Alternative Pathway C3 Research Articles

Purification and characterization of a membrane protein (gp45-70) that is a cofactor for cleavage of C3b and C4b.

Based on preliminary evidence indicating that a cell-associated protein of U937 (a human monocyte-like cell line) possessed cofactor activity and was not the C3b/C4b receptor, we sought to further characterize this protein. A sequential four-column purification procedure was devised that includes C3(H2O) affinity chromatography to isolate in reasonable yields and purity a cell-associated protein of U937 and several other human cell lines. Based on its pattern and Mr on SDS-PAGE, acidic pI, and ligand specificity, it is identical to a recently described C3(H2O) or C3b-binding membrane glycoprotein of human PBL and cell lines; having no presently identified function, it was termed gp45-70. After purifying this protein, we determined its functional capabilities and compared them to those of the other complement proteins with regulatory activity directed at components comprising the C3 convertases. This protein was the most efficient (50 times that of H) yet-described cofactor for the I-mediated first cleavage of C3b. It also was a cofactor for the first cleavage of C4b, but was not as efficient as C4bp. The second cleavage of C3b and C4b was not efficiently mediated. It had no ability to accelerate decay in the classical or alternative pathway C3 convertases. Based on this unique activity profile and ability to be surface labeled, we have renamed this molecule membrane cofactor protein (MCP). We suggest that this protein plays a major role in preventing autologous complement activation.

Plasma separation using a hollow fiber membrane device.

A disposable hollow fiber device was evaluated by collecting approximately 550 ml of normal donor plasma (n = 43) and by performing sham (n = 10) and therapeutic (n = 12) plasma exchanges. Blood was processed at 70 ml per min, and plasma flux averaged 23 (collection) and 25 (exchange) ml per min (mean separation efficiencies of 52 and 60%, respectively). The procedures were tolerated well by all donors and patients. The plasma hemoglobin concentration in separated plasma averaged 1 mg per dl, and cell contamination was negligible (mean of 1, 3, and 6 RBCs, platelets and WBCs/microliter, respectively). There was no evidence of in vivo classical or alternative pathway complement activation as assessed by total hemolytic complement generation (CH50), alternative pathway hemolytic activity (AP50), C3 conversion, or C5 activation, nor were unexpected changes seen in the results of laboratory tests performed after the procedure. Sieving coefficients during sham plasma exchange averaged as follows: albumin, 1.03; IgM, 1.0, IgG, 1.0; IgA, 0.98; factor V, 1.07; factor VII, 0.89; factor VIII, 1.05; and factor IX, 1.19. The device appears to be useful for separation of cell-free plasma from blood during therapeutic plasma exchange procedures.

Structural and functional studies on the human C3b/C4b receptor (CR1) purified by affinity chromatography using a monoclonal antibody.

A procedure was devised that has several advantages over previously described methods to purify CR1 from both erythrocytes (E) and the HL-60 promyelocytic cell line. Using a monoclonal antibody immunoaffinity column, CR1 was purified to homogeneity as assessed by silver staining and 2-D gel analysis. Protein purified by this method comigrates on SDS-PAGE with 125I surface-labeled CR1 isolated by immunoprecipitation or iC3-Sepharose affinity chromatograhy and can be specifically immunoblotted with a second monoclonal anti-CR1 antibody. Employing this method, CR1 can be purified to homogeneity in amounts adequate for both functional studies and biochemical microanalysis. Purified E CR1 is functionally active as assessed by its ability to specifically rebind to an iC3-Sepharose affinity column, act as a cofactor for I-mediated cleavage of C3b to C3c and C3d, g and to accelerate decay of both the classical and alternative pathway C3 convertases. Its specific activity is similar to that of CR1 purified by a method not employing the potentially denaturing washing and eluting conditions of immunoaffinity chromatography. The pIs of the two major E CR1 allotypes are both approximately 7.1. Using pooled human E CR1, an amino acid composition was derived which revealed a relatively high proline content. This has also been found in two functionally related and genetically linked complement-regulatory proteins, H and C4-binding protein, NH2-terminal sequencing of E CR1 and HL-60 CR1 was unsuccessful indicating that the NH2-terminus is blocked.

Experimental Bacteroides fragilis Bacteremia in a Primate Model

Experimental Bacteroides fragilis bacteremia was studied in subhuman primates. Following intravenous infusion of viable B. fragilis there was an exponential clearance of organisms from the bloodstream. The major clearance organ was the liver, which accumulated 68.2% of the total inoculum. The most efficient clearance was exhibited by the spleen, with uptake of 1.16% gm tissue. Hemodynamic studies revealed no significant changes in heart rate, mean arterial pressure, or cardiac output following B. fragilis infusion. Complement activity as measured by CH50, alternative pathway hemolytic activity, granulocyte aggregometry, C4, C3, properdin, and Factor B levels were similarly unaffected by infusion of B. fragilis. In contrast, profound hemodynamic changes and a consistent decrease in complement activity was noted after challenge with S. minnesota. The results of this study suggest that B. fragilis bacteremia has a minor role in producing the acute hemodynamic changes associated with the septic shock syndrome.

Activation of factor B of the complement system by kallikrein and its light chain

The cleavage of factor B, a protein of the alternative pathway of complement, by kallikrein was studied. Like factor D, kallikrein can cleave B to generate the alternative pathway C3 convertase C3bBb. When this convertase was formed on erythrocytes previously coated with C3b, lysis was observed indicating that a functionally active C3 convertase was formed. B was also cleaved by kallikrein in the presence of fluid phase C3b, and this resulted in B fragments comparable in size to those generated in the presence of D. The capacity of kallikrein to cleave B is localised in the light chain of the kallikrein molecule, which is the same chain of kallikrein that is responsible for its other enzymatic activities. Since on a molar basis D is much more active then kallikrein in cleaving B, a physiological role for B activation by kallikrein is only likely under certain conditions, and still has to be established.

Complement activation by antigenic fractions of Entamoeba histolytica.

Complement (C) activation induced by Entamoeba histolytica in normal non-immune human serum was studied by testing in vitro the ability of different fractions of the trophozoites to cause C3 breakdown. Whole trophozoites were found to activate both alternative and classical C pathways. The antibody-independent classical pathway (MgEGTA inhibitable) C activating capacity was found to greatly increase after disruption of the cell membrane by sonication. Subsequent analysis after differential centrifugation and ion exchange chromatography of the membrane/particulate fractions showed, that this activity was not due to DNA, which has been shown to have the similar characteristics, but to other, as yet unidentified components. A rather homogenous membrane fraction obtained by elution with 0.4 M Tris-HCl, pH 8.1 (described as 4M) and some cytoplasmic constituents obtained after gel chromatography retained a moderate degree of alternative pathway C3 activating capacity seen with intact trophozoites. Thus, it seems, that serum contact to the outer surface of E. histolytica trophozoites leads to C activation via both pathways with cell death as the result and to subsequent release of more efficiently classical pathway activating components. These phenomena probably have an important role in the inflammatory process in invasive amoebiasis.

Inhibition of human and guinea pig complement by heparin fractions differing in affinity for antithrombin III or in average molecular weight.

Heparin comprises a mixture of structurally related molecules. Affinity for antithrombin III (AT III) is a prerequisite for its anticoagulant activity, which also is dependent on its molecular weight. In this study heparin fractions prepared by affinity chromatography on immobilized AT III and by gel filtration chromatography were compared for their ability to inhibit complement mediated haemolysis and both classical and alternative pathway C3 activation as measured by crossed immunoelectrophoresis. In normal human serum, inhibition of haemolysis and of heat-aggregated IgG (HAGG) as well as zymosan induced C3 activation by heparin was found to be independent of its AT III affinity and of its molecular weight (range 4800-17,000), on a weight basis. In guinea pig serum similar results were obtained for inhibition of HAGG induced C3 activation, but inhibition of haemolysis showed a marked molecular weight dependency and was also reduced for the fraction with low affinity for AT III. This may reflect a species difference in the haemolytic action of human and guinea pig complement. It is concluded that inhibition of human complement by heparin is independent of its anticoagulant activity and of its size and it is suggested that a fraction of heparin with reduced risk for bleeding and platelet aggregation is a potential anti-inflammatory agent.

Inhibition of complement activation on the surface of cells after incorporation of decay-accelerating factor (DAF) into their membranes.

Decay-accelerating factor (DAF), extracted from the stroma of human erythrocytes, was purified to homogeneity and incorporated into the membrane of sheep red cell complement intermediates, where its functional properties were analyzed. Incorporation of DAF into the cell membranes was temperature dependent, took place on pronase- or trypsin-treated erythrocytes, and did not depend on prior deposition of antibody, C1 or C4. Serum lipoproteins (high and low density) effectively inhibited DAF incorporation, but had no effect on the activity of DAF after its association with the cell membrane. The incorporated DAF could not be removed from the red cell surface by repeated washings in the presence of high salt concentration but was solubilized when the stroma were extracted with 0.1% Nonidet P-40. The presence of DAF in the membrane of EA did not affect the deposition of C1 and C4, but as few as 10(2) DAF molecules per cell profoundly inhibited the assembly of C3 and C5 convertases of both the classical and alternative pathways. The DAF inhibitory effect on EAC14 or EAC43 was not overcome by supplying an excess of C2 or factor B, but the alternative pathway C3 convertase could be assembled in the presence of Ni++, or nonphysiological concentrations of Mg++, which enhances the binding affinity of factor B for C3b. The DAF effect on EAC14 or EAC143 was entirely reversed by treating the cells with specific anti-DAF antibodies, showing that DAF did not alter the structure of C4b or C3b. Taken together, the experimental evidence suggests that DAF interacts directly with membrane-bound C3b or C4b and prevents subsequent uptake of C2 and factor B. DAF can function only within the cell membrane. Indeed, the decay dissociation of the C4b2a enzyme on DAF-containing sheep intermediates was not changed by varying the cell concentration. DAF-treated EA had no influence on the decay of nontreated EAC142 present in the same mixture. Moreover, the inhibitory activity of intact human erythrocytes on C4b2a was not blocked by antibodies to DAF, but was abolished by antibodies to the C3b/C4b receptor (CR1). When incorporated into the membrane of rabbit erythrocytes, human DAF inhibited their lysis by human complement. In conclusion, on the basis of these and previous results, it appears that DAF plays a central role in preventing the amplification of the complement cascade on host cell surfaces.(ABSTRACT TRUNCATED AT 400 WORDS)

Complement-dependent cellular cytotoxicity due to alternative pathway C3 activation by the target cell membrane

The cytotoxic activity of human blood lymphocytes toward Raji cells was strongly elevated when human serum (HS) was included in the cytotoxicity assay. This phenomenon also occurred when the effector cells were activated by interferon (IFN). Hypogammaglobulinemic serum (HyS) and heat-inactivated serum could also augment cytotoxicity, but C3-depleted serum was inefficient. IFN treatment of Raji cells decreased their sensitivity to lysis and this effect was counteracted by addition of HS to the system. It is likely that C3 activation by, and deposition on, Raji cells when used as targets for cytotoxicity facilitate their recognition and lysis by lymphocytes. These events may represent one mechanism operating in the natural killing phenomenon.

Protection of the classical and alternative complement pathway C3 convertases, stabilized by nephritic factors, from decay by the human C3b receptor.

Formation and function of the classical (C4b,2a) and alternative (C3b,Bb) complement pathway C3 convertases are regulated by the intrinsic lability of the enzymes, extrinsic decay by C4bp and H, cleavage of C4b and C3b by I, and by the inhibitory action of the C3b receptor molecule (CR1). Binding of C4 nephritic factor (C4Nef) to C4b and of C3 nephritic factor (C3Nef) to C3b stabilizes the C3 convertases and bypasses inactivation by C4bp, H and/or I. In the present study, binding of C4Nef to the classical C3 convertase was found to prevent decay of C4b,2a by inputs of CR1 that were at least 15 times the amount of CR1 which inactivated 50% unstabilized classical pathway C3 convertase sites in 2.5 min. CR1 could however inhibit lysis of C4b,2a(C4Nef)-bearing cells in a dose-dependent manner. The latter inhibitory effect was directed at the interaction of C5 with the C5 convertase, most likely at C5 binding to cell-bound C3b. In an analogous manner to C4Nef in the classical pathway, stabilization of alternative pathway C3b,Bb convertase sites by C3Nef resulted in a relative protection of C3 convertase sites from decay by CR1. Thus, C4Nef and C3Nef can bypass all mechanisms susceptible to regulate function of the classical and alternative pathway C3 convertases. Because CR1 is essential for degradation of C3b bound to immune complexes in whole blood, stabilization of C4b,2a and C3b,Bb by C4Nef and C3Nef may alter in vivo processing of immune complexes in patients with nephritic factors.

Phagocytosis by human monocytes of particles activating the alternative pathway of complement.

The phagocytosis of particles activating the alternative pathway of complement by human monocytes cultured under serum-free conditions was studied. In contrast to native zymosan particles, which were easily ingested, rabbit erythrocytes and agarose beads had to be coated with C3b or C3bi to be engulfed by the monocytes. The binding and ingestion by monocytes of particles coated with C3bi were greater than for the same particles coated with the equivalent amount of C3b. The binding and uptake of rabbit erythrocytes and agarose beads were proportional to the amount of C3b or C3bi on the particles. In contrast to the complement activator particles, C3b- and C3bi-coated sheep erythrocytes, which are non-activators, were not ingested by the monocytes, although attachment to the monocytes took place. The presence of methylamine or cobra venom factor, which are complement inhibitors, strongly reduced the ingestion of native zymosan by the monocytes, whereas the uptake of C3b- or C3bi-coated zymosan particles were only weakly affected. This suggests that the binding of native zymosan to monocytes is sensitive to interference from a cell-derived alternative pathway C3 convertase (C3bBb). Binding and uptake of activators by human monocytes via complement receptor(s) are discussed.

The ability of H to modulate C5 utilization by the cell-bound alternative pathway C5 convertase differs on sheep and rabbit erythrocytes

The ability of H to inhibit formation of the alternative pathway C3 convertase differs on the non-activating cells sheep erythrocytes (Es) and on the activating cells rabbit erythrocytes (Er) or desialated sheep erythrocytes (Edes). C3 convertase sites are converted to C5 convertase sites by deposition of additional C3b molecules and C5 binds to cell-bound C3b in a reaction that is inhibited by H. C5 convertase sites P(C3b)nBb were formed on Es, Er, Edes and revealed by incubation of the cells with purified C5 and with rat serum treated with KSCN and hydrazine. When incremental amounts of H were added to the reaction, a dose-dependent inhibition of C5 utilization was observed. The amount of H that inhibited 50% of C5 utilization was the same for a given cell regardless of the number of C5 convertase sites that were expressed on the cell. However the 50% inhibitory dose of H was 92 ng/107 EsP(C3b)nBb, 87 ng/107 EdesP(C3b)nBb while it was at least 380 ng/107 ErP(C3b)nBb. These results suggest that H competes with C5 for uptake within the C5 convertase site and that the regulatory effect of H on C5 cleavage depends on the surface to which the convertase is bound.

Affected erythrocytes of patients with paroxysmal nocturnal hemoglobinuria are deficient in the complement regulatory protein, decay accelerating factor.

Paroxysmal nocturnal hemoglobinuria (PNH) is an acquired defect of bone marrow stem cells in which the affected clones produce erythrocytes (also granulocytes and platelets) with membranes that are abnormally sensitive to complement-mediated lysis. Abnormal erythrocytes (E) from patients with PNH (PNH-E) are 3-5 times more sensitive (type II PNH-E) or 15-25 times more sensitive (type III PNH-E) to lysis in vitro by human complement than normal E from unaffected individuals and the functionally normal E that arise from unaffected clones and the functionally normal E that arise from unaffected clones in PNH patients (type I PNH-E). After complement activation by either the classical or alternative pathway, abnormal amounts of C3b are deposited on the membranes of PNH-E compared with normal E, suggesting that the PNH-E membrane cannot regulate the events responsible for C3b deposition. Two proteins that decrease the stability of the classical and alternative pathway C3 convertases on target cells have been isolated from normal human E stroma: the 70,000 Mr decay accelerating factor of stroma (DAF) and the 250,000 Mr C3b receptor (C3bR). Specific immune precipitates of solubilized membranes from 125I-surface-labeled normal E demonstrate both proteins. In contrast, specific immune precipitates of PNH-E from three patients show C3bR but are deficient in DAF; type II PNH-E are relatively deficient and type III PNH-E are totally deficient in DAF. Antibody that neutralizes the activity of isolated DAF is adsorbed by intact normal E under conditions in which it is weakly adsorbed by type II PNH-E and not adsorbed by type III PNH-E. The deficiency of DAF antigen in PNH-E, as assessed by lack of immunoprecipitation and antibody adsorption, could explain the abnormal sensitivity of PNH-E to complement-mediated lysis and suggests that DAF may protect the membranes of normal E from damage resulting from autologous complement activation.

The C3/C5 convertase of the alternative pathway of complement: Stabilization and restriction of control by lanthanide ions

The alternative pathway C3 convertase (C3b,Bb) is a Mg-dependent, labile enzyme with a t/2 of 3 min at 37°C and of 14 min at 24°C (at half physiological ionic strength). To stabilize the enzyme. metal ions of the lanthanide series were tested. Formation and decay of the enzyme as well as binding of radiolabeled Factor B, Factor H or properdin to C3b were measured using C3b-bearing sheep erythrocytes (EC3b). Binding of Factor B to EC3b in presence of 40 μ M gadolinium (Gd) was two to three times greater than in presence of 1 m M Mg. Binding of Factor H and of properdin to EC3b was partially inhibited by Gd. Although it enhanced Factor B uptake by EC3b, Gd was unable to substitute for Mg in enzyme formation by Factor D and completely inhibited (at 10 μ M) Mg-dependent enzyme activation. However, the preformed enzyme was not inhibited by Gd. Instead, exposure of EC3b,Bb to 40–100 μ M Gd increased the t/2 at 37°C from 3 min to 12–28 min, and at 24°C from 14 min to 32min. The slow decay of the enzyme correlated with slow release of Bb. Similar enzyme stabilization was observed using terbium, ytterbium, dysprosium and lanthanum. The Gd stabilized enzyme was also less susceptible to control by Factor H and properdin than the unstabilized enzyme. Furthermore, Gd protected surface bound-C3b from being cleaved by Factor I. The Gd effects were instantaneously reversed upon addition of 10 m M EDTA. Thus, Gd is able to stabilize preformed C3b,Bb and to render the enzyme refractory to control by Factors H and I.

Inhibition of alternative pathway factor D by factor B-related synthetic hexapeptides.

Hexapeptides mimicking the partial amino acid sequence of factor B surrounding the bond that is cleaved by factor D have been synthesized. These peptides have been assessed for their ability to inhibit factor D enzymatic activity and for their susceptibility to serine proteases. The synthetic peptides were cleaved by bovine trypsin and C1s but not by alpha-thrombin and factor D. The peptides inhibited factor B cleavage and fluid-phase or cell-bound alternative pathway C3 convertase activation by factor D. Altogether, these results suggest that peptides analogous to factor B specifically inhibit factor D enzymatic activity. Thus, they constitute an interesting tool for study of alternative pathway activation and can be of use when attempting to manipulate this pathway, since factor D is an essential component for alternative pathway initiation and amplification.

Complement receptor is an inhibitor of the complement cascade.

A glycoprotein from the membrane of human erythrocytes has been identified as a receptor for C3b (CR1). It promotes the dissociation of the alternative pathway C3 convertase C3b,Bb and the cleavage of C3b by C3b/C4b inactivator. We find that CR1 also inactivates the C3 and C5 convertases of the classical pathway. CR1 inhibits the consumption of C3 by C3 convertase EAC142 and enhances the decay of C4b,2a sites. On a weight basis, CR1 is approximately 5-10 times more active than C4 binding protein, a serum inhibitor of C4b,2a. The binding of 125I-CR1 to EAC14 cells is inhibited by C2. Therefore, it is likely that CR1 and C2 compete for a site on C4b. CR1 inhibited C5 convertase even more effectively, but had no effect on the assembly of the late complement components. At high concentrations, CR1 alone has no irreversible effects on cell-bound C4b. In the fluid phase, CR1 can function as a cofactor for the cleavage of the alpha' chain of C4b by C3b/C4b inactivator. A well-known function of CR1 is to promote adherence of microbes or immune complexes bearing C3b and C4b to cells. This interaction could result in a microenvironment damaging to the plasma membrane of the responding cell because the extrinsic C3b and C4b fragments can serve as additional sites of assembly of enzymes of the cascade. We therefore wish to propose that CR1 on the surface of cells supplies an increased local concentration of a strong inhibitor of the amplifying enzymes of the complement system and provides cells with a mechanism for circumventing damage when they bind C3b- and C4b-bearing substrates.

Treatment of human complement components C4 and C3 with amines or chaotropic ions. Evidence of a functional and structural change that provides uncleaved C4 and C3 with properties of their soluble activated froms, C4b and C3b.

Treatment of human components C4 and C3 with amines like hydrazine, ammonium hydroxide, and neutral ammonium salts or with chaotropic salts like KSCN and NaBr leads to complete loss of haemolytic activity. The pretreated components are, however, still active in formation of soluble C3 convertases. This activity pattern is reminiscent of the activities of C4 and C3 that have been activated by cleavage in the fluid phase. Indeed, the antigenic properties of pretreated C4 and C3 are similar to soluble C4b and C3b. The polypeptide chain structure of pretreated C4 and C3, is, however, identical to that of the untreated components when investigated by SDS gel electrophoresis. Pretreatment even reduces greatly the susceptibility of C4 to cleavage by C1s and of C3 to cleavage by classical and alternative pathway C3 convertases. Pretreated components have lost the ability to combine with EAC1 and EAC142, respectively; this fact explains their failure to exhibit haemolytic activity. In serum, pretreated C4 and C3 are cleaved in a manner similar to C4b and C3b. Amines and chaotropic ions cause the same functional and structural alterations, which are best explained by assumption of a conformational change. A similar transformation can also occur in C4 and C3 during preparation or storage.

Nephritic factor of the classical pathway of complement: immunoglobulin G autoantibody directed against the classical pathway C3 convetase enzyme.

A factor, functionally characterized by its capacity to stabilize the normally labile classical pathway C3-converting complex of the classical pathway of complement, has been isolated from the serum of one patient with a case of acute glomerulonephritis, subsequent to a cutaneous infection. The factor confers long-lived stabilization of classical pathway C3 convertase complexes formed both in the solid (sensitized sheep erythrocytes bearing activated C1 and the classical pathway C3 convertase) and fluid phase. The half-life of such stabilized C3-cleaving enzymes extended beyond several hours at 37 degrees C. The stabilizing activity was associated with a protein fraction immunochemically identified as immunoglobulin (Ig)G, a sizeable population of which exhibited a gamma chain of 60,000 daltons. The IgG-associated stabilizing activity was found to bind to the classical pathway C3 convertase enzyme via a fragment bearing the antigen-binding site of the molecule [F(ab)(2) and F(ab)]. Such binding was demonstrable for classical pathway and not for alternative pathway C3 convertase. Thus, the stabilizing factor behaves like an autoantibody to the C3-converting complex of the classical pathway of complement. The binding of the antibody to the enzyme affords protection of the latter against decay-degradation. By analogy with the nephritic factor of the alternative pathway situation where IgG autoantibodies specifically bind to alternative pathway C3 convertase enzymes and protect them from degradation, the functionally unusual IgG in our patient was designated as the nephritic factor of the classical pathway. Indirect evidence suggests that nephritic factor of the classical pathway-IgG might be of the IgG3 subclass.

The Alternative Pathway C3/C5 Convertase: Chemical Basis of Factor B Activation

The structural basis of activation of the alternative pathway C3 convertase was explored. For this purpose a modified isolation procedure of the activating enzyme, Factor D, was elaborated. The procedure affords a 70,000-fold purification of the enzyme with a 20% yield. A simple assay was designed for the quantitation of both Factor D and Factor B activity. On the basis of activity measurements and amino acid analysis, Factor D concentration in plasma was estimated to be 1 microgram/ml. Highly purified Factor D was used to activate Factor B in the presence of C3b and Mg++. The resulting fragments, Ba and Bb, were characterized with respect to their circular dichroism spectra, amino acid compositions, reactive sulfhydryl groups, and partial amino- and carboxy-terminal sequences. The results indicate that the Ba fragment constitutes the amino-terminal region and the Bb fragment the carboxy-terminal region of Factor B. The bond in Factor B that is cleaved by Factor D is proposed to be an arginyl-lysine bond.

Reaction mechanisms of beta1H globulin.

The reaction mechanisms of beta1H were studied. The generation of alternative pathway C3 and C5 convertases on the cell surface as well as in the fluid phase was inhibited by beta1H globulin. The cell preparation bearing the C3b site could bind beta1H with little effect on the C3b hemolytic activity. Bound beta1H was dissociated by the action of C3bINA and C3bINA-treated C3b bearing cell did not bind beta1H anymore. Cell-bound beta1H was also dissociated by the action of B (or Bb). From these and other results, the following conclusions were obtained. The C3b site-bearing cell could bind beta1H on the C3c region of C3b molecules facilitating the C3bINA action on C3b, and beta1H shared the same binding site with B (or Bb) inhibiting the generation of the alternative pathway convertases competitively.