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PubMed1991

Absence of complement-mediated events after protamine reversal of heparin anticoagulation.

Protamine reversal of heparin anticoagulation is associated with adverse hemodynamic effects that may be attenuated with protamine pretreatment (PP). This study assesses the role of complement activation during these phenomena in adult cardiac surgery patients. Sixteen individuals undergoing cardiopulmonary bypass were given intravenous normal saline or protamine (2 mg/kg) as a randomized pretreatment prior to undergoing heparin anticoagulation (400 IU/kg), coronary artery revascularization, and subsequent reversal of the anticoagulated state with protamine (4 mg/kg). Blood pressure, pulmonary artery diastolic pressure (PAD), heart rate, and cardiac output (CO) were measured during and after pretreatment, prior to heparin reversal by protamine, and for 10 min after reversal. Total hemolytic complement (CH50), C3 conversion to C3b, C3a/C5a, platelet count, and white blood cell count (WBC) were also measured at the same time periods. No significant correlation existed between complement activation and hemodynamic events, as might have been evident by decreased CH50, increased C3 conversion to C3b, or elevations in C3a/C5a levels. PP significantly prevented the CO decrease occurring at 1 and 3 min following heparin reversal by protamine (-0.8 and -1.4 liters/min vs 0.1 and -0.2 liters/min, P less than 0.05 and P less than 0.01, respectively). Reversal hypotension was less with PP, although PAD fell equally in both groups. WBC decreases after heparin reversal were less after PP (-25% vs -7%, P = 0.06). These data support the conclusion that, contrary to earlier reports, adverse hemodynamic and hematologic responses accompanying protamine reversal of heparin anticoagulation do not appear to be correlated with activation of complement. In fact, those patients having the greatest C3a generation exhibited the least hemodynamic changes.

Adult↗
PubMed1992

Ultrastructures and interactions of complement factors H and I.

The human complement regulatory protein, factor H, was examined by high resolution transmission electron microscopy. Results of electron microscopy confirm hydrodynamic analysis and indicate that factor H is a monomer of M(r) approximately 155,000. Factor H is an extended flexible molecule with a contour length of 495 A and a cross-sectional diameter of 34 A. Most images of factor H indicate that its polypeptide chain typically folds back on itself with the result that the average length of a factor H molecule is about half its contour length. Only one end of factor H associates with C3b. When bound to C3b, factor H still shows considerable conformational flexibility. Factor I is a bilobal protein of 130 A in length, and its two globular parts have maximal diameters of 54 and 49 A. The results establish that factor I is a two domain protein where the smaller subunit is a protease and the larger one is involved with binding C3b. Factor I binds C3b with a one-to-one stoichiometry in an ionic strength-dependent fashion. In the absence of sodium chloride an affinity constant of 5.7 x 10(5) M-1 was determined for factor I interaction with C3b. Whereas the Scatchard plot of factor I binding to C3b in the absence of factor H is linear, in the presence of factor H a curvilinear graph is obtained. The strong binding sites on C3b for factor I have an affinity at least 15-fold higher in the presence of factor H than in its absence. The results of both electron microscopy and binding studies were combined to compose a scheme envisioning how factors H and I cooperate for the processing of C3b.

Binding Sites↗
PubMed1991

Preferential inactivation of the C5 convertase of the alternative complement pathway by factor I and membrane cofactor protein (MCP).

Human C3b bound to the ghost of sheep erythrocytes (E*) via activation of the alternative complement pathway (E*AC3b) consists of four major constituents on SDS-PAGE of 350, 260, 210 and 180 kDa. 350 kDa C3b is a dimeric form of C3b in which the alpha' chain of one C3b binds covalently to that of the other C3b. This complex is presumed to serve as a core for the alternative pathway C5 convertase. The other C3b populations are monomers complexed with membrane proteins or sugars. Using E*AC3b (C3b labeled) as a substrate, we have investigated functional properties of membrane cofactor protein (MCP), which is an integral membrane protein with C3b-binding and factor I-dependent cofactor activities. In conjunction with factor I, MCP was found to degrade the protein-bound C3b preferentially including the 350 kDa dimer. There was a similar but lesser tendency of this selective cleavage of C3b-dimer by CR1 but not by factor H or C4bp. In contrast to CR1 and factor H, detergent solubilization of EAC3b was required for MCP to fully express its cofactor activity for this selective degradation of C3b. We next separated the C3b dimer from the monomers and assessed their ability to assemble the alternative C5 convertase. The C3b dimer but not the monomers expressed C5 convertase activity following the addition of factors B and D, C5 and Ni2+. Kinetic analysis of the degradation of the C3b dimer by MCP and factor I suggested that only one C3b was efficiently converted to C3bi and this occurred concomitant with a decrease in C5 convertase activity. These results suggest that MCP has the ability to more efficiently interact with protein-bound C3b and that this may relate as well to its preferential ability to irreversibly inactivate the C5 convertase.

Antigens, CD↗
PubMed1982

Antibody restores human alternative complement pathway activation by mouse erythrocytes rendered functionally deficient by pretreatment with pronase.

Activation of the human alternative pathway of complement (C) by surfaces requires the initial deposition of C3b by fluid-phase C3 convertase and sustained C3 cleavage by C3 convertases fixed to the surface. Nonactivating particles have previously been characterized by an inability to sustain the function of C3 convertases on their surfaces because these sites were susceptible to the regulatory action of the control proteins. Pronase converts the mouse erythrocyte (E) from an activator to a nonactivator by markedly decreasing the ability of the cell to affix C3b generated by a fluid-phase C3 convertase; this conversion is unrelated to the action of the control proteins on bound C3b. The capacity of antibody and its F(ab')2 or Fab' fragments to restore the alternative pathway-activating function of pronase-treated mouse E indicates that the contribution of antibody is mediated by its combining site without a requirement for bridging or for the Fc portion. The fab'-dependent activation of the alternative pathway of C relates to the deposition of C3b on the particle surface, which is a first and continuing step in alternative pathway activation. The antibody effect is not directed to the action of the regulatory proteins. Thus, particle-dependent activation of the alternative C pathway has been shown for the first time to be abolished and restored by cell surface-directed mechanisms that function independently of the regulatory proteins.

Animals↗
PubMed1978

Regulation by membrane sialic acid of beta1H-dependent decay-dissociation of amplification C3 convertase of the alternative complement pathway.

Sheep erythrocytes in their native state did not activate the alternative complement pathway, as measured by lysis in dilutions of normal human serum containing [ethylenebis(oxyethylenenitrilo)] tetraacetic acid but acquired this capacity after membrane sialic acid residues had been removed (by sialidase) or modified (by NaIO(4)). Activation of the alternative pathway by sheep erythrocytes required removal or modification of at least 40% of the membrane sialic acid to reach threshold, and it increased proportionately when larger amounts of sialic acid had been affected. Studies with isolated proteins of the alternative pathway demonstrated that the altered erythrocyte membranes resembled natural activators in protecting bound C3b from inactivation by C3b inactivator and beta1H and protecting bound amplification C3 convertase (C3b,Bb) from decay-dissociation by beta1H. A 1% decrease in intact sialic acid was associated with a 1% decrease in beta1H activity in decay-dissociation of membrane bound C3b,Bb. Because removal of the C8 and C9 carbon atoms from the polyhydroxylated side chain of sialic acid by oxidation with NaIO(4) was functionally equivalent to removal of the entire sialic acid moiety, secondary effects of the latter reaction, such as diminution of the negative charge of the membrane or exposure of penultimate galactose residues, were not considered to be responsible for the altered activity of beta1H. These studies suggest that facilitation, by membrane sialic acid residues, of the interaction between bound C3b and beta1H is essential to prevent the particle from effectively activating the alternative pathway.

Animals↗
PubMed1983

Inhibition of classical C5 convertase in the complement system by factor H.

This paper described the influence of factor H on the haemolytic activity of the classical C5 convertase. Factor H showed little effect on the interaction of C5 with EAC1,4b,2a,3b cells bearing low numbers of C3b sites, but displayed the inhibitory effect on the interaction of C5 with the intermediate cells bearing high numbers of C3b sites. The higher the number of C3b sites on the cells, the greater the degree of the inhibition by factor H. The inhibition by factor H was accompanied by the inhibition of consumption of C5 from the fluid phase, indicating that factor H inhibits the activity of C5 convertase, not the binding of activated C5 to the cells.

Animals↗
PubMed1985

Species specificity of recognition by the alternative pathway of complement.

The recognition function of the alternative complement pathway was studied with isolated human and rabbit components. Zymosan and homologous and heterologous erythrocytes were used as representative activators or nonactivators. The binding affinity of Factor B and Factor H for particle-bound C3b was measured. In both species, the average affinity of Factor H for bound C3b on homologous cells (nonactivators) was eight to 10 times higher than on zymosan particles (activators). The interaction between Factor H and C3b on rabbit erythrocytes was species-specific: rabbit Factor H bound strongly to rabbit C3b on rabbit erythrocytes and also on human erythrocytes, which are nonactivators for the rabbit alternative pathway. Human Factor H bound strongly to human C3b on human erythrocytes but seven times weaker on rabbit erythrocytes, which are activators of the human alternative pathway. No substantial differences were found in the binding of Factor B to bound C3b regardless of the nature of the particle to which C3b was bound. The results indicate that in the two species studied, the molecular mechanism of recognition is analogous and that recognition is species-specific.

Animals↗
PubMed1981

The bovine complement system.

Methods were developed for titrating bovine C3b-inactivator, C2, C3 and C4 by non-hemolytic means, and for assaying by hemolysis all the components of the bovine classical complement system except C2. All components were detected at serum dilutions above 1:1000, and some at dilutions above 1:100,000. C1, C4, C5, C7 and C9 titers were very high in adult bovine serum, and C2 and C8 were relatively low. C1, C2, and C8 were quite heat-labile at 56 degrees C, and C7 was moderately so, while C3 amd C6 titers increased after heating due to inactivation of a heat-labile inhibitor. Fetal bovine serum contained approximately 1-3% of adult levels of conglutinin, C1 and C6, and 5-50% of adult levels of the remaining components except C3. C3 antigen was found, but C3 functional activity was undetectable in most fetal bovine sera, though present at low levels in a few.

Animals↗
PubMed1999

Amplification of the antibody response by C3b complexed to antigen through an ester link.

Complement C3 has been described as playing an important role in the cell-mediated immune response. C3b has the capacity to covalently bind Ag and then to stimulate in vitro Ag presentation to T lymphocytes. To verify this observation in vivo, we prepared and purified covalent human C3b-Ag complexes using lysozyme (HEL) as Ag. The characterization of these HEL-C3b complexes indicates that they are representative of those susceptible to be generated in physiological conditions. Mice were immunized with 0.1 to 0.6 microgram of either free HEL, HEL + C3b, HEL-C3b, or HEL + CFA. Response was assessed after two i.p. injections by quantification of specific Ab production. Immunization with either HEL-C3b complexes or HEL + CFA leads to anti-HEL IgG production whereas free HEL or HEL + C3b was ineffective. Either HEL-C3b or HEL + CFA immunizations led to a similar Ig subclass patterns, including IgG1, IgG2a, IgA, and IgM. Our experiments provide the first evidence for modulation of specific Ab response by C3b when it is bound to Ag through a physiological-like link. Taken together with previous data concerning Ab response following recombinant HEL-C3d immunization, cellular events such as processing of C3b-Ag by APC and recognition by T lymphocytes, this present result underlines the importance of C3b and its fragments in stimulation of the immune system, through the multiplicity and complementarity of its interactions.

Animals↗
PubMed1986

A reexamination of the role of magnesium in the human alternative pathway of complement.

The formation of the alternative-pathway C3 convertase has been previously suggested to have an absolute requirement for Mg2+, especially at the level of complex formation between C3b and factor B (B). In the course of defining spectral probes that could be used to monitor the C3b-B interaction (e.g. 1-anilino-8-naphthalene sulfonic acid fluorescence and near-u.v. circular dichroism) we observed that the signal change reporting on this binding was not completely reversed upon addition of excess ethylene-diaminetetraacetic acid (EDTA). Using sucrose gradient ultracentrifugation, we have directly demonstrated a Mg2+-independent C3b-B complex in the fluid phase. B thus bound was not only susceptible to specific proteolytic activation by factor D, but the resulting C3bBb enzyme was able to convert native C3 to C3b. Interestingly, we were unable to detect Mg2+-independent specific binding of 125I-B to C3b which was particle-bound. Using a sensitive hemolytic assay, however, we estimated that the functional activity of B with surface-bound C3b is 80-fold greater in the presence of physiological Mg2+ (0.5 mM) than in 2 mM EDTA. In contrast, the fluid-phase association is estimated to differ less than three-fold under the same conditions. These data demonstrate that the requirement for Mg2+ in the formation of the fluid-phase alternative-pathway C3 convertase is not absolute. Furthermore, they suggest a difference in the stable functional properties of fluid-phase and surface-bound C3b.

Centrifugation, Density Gradient↗
PubMed2002

Structural aspects and design of low-molecular-mass complement inhibitors.

We present a mini-review on the structure-based design of three promising complement inhibitors. Firstly, we review compstatin, a 13-residue cyclic peptide that binds to C3 and inhibits the cleavage of C3 to C3a and C3b. Secondly, we review a six-residue cyclic peptide that binds to C5aR and antagonizes the binding of C5a to its receptor C5aR. Finally, we review three small molecules that bind to Factor D and inhibit the enzymic action of Factor D, during which Factor D proteolytically cleaves Factor B in complex with C3 or C3b.

Animals↗
PubMed1994

All forms of human IgA antibodies bound to antigen interfere with complement (C3) fixation induced by IgG or by antigen alone.

Polyclonal human secretory IgA1 and IgA2 antibodies to a bacterial protein antigen Streptococcus mutans AgI/II, and polyclonal human serum IgA1 and IgA2 antibodies to staphylococcal alpha-toxin, were found to interfere with antigen-mediated C3b fixation. In fluid phase, immune complexes of antigen and IgA failed to fix C3b, whereas antigen-IgG complexes did fix C3b. Partial removal of glycan chains with Streptococcus mitis SK96 glycosidases diminished the capacity of IgA antibodies to interfere with antigen-mediated C3b fixation by the alternative complement pathway. The authors conclude that native serum or secretory IgA antibodies suppress C3b fixation, and that the glycan chains play a significant role in maintaining this property.

Antibodies, Anti-Idiotypic↗
PubMed1997

Activation of the alternative pathway of complement by calcium-loaded erythrocytes resulting from loss of membrane phospholipid asymmetry.

The aminophospholipids phosphatidylserine (PS) and phosphatidylethanolamine (PE) are exposed on the outer membrane leaflet of deoxygenated and irreversibly sickled erythrocytes and senescent normal cells. PS exposure on erythrocytes results in the expression of procoagulant activity for the conversion of prothrombin to thrombin. Because liposomes or vesicles composed of aminophospholipids can activate the alternative pathway of complement, the possibility that increased exposure of PS and PE on intact erythrocytes would also make them capable of activating the alternative pathway was examined. Loss of normal membrane phospholipid asymmetry was induced by incubation of erythrocytes with calcium (Ca2+) and the calcium ionophore A23187. PS exposure on 60% of erythrocytes was confirmed by binding of fluorescein isothiocyanate-conjugated annexin V. Expression of procoagulant activity, measured with the Russell's viper venom clotting assay, was significantly increased on the Ca2+/A23187-treated erythrocytes. In addition, the erythrocytes became capable of activating the alternative pathway of complement, as judged by an increase in cell-bound C3b after incubation with serum and a decrease in alternative pathway hemolytic activity of the serum. The effect could be reversed by incubation of the Ca2+/A23187-treated erythrocytes under conditions that induced recovery of normal membrane phospholipid asymmetry. In contrast, tetrathionate-treated erythrocytes showed no increase in binding of annexin V and no procoagulant activity and failed to activate the alternative pathway of complement. These findings demonstrate that loss of phospholipid asymmetry in erythrocytes not only results in expression of procoagulant activity but also renders the cells capable of activating the alternative pathway of complement.

Annexin A5↗
PubMed2006

Complement activation is involved in biological responses to leukocyte adsorptive apheresis.

We examined the effects of complement activation on the biological responses of cellulose acetate (CA) beads. Peripheral blood containing the complement activation inhibitor nafamostat mesilate (NM) or heparin was incubated with CA beads in vitro. Thereafter, the fraction of adsorbed granulocytes as well as the generation of complement activation fragments (C3a and C5a) and interleukin 1 receptor antagonist (IL-1ra) were measured. Granulocyte adsorption, complement activation, and IL-1ra release were significantly inhibited in the presence of NM. Adsorption was significantly increased onto CA beads pretreated with plasma containing heparin even in the presence of NM and adding C3a or C5a enhanced IL-1ra release. These results suggested that bound complement fragment (e.g., C3b) on CA beads plays a central role in granulocyte adsorption to CA beads and that C3a and C5a augment the release of anti-inflammatory substances. We therefore conclude that complement activation is involved in these biological responses of leukocyte apheresis.

Adsorption↗
PubMed2004

Rituximab infusion promotes rapid complement depletion and acute CD20 loss in chronic lymphocytic leukemia.

Complement plays an important role in the immunotherapeutic action of the anti-CD20 mAb rituximab, and therefore we investigated whether complement might be the limiting factor in rituximab therapy. Our in vitro studies indicate that at high cell densities, binding of rituximab to human CD20(+) cells leads to loss of complement activity and consumption of component C2. Infusion of rituximab in chronic lymphocytic leukemia patients also depletes complement; sera of treated patients have reduced capacity to C3b opsonize and kill CD20(+) cells unless supplemented with normal serum or component C2. Initiation of rituximab infusion in chronic lymphocytic leukemia patients leads to rapid clearance of CD20(+) cells. However, substantial numbers of B cells, with significantly reduced levels of CD20, return to the bloodstream immediately after rituximab infusion. In addition, a mAb specific for the Fc region of rituximab does not bind to these recirculating cells, suggesting that the rituximab-opsonized cells were temporarily sequestered by the mononuclear phagocytic system, and then released back into the circulation after the rituximab-CD20 complexes were removed by phagocytic cells. Western blots provide additional evidence for this escape mechanism that appears to occur as a consequence of CD20 loss. Treatment paradigms to prevent this escape, such as use of engineered or alternative anti-CD20 mAbs, may allow for more effective immunotherapy of chronic lymphocytic leukemia.

Antibodies, Monoclonal↗
PubMed1992

Cell-surface regulation of the human alternative pathway of complement. Sheep but not rabbit erythrocytes express factor I-dependent cofactor activity.

When incubated in normal human serum, rabbit erythrocytes are haemolysed as a result of activation of the alternative pathway of complement (APC), but sheep erythrocytes do not spontaneously activate the human APC under physiological conditions. The mechanism for this difference has been attributed to differences in the relative affinity of membrane-bound C3b for its natural ligands, factor B and factor H, that favour the formation and stability of the APC C3 convertase on rabbit erythrocytes and inhibit convertase activity on sheep erythrocytes. Previous studies have also suggested that factor I inactivated C3b on sheep erythrocytes more effectively than on rabbit erythrocytes. Further, sheep erythrocytes have recently been shown to have a membrane protein that associates non-covalently with cell-bound C3b, but rabbit erythrocytes lack a predominant C3b binding protein. Together, these results suggested the possibility that sheep but not rabbit erythrocytes have a membrane constituent with factor I cofactor activity. To investigate this hypothesis, rabbit and sheep erythrocytes bearing radiolabelled C3b were treated either with factor I or with factor I and factor H, and conversion to iC3b was assessed by autoradiography. Factor I caused a concentration-dependent conversion of C3b to iC3b on sheep erythrocytes; however, only trace amounts of C3b on rabbit erythrocytes were degraded even when high concentrations of factor I (83 micrograms/ml) were used. While C3b on rabbit erythrocytes was converted to iC3b by the combination of factor H and factor I, much less factor H was required for the same degree of conversion of C3b on sheep erythrocytes. Treatment of sheep erythrocytes with neuraminidase had no effect on either factor I-dependent cofactor activity or the capacity of factor H to serve as a factor I cofactor. Sheep erythrocytes did not manifest decay accelerating activity, however, suggesting that the factor I cofactor constituent is a functional analogue of the human membrane cofactor protein.

Animals↗
PubMed1995

Human factor H and C4b-binding protein serve as factor I-cofactors both encompassing inactivation of C3b and C4b.

Human factor H in the complement (C) system has been characterized as a decay-accelerator for the alternative C pathway C3 convertase and a cofactor for factor I-mediated inactivation of C3b. The current concept is that it does not serve as a C4b-inactivating cofactor. In the present study, we demonstrated that in fluid-phase, factor H and Factor I can cleave methylamine-treated C4(C4ma), a C4b analogue, to C4d, regardless of its isotype. The buffer pH and ionic strength were critical factors for the C4ma cleavage, which proceeded at around pH 6.0 and low conductivity around 3.0 mS. Similar results were obtained with fluid-phase C4b. Cell-bound C4b, however, did not undergo factor I-mediated inactivation by factor H. Hence, all of the human cofactors reported to date can mediate factor I-mediated cleavage of both C3b and C4b at least in the fluid-phase.

Carrier Proteins↗
PubMed1984

Lysine residues, but not carbohydrates, are required for the regulatory function of H on the amplification C3 convertase of complement.

Lysine epsilon-amino groups of human factor H were selectively converted to guanidino groups by treatment with 0.1 M O-methylisourea at pH 10.4. Guanidination resulted in a dose-dependent decrease in the capacity of the regulatory protein to accelerate decay dissociation of P-stabilized amplification C3 convertase sites, to serve as a co-factor for cleavage of cell-bound C3b by I, and to compete for binding of 125I-untreated H to C3b. Modification of approximately 75% lysine epsilon-amino groups suppressed 97% of H functional activity. Biochemical analysis of native H demonstrated a total carbohydrate content of 18.5% (w/w) and the presence in the molecule of 11 biantennary oligosaccharidic chains of the N-acetyl-lactosaminic type. Total desialation of H by using Clostridium perfringens neuraminidase, and total deglycosylation of desialated H by using beta-endo-N-acetylglucosaminidase resulted in a 1.5- to 2-fold increase in H activity on a weight basis. Deglycosylation did not alter the capacity of H to discriminate between activating and nonactivating surfaces of the alternative pathway. Thus, lysine residues are important determinants of the binding capacity of H for cell-bound C3b, whereas the carbohydrate portion of the molecule is not required for the regulatory function of the protein on the amplification C3 convertase.

Acetylglucosamine↗