC, blocking C3a formation, would additionally prevent monocyte activation.

Methods and Results—SECC was established by recirculating heparinized whole blood from human volunteers on a membrane oxygenator. CAB-2, a chimeric protein constructed from genes encoding the complement regulatory proteins CD46 and CD55, inactivates the C3/C5 convertases and blocks in vitro generation of C3a, C5a, and C5b-9. CAB-2 was used in 4 experiments at a final concentration of 300 µg/mL and 4 experiments at 30 µg/mL; 4 control runs used vehicle alone. Samples were assayed for C3a and C5b-9, monocyte activation (CD11b upregulation), PMN activation (CD11b upregulation and elastase release), and platelet activation (P-selectin expression and monocyte-platelet conjugate formation). CAB-2 at both doses significantly inhibited formation of C3a and C5b-9 during SECC. High-dose CAB-2 significantly blocked monocyte and PMN CD11b upregulation and PMN elastase release. CAB-2 also inhibited formation of platelet activation–dependent monocyte–platelet conjugates.

Conclusions—Blockade of complement activation early in the common pathway inhibited monocyte CD11b upregulation during SECC, suggesting that early complement components contribute most to monocyte activation during SECC. As expected, PMN and platelet activation were blocked by terminal complement inhibition. This investigation further elucidates the relation between complement and blood cell activation during simulated cardiopulmonary bypass.

Key Words: extracorporeal circulation • cell adhesion molecules • leukocytes • platelets

	Introduction

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Cardiopulmonary bypass (CPB) is associated with an inflammatory response consisting of humoral and cellular changes that contribute to tissue injury. Complement activation is a potential contributor to this inflammatory response,^{1 2} but the relation between specific complement components and the activation of inflammatory or coagulant pathways has been difficult to ascertain. Simulated extracorporeal circulation (SECC) has been used extensively in vitro to model inflammatory changes produced during in vivo CPB.^{3 4} SECC activates complement (C3a and C5b-9 formation), platelets (increased CD62P-positive platelets and monocyte-platelet conjugate formation), and leukocytes [neutrophil (PMN) and monocyte CD11b upregulation and PMN elastase secretion] comparable to in vivo bypass.⁵ We previously demonstrated that inhibiting formation of the late complement components, C5a and C5b-9, during SECC prevents PMN and platelet activation but did not clearly inhibit monocyte activation.⁴

In the present study, we used complement activation blocker-2 (CAB-2) to explore the role of C3a formation in monocyte activation during SECC. CAB-2, the product of a chimeric gene constructed from the genes encoding human membrane cofactor protein (MCP, CD46) and human decay accelerating factor (DAF, CD55), is a soluble, glycosylated, 110-kDa protein whose chimeric nature is confirmed by reactivity with both MCP- and DAF-specific antibodies.⁶ MCP cleaves complement factors C3b and C4b to their inactive forms, iC3b and iC4b, respectively. The complement regulatory activity of DAF results from its ability to dissociate the C3 and C5 convertase subunits, thus downregulating formation of C3a and C5a, respectively. The chimeric CAB-2 product combines both of these activities and thus inactivates both classic and alternative C3/C5 convertases through proteolysis of C3b (through MCP) and enhancement of convertase decay (through DAF),⁶ thereby inhibiting formation of C3a, C5a, and C5b-9 in vitro and in vivo.

The soluble activities of CAB-2 have IC₅₀ values nearly identical to soluble MCP (sMCP) and soluble DAF (sDAF). However, against cell-associated convertases, CAB-2 has greater activity than the parent proteins combined,⁶ with 150-fold more MCP activity and 10-fold more DAF activity against classical pathway-mediated sheep red blood cell hemolysis. Blockade of alternative pathway hemolysis was similarly greater with CAB-2 than that seen with a mixture of sMCP and sDAF in both a Forssman shock model (guinea pig) and during in vitro generation of human C3a. Thus CAB-2 during SECC should block both alternate and classical complement activation, with abrogation of C3a formation together with later complement components. We have now examined the complement regulatory activity of this chimeric protein in human blood to determine its relation to leukocyte and platelet activation on SECC.

	Methods

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Extracorporeal Circuit Preparation
Identical to previous studies,⁴ extracorporeal circuits were assembled with the use of a pediatric membrane oxygenator (VP CML Plus, Cobe Cardiovascular) primed with 600 mL of lactated Ringers containing dextrose (4.0 g/L), mannitol (4.0 g/L), and porcine heparin (5 U/mL). The prime was circulated at 1.5 L/min with a minimally occlusive roller pump with sweep gas flow (95% oxygen and 5% carbon dioxide) at 0.25 L/ min. The pH, PO₂, and perfusate temperature were maintained at a pH of 7.30 to 7.45 and a PO₂ >150 mm Hg.

Extracorporeal Circuit Operation and Sampling
After approval by the Yale Human Investigation Committee and informed consent, blood (500 mL) was drawn over 5 minutes from healthy volunteers receiving no medications into a transfer pack containing porcine heparin (5 U/mL final concentration). Purified CAB-2, supplied by Cytomed, Inc,⁶ or vehicle alone was then added to the transfer pack immediately before blood addition to the circuit. As blood was introduced to the circuit reservoir, 400 mL of prime fluid was simultaneously withdrawn to yield a final circuit volume of 700 mL and a mean hematocrit of 25±3% (SD). The blood/prime combination was recirculated and mixing accomplished within 2 minutes (time 0). The circuit was cooled to 27°C over 5 minutes, maintained for 60 minutes, then rewarmed to 37°C for an additional 30 minutes (total recirculation time 90 minutes). These temperatures simulate the in vivo conduct of CPB at this and other institutions. Four experiments were performed with 30 µg/mL CAB-2, 4 with 300 µg/mL CAB-2, and 4 with vehicle alone. Blood samples were drawn at 0, 5, 15, 30, 45, 60, 75, and 90 minutes of recirculation. Plasma samples for C3a and C5b-9 assays were snap-frozen in liquid nitrogen and stored at -70°C until assayed. Plasma samples for neutrophil elastase-antitrypsin complex were anticoagulated with EDTA, snap-frozen, and stored at -70°C. Whole blood samples for flow cytometric studies were fixed in 1% (final concentration) paraformaldehyde in PBS.⁷ An additional blood sample was drawn into 5 mmol/L EDTA at 0, 30, and 90 minutes for a complete blood count and differential.

Flow Cytometry
Whole blood samples were fixed for 60 minutes at 4°C followed by addition of 1:8 vol/vol of Tris-glycine as previously described.⁸ Samples were washed and resuspended in Tyrode's-HEPES buffer and divided into aliquots for labeling with monoclonal antibodies (mAb) at 4°C for 20 minutes, then washed and resuspended in Tyrode's-HEPES buffer for FACS analysis. For determination of the percentage of leukocytes with bound platelets and leukocyte activation, samples were labeled with (a) FITC–anti-CD45 and phycoerythrin (PE)–anti-GPIIb/IIIa and (b) FITC-anti-CD45 and PE-anti-CD11b, respectively as previously described.⁸ For determination of platelet activation (CD62P+platelets), samples were labeled with (c) FITC–anti-gpIIb/IIIa and PE–anti-CD62P as previously detailed.⁸

Samples were analyzed on a FACScan flow cytometer (Becton-Dickinson). Leukocyte measurements were performed by live gating on FITC-positive, leukocyte-sized events, with mean CD11b fluorescence and monocyte-platelet conjugates determined as previously described.⁵ Platelet analysis was accomplished by acquisition of FITC-positive, platelet-sized events and the percentage of CD62P⁺ platelets determined as previously described.⁵ An isotype-matched (PE-conjugated) control mAb set the threshold (99% of events below threshold) for both P-selectin expression and leukocyte-platelet conjugates.

Plasma Assays
C3a and C5b-9 levels were measured by ELISA (Quidel) according to the manufacturer's instructions. Neutrophil elastase:1-antitrypsin complexes levels were measured by capture ELISA as follows (all incubations at room temperature). Nunc Maxi Sorp immunoplates (Nunc) were coated with 2.5 µg/mL mAb to -elastase (The Binding Site) in 0.1 mol/L carbonate/bicarbonate buffer (pH 9.6) at 100 µL/well, incubated x23 hours, and washed x4 with PBS/Tween 20. The plates were loaded with human elastase:1-antitrypsin standards (Sigma) or SECC samples in duplicate (100 µL/well) and incubated x2 hours, washed with PBS/Tween 20, then incubated with horseradish peroxidase–sheep human antitrypsin conjugate (The Binding Site) diluted 1:5000 in PTG (1x PBS, 0.02% Tween 20, 0.2% gelatin) for 1 hour. TMB substrate (100 µL/well) diluted to 0.1 mg/mL in 0.11 mol/L NaOAc, pH 5.5, and 0.003% H₂O₂ in 1x PBS (Sigma) was added and incubated x3 minutes. The reaction was stopped with 100 µL/well of 3 mol/L H₂SO₄, and the plates were scanned for optical density (450 nm).

CAB-2–Mediated Inhibition of Monocyte CD11b Upregulation by In Vitro Complement Activation
Complement was activated in human serum by addition of Zymosan (Sigma) similar to the method described by Stahl et al.⁹ Briefly, human blood was clotted on ice to preserve complement activity, serum was harvested, and aliquots were incubated with CAB-2 at 3 mg/mL or PBS as a control. Zymosan (10 mg) was added to serum (2 mL), incubated x2 hours (37°C), the sample was centrifuged, and supernatant serum (50 µL) was added to 150 µL of heparinized autologous whole blood. Whole blood samples were incubated at 37°C x1 hour, then fixed in 1% paraformaldehyde and monocyte CD11b expression measured as above. Zymosan-treated serum samples were assayed for C3a and C5b-9 as above.

Statistics
C3a and C5b-9 levels are reported as mean±SEM of the absolute values. Other data are presented normalized to 100% of the baseline value¹⁰ and reported as mean±SEM. Statistical analysis was performed with Stratigraphics software (Manugistics) with multivariate ANOVA for repeated measurements over time, with significance at P0.05 on both the absolute values for all variables and the normalized values (eg, the percentage of the time 0 value), with complete concordance of results.

	Results

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Complement Activation
Control SECC (vehicle alone) produced significant (P<0.01) complement activation, with C3a levels rising from 699±233 ng/mL to 4887±666 ng/mL and C5b-9 levels from 63±28 ng/mL to 793±192 ng/mL, both peaking after 90 minutes of recirculation (Figure 1, A and B, respectively, and Table 1). Recirculation with CAB-2 inhibited C3a generation in a dose-dependent fashion, with peak levels of 2188±173 ng/mL (90 minutes) and 695±242 ng/mL (90 minutes) at 30 µg/mL and 300 µg/mL of CAB-2, respectively (P<0.01 for both compared with control, Figure 1A). Similarly, C5b-9 formation during SECC was significantly inhibited by CAB-2, with peak levels of 269±33 ng/mL and 111±31 ng/mL after 90 minutes for 30 µg/mL and 300 µg/mL CAB-2, respectively (P<0.01 for both vs control, Figure 1B).

View larger version (19K): [in this window] [in a new window]   Figure 1. C3a and C5b-9 during SECC. Levels of C3a (A) and C5b-9 (B) were measured in plasma at the time points indicated. Mean±SEM from 4 control experiments (•), 4 experiments with 30 µg/mL CAB-2 (), and 4 experiments with 300 µg/mL CAB-2 () are shown. CAB-2 significantly inhibited C3a and C5b-9 generation (P<0.01 for both CAB-2 doses).

View this table: [in this window] [in a new window]   Table 1. Effect of CAB-2 on Peak Levels (Mean±SEM) of Selected Variables During SECC

Leukocyte Activation
Control SECC resulted in significant monocyte activation, as measured by CD11b upregulation (Figure 2). Monocyte CD11b began to increase while the blood was maintained at 27°C (60 minutes), then increased dramatically during the 30 minutes at 37°C, peaking after 90 minutes total at 353±107% of baseline (P<0.01). CAB-2 addition at 300 µg/mL significantly blunted monocyte CD11b upregulation, which peaked at only 126±21% of baseline at 60 minutes; in particular, the late increase with rewarming to 37°C was not seen with CAB-2 addition (P<0.05, Figure 2). CAB-2 at 30 µg/mL did not significantly inhibit monocyte CD11b upregulation, with levels reaching 150±36% of baseline at 60 minutes (P=0.087, Figure 2).

View larger version (16K): [in this window] [in a new window]   Figure 2. Monocyte CD11b during SECC. CD11b fluorescence density was measured on monocytes at the time points indicated, expressed as a percentage of the baseline fluorescence. Mean±SEM from 4 control experiments (•), 4 experiments with 30 µg/mL CAB-2 (), and 4 experiments with 300 µg/mL CAB-2 () are shown. CAB-2 at 300 µg/mL but not 30 µg/mL significantly inhibited the increase in monocyte CD11b (P<0.01).

As previously demonstrated, control SECC activated PMN, with CD11b levels peaking at 301±135% of baseline (P<0.01, Figure 3A) and elastase:1-antitrypsin complex levels at 982±132% of baseline (P<0.01, Figure 3B), both at 90 minutes. CAB-2 at 300 µg/mL significantly inhibited PMN activation, with CD11b levels peaking at only 116±12% of baseline after 45 minutes (P<0.05, Figure 3A). Elastase:1-antitrypsin complex levels at the higher CAB-2 dose were also blunted, reaching only 289±86% of baseline compared with the 9-fold increase in control experiments at 90 minutes (P<0.01, Figure 3B). CAB-2 at 30 µg/mL did not significantly inhibit PMN CD11b upregulation, with levels reaching 136±13% of baseline at 45 minutes (P=0.06). Similarly, elastase:1-antitrypsin complex levels reached 787±158% of baseline at 90 minutes (P>0.10), not significantly different from control SECC.

View larger version (20K): [in this window] [in a new window]   Figure 3. PMN CD11b and neutrophil elastase: 1-antitrypsin complex levels during SECC. PMN CD11b fluorescence density (A) and plasma elastase:1-antitrypsin (B) were measured at the time points indicated, both expressed as a percentage of baseline fluorescence. Mean±SEM from 4 control experiments (•), 4 experiments with 30 µg/mL CAB-2 (), and 4 experiments with 300 µg/mL CAB-2 () are shown; 300 µg/mL but not 30 µg/mL CAB-2 inhibited the PMN CD11b increases (P<0.05) and elastase:1-antitrypsin complex formation (P<0.01).

Platelet Activation
The percentage of circulating P-selectin–positive platelets increased significantly but only modestly during control SECC, peaking at 90 minutes at 136±10% of baseline (P<0.05, Table 1). CAB-2 addition did not significantly block the increase in circulating activated platelets [peak levels of 131±24% (30 minutes) and 111±13% (90 minutes) for 30 µg/mL and 300 µg/mL CAB-2, respectively, P>0.1 for both, Table 1]. However, the percentage of monocytes binding activated platelets is perhaps a more sensitive⁸ and biologically relevant marker¹¹ of platelet activation in whole blood. Monocyte-platelet binding increased significantly during control SECC (P<0.01), with the percentage of monocyte-platelet conjugates peaking at 283±54% of baseline after 90 minutes of recirculation (Figure 4). CAB-2 at 300 µg/mL significantly inhibited this measure of platelet activation (P<0.01, Figure 4), with the percentage of monocyte-platelet conjugates peaking at only 139±31% of baseline after 90 minutes. Similarly, 30 µg/mL CAB-2 inhibited monocyte-platelet conjugate formation, peaking at only 158±22% of baseline at 90 minutes (P<0.05). PMN-platelet binding, a less robust marker of platelet activation,¹² was not significantly inhibited by CAB-2 at either dose (Table 1).

View larger version (17K): [in this window] [in a new window]   Figure 4. Platelet-monocyte conjugate formation during SECC. Percentage of monocytes with binding platelets was measured at time points indicated. Mean±SEM from 4 control experiments (•), 4 experiments with 30 µg/mL CAB-2 (), and 4 experiments with 300 µg/mL CAB-2 () are shown. Both 300 µg/mL and 30 µg/mL CAB-2 inhibited formation of platelet-monocyte conjugates during SECC (P<0.01 and P<0.05, respectively).

Cell Counts
The monocyte count decreased during control SECC, reaching a nadir of 71±6% of baseline (P<0.01). CAB-2 addition did not preserve monocyte counts that fell to 81±9% and 80±4% of baseline for CAB-2 at 30 and 300 µg/mL, respectively (P>0.05 for both, Table 1). PMN counts similarly decreased during control SECC, reaching 71±5% of baseline at 75 minutes of recirculation (P<0.01). In contrast to monocytes, CAB-2 addition significantly preserved circulating PMN, with numbers decreasing to only 85±2% and 86±2% of baseline for CAB-2 at 30 and 300 µg/mL, respectively (P<0.05 for both, Table 1). Platelet counts also decreased during control SECC, reaching a nadir of 76±8% of baseline after 60 minutes of recirculation (P<0.01), but this decrease was not affected by CAB-2, with platelet counts falling to 85±4% and 86±3% of baseline after 60 minutes with CAB-2 at 30 and 300 µg/mL, respectively (P>0.05 for both).

Zymosan-Stimulated Monocyte CD11b Upregulation In Vitro
Zymosan was used to stimulate complement activation in serum incubated in diluent or CAB-2. Because zymosan-induced C3a levels were 10-fold higher than during SECC (data not shown), the CAB-2 dose in these in vitro studies was increased to 3 mg/mL, 10-fold higher than the highest CAB-2 dose used during SECC. CAB-2 at 3 mg/mL significantly inhibited monocyte CD11b upregulation in whole blood exposed to zymosan-activated serum. Monocyte CD11b expression (in arbitrary fluorescence units) was 113±36 for untreated serum added to blood, 364±4.9 for diluent-incubated, zymosan-treated serum, and 132±25.5 for CAB-2-incubated, zymosan-treated serum (mean±SD for 3 experiments, P<0.01 by paired t test, Table 2).

View this table: [in this window] [in a new window]   Table 2. Whole Blood Monocyte CD11b (Mean±SEM) After Addition of Zymosan-Activated Serum: Effect of CAB-2

	Discussion

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Complement activation is known to participate in the pathophysiology of CPB,¹ yet, the relation between the generation of specific complement components and cellular activation has been difficult to delineate. In this study we demonstrated that early complement blockade, preventing generation of C3 cleavage products and subsequent mediators, decreases monocyte, PMN, and platelet activation in simulated bypass. In earlier work, we used an anti-human C5 mAb to block C5a and C5b-9 formation during SECC. Simultaneous with inhibition of these terminal complement components but with persistent C3a formation, we demonstrated inhibition of platelet and PMN but not monocyte activation. From this earlier study, we concluded that terminal complement components were major contributors to the platelet and PMN activation induced during SECC. The current study confirms our earlier findings of an association between complement activation and activation of platelets and PMN during SECC. In addition, we demonstrate the added ability of earlier complement inhibition, blocking C3 cleavage, to effectively prevent in vitro monocyte activation, as manifest by CD11b upregulation.

C3a and C5a are broadly defined as anaphylatoxins,¹³ and C5a produces PMN activation¹⁴ and secretion of lysosomal enzymes¹⁵ ; the role of C3a in inflammatory conditions is less well understood. However, the presence of a receptor for C3a (C3aR) on monocytes has recently been demonstrated¹⁶ ; C3a binding induces monocyte calcium flux¹⁶ and causes adherent monocytes to synthesize tumor necrosis factor- and interleukin-1ß.¹⁷ Ligation of either the C3aR or the iC3b receptor, CD11b/CD18, on monocytes induces nuclear translocation of nuclear factor-B,¹⁸ with subsequent production of tumor necrosis factor- and interleukin-1ß. However, ours is the first study to implicate C3 cleavage products in the upregulation of monocyte CD11b. The ability of CAB-2 to inhibit monocyte CD11b upregulation was confirmed with the use of zymosan-activated serum, a less complex system than SECC. In those experiments, CAB-2-incubation prevented monocyte CD11b upregulation produced by addition of zymosan-treated serum to whole blood.

CD11b/CD18 is a ß2-integrin on monocytes and PMN, whose activation-dependent upregulation permits its binding to endothelial cell intracellular adhesion molecule-1 as a prelude to vascular egress.^{19 20} As noted earlier, monocyte CD11b also serves as a receptor for iC3b, facilitating binding and phagocytosis of complement-opsonized particles.²¹ Thus it is not surprising that a product released early in complement activation should stimulate upregulation of this monocyte integrin. Complement components C3a, iC3b, and C3c stimulate prostaglandin release by monocytes²² ; blockade of one or all of these early components may be important in preventing CD11b upregulation on CPB. Although monocyte CD11b upregulation was demonstrated in both the present study, our previous SECC investigation,⁴ and during in vivo⁵ CPB, earlier work using a different SECC system²³ did not find monocyte CD11b upregulation to be caused by SECC itself. Monocyte CD11b upregulation in that study²³ was found to be more dependent on temperature change, with higher CD11b levels at 37°C; this finding is consistent with the CD11b increase noted during rewarming in the present study. On the basis of these studies, it is reasonable to postulate that monocyte CD11b upregulation on SECC is predominantly a consequence of early complement activation and is potentiated at 37°C compared with 27°C. The role of activated monocytes in complications of CPB is not well defined but likely includes both proinflammatory effects through monocyte cytokine synthesis¹⁸ and prothrombotic potential through tissue factor expression.²⁴

Activated PMN can induce tissue injury by local release of toxic oxygen species and granule contents including elastase. In addition to facilitating transendothelial migration, PMN CD11b also amplifies the inflammatory response, with oxidative burst activity linked to receptor occupancy.^{25 26} Studies probing for a C3a receptor on PMN have produced conflicting results,¹⁶ and functional effects of C3a on PMN are unclear.²⁷ Indeed, C3a-induced stimulation of PMN may be secondary to eosinophil activation in blood.²⁸ By contrast, C5a is a potent PMN activator, producing significant CD11b upregulation.²⁰ On the basis of our previous work,⁴ C3a formation does not cause significant PMN CD11b upregulation during SECC. PMN elastase, not measured in our earlier work, was significantly decreased in this study by CAB-2 addition. Both C3a¹³ and contact activation²⁹ stimulate elastase release; the decrease in elastase measured here may also result in part from inhibition of C3a or one of the contact activation pathway components. Potent inhibition of contact activation during SECC has been shown to inhibit neutrophil elastase release^{30 31} despite ongoing complement activation. It is possible that products of both contact and complement activation play a synergistic role in PMN activation during CPB, and inhibition of either pathway reduces PMN activation.

Complement component C5b-9 induces platelet P-selectin expression.³² Both CAB-2 doses significantly inhibited monocyte-platelet conjugate formation, a consequence of platelet P-selectin expression in whole blood.⁸ P-selectin mediates activated platelet binding to monocytes/PMN through P-selectin glycoprotein ligand (PSGL-1),³³ and the platelet-monocyte conjugate has the potential for both procoagulant²⁴ and proinflammatory¹¹ effects. In vivo studies have demonstrated increases in leukocyte-platelet conjugates in both stable³⁴ and unstable³⁵ coronary artery disease and during CPB.⁵ In both in vitro⁸ and in vivo^{5 36 37} studies of platelet activation, the monocyte-platelet conjugate formation consistently exceeds PMN-platelet conjugates; this may partly result from higher surface PSGL-1 density on the monocyte (Rinder, unpublished observations). Furthermore, potent activation of PMN but not monocytes decreases P-selectin–dependent PMN-activated platelet binding¹² through PSGL-1 clustering, thereby reducing its binding availability.³⁸ SECC-induced formation of platelet-PMN conjugates was blunted by CAB-2, but this decrease did not reach statistical significance. Although the increase in unbound P-selectin–positive platelets was also not inhibited by CAB-2, this may be partly due to the modest level of platelet activation produced by control SECC in this study, with P-selectin–positive platelets increasing to only 130% of baseline. As an alternative explanation, the degree of C5b-9 inhibition by high-dose CAB-2 (77%) was less than demonstrated in our previous study with an anti-C5 mAb (>90%). It is possible that the small amounts of C5b-9 still formed in the present study may, in part, be responsible for the lack of inhibition of platelet P-selectin expression on SECC.

In conclusion, this study extends in vitro (SECC) investigations of the role of C3a in monocyte activation; early complement blockade has a significant role in preventing monocyte CD11b upregulation induced during the rewarming phase of extracorporeal circulation. Blockade of early (C3a) and late (C5b-9) complement components effectively blunts monocyte, neutrophil, and platelet activation under conditions that simulate human in vivo CPB.

	Acknowledgments

 
This study was supported by NIH grants HL-47193 (B.R.S.) and HL-02668 (H.M.R.) and Chiron Corp and Cytomed Inc. Dr Rinder is a recipient of an American Heart Association Clinician-Scientist Award.

Received December 22, 1998; revision received March 29, 1999; accepted April 9, 1999.

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