Complex C5b-9 Research Articles

Complement Proteins C5b-9 Induce Secretion of High Molecular Weight Multimers of Endothelial von Willebrand Factor and Translocation of Granule Membrane Protein GMP-140 to the Cell Surface

The effect of immune activation of the serum complement system on the secretory response of human endothelial cells was examined. Exposure of antibody sensitized cultured umbilical vein endothelial cells to human serum resulted in secretion of very high molecular weight multimers of von Willebrand factor which coincided with new surface expression of the intracellular granule membrane protein GMP-140. This response required complement activation through deposition of C5b-9 and was not observed with cells exposed to antibody plus C8-deficient serum or to membrane C5b-8 (in the absence of C9). This C5b-9-induced secretion was observed with minimal cell lysis, as assessed by the release of lactic dehydrogenase. Delayed addition of C8 and C9 to cells exposed to antibody plus C8-deficient serum revealed a rapid decay of membrane C8 binding sites accompanied by loss of the secretory response, suggesting a process of removal or inactivation of nascent C5b67 complexes deposited on the endothelial surface. Membrane assembly of C5b-9 complexes caused an increase in endothelial cytosolic [Ca2+], due to influx across the plasma membrane. This C5b-9-dependent increase in cytosolic [Ca2+] and concomitant von Willebrand factor secretion were both abolished by removal of external calcium. In addition to being linked to the level of external Ca2+, the C5b-9-induced secretory response was partially inhibited by the protein kinase inhibitor, sphingosine. The capacity of the C5b-9 proteins to stimulate endothelial cells to secrete a platelet adhesive protein provides one mechanism for increased platelet deposition at sites of inflammation, and suggests the potential for other functional changes in endothelium exposed to C5b-9 during intravascular complement activation.

Effect of complement on the lateral mobility of erythrocyte membrane proteins. Evidence for terminal complex interaction with cytoskeletal components.

The lateral mobilities of erythrocyte membrane proteins and terminal complement complexes (TCC) were measured on C-treated erythrocyte ghosts by the technique of fluorescence redistribution after photobleaching. Results showed that the lateral diffusion coefficient of the bulk membrane proteins decreased with the assembly of TCC on the membrane at low C dose and was significantly reduced with assembly of the full membrane attack complex (C5b-9), even in the absence of cell lysis. At high serum doses, the mobility of the membrane proteins increased slightly above that of the control cells. The diffusion coefficients of the TCC on the erythrocyte membrane range from 1.18 to 4.37 x 10(-11) cm2/s, values characteristic of anchored membrane proteins. Spectrin-depletion of the C-lysed erythrocytes results in 25- and 45-fold increases in the diffusion coefficients of the membrane proteins and the C5b-9 complex, respectively. Conversely, oxidative cross-linking of spectrin by diamide reduced the diffusion coefficients of both membrane and C proteins. These studies indicate that the deposition of TCC on an erythrocyte can result in a substantial change in the physical and structural properties of the target membrane, aside from the creation of functional lesions. The low mobilities of the terminal complexes on the target membrane suggest possible interactions with cytoskeletal elements or with anchored membrane proteins.

Cells carrying C5b-9 complement complexes in human atherosclerotic wall.

Fibrous plaques and intimal thickenings of 5 femoral and 5 iliac human arteries obtained at surgery were processed for indirect and double-labeling immunoelectron microscopy using an affinity purified rabbit IgG anti-C5b-9 neoantigen and the EBM 11 monoclonal antibody anti-human macrophages. The C5b-9 complexes were localized in intact cells, disintegrated cells and cell debris enmeshed in the connective tissue matrix. Some of the cell debris bearing C5b-9 deposits was found to be of macrophage origin. Endocyted or exocyted pieces of membrane with pore-forming C5b-9 complexes were also identified. Damage of cells by complement in atherosclerotic lesions may contribute to atherogenesis.

Amastigotes of Trypanosoma cruzi escape destruction by the terminal complement components.

We studied the effect of complement on two life cycle stages of the protozoan parasite Trypanosoma cruzi: epimastigotes, found in the insect vector, and amastigotes, found in the mammalian host. We found that while both stages activate vigorously the alternative pathway, only epimastigotes are destroyed. The amounts of C3 and C5b-7 deposited on the amastigotes were similar to those bound to the much larger epimastigotes. Binding of C9 to amastigotes was four to six times less than binding to epimastigotes, resulting in a lower C9/C5b-7 ratio. Although a fairly large amount of C9 bound stably to amastigotes, no functional channels were formed as measured by release of incorporated 86Rb. The bound C9 had the characteristic properties of poly-C9, that is, it expressed a neo-antigen unique to poly-C9, and migrated in SDS-PAGE with an apparent Mr greater than 10(5). The poly-C9 was removed from the surface of amastigotes by treatment with trypsin, indicating that it was not inserted in the lipid bilayer. Modification of amastigote surface by pronase treatment rendered the parasites susceptible to complement attack. These results suggest that amastigotes have a surface protein that binds to the C5b-9 complex and inhibits membrane insertion, thus protecting the parasites from complement-mediated lysis.

Toxoplasma gondii: mechanism of resistance to complement-mediated killing.

Tachyzoites of the obligate intracellular protozoan Toxoplasma gondii are resistant to lysis in non-immune human serum. We have examined the mechanism of this serum resistance in RH and P strain organisms, which differ markedly in virulence, but are equally resistant to serum killing. Rapid, but limited, activation of the alternative complement pathway occurred in non-immune human serum, with deposition of equivalent amounts of C3 on the two strains. C component C3 bound covalently to parasite acceptor molecules via an ester linkage. The predominant form of C3 was iC3b which cannot participate in formation of a lytic C5b-9 complex. Multiple membrane constituents of the tachyzoite of T. gondii may serve as acceptors for the limited amount of C3 deposited during incubation in non-immune serum. When tachyzoites were presensitized with the lytic anti-p30 mAb 7B8, new amide-linked C3-acceptor complexes formed. Nearly equivalent C3 binding but a threefold enhancement of 125I-C9 binding occurred when mAb 7B8 pre-sensitized tachyzoites were compared to native organisms. These results indicate that tachyzoites of T. gondii are serum resistant because of failure to activate C efficiently. Presensitization with a lytic mAb alters the site of complement deposition and augments C5b-9 formation.

Lytic rabbit IgG for tissue culture trypomastigotes of Trypanosoma cruzi alters the extent and form of complement deposition

Infective and vertebrate stages of Trypanosoma cruzi are resistant to lysis by the alternative pathway of complement. To further elucidate the mechanism of complement evasion and to study how some immune sera render the infective stage sensitive to lysis, we compared the interaction of complement components C3 and C9 with the surface of complement susceptible, vector stage epimastigotes and vertebrate stage trypomastigotes of T. cruzi. Our studies showed that, upon incubation in human serum, complement resistant tissue culture trypomastigotes (TCT) bound five- to eightfold less C3 or C9 than complement sensitive epimastigotes (Epi). C3 bound to Epi is mainly in the hemolytically active C3b form, while TCT bear predominantly the hemolytically inactive iC3b fragment, which cannot participate in C5 convertase formation or lead to deposition of the lytic C5b-9 complex. Three- to sixfold more C3 and two- to threefold more C9 were deposited on TCT when lytic rabbit immune IgG with broad specificity was used to sensitize the parasites, and nearly one-half of bound C3 was present as C3b. In contrast, a comparison of three different sources of IgG from immune human serum showed a less clear correlation between the titer or specificity of anti- T. cruzi antibody, enhancement of C3 or C9 deposition, change in the form of bound C3, or killing. These results show that lytic rabbit IgG for T. cruzi changes the form and amount of bound complement components in anticipated fashion, but that human immune IgG does not give predictable changes in the extent or form of C3 or C9 deposition.

Assembly of complement components C5b-8 and C5b-9 on lipid bilayer membranes: visualization by freeze-etch electron microscopy.

We have visualized by freeze-etch electron microscopy the macromolecular complexes of complement, C5b-8 and C5b-9, respectively, assembled on synthetic phospholipid bilayers. These complexes were formed sequentially by using purified human complement components C5b-6 followed by C7, C8, and C9. Complexes of C5b-8 were observed on the external surface (ES) of vesicles as 12-nm particles that tended to form polydisperse aggregates. The aggregates were sometimes of a regular chainlike structure containing varying numbers of paired subunits. Etching of vesicles containing C5b-9 complexes revealed on the ES large rings of approximately 27-nm outer diameter. One or two knobs usually were attached to the perimeter of the rings. Splitting of the membrane resulted in partitioning of the C5b-9 with the outer leaflet. Thus, round holes of approximately 17-nm diameter were present in the protoplasmic face (PF), and raised circular stumps of a matching size were present on the exoplasmic face (EF) of C5b-9 vesicles. C5b-9 complexes were frequently localized in regions of the lowest lipid order. That is, in micrographs of the EF and ES, single C5b-9 complexes were located where the ripples of the P beta' phase bend or reach a dead end, and linear arrays of C5b-9 complexes outlined disclination-like structures in the lattice; the holes in the PF mirrored this distribution. The membrane immediately surrounding C5b-9 rings was often sunk inwardly over an area much larger than that of the ring itself.(ABSTRACT TRUNCATED AT 250 WORDS)

Complement fixation by pemphigus antibody. V. Assembly of the membrane attack complex on cultured human keratinocytes.

Previous studies have shown that pemphigus vulgaris (PV) IgG will fix early complement components (C1q, C4, and C3) to cultured murine epidermal cell surfaces and that PV IgG and complement alter epidermal cell membrane integrity. The present study was undertaken to determine if assembly of terminal complement components (C5, C6, C7, C8, and C9) and expression of C5b-9 neoantigens occur when PV IgG interacts with human keratinocyte (HuK) cell surface antigens in the presence of a source of complement. Monoclonal antibodies specific for C5, C6, C7, C8, C9, and C5b-9 neoantigens were screened for reactivity to the individual complement components in an assembled complex of human C5b-9 on rabbit red blood cell ghosts. Monoclonal antibodies (tissue culture supernatants) that bound to antigenic determinants accessible in the C5b-9 complex were selected for this study using immunofluorescence methods. HuK treated with PV IgG fixed C5, C6, C7, C8, C9, and C5b-9 neoantigens in a characteristic speckled pattern, while normal IgG did not. Heat inactivation or EDTA treatment of the complement source, or substitution of C2-depleted serum abolished C5, C6, C7, C8, C9, and C5b-9 neoantigen staining. PV IgG and complement also resulted in significant cytotoxicity to cell membranes as assessed using an ethidium bromide-fluorescein diacetate assay. These results suggest that PV IgG will activate the membrane attack complex of the complement system on HuK cell surfaces, resulting in cytotoxicity to cell membranes, further implicating complement in the pathogenesis of pemphigus.

Complement proteins C5b-9 cause release of membrane vesicles from the platelet surface that are enriched in the membrane receptor for coagulation factor Va and express prothrombinase activity.

We have investigated the composition and function of membrane microparticles released from platelets exposed to the C5b-9 proteins of the complement system. Gel-filtered human platelets were incubated with sub-lytic amounts of the purified C5b-9 proteins and the distribution of surface antigens was analyzed using monoclonal antibodies and flow cytometry. C5b-9 assembly caused secretory fusion of the alpha-granule membrane with the plasma membrane and the release of membrane vesicles (approximately 0.1-micron diameter) that contained the plasma membrane glycoproteins (GP) GP Ib and GP IIb-IIIa as well as the alpha-granule membrane protein GMP-140. These microparticles were highly enriched in the C9 neoantigen of the C5b-9 complex. The apparent surface density of C5b-9 on the microparticles was approximately 10(3)-fold higher than on the platelet itself, suggesting that the vesicles were selectively shed from the plasma membrane at the site of C5b-9 insertion. C5b-9 induced the expression of an activation-dependent epitope (recognized by monoclonal antibody, PAC1) in GP IIb-IIIa on the platelet surface but not in GP IIb-IIIa on the microparticles. The surface of the microparticles was also highly enriched in alpha-granule-derived coagulation factor V (or Va), accounting for nearly half of all the membrane-bound factor V detected. The number of potential membrane binding sites for factor Va was probed by adding saturating concentrations of factor Va light chain. Under these conditions, the density of factor Va binding sites on the microparticle surface exceeded that on the C5b-9-treated platelet by three to four orders of magnitude. Moreover, the microparticles provided most of the membrane surface for conversion of prothrombin to thrombin by VaXa. These studies demonstrate that the microparticles shed by C5b-9-treated platelets (and not the platelets themselves) provide the principal binding sites for coagulation factor Va and the principal catalytic surface for the prothrombinase complex. Platelet-derived microparticles formed during complement activation in vivo could provide a membrane surface that facilitates the assembly and dissemination of procoagulant enzyme complexes.

Relative inefficiency of terminal complement activation.

The efficiency of generation of fluid-phase SC5b-9 and membrane C5b-9(m) complexes relative to cleavage of C3 and C5 was studied. Fluid-phase C activation was induced through addition of purified bacterial Ag to human serum. Sephadex beads were used as particulate activators of the alternative pathway. Rabbit or antibody-coated sheep or human E were used to study formation of cytolytic C5b-9(m) complexes. The molar ratios of C3a:C5a generated in the model systems were found to be in the range of 60 to 200:1 in the case of soluble immune complex activators, and 70 to 150:1 with particulate activators and cells. The efficiency of C5 cleavage relative to C3 cleavage increased on surfaces with the density of antibody and/or C3b-binding sites. With soluble immune complexes, the efficiency of subsequent SC5b-9 generation displayed wide variations dependent on Ag and donor with molar ratios of C5a:SC5b-9 ranging from 30:1 for teichoic acid and sometimes approaching 1:1 for streptolysin-O. In contrast, activation on particles or cells always led to C5a:C5b-9 (calculated as the sum of generated moles SC5b-9 and C5b-9(m] ratios approaching 1:1. Hence, there is an overall inefficiency of terminal sequence activation in the C cascade due first to a dissociation at the level of C5 convertase formation/C5-cleavage and second, to a frequent inefficiency of C5b-utilization in the fluid-phase. The results provide an explanation for the very low levels of SC5b-9 found in plasma of healthy individuals and in patients with C-consuming immune complex disease.

Complement induces a transient increase in membrane permeability in unlysed erythrocytes.

The effects of low concentrations of human serum on antibody-sensitized sheep erythrocytes (EA) were studied. We report that exposure to low concentrations of serum induced a large but transient increase in the membrane permeability of those EA that do not lyse. This change in the permeability of the erythrocyte membrane resulted in net uptake of Na+ and decrease in cell K+, without affecting the total internal cation content. Although exposure to serum also allowed for net uptake of larger molecules like L-glucose, it did not lead to cell swelling. Experiments with sera genetically deficient in one of the terminal complement components showed that C8, but not C9, was required to produce the observed change in membrane permeability. Therefore, we propose that the C5b-8 complex can mediate the transient increase in permeability observed in unlysed erythrocytes during complement activation by whole serum.

Comparison between complement and melittin hemolysis: anti-melittin antibodies inhibit complement lysis

A comparison is made between the hemolytic actions of melittin and the ninth component of complement (C9). Melittin and C9 produce "pores" of similar effective radius in erythrocytes under standardized conditions, and their hemolytic action is suppressed by metal ions at similar concentrations, suggesting a common mechanism. Polyclonal anti-melittin immunoglobulin G (IgG) produced in rabbits retards hemolysis mediated by human C9 in a specific manner. Such antibodies react in several immunoassays with human and monkey C9 but not with C9 from lower animals, and no inhibition of lysis mediated by C9 molecules from these animals is observed. Thus, it is unlikely that anti-melittin IgG reacts with a structural element, such as an amphipathic helix, on human C9 since such structures are also predicted to exist in other C9 molecules. Human C9 and melittin block cross-reactivity in a dose-dependent manner, and anti-melittin IgG recognizes an epitope located between amino acid residues 245 and 390 of human C9 on "Western" blots. Comparison of the melittin and human C9 sequences indicates two regions of complete homology, a tetrapeptide at positions 292-295, and a pentapeptide at positions 527-531 in human C9, corresponding to residues 8-16 in melittin. Inhibition of hemolysis is not caused by blocking of C9 binding to the C5b-8 complex; rather the antibody must dissociate from the bound C9 before lysis ensues, indicating that it interferes with a postbinding event. It is proposed that anti-melittin binds to a conformational epitope on native, folded human C9 and thereby retards unfolding of the molecule, which is required for membrane insertion and hemolysis.

Complement-independent lysis of human red blood cells by cold hemagglutinins.

Hemolysis mediated by human antibodies is generally ascribed to the attack of red blood cells (RBC) by complement. We here extend earlier in vitro observations which indicate that potent cold agglutinins can directly cause lysis of RBC without the participation of complement. We have noted that EDTA plasma taken from patients with cold agglutinin disease is frequently reddish if the plasma is not immediately separated from the cells at 37 degrees C. Moreover, eluates prepared in such cases from plasma or heat-inactivated serum (30 min at 56 degrees C) by absorption (at 4-20 degrees C) and elution (at 37 degrees C) are usually contaminated with hemoglobin, and a large number of RBC used for absorption is lost during the procedure. To characterize this phenomenon further, we examined the effect of different hemagglutinating antibodies in vitro on normal RBC in the absence of complement. Hemolysis (5-17%) of RBC only occurred after treating the cells with potent antibodies at low temperatures (0-20 degrees C). This hemolysis increased 2- to 3-fold when the RBC were treated with an enzyme and decreased with rising temperature. Unlike cells hemolyzed by complement activation, no C5b-9 complexes could be detected on RBC damaged by this mechanism.

Rapidly Progressive Glomerulonephritis: Classification, Pathogenetic Mechanisms, and Therapy

Immunopathologic studies over the past two decades have demonstrated that rapidly progressive glomerulonephritis (RPGN) can result from glomerular deposition of anti-GBM antibody, immune complexes, or from some as yet undefined mechanism that does not involve glomerular antibody deposition. The latter process may be cell mediated and resembles a small vessel vasculitis. Most cases of idiopathic RPGN are not accompanied by pathogenic glomerular immunoglobulin deposition. Recent experimental studies of immune mechanisms of glomerular injury have identified several new processes that can induce damage to the capillary wall sufficient to result in crescentic glomerulonephritis (GN). These include direct effects of anti-GBM antibody alone and of the complement C5b-9 (membrane attack) complex, nephritogenic effects of inflammatory effector cells that involve reactive oxygen species and glomerular halogenation, and injury mediated by sensitized lymphocytes independently of antibody deposition. Macrophages have been shown to participate in both intracapillary and extracapillary fibrin deposition and crescent formation as well as to mediate capillary wall damage. The role of resident glomerular cells and cell-cell interactions in glomerulonephritis is still under active investigation. Despite these several advances in understanding immune injury to the glomerulus, therapy for RPGN remains largely empiric. Although the prognosis in RPGN has clearly improved over time, no form of disease-specific therapy has been clearly shown yet to be beneficial in a controlled study. Interpretation of the existing literature on therapy is complicated by the availability of only historical rather than concurrent controls, lack of attention to several variables known to affect disease outcome, and uncertainty regarding bias in favor of reporting positive results. Available data suggests that optimal outcomes may be achieved in anti-GBM nephritis by treatment with steroids, immunosuppression and plasma exchange, particularly when therapy is directed at patients with mild but rapidly progressive disease before oliguria or severe azotemia develop. Pulse steroids are probably the most cost-effective therapy for the idiopathic form of RPGN, but treatment with cytotoxic agents should be considered if clinical or histologic evidence of vasculitis is present.

The cytolytic C5b-9 complement complex: feedback inhibition of complement activation.

We describe a regulatory function of the terminal cytolytic C5b-9 complex [C5b-9(m)] of human complement. Purified C5b-9(m) complexes isolated from target membranes, whether in solution or bound to liposomes, inhibited lysis of sensitized sheep erythrocytes by whole human serum in a dose-dependent manner. C9 was not required for the inhibitory function since C5b-7 and C5b-8 complexes isolated from membranes were also effective. No effect was found with the cytolytically inactive, fluid-phase SC5b-9 complex. However, tryptic modification of SC5b-9 conferred an inhibitory capacity to the complex, due probably to partial removal of the S protein. Experiments using purified components demonstrated that C5b-9(m) exerts a regulatory effect on the formation of the classical- and alternative-pathway C3 convertases and on the utilization of C5 by cell-bound C5 convertases. C5b-9(m) complexes were unable to inhibit the lysis of cells bearing C5b-7(m) by C8 and C9. Addition of C5b-9(m) to whole human serum abolished its bactericidal effect on the serum-sensitive Escherichia coli K-12 strain W 3110 and suppressed its hemolytic function on antibody-sensitized, autologous erythrocytes. Feedback inhibition by C5b-9(m) represents a biologically relevant mechanism through which complement may autoregulate its effector functions.

Deposition of terminal C5b-9 complement complexes on erythrocytes and leukocytes during cardiopulmonary bypass.

Hemolysis, leukopenia, a hemostatic deficit, and nonspecific systemic reactions collectively known as the postperfusion syndrome develop in patients who undergo cardiopulmonary bypass. We now report that terminal C5b-9 complement complexes are deposited on erythrocytes and polymorphonuclear neutrophilic leukocytes during cardiopulmonary bypass. Plasma samples taken from 48 unselected patients during and at the end of cardiopulmonary bypass contained raised levels of fluid-phase SC5b-9 complement complexes, indicating that the complement sequence had been activated to completion. Various degrees of overt intravascular hemolysis were observed in all the patients, and lysed erythrocyte membranes were recovered from the blood samples. Immunoassays performed with use of antibodies to C5b-9 neoantigens demonstrated the presence of C5b-9 on red-cell ghosts but not on intact erythrocytes. The appearance of ghosts carrying C5b-9 always coincided with hemolysis. Furthermore, granulocytes isolated from 20 patients during bypass were all found to carry C5b-9 complexes, whereas cells isolated before or 24 hours after surgery carried no C5b-9. The neoantigen-positive material present in detergent extracts of granulocytes sedimented in a broad peak (25 to 40 sedimentation coefficient [S]) in sucrose-density gradients, exactly as did pore-forming C5b-9 complexes. Deposition of C5b-9 on blood cells during cardiopulmonary bypass may be partly responsible for the hemolysis and may augment granulocyte activation by the stimulation of arachidonate metabolism in those cells.

Restriction of cell lysis by homologous complement: II. Protection of erythrocytes against lysis by newly activated complement

Our previous work revealed that homologous complement (C) was ineffective in lysing antibody-sensitized erythrocytes (EA) even at high concentrations. It was also shown that activation of complement on homologous EA resulted in the binding of C9 and the formation of EA bearing complement proteins C1 through C9 (EAC1–9), yet few hemolytic sites were formed. Instead, as shown here, the formation of homologous EAC1–9 caused the cells to become resistant to lysis even by heterologous complement during a second incubation. In contrast, when homologous EAC1–8 were produced by incubating EA with C9-depleted serum, such intermediates were not protected against lysis by heterologous complement during a second incubation. Furthermore, homologous C9 on EAC1–9 was able to reduce the hemolytic efficiency of heterologous complement without blocking C activation and the formation of new C5b-9 complexes. Protection was not modified when homologous EAC1–9 were produced in one step, by incubation of EA with serum, or sequentially by adding C9 to EAC1–8. The minimum number of 9-sites required to confer a protective effect on EAC1–9 was less than 200 per cell. Thus, in addition to its known effect in heterologous cell killing, homologous C9 is capable of protecting homologous cells against inadvertent complement lysis.

Restriction of Cell Lysis by Homologous Complement: II. Protection of Erythrocytes Against Lysis by Newly Activated Complement

Our previous work revealed that homologous complement (C) was ineffective in lysing antibody-sensitized erythrocytes (EA) even at high concentrations. It was also shown that activation of complement on homologous EA resulted in the binding of C9 and the formation of EA bearing complement proteins C1 through C9 (EAC1-9), yet few hemolytic sites were formed. Instead, as shown here, the formation of homologous EAC1-9 caused the cells to become resistant to lysis even by heterologous complement during a second incubation. In contrast, when homologous EAC1-8 were produced by incubating EA with C9-depleted serum, such intermediates were not protected against lysis by heterologous complement during a second incubation. Furthermore, homologous C9 on EAC1-9 was able to reduce the hemolytic efficiency of heterologous complement without blocking C activation and the formation of new C5b-9 complexes. Protection was not modified when homologous EAC1-9 were produced in one step, by incubation of EA with serum, or sequentially by adding C9 to EAC1-8. The minimum number of 9-sites required to confer a protective effect on EAC1-9 was less than 200 per cell. Thus, in addition to its known effect in heterologous cell killing, homologous C9 is capable of protecting homologous cells against inadvertent complement lysis.

Release of granulocyte proteins during cardiopulmonary bypass: effect of different pharmacological interventions.

Leukopenia and anaphylatoxin formation occur during cardiopulmonary bypass1,2. Both C3a and C5a are converted to their respective desArg forms by plasma carboxypeptidase, but C5a-desArg still retains its capacity to activate leukocytes2-6. After C5 cleavage C5b-9 complexes are generated on target membranes or SC5b-9 complexes in the fluid phase. Recent studies demonstrated deposition of C5b-9 on blood cells during cardiopulmonary bypass. It was suggested that C5b-9 complexes may be partly responsible for the hemolysis and may augment granulocyte activation by the stimulation of arachidonate metabolism in those cells7.KeywordsCardiopulmonary BypassGranulocyte ElastaseElastase ReleaseLactoferrin LevelGranulocyte ActivationThese keywords were added by machine and not by the authors. This process is experimental and the keywords may be updated as the learning algorithm improves.

Structure and function of C8 in the membrane attack sequence of complement.

Complement-mediated cell lysis occurs as a result of interactions between complement proteins C5b, C6, C7, C8, and C9 to produce the membrane attack complex C5b-9 (Muller-Eberhard 1986): $$ C5b\;\xrightarrow{{C6}}\;C5b - 6\;\xrightarrow{{C7}}\;C5b - 7\xrightarrow{{C8}}\;C5b - 8\;\xrightarrow{{nC9}}\;C5b - 9 $$ Assembly of C5b-9 begins with proteolytic conversion of C5 to C5b by the C5 convertases formed as a consequence of complement activation. Development of a transient binding site for C6 leads to formation of a stable C5b-6 dimer. Subsequent binding of C7 and formation of C5b-7 coincides with the expression of a high-affinity lipid-binding site that mediates a strong but noncovalent interaction between the nascent complex and target membranes. Binding of C8 yields the tetramolecular C5b-8 complex. Although capable of slowly lysing erythrocytes and some nucleated cells, C5b-8 functions primarily as a receptor for C9 and thereby mediates formation of the more lytically effective C5b-9 complex. The number of C9 molecules per complex differs depending on C9 input and conditions of formation. The ultrastructure varies accordingly from what are functional lesions with one or a few C9s to highly organized porelike structures formed by polymerization of as many as 16 C9s per C5b-8. Facts and controversies about the function of C9 and the stoichiometry, structure, and mechanism of action of C5b-9 are summarized in other reports (Podack 1986; Muller-Eberhard 1986; Esser 1987; Stanley, this volume).