Identification of the beta-endorphin-binding subunit of the SC5b-9 complement complex: S protein exhibits specific beta-endorphin-binding sites upon complex formation with complement proteins

Beta-Endorphin has been reported to specifically interact with SC5b-9 complement complexes via non-opioid binding sites. Covalent cross-linking of [125I]beta H-endorphin to SC5b-9 and analysis of the cross-linking products by gel electrophoresis and subsequent autoradiography revealed a single specifically labelled species which was identical with the S protein subunit of the complement complex. In contrast to SC5b-9, no cross-linking of labelled beta-endorphin to subunits of C5b-9(m) could be observed, indicating that beta-endorphin binding to SC5b-9 was mediated exclusively via S protein. Beta-Endorphin binding to SC5b-9 was compared with binding to purified S protein. Whereas beta-endorphin binding to purified S protein was only modest, complex formation of S protein with complement proteins led to a strong increase in beta-endorphin-binding site concentration, compatible with the exposure of primarily cryptic beta-endorphin-binding sites on S protein.     

Characterization and identification of heparin-induced nonopioid-binding sites for beta-endorphin in human plasma

We have characterized the specific binding of human beta-endorphin (1-31) to novel binding sites which are formed in human plasma or serum in the presence of heparin. The formation of the binding sites is temperature-dependent and does not occur in the presence of other anticoagulants, such as sodium-EDTA, sodium-oxalate, or sodium-citrate. The specific binding of 125I-beta H-endorphin to heparin-induced binding sites in human plasma is saturable and reversible. It is not inhibited by morphine or naloxone or by various opioid peptides which share their NH2-terminal opioid-active sequence with beta H-endorphin. In contrast, binding is inhibited by the COOH-terminal beta H-endorphin fragment Gly-Glu indicating that binding is to nonopioid sites. Electroimmunoprecipitation techniques revealed that these binding sites are identical with S protein/vitronectin or derivatives thereof. S protein is a plasma alpha 1-glycoprotein involved in attachment and spreading of cells and also in blood coagulation and complement activation. It is possible that the interaction of beta-endorphin with S protein is of physiological significance.     

Interaction of human beta-endorphin with the terminal SC5b-9 and "preterminal" SC5b-7 and SC5b-8 complexes of human complement

Human beta-endorphin (beta H-EP) is demonstrated to bind to the "preterminal" SC5b-7 and SC5b-8 complexes and to the terminal SC5b-9 complex of human complement. Detailed binding studies revealed saturability, reversibility and structural specificity of the beta H-EP interaction with high or low affinity non-opiate binding sites on SC5b-7 and SC5b-9 complexes. The high affinity binding sites seem to be located predominantly on C5b, C6 or C7 subunits of the complexes.     

Terminal membrane C5b-9 complex of human complement: transition from an amphiphilic to a hydrophilic state through binding of the S protein from serum

The membrane-damaging C5b-9(m) complex of complement is a cylindrically structured, amphiphilic molecule that is generated on a target membrane during complement attack. Isolated C5b-9(m) complexes are shown here to possess the capacity of binding a protein, termed "S"-protein, that is present in human plasma. Binding of this protein apparently shields the apolar surfaces of C5b-9(m), since the resulting "SC5b-9(m)" complex is hydrophilic and no longer aggregates in detergentfree solution. Dispersed SC5b-9(m) complexes exhibit an apparent sedimentation coefficient of 29S in sucrose density gradients, corresponding to a molecular weight of approximately 1.4 million. SDS PAGE analyses indicate binding of 3-4 molecules of S-protein per C5b-9(m) complex. These data are consistent with a monomer nature and molecular weight of 1-1.1 million of the C5b-9(m) complex. Ultrastructural analysis of SC5b- 9(m) shows preservation of the hollow cylindrical C5b-9(m) structure. Additional material, probably representing the S-protein itself, can be visualized attached to the originally membrane-embedded portion of the macromolecule. The topography of apolar surfaces on a molecule thus appears directly probed and visualized through the binding of a serum protein.     

The human complement regulatory protein CD59 binds to the alpha-chain of C8 and to the "b"domain of C9.

The erythrocyte membrane inhibitor of the human terminal complement proteins, surface antigen CD59, has previously been shown to enter into a detergent-resistant complex with either the membrane-bound complex of C5b-8 or C5b-9 (Meri, S., Morgan, B. P., Davies, A., Daniels, R. H., Olavesen, M. G., Waldmann, H. and Lachmann, P. J. (1990) Immunology 71, 1-9; Rollins, S. A., Zhao, J., Ninomiya, H., and Sims, P. J. (1991) J. Immunol, 146, 2345-2351). In order to further define the interactions that underlie the complement-inhibitory function of CD59, we have examined the binding interactions between 125I-CD59 and the isolated components of human complement membrane attack complex, C5b6, C7, C8, and C9. By density gradient analysis, we were unable to detect interaction of 125I-CD59 with any of these isolated complement components in solution. Specific binding of 125I-CD59 to C8 and C9 was detected when these human complement proteins were adsorbed to either plastic or to nitrocellulose, suggesting that a conformational change that accompanies surface adsorption exposes a CD59-binding site that is normally buried in these serum proteins. The binding of 125I-CD59 to plastic-adsorbed C8 and C9 was saturable and competed by excess unlabeled CD59, with half-maximal binding observed at 125I-CD59 concentrations of 80 and 36 nM, respectively. No specific binding of 125I-CD59 was detected for surface-adsorbed human C5b6 or C7 nor was such binding observed for C8 or C9 isolated from rabbit serum. Binding of CD59 to human C8 and C9 was not mediated by the phospholipid moiety of CD59, implying association by protein-protein interaction. In order to further define the binding sites for CD59, ligand blotting with 125I-CD59 was performed after separation of C8 into its noncovalently associated subunits (C8 alpha-gamma and C8 beta) and after alpha-thrombin digestion of C9. These experiments revealed specific and saturable binding of 125I-CD59 to C8 alpha-gamma subunit (half-maximal binding at 75 nM), but not to C8 beta, and specific and saturable binding to the 37-kDa fragment (C9b) of thrombin-cleaved C9 (half-maximal binding at 35 nM), but not to the 25-kDa C9a fragment. Partial reduction of C8 alpha-gamma revealed that only C8 alpha polypeptide exhibited affinity for CD59, and no specific binding to the C8 gamma chain was detected.(ABSTRACT TRUNCATED AT 400 WORDS)     

The ninth component of human complement (C9). Functional activity of the b fragment

The domain structure of human complement protein C9 was investigated by determining the functional activities of the NH2-terminal (C9a) and COOH-terminal (C9b) fragments obtained by cleavage of C9 with alpha-thrombin. The two fragments were separated by preparative sodium dodecyl sulfate-polyacrylamide gel electrophoresis and renatured by dialysis against buffers containing zwitterionic detergents. The C9b fragment produced membranolytic activities in three independent assays. First, it produced single, ion-conducting channels of varying conductances in planar lipid membranes. Most of the channels had an average conductance of 11 picoSiemens and an average lifetime of about 30 s. The channels showed lipid specificity and a 3-fold preference for conducting K+ over Na+. Second, the fragment also caused specific marker release from liposomes which was inhibitable by a C9b-specific monoclonal antibody, and third, it lysed erythrocytes in the absence of a fully assembled C5b-8 complex. The isolated C9a fragment did not produce single channels in planar lipid membranes but was also effective in releasing markers from liposomes and in lysing erythrocytes. Secondary structure predictions indicate the presence of several amphiphilic, "surface-seeking" segments in the primary structure of C9 which are mainly alpha-helices in C9b and beta-sheets in C9a. These results may indicate the presence of surface-binding domains in the NH2-terminal half and channel-forming domains in the COOH-terminal portion of native, monomeric C9.     

Studies of the association of the eighth and ninth components of human complement within the membrane-bound cytolytic complex

The association of the eighth (C8) and ninth (C9) components of human complement within membrane-bound C5b-9 was investigated using the photosensitive cross-linking reagent N-succinimidyl-6-(4'-azido-2'-nitrophenylamino)hexanoate. Reaction of this reagent with either the purified alpha-gamma or beta subunit of C8 resulted in the introduction of 6-8 mol/mol of photosensitive 6-(4'-azido-2'-nitrophenylamino)hexanoate (ANH) as an intrinsic ligand on each protein. The resulting ANH-(alpha-gamma) or ANH-(beta) was capable of recombining with equimolar amounts of beta or alpha-gamma, respectively, to yield ANH-C8. Parallel modifications of purified C9 resulted in incorporation of 3-4 mol/mol of ANH-ligand. Both ANH-C8 and ANH-C9 retained their ability to incorporate into C5b-9. Two approaches were used to determine the proximity of C8 subunits to C9 within C5b-9. In one, the complex was assembled on erythrocytes by incubating EAC1-7 cells separately with each form of ANH-C8 and subsequently saturating with 125I-C9. After lysis, membranes were irradiated, solubilized, and analyzed by gel electrophoresis. Cross-linking was assessed by a shift in electrophoretic mobility of 125I-C9 to a higher molecular weight. Results using either form of ANH-C8 in C5b-9 showed that, although at least 30% was involved in cross-linking, none was cross-linked to C9. Similar results were obtained using a second approach in which cross-linker and radiolabel were transposed between C8 and C9. Here, EAC1-7 cells were incubated first with 125I-C8 containing either 125I-(alpha-gamma) or 125I-(beta) and subsequently with ANH-C9. Although at least 48% of ANH-C9 in C5b-9 was involved in cross-linking in these experiments, no cross-linking to either subunit of C8 was detected. These results suggest that C8 is not in close physical association with C9 within membrane-bound C5b-9.     

A novel beta-endorphin binding protein. Complement S protein (= vitronectin) exhibits specific non-opioid binding sites for beta-endorphin upon interaction with heparin or surfaces

Human beta-endorphin (1-31) (beta H-endorphin) was found to specifically interact with purified complement S protein from human plasma. As found by chemical cross-linking beta H-endorphin bound to both, the 65- and 75-kDa molecular mass forms of S protein. The interaction of S protein with heparin as well as the adsorption of S protein to surfaces led to an almost 10-fold increase of specific binding which was due to the exposure of further beta H-endorphin-binding sites. The interaction of beta H-endorphin with S protein bore characteristics of a ligand-receptor interaction, such as time dependence, reversibility, high affinity, saturability, and structural specificity and was mediated through the non-opioid COOH terminus of the beta H-endorphin molecule. beta H-Endorphin binding to S protein was observed at physiological pH or cation concentrations, indicating that the interaction may well occur in vivo. Our results provide conclusive evidence that interactions of S protein with very different effectors led to similar conformational changes which uniformly resulted in exposure of a highly specific beta H-endorphin binding domain on S protein. With S protein as major beta H-endorphin-binding protein in the periphery, the molecular basis of a widespread system of humoral target sites of the neuroendocrine effector appears to be established. In view of S protein involvement in processes of inflammation and wound repair and beta-endorphin effects on immunocompetent cells, the demonstrated S protein-beta H-endorphin interaction appears to be of considerable functional significance.     

Molecular composition of the terminal membrane and fluid-phase C5b-9 complexes of rabbit complement. Absence of disulphide-bonded C9 dimers in the membrane complex

The terminal membrane C5b-9(m) and fluid-phase SC5b-9 complexes of rabbit complement were isolated from target sheep erythrocyte membranes and from inulin-activated rabbit serum respectively. In the electron microscope, rabbit C5b-9(m) was observed as a hollow protein cylinder, a structure identical with that of human C5b-9(m). Monodispersed rabbit C5b-9(m) exhibited an apparent sedimentation coefficient of 29 S in deoxycholate-containing sucrose density gradients, corresponding to a composite protein-detergent molecular-weight of approx. 1.4 X 10(6). Protein subunits corresponding to human C5b-C9 were found on sodium dodecyl sulphate/polyacrylamide-gel electrophoresis. By densitometry, there were consistently six molecules of monomeric C9 present for each monomeric C5b-8 complex. Fluid-phase rabbit SC5b-9 was a hydrophilic 23 S ma macromolecule that differed in subunit composition from its membrane counterpart in that it contained S-protein and only two to three molecules of C9 per monomer complex. The data are in accord with the previous report on human C5b-9 that C5b-9(m) contains more C9 molecules than SC5b-9 [Ware & Kolb (1981) Proc. Natl. Acad. Sci. U.S.A. 78, 6426-6430]. They corroborate the previous molecular-weight estimate of approx. 10(6) for C5b-9(m) and thus support the concept that the fully assembled, unit lesion of complement is a C5b-9 monomer [Bhakdi & Tranum-Jensen (1981) Proc. Natl. Acad. Sci. U.S.A. 78, 1818-1822]. They also show that C9 dimer formation is not required for assembly of the rabbit C5b-9(m) protein cylinder, or for expression of its membrane-damaging function.     

Endotoxin-induced formation of specific β-endorphin binding sites in human serum

Specific binding of human β-endorphin is demonstrated to sites that are present in endotoxin-treated, but not in native, human serum. Binding is saturable and reversible and is mediated by a nonopioid segment of β_H-endorphin to sites that are located on the terminal SC5b-9 complement complex. Since endotoxemia causes both the secretion of β-endorphin into blood (1) and the formation of specific β-endorphin binding sites therein, both effects might be elements of a common physiological process.     

Evaluation of antisperm complement-dependent immune mediators in human ovarian follicular fluid

A "sandwich"-type radiolabeled antiglobulin assay using monoclonal anti-C5b-9 neoantigen and polyclonal anti-C5b-9 was used to evaluate the presence of terminal C complexes (SC5b-9 or MC5b-9) in the sera and ovarian follicular fluid (FF) from 45 infertile women. FF SC5b-9 was detectable in all clinical diagnostic categories. The mean SC5b-9 levels in FF and sera were 399 ng/ml (range 75 to 1350 ng/ml) and 798 ng/ml (range 0 to 2700 ng/ml), respectively. Twelve (26.6%) of the 45 FF samples had normal hemolytic C activity, and all FF (n = 44) samples initiated C8/C9-dependent lysis of sensitized sheep E coated with human C1-7. Human plasma IgG antisperm antibodies (ASA) were capable of activating C in 31 (72%) of 43 FF samples as detected by their ability to deposit MC5b-9 on human sperm. Sera from infertile women with ASA in their sera and FF impaired human sperm binding to human zona pellucida and binding and penetration of zona-free hamster oocytes in vitro. The discovery of SC5b-9 and MC5b-9 in ovarian FF implies that the interaction of ASA and C could have a deleterious effect on sperm during in vivo and in vitro sperm-egg interactions in women with antisperm antibodies.     

Identification of the C3b receptor-binding domain in third component of complement

We report here that complement receptor type one (CR1) binds to a region of C3b that is contained within the NH2 terminus of the alpha' chain. In an enzyme-linked immunosorbent assay, CR1 bound to C3b, iC3b, and C3c but not to C3d, and this binding was inhibited by soluble C3b and C3c. Further attempts to generate a small C3 fragment capable of binding CR1 were unsuccessful. However, elastase degradation of C3 generated four species of C3c (C3c I-IV), two of which bound CR1. NH2-terminal sequence analysis and sodium dodecyl sulfate-gel electrophoresis of the C3cs indicated that the beta chains and the 40,000-dalton COOH-terminal alpha' chain fragments were identical; the NH2-terminal alpha' chain fragments of C3c I-IV varied from 21,000 to 27,000 daltons and accounted for the differential binding to CR1. C3c-I and II, which do not bind CR1, were missing 8 and 9 residues from the NH2 terminus of the alpha' chain when compared with the intact alpha' chain of C3b. C3c-III and IV, which bind CR1, had NH2 termini identical to the intact NH2-terminal alpha' chain of C3b. Using iodinated concanavalin A and endoglycosidase H, we showed that the NH2-terminal alpha' chains of C3c-I and III were glycosylated, while C3c-II and IV were not. Therefore, these data indicated that the amino terminus of the NH2-terminal alpha' chain fragment of C3c was responsible for binding CR1 while the COOH terminus of this fragment was not involved since the presence or absence of this region in C3c did not affect CR1 binding to C3c. Subsequently, two peptides were synthesized from the NH2-terminal alpha' chain fragment of C3c: X42, 42 residues in length from the NH2 terminus and C30, 30 residues in length from the COOH terminus. X42 inhibited binding of CR1 to C3b, and this effect was also observed with antipeptide antibodies against the X42 peptide. The C30 and other C3-derived peptides and antipeptide antibodies had no effect on the binding of CR1 to C3b.     

Complement proteins C5b-9 initiate secretion of platelet storage granules without increased binding of fibrinogen or von Willebrand factor to newly expressed cell surface GPIIb-IIIa

The functional and conformational activation of cell surface glycoproteins IIb-IIIa (GPIIb-IIIa) was probed in platelets stimulated to secrete by complement proteins C5b-9. Gel-filtered human platelets exposed to the purified human C5b-9 proteins exhibited non-lytic secretory release of both alpha- and dense granule storage pools with only a small increase in total binding of 125I-fibrinogen (less than 3000 molecules/cell) to the cell surface. By contrast to ADP- or thrombin-activated platelets, increased 125I-fibrinogen bound to C5b-9 platelets was not inhibited by Arg-Gly-Asp-containing peptides, suggesting that the high affinity membrane receptor for fibrinogen is not expressed under these conditions. C5b-9-stimulated platelets also failed to bind 125I-von Willebrand factor (less than 1 ng/10(8) platelets), confirming that the adhesive protein receptor function of cell surface GPIIb-IIIa is not expressed in these cells. Although specific binding of 125I-fibrinogen or 125I-von Willebrand factor did not significantly increase after C5b-9 assembly, these proteins elicited de novo expression of the GPIIb-IIIa activation-associated epitope recognized by monoclonal antibody PAC-1, and binding of this antibody to C5b-9 platelets was fully competed by Arg-Gly-Asp-containing peptides. These data suggest that the metabolic events which trigger granule secretion after C5b-9 insertion into the plasma membrane cause cell surface GPIIb-IIIa to be expressed in an activation-associated but functionally incompetent conformation.     

Opioid and non-opioid binding of beta-endorphin to bovine adrenal medullary membranes

Binding of human beta-endorphin (beta h-EP) to bovine adrenal medullary membranes was characterized using [125I]Tyr27-beta h-EP [( 125I]beta h-EP) as a primary ligand. The specific binding of [125I]beta h-EP was time-dependent, saturable and stereospecific. Analysis of a saturation isotherm revealed two apparent classes of specific binding sites with dissociation constants of 2.4 and 34 nM. The extent of maximum inhibition of specific [125I]beta h-EP binding by either levorphanol, morphine, naloxone, dynorphin A (1-13) or D-Ala2-D-Leu5-enkephalin was similar to each other and remained partial (60-70%). Levorphanol eliminated the high affinity component but showed no effect on the low affinity component of [125I]beta h-EP binding. beta h-EP(1-31) displaced completely the [125I]beta h-EP binding. However, beta h-EP(1-23) only partially (approximately 80%) inhibited the [125I]beta h-EP binding. beta h-EP(6-31) showed inhibitory activity on [125I]beta h-EP binding. These results suggest that [125I]beta h-EP binding to bovine adrenal medullary membranes consists of a high affinity opioid-sensitive component and a low affinity non-opioid component. The non-opioid component of [125I]beta h-EP binding may be related to COOH-terminal of the beta h-EP molecule.     

Activation of the human complement system by cholesterol-rich and PEGylated liposomes-modulation of cholesterol-rich liposome-mediated complement activation by elevated serum LDL and HDL levels

Intravenously infused liposomes may induce cardiopulmonary distress in some human subjects, which is a manifestation of "complement activation-related pseudoallergy." We have now examined liposome-mediated complement activation in human sera with elevated lipoprotein (LDL and HDL) levels, since abnormal or racial differences in serum lipid profiles seem to modulate the extent of complement activation and associated adverse responses. In accordance with our earlier observations, cholesterol-rich (45 mol% cholesterol) liposomes activated human complement, as reflected by a significant rise in serum level of S-protein-bound form of the terminal complex (SC5b-9). However, liposome-induced rise of SC5b-9 was significantly suppressed when serum HDL cholesterol levels increased by 30%. Increase of serum LDL to levels similar to that observed in heterozygous familial hypercholesterolemia also suppressed liposome-mediated SC5b-9 generation considerably. While intravenous injection of cholesterol-rich liposomes into pigs was associated with an immediate circulatory collapse, the drop in systemic arterial pressure following injection of liposomes preincubated with human lipoproteins was slow and extended. Therefore, surface-associated lipoprotein particles (or apolipoproteins) seem to lessen liposome-induced adverse haemodynamic changes, possibly as a consequence of suppressed complement activation in vivo. PEGylated liposomes were also capable of activating the human complement system, and the presence of surface projected methoxypoly(ethylene glycol) chains did not interfere with generation of C3 opsonic fragments. We also show that poly(ethylene glycol) is not responsible for PEGylated liposome-mediated complement activation. The net anionic charge on the phosphate moiety of the phospholipid-mPEG conjugate seemed to play a critical role in activation of both the classical and alternative pathways of the complement system.     

Binding site for vitamin K-dependent protein S on complement C4b-binding protein

Half of the protein S in plasma is present as a complex with a C4b-binding protein (C4bp), a complement component (Mr 570,000). In this study, the protein S-binding site on C4bp was examined by using monoclonal anti-C4bp-IgGs. C4bp was cleaved by chymotryptic digestion into seven NH2-terminal arm fragments (Mr 48,000) and a COOH-terminal core fragment (Mr 160,000). The COOH-terminal fragment inhibited the cofactor activity of protein S and its binding to C4bp in a dose-dependent manner. A monoclonal anti-C4bp-IgG (MFbp16), which binds to the COOH-terminal fragment, inhibited the binding of protein S to C4bp. The chymotryptic digest of the reduced and carboxymethylated COOH-terminal fragment was subjected to MFbp16-Sepharose 4B column affinity chromatography, and a peptide of Mr 2,500 was obtained. Protein S bound to the Mr 2,500 peptide, and this binding was inhibited by C4bp in a dose-dependent manner. The sequence of this peptide corresponded to Ser447-Tyr467 near the COOH terminus of the C4bp subunit. MFbp16, which bound to Mr 570,000 C4bp (C4bp-high), did not bind to Mr 510,000 C4bp (C4bp-low) in human plasma that does not form a complex with protein S. This suggests that C4bp-low lacks the protein S-binding site present in the COOH-terminal region of C4bp-high. Since C4bp-low also dissociates into identical subunits when reduced, the interchain disulfide bond region that links the seven subunits of C4bp appears to be closer to the NH2-terminal end than the protein S-binding site.     

Evidence that C5b recognizes and mediates C8 incorporation into the cytolytic complex of complement

The aim of this study was to identify constituents of the intermediate C5b-7 complex of human complement that mediate binding of C8 and formation of C5b-8. Analysis of interactions between purified C8 and C5, C6, or C7 indicate that C5 and C8 associate to form a dimer in solution. This interaction is specific and involves a single C5 binding site located on the beta-subunit of C8. Simultaneous interaction of C8 with C5 and C9 in solution suggests that during assembly of the cytolytic C5b-9 complex on membranes, C8 binds to C5b-7 through association of beta with C5b, after which C9 associates through interaction with the previously identified C9-specific site on the alpha-subunit. Other evidence of interaction with C5b was provided by the fact that C8 can bind purified C5b6. Also, in situ cross-linking experiments showed that within C5b-8, the beta-subunit is in close proximity to C5b. These results indicate that C8 binding to C5b-7 is mediated by a specific C5b recognition site on beta, thus explaining the requirement for this subunit in C5b-8 formation. They also reveal that C5b contains a specific site for interaction with beta.     

Causative factors behind poloxamer 188 (Pluronic F68, Flocor)-induced complement activation in human sera. A protective role against poloxamer-mediated complement activation by elevated serum lipoprotein levels

Poloxamer 188 is a complex polydisperse mixture of non-ionic macromolecules. Adverse non-IgE-mediated hypersensitivity reactions occur in some individuals following intravenous injection of poloxamer 188-based pharmaceuticals, presumably via complement activation. Here we have delineated potential causal chemical and biological interactive factors behind poloxamer 188-induced complement activation in human serum specimens. We identified the molecular constituents inherent in poloxamer 188 preparations and studied their effect on generation of the two complement split products, SC5b-9 and Bb. Poloxamer 188 activated complement at sub-micellar concentrations and the results indicated the potential involvement of all three known complement activation pathways. The poloxamer-induced rise of SC5b-9 in human sera was abolished in the presence of a recombinant truncated soluble form of complement receptor type 1, thus confirming the role of C3/C5 convertases in the activation process. Poloxamer 188-mediated complement activation is an intrinsic property of these macromolecules and was independent of the degree of sample polydispersity, as opposed to other non-polymeric constituents. Poloxamer 188 preparations also contained unsaturated chains of diblock copolymers capable of generating SC5b-9 in human sera; this effect was terminated following the removal of double bonds by catalytic hydrogenation. By quasi-elastic light scattering, we established interaction between poloxamer and lipoproteins; interestingly, poloxamer-induced rise in SC5b-9 was significantly suppressed when serum HDL and LDL cholesterol levels were increased above normal to mimic two relevant clinical situations. This observation was consistent with previously reported data from patients with abnormal or elevated lipid profiles where no or poor complement activation by poloxamer 188 occurred. Our findings could provide the basis of novel approaches to the prevention of poloxamer-mediated complement activation.     

Platelet-activating factor and kinin-dependent vascular leakage as a novel functional activity of the soluble terminal complement complex

The infrequent occurrence of septic shock in patients with inherited deficiencies of the terminal complement components experiencing meningococcal disease led us to suspect that the terminal complement complex is involved in vascular leakage. To this end, the permeabilizing effect of the cytolytically inactive soluble terminal complement complex (SC5b-9) was tested in a Transwell system measuring the amount of fluorescein-labeled BSA (FITC-BSA) leaked through a monolayer of endothelial cells. The complex caused increased permeability to FITC-BSA after 15 min as opposed to the prompt response to bradykinin (BK). The effect of SC5b-9 was partially reduced by HOE-140 or CV-3988, two selective antagonists of BK B2 and platelet-activating factor receptors, respectively, and was completely neutralized by the mixture of the two antagonists. Also, DX-88, a specific inhibitor of kallikrein, partially inhibited the activity of SC5b-9. The permeabilizing factor(s) released after 30 min of incubation of endothelial cells with SC5b-9 caused a prompt leakage of albumin like BK. Intravital microscopy confirmed both the extravasation of circulating FITC-BSA across mesenteric microvessels 15 min after topical application of SC5b-9 and the complete neutralization by the mixture of HOE-140 and CV-3988. SC5b-9 induced opening of interendothelial junctions in mesenteric endothelium documented by transmission electron microscopy.     

Complement pores in erythrocyte membranes: Analysis of C8/C9 binding required for functional membrane damage

The number of membrane-bound terminal complement proteins (C5b-9) required to generate a functional pore in the human erythrocyte membrane ghost has been determined. Resealed erythrocyte ghost membranes (ghosts) were treated with human complement proteins C5b6, C7, ¹³¹I-C8, and ¹²⁵I-C9 under non-lytic conditions. Following C5b-9 assembly, sucrose-permeant ghosts were separated from C5b-9 ghosts that remained impermeant to sucrose by centrifugation over density barriers formed of 43% (w/v) sucrose. Analysis of ¹³¹I-C8 and ¹²⁵I-C9 bound to sucrose-permeant and sucrose-impermeant subpopulations of C5b-9 ghosts revealed: 1. Sucrose-permeant C5b-9 ghosts show increased uptake of both ¹³¹I-C8 and ¹²⁵I-C9 as compared to ghosts that remain impermeant to sucrose. Ghosts with less than 300 molecules ¹³¹I-C8 bound remain impermeant to sucrose, irrespective of the total C9 input, or, the multiplicity of C9 uptake by membrane C5b-8. 2. In the presence of excess ¹²⁵I-C9, the ratio of ¹²⁵I-C9/¹³¹I-C8 bound to membrane C5b67 is 3.2 ± 0.8 (mean ± 2 S.D.), suggesting an average stoichiometry of 3 C9 per C5b-8. Under these conditions, the ratio of ¹²⁵I-C9/¹³¹I-C8 bound to sucrose-permeant ghosts (3.3 ± 0.7) does not significantly differ from the ratio bound to sucrose-impermeant ghosts (2.9 ± 0.6). 3. With limiting C9 input, the threshold of total C5b-8 uptake required for sucrose permeability increases significantly above 300 per cell when the ratio of bound ¹²⁵I-C9/¹³¹I-C8 is decreased below unity. In the complete absence of C9, 11 700 C5b-8 complexes are bound to sucrose-permeant ghosts. It is concluded that more than 300 C5b-9 complexes must bind to the human erythrocyte to form a sucrose-permeant lesion. Although the binding of one C9 per C5b-8 is critical to the pore-forming activity of these proteins, the binding of additional molecules of C9 to each complex (C9/C8 > 1) does not significantly alter the threshold of total C5b-9 uptake required for lesion formation.