Relation of putative thioester bond in C3 to activation of the alternative pathway and the binding of C3b to biological targets of complement.

The reaction of [14C]methylamine with native human C3 led to the stoichiometric incorporation of methylamine, loss of hemolytic activity, and the concomitant exposure of a sulfhydryl group that could be labeled with [14C]iodoacetamide. Both labeled sites were located in the C3d portion of the alpha-chain, which is known to contain the metastable binding of C3b. The methylamine-modified C3 [C3(CH3NH2)] was shown to exhibit many of the functional properties of C3b, although the C3a portion of the molecule remained covalently attached. C3(CH3NH2) bound Factor B and beta 1H, and could be cleaved by C3b inactivator in the presence of beta 1H. C3(CH3NH2) added to human serum caused activation of the alternative pathway and consumption of C3. In presence of Factors B and D and Mg++, C3(CH2NH2) formed a C3 convertase. The convertase-forming material could be removed from solution by anti-C3a Sepharose and the preformed convertase was completely inhibited by purified antibody to C3a. This antibody did not affect the function of the C3 convertase that contained C3b. Similar functional properties were exhibited by C3 exposed for short periods of time to relatively low concentrations of chaotropic reagents, such as KSCN or guanidine. These results suggest that the initial C3 convertase of the alternative pathway may be formed from native C3, without proteolysis, by the attack of a variety of nucleophiles including water. The C3 convertase formed from this altered C3 then generates by proteolytic cleavage the initial metastable C3b that is capable of attaching to receptive surfaces. Conversion of C3 to C3b exposes one sulfhydryl residue as does modification of C3 with methylamine. When the C3d portion of C3b bound to zymosan particles via the metastable binding site was treated with radiolabeled methylamine, the fragment was released from the particles in radiolabeled form. These findings are consistent with the concept that native C3 contains an active carbonyl group, probably in the form of a thioester, which can either react with water to form functionally C3b-l;ike C3 or, upon enzymatic conversion of C3 to C3b, allows C3b to form an ester bond with hydroxyl groups on the target surface.

Breakdown of C3 after complement activation. Identification of a new fragment C3g, using monoclonal antibodies.

The physiological breakdown of C3 has been studied using monoclonal anti-C3 antibodies, and it has been found that the later stages of this process--the breakdown of C3bi--is more complex than had previously been recognized. C3bi is the reaction product produced from C3b by the action of factor I which, in the presence of factor H, produces a double cleavage in the alpha chain of C3b. It is here reported that, both on cells and in the fluid phase, the breakdown of C3bi in serum gives rise to two products: C3c and the product previously described as alpha 2D, which we now propose to designate C3d,g. Alpha 2D differs from C3d in that it contains an additional fragment of approximately 8,000 mol wt that carries the antigenic determinant for the clone 9 monoclonal anti-C3 antibody. C3g cannot be precipitated by anti-C3 antisera and therefore behaves as a uni- or bideterminant antigen. The cleavage of C3d,g to C3d and C3g does not occur in sterile serum. It is also still uncertain what enzyme cleaves C3bi to C3c and C3d,g in plasma. Plasmin can do so in vitro, but plasminogen-depleted serum can still produce the cleavage. The antigenic determinant recognized by clone 9 in C3 is not exposed in C3 or C3b, but appears as a neoantigen in C3bi (and in C3d,g). Anti-C3g therefore is a potentially useful ligand for detecting complement-activation products. C3g represents a new, highly anionic C3 fragment and seems not to be identical with the C3e fragment described by others.

Formation of the initial C3 convertase of the alternative complement pathway. Acquisition of C3b-like activities by spontaneous hydrolysis of the putative thioester in native C3.

Activation of the alternative pathway of complement commences with the formation of an initial fluid-phase C3 convertase. Treatment of C3 with the nucleophilic reagent methylamine has previously been shown to result in the cleavage of an intramolecular thioester bond and to induce C3b-like properties, including the ability to form a fluid-phase C3 convertase. This report examines the hypothesis that spontaneous hydrolysis of the thioester generates a derivative of C3 that is responsible for the formation of the initial C3 convertase of the alternative pathway. The rate of spontaneous decay of C3 hemolytic activity in buffer was found to be between 0.2 and 0.4%/h. In the presence of other alternative pathway proteins, the rate of inactivation was 1%/h. The rate of spontaneous inactivation was greatly accelerated by low concentrations of chaotrophic agents such as KSCN or guanidine. Liberation of a sulfhydryl group, not present in native C3, correlated with loss of hemolytic activity, indicating that exposure to chaotropic agents resulted in thioester hydrolysis. Unlike native C3, C3 bearing a single reactive sulfhydryl group was capable of generating fluid-phase C3 convertase with Factors B, D, and P and was cleaved by Factor I (C3b inactivator) in the presence of Factor H (beta 1H). The fragmentation patterns indicated that the C3a domain was covalently associated with the functionally C3b-like C3. Organomercurial agarose was employed for the rapid removal of sulfhydryl-bearing, hemolytically inactive forms of C3 and C3b from native hemolytically active C3.

Human complement C3b inactivator: isolation, characterization, and demonstration of an absolute requirement for the serum protein beta1H for cleavage of C3b and C4b in solution.

The complement regulatory enzyme, C3b inactivator (C3bINA), has been purified from human serum by affinity chromatography on an anti-C3bINA Sepharose column. Subsequent chromatography on DEAE-cellulose and removal of IgG with anti-IgG Sepharose resulted in a product which was found to be homogeneous by polyacrylamide gel electrophoresis at pH 8.9 and by sodium dodecyl sulfate polyacrylamide gel electrophoresis. The molecule is composed of two disulfide bonded polypeptide chains with mol wt of 50,000 and 38,000 daltons. Human CobINA was found to be a glycoprotein containing at least 10.7% carbohydrate and to have a normal serum concentration of 34 +/- 7 mug/ml (mean +/- 1 SD). Highly purified C3bINA cleaved neither free C3b nor free C4b if trace amounts of contaminating beta1H were removed from these proteins with anti-beta1H Sepharose. However, in the presence of highly purified beta1H and C3bINA, both C3bIna, both C3b and C4b were cleaved. Incubation of native C3 or C4 with C3bINA and beta1H had no effect on their cleaved. Incubation of native C3 or C4 with C3bINA and beta1H had no effect on their structure. The action of C3bINA and beta1H on C3b produced two fragments of the alpha1-chain which did not dissociate without reduction of the molecule. These fragments have mol wt of 67,000 and 40,000 daltons. The action of C3bINA and beta1H on C4b resulted in cleavage of the alpha'-chain giving rise to the 150,000-dalton C4c and the 49,000-dalton C4d fragments which dissociated without reduction. To produce from C3b the immunochemically defined C3c and C3d, fragments, the action of an additional serum enzyme appears to be required, the effect of which can be mimicked by trypsin.

Paroxysmal nocturnal hemoglobinuria: deficiency in factor H-like functions of the abnormal erythrocytes.

Erythrocytes from patients with paroxysmal nocturnal hemoglobinuria (PNH) contained a subpopulation that lacked membrane-associated Factor H-like activity present on normal human erythrocytes. Initial deposition of C3b on the erythrocytes was effected using a fluid phase C3 convertase. The cells were then treated with fluorescein-labeled C3 and the cell-bound C3 convertase. Analysis utilizing the fluorescence-activated cell sorter revealed two distinct cell populations, one of which was highly fluorescent, indicating a large number of C3b molecules per cell. Only this population (43%) was susceptible to lysis (44%) when exposed to acidified serum before C3b deposition. The less fluorescent population resembled normal human erythrocytes and was not affected by prior treatment with acidified serum. Since C3b deposition occurred almost exclusively on the complement-sensitive cells in the PNH erythrocyte population, these cells could be examined for the Factor H-like regulatory activities without prior isolation. These functions include enhancement of inactivation of erythrocyte-bound C3b by Factor I and acceleration of the decay of erythrocyte-bound C3 convertase, C3b,Bb. It was found that C3b on PNH erythrocytes was 100-fold less susceptible to inactivation by Factor I than C3b on normal human erythrocytes. The half-life at 22 degrees C of C3b,Bb on PNH erythrocytes was threefold greater than on normal human erythrocytes and similar to that of the enzyme bound to particles that do not possess Factor H-like activity. These observations suggest that the abnormal susceptibility of PNH erythrocytes to lysis by complement is due to a functional deficiency in one or more of the Factor H-like proteins present on normal human erythrocytes.

Binding of complement factor H to loop 5 of porin protein 1A: a molecular mechanism of serum resistance of nonsialylated Neisseria gonorrhoeae.

Neisseria gonorrhoeae isolated from patients with disseminated infection are often of the porin (Por1A) serotype and resist killing by nonimmune normal human serum. The molecular basis of this resistance (termed stable serum resistance) in these strains has not been fully defined but is not related to sialylation of lipooligosaccharide. Here we demonstrate that Por1A bearing gonococcal strains bind more factor H, a critical downregulator of the alternative complement pathway, than their Por1B counterparts. This results in a sevenfold reduction in C3b, which is >75% converted to iC3b. Factor H binding to isogenic gonococcal strains that differed only in their porin serotype, confirmed that Por1A was the acceptor molecule for factor H. We identified a surface exposed region on the Por1A molecule that served as the binding site for factor H. We used gonococcal strains with hybrid Por1A/B molecules that differed in their surface exposed domains to localize the factor H binding site to loop 5 of Por1A. This was confirmed by inhibition of factor H binding using synthetic peptides corresponding to the putative exposed regions of the porin loops. The addition of Por1A loop 5 peptide in a serum bactericidal assay, which inhibited binding of factor H to the bacterial surface, permitted 50% killing of an otherwise completely serum resistant gonococcal strain. Collectively, these data provide a molecular basis to explain serum resistance of Por1A strains of N. gonorrhoeae.

A novel sialic acid binding site on factor H mediates serum resistance of sialylated Neisseria gonorrhoeae.

Factor H (fH), a key alternative complement pathway regulator, is a cofactor for factor I-mediated cleavage of C3b. fH consists of 20 short consensus repeat (SCR) domains. Sialic acid binding domains have previously been localized to fH SCRs 6-10 and 13. To examine fH binding on a sialylated microbial surface, we grew Neisseria gonorrhoeae in the presence of 5'-cytidinemonophospho-N-acetylneuraminic acid, which sialylates lipooligosaccharide and converts to serum resistance gonococci previously sensitive to nonimmune serum killing. fH domains necessary for binding sialylated gonococci were determined by incubating organisms with recombinant human fH (rH) and nine mutant rH molecules (deletions spanning the entire fH molecule). rH and all mutant rH molecules that contained SCRs 16-20 bound to the sialylated strain; no mutant molecule bound to serum-sensitive nonsialylated organisms. Sialic acid was demonstrated to be the fH target by flow cytometry that showed a fourfold increase in fH binding that was reversed by neuraminidase-mediated cleavage of sialic acid off gonococci. Functional specificity of fH was confirmed by decreased total C3 binding and almost complete conversion to iC3b on sialylated gonococci. Sialic acid can therefore bind fH uniquely through SCRs 16-20. This blocks complement pathway activation for N. gonorrhoeae at the level of C3.

Binding of C4b-binding protein to porin: a molecular mechanism of serum resistance of Neisseria gonorrhoeae.

We screened 29 strains of Neisseria gonorrhoeae and found 16/21 strains that resisted killing by normal human serum and 0/8 serum sensitive strains that bound the complement regulator, C4b-binding protein (C4bp). Microbial surface-bound C4bp demonstrated cofactor activity. We constructed gonococcal strains with hybrid porin (Por) molecules derived from each of the major serogroups (Por1A and Por1B) of N. gonorrhoeae, and showed that the loop 1 of Por1A is required for C4bp binding. Por1B loops 5 and 7 of serum-resistant gonococci together formed a negatively charged C4bp-binding domain. C4bp-Por1B interactions were ionic in nature (inhibited by high salt or by heparin), whereas the C4bp-Por1A bond was hydrophobic. Only recombinant C4bp mutant molecules containing the NH2-terminal alpha-chain short consensus repeat (SCR1) bound to both Por1A and Por1B gonococci, suggesting that SCR1 contained Por binding sites. C4bp alpha-chain monomers did not bind gonococci, indicating that the polymeric form of C4bp was required for binding. Using fAb fragments against C4bp SCR1, C4bp binding to Por1A and Por1B strains was inhibited in a complement-dependent serum bactericidal assay. This resulted in complete killing of these otherwise fully serum resistant strains in only 10% normal serum, underscoring the importance of C4bp in mediating gonococcal serum resistance.

Specific deposition of complement protein C3b on abnormal PNH erythrocytes permits their separation by partitioning. Possible general approach for isolation of specific cell populations.

The deposition of complement proteins on a cell surface has previously been shown to reduce the cell's partition ratio in a two-polymer aqueous phase system. This phenomenon has now been extended to segregate, by partitioning, subpopulations of erythrocytes from patients with paroxysmal nocturnal hemoglobinuria (PNH). Purified components of the complement system were employed to deposit the protein C3b specifically on abnormal erythrocytes which lacked the membrane-associated complement regulatory protein DAF. As few as 2100 C3b/cell reduced the partition ratio and 24,000 C3b/cell resulted in resolution of the C3b-bearing and non-bearing human red cells. It was found that the proportion of cells separated did not equal the proportion of cells lysed by complement in the acidified serum lysis test when blood from three of the five patients was examined. The results indicate that the defect giving rise to DAF- cells may be, but is not necessarily, coexpressed with defects affecting other membrane-associated regulatory factors. A broader application of the method using monoclonal antibodies to direct purified complement components to specific cell populations should permit their isolation in large quantities.

Identification of multiple sites of interaction between heparin and the complement system.

Many diverse effects of heparin on the complement system have been reported. In only a few cases have the sites or the mechanisms of these effects been identified. In order to understand these results we sought to comprehensively analyze which complement proteins interact with heparin and which do not. Purified components of the classical, alternative and terminal pathways of complement were radiolabeled and their affinity for heparin determined. Affinity chromatography of normal human serum on heparin-agarose allowed a complete analysis of complement proteins and confirmed the results obtained with radiolabeled purified components. Of the 22 complement proteins examined, 13 bound heparin (C1q, C2, C4, C4bp, C1INH, B, D, H, P, C6, C8, C9, and vitronectin) while 9 did not bind heparin (C1r, C1s, C3, Factor I, C5, C7, C3b, Ba and Bb). These observations help explain the many effects heparin has on the complement system and they identify the proteins which need to be examined in order to explain these effects.

Tyrosine is a potential site for covalent attachment of activated complement component C3.

Activation of C3 results in the generation of metastable C3b, which has been shown to preferentially react with the hydroxyl groups of carbohydrates and with specific serine and threonine residues in proteins. In this study we have examined the reactivity of metastable C3b with the third type of hydroxyl group present in proteins, tyrosine (Tyr). The results demonstrated that Tyr reacts with the thioester of metastable C3b and that this reactivity was 11-fold better than that of threonine, 47-fold better than serine and 50-fold better than the reactivity of carbohydrates. Model peptides containing Tyr showed even higher reactivity than free Tyr, demonstrating that incorporation into peptide structures does not block C3b attachment. The site of attachment was found to be in the alpha'-chain of C3b and the bond was hydroxylamine sensitive, indicating an ester linkage. The stability of the C3b-Tyr complex was measured under physiological conditions (pH 7.4, 37 degrees C) and compared to the stability of other C3b complexes. C3b-Tyr decayed 50% in 19 hr at 37 degrees C, but C3b bound to a Tyr-containing peptide was more stable, exhibiting a t1/2 of 53 hr. The ester linked complexes C3b-IgG and C3b-glycerol were less stable each exhibiting a t1/2 of approximately 8 hr. As yet, only two specific C3b attachment sites on proteins have been identified, Ser1217 in C4b and Thr144 in IgG1. The present evidence demonstrates that Tyr residues are highly reactive and that the C3b-Tyr linkage is stable. The findings suggest that complexes involving tyrosine residues as the site of attachment will also be found.

The contrasting mechanisms of serum resistance of Neisseria gonorrhoeae and group B Neisseria meningitidis.

Neisseria gonorrhoeae and Neisseria meningitidis have evolved intricate mechanisms to evade complement-mediated killing. Sialylation of gonococcal lipooligosaccharide (LOS) results in conversion of previously serum sensitive strains to unstable serum resistance, which is mediated by factor H binding. Porin (Por) is also instrumental in mediating stable serum resistance in gonococci. The 5th loop of certain gonococcal PorlAs binds factor H, which efficiently inactivates C3b to iC3b. Factor H glycan residues may be essential for factor H binding to certain Por1A strains. Por1A strains can also regulate the classical pathway by binding to C4b-binding protein (C4bp) probably via the 1st loop of the Por molecule. Certain serum resistant Por1 B strains can also regulate complement by binding C4bp through a loop other than loop 1. Purified C4b can inhibit binding of C4bp to Por 1B, but not Por1A, suggesting different binding sites on C4bp for the two Por types. Unlike serum resistant gonococci, resistant meningococci have abundant C3b on their surface, which is only partially processed to iC3b. The main mechanism of complement evasion by group B meningococci is inhibition of membrane attack complex (MAC) insertion by their polysaccharide capsule. LOS structure may act in concert with capsule to prevent MAC insertion. Meningococcal strains with Class 3 Por preferentially bind factor H, suggesting Class 3 Por acts as a receptor for factor H.

Biologically active recombinant human complement factor H: synthesis and secretion by the baculovirus system.

A complete cDNA clone (4.3 kb) encoding human complement factor H (hCFH; 155 kDa) has been cloned into the pVL1393 baculovirus expression vector and transfected into Spodoptera frugiperda Sf9 insect cells. A biologically active (92 +/- 15%) 140-kDa protein was secreted into the medium with a yield of more than 5 mg/liter. This is the first report of synthesis of biologically active recombinant hCFH in a heterologous system.

Spontaneous thioester bond formation in alpha 2-macroglobulin, C3 and C4.

Purified alpha 2-macroglobulin and complement proteins C3 and C4 were treated with ammonia to break their intramolecular thioester bonds and reform the original free cysteinyl and glutamyl side chains. When this reaction was performed at low temperature a conformational intermediate was trapped which lacked a thioester, but which could refold to the native structure and spontaneously reform the thioester and full biological function. The findings suggest that these proteins may undergo spontaneous post-translational self-modification forming the thioesters without involvement of enzymes or high energy metabolites such as ATP.

Analysis of the recognition mechanism of the alternative pathway of complement by monoclonal anti-factor H antibodies: evidence for multiple interactions between H and surface bound C3b.

The ability of the alternative pathway of complement to discriminate targets as either activators or non-activators is mediated by different binding properties of factor H to surface-associated C3b molecules. In the present study we have probed the interaction between H and C3b using five anti-H mAb. The binding sites of the mAb were mapped by Western blotting using both recombinant and trypsin-generated H fragments. Two mAb bound to CCP1 (90X, 196X), two to CCP5 (MRC OX24, 86X) and one to CCP8-15a (131X). At a molar ratio 2:1 of 125I-H:mAb all tested mAb enhanced binding of H to both activator- and non-activator-bound C3b. At higher concentrations two mAb had an inhibitory effect on H binding to surface-associated C3b (OX24, 131X). Thus the mAb 131X inhibits H binding to surface-bound C3b but unlike OX24 it does not bind to the previously described C3b binding site within or near CCP4-5. These results indicate that there is an additional interaction site on factor H for surface-bound C3b.

A fluorimetric assay for native C3. The hemolytically active form of the third component of human complement.

The content of native C3 in samples of purified C3 may be accurately determined using the fluorescent probe ANS (8-anilino-1-napthalene sulfonate). The assay is based on the 11.5-fold increase in fluorescence intensity of ANS which accompanies proteolytic conversion of native C3 to C3b. The assay may be performed in the presence of hemolytically inactive derivatives of C3 such as C3b and C3(H2O). It exhibits the unique feature of being independent of protein concentration and it does not require a C3 standard, other purified complement components, C3 depleted serum, cells or cell-bound intermediate complexes, such as EAC142. A method utilizing cation exchange chromatography (Mono S, Pharmacia) is also described for the rapid (30 min) analytical or preparative separation of native C3 from inactive forms of C3 and from C3 fragments.

Investigation of mechanism-based inhibitors of complement targeting the activated thioester of human C3.

An intramolecular thioester bond in complement protein C3 is vital for covalent attachment of C3b (the proteolytically activated form of C3) to biological surfaces and for activation of the complement system. Proteolytic removal of C3a from C3 activates the thioester in the C3b fragment. Activated C3b primarily forms ester bonds with hydroxyl groups of carbohydrates on complement activating surfaces, but it has also been shown to react with the hydroxyl group of tyrosine and with specific Ser and Thr residues on IgG and on complement protein C4b. To examine the reactivity of the thioester, several families of hydroxylated compounds were examined. Reactivity of a series of substituted phenols varied over two orders of magnitude and demonstrated a linear correlation between reactivity and the Hammett substituent constants. Hydroxylated drugs including members of the L-DOPA/epinephrine family and hydroxamic acids also were examined. Compounds were identified that were 20,000 times more reactive than carbohydrates. These compounds were found to inhibit both the classical and alternative pathways of complement activation. Although the specificity of the thioester for its natural biological targets appears to be determined by many structural features, the data presented here demonstrate that increasing the nucleophilic character of the target hydroxyl group can increase the potency of a synthetic inhibitor many orders of magnitude.

Inhibition of complement by covalent attachment of rosmarinic acid to activated C3b.

Rosmarinic acid has been reported to inhibit complement activation in vivo as well as in vitro. Previous studies suggested that the inhibitory effect was due to inhibition of C3/C5 convertases, but inhibition of C3b attachment would yield the same results. Recent work in our laboratory demonstrated that compounds with polyhydroxylated phenyl rings are highly reactive with the thioester bond in nascent C3b. These compounds block complement activation by preventing attachment of C3b to the activating surface. Because rosmarinic acid contains two 3,4-dihydroxyphenyl groups, the current study was undertaken to re-examine the mechanism of inhibition by analyzing the effect of rosmarinic acid on C3b attachment. In assays using purified complement proteins, rosmarinic acid inhibited covalent attachment of C3b to cells with an 1C50 = 34 microM. Inhibition of C5 convertase activity required 1500 microM rosmarinic acid, and no significant inhibition of the C3 convertase enzyme, which produces C3b from C3, was observed at 10,000 microM. In hemolytic assays using human serum, rosmarinic acid was shown to inhibit activation of both the classical (IC50 = 180 microM) and the alternative (IC50 = 160 microM) pathways of complement. Rosmarinic acid concentrations up to 10,000 microM did not cause direct inactivation of C3. Radioiodination of rosmarinic acid was used to demonstrate covalent activation-dependent incorporation of rosmarinic acid specifically into the thioester-containing alpha'-chain of nascent C3b. These findings indicate that inhibition of complement activation by rosmarinic acid is due to the reaction of rosmarinic acid with the activated thioester of metastable C3b, resulting in covalent attachment of the inhibitor to the protein.

Initiation of the alternative complement pathway due to spontaneous hydrolysis of the thioester of C3.

Initiation of the alternative pathway of complement involves the random attachment of C3b on all biological particles in contact with plasma. This process requires continuous proteolytic generation of metastable C3b. The responsible protease arises as a result of conformational changes in C3 that occur upon thioester hydrolysis. The product, C3(H2O), is functionally C3b-like and forms a C3 convertase with Factors B and D. The resulting biomolecular enzyme, C3(H2O),Bb, is a serine protease that cleaves C3 generating metastable C3b. The rate of spontaneous generation of C3(H2O) under physiological conditions is between 0.2 and 0.4%/hr. C3(H2O),Bb produces only three to five metastable C3b molecules per enzyme before being inactivated by regulatory proteins. The thioester bond in metastable C3b has been estimated to be 10(10) times more reactive than that in native C3 and mediates attachment of C3b to biological particles. Once bound, C3b is subject to two competing processes: (1) inactivation and (2) a chain reaction-like amplification process that can deposit large numbers of C3b molecules on the particles within a very short period of time after the initial C3b binds. On activators of the alternative pathway, inactivation of C3b is restricted and amplification of C3b results in activation of the cytolytic pathway of complement.

Structure/function of C5 convertases of complement.

C5 convertases are serine proteases that cleave both C3 and C5. Alternative pathway C3/C5 convertases formed with monomeric C3b (C3b,Bb) because of their weak interaction with C5 primarily cleave C3 thereby opsonizing the cell surface with C3b. In contrast, C3/C5 convertases formed with a high density of C3b/cell exhibit higher affinities for C5 as indicated by Km values well below the physiological concentration of C5 in blood. These C3/C5 convertases bind C5 efficiently and cleave it at a velocity approaching Vmax thereby switching the enzyme from C3 cleavage to production of the cytolytic C5b-9 complex. Studies of the structure of C3/C5 convertases have postulated that C4b-C3b and C3b-C3b dimers from high affinity C5 binding sites while indel studies have shown two binding sites in C5 for the convertase in addition to the C5 cleavage site. Together, these studies indicate that with increasing deposition of C3b on the surface, C3b complexes are formed which through multivalent attachment bind the substrate C5 with higher affinities, thereby converting the low affinity C3/C5 convertases to high affinity C5 convertases. The process underlying the formation of high affinity C5 convertases during complement activation is discussed.