Properdin-Mediated C5a Production Enhances Stable Binding of Platelets to Granulocytes in Human Whole Blood.

Enhanced levels of platelet/granulocyte aggregates (PGAs) are found in patients suffering from many different inflammatory vascular diseases, and their formation in animal models of vascular disease is associated with increased thromboinflammation and worsened outcomes. The complement system, a part of the innate immune system, influences PGA formation, but the mechanisms for its effects are unknown. In this study, we have defined complement-mediated mechanisms that enhance PGA formation in human whole blood stimulated with thrombin receptor-activating peptide (TRAP) using ex vivo flow cytometry assays. We demonstrate that physiological properdin, a positive regulator of complement alternative pathway activity, increases PGA formation when added to TRAP-stimulated blood. All physiological properdin forms increase PGA formation, but properdin tetramers are the most efficient at increasing complement activity and PGA formation. Inhibition of endogenous properdin, either circulating in the blood or produced locally by leukocytes, impairs TRAP-mediated PGA formation to the same level as specific inhibition of either the alternative or classical pathway. Additionally, blocking the interaction of C5a with its cellular receptor prevents properdin-mediated increases in PGA formation. Adding either properdin tetramers or C5a to whole blood increases CD11b expression on granulocytes, and this increase is prevented by blockade of the C5a-C5a receptor axis. Finally, we demonstrate that the effects of properdin on PGA formation are tightly regulated by Factor H. Cumulatively, our data indicate that properdin enhances PGA formation via increased production of C5a, and that inhibition of properdin function has therapeutic potential to limit thromboinflammation in diseases characterized by increased PGA formation.

Purification and functional analysis of human properdin.

Properdin is a member of the alternative pathway of complement. It is unique in that it is the only known positive regulator of the complement system. Properdin can stabilize and promote complement activation, but in addition is also capable of initiating the alternative pathway, making it a unique protein. This chapter focuses on the methods required for purifying human properdin and testing its functionality.

An evaluation of the role of properdin in alternative pathway activation on Neisseria meningitidis and Neisseria gonorrhoeae.

Properdin, a positive regulator of the alternative pathway (AP) of complement is important in innate immune defenses against invasive neisserial infections. Recently, commercially available unfractionated properdin was shown to bind to certain biological surfaces, including Neisseria gonorrhoeae, which facilitated C3 deposition. Unfractionated properdin contains aggregates or high-order oligomers, in addition to its physiological "native" (dimeric, trimeric, and tetrameric) forms. We examined the role of properdin in AP activation on diverse strains of Neisseria meningitidis and N. gonorrhoeae specifically using native versus unfractionated properdin. C3 deposition on Neisseria decreased markedly when properdin function was blocked using an anti-properdin mAb or when properdin was depleted from serum. Maximal AP-mediated C3 deposition on Neisseriae even at high (80%) serum concentrations required properdin. Consistent with prior observations, preincubation of bacteria with unfractionated properdin, followed by the addition of properdin-depleted serum resulted in higher C3 deposition than when bacteria were incubated with properdin-depleted serum alone. Unexpectedly, none of 10 Neisserial strains tested bound native properdin. Consistent with its inability to bind to Neisseriae, preincubating bacteria with native properdin followed by the addition of properdin-depleted serum did not cause detectable increases in C3 deposition. However, reconstituting properdin-depleted serum with native properdin a priori enhanced C3 deposition on all strains of Neisseria tested. In conclusion, the physiological forms of properdin do not bind directly to either N. meningitidis or N. gonorrhoeae but play a crucial role in augmenting AP-dependent C3 deposition on the bacteria through the "conventional" mechanism of stabilizing AP C3 convertases.

Native polymeric forms of properdin selectively bind to targets and promote activation of the alternative pathway of complement.

Properdin, a positive regulator of the complement system, has recently been reported to bind to certain pathogenic microorganisms, to early or late apoptotic and necrotic cells, and to particular live human cell lines, thus providing a platform for de novo convertase assembly and complement activation. These studies, with some contradictory results, have been carried out with purified properdin, which forms a series of oligomers of a ∼53,000 Mr subunit, assembling into dimers (P2), trimers (P3), tetramers (P4) and higher forms (P(n)). The P(n) forms have been shown to likely be an artefact of purification that results from procedures including freeze-thawing of properdin. In this study we isolated the individual natural forms of properdin (P2, P3, and P4) and separated them from the P(n) forms present in purified frozen properdin using ion exchange and/or size exclusion chromatography. We analyzed the ability of each form to bind to live or necrotic Jurkat and Raji cells, rabbit erythrocytes (E(R)), and zymosan by FACS analysis. While the unseparated properdin and the purified P(n) forms bound to all the surfaces except E(R), the physiological P2-P4 forms specifically bound only to zymosan and to necrotic nucleated cells. Our results indicate that aggregated P(n) present in unseparated properdin may bind non-specifically to some surfaces and should be separated before analysis in order to obtain meaningful results. Finally, we have determined for the first time that the physiological forms of human properdin can selectively recognize surfaces and enhance or promote complement activation, which is in agreement with the reported role for properdin as a complement initiator.

The fractionation of human plasma proteins. I. Affinity purification of human complement properdin.

A method was developed for the affinity purification of human complement properdin. The preparation is part of an integrated scheme in which over 20 human plasma proteins can be recovered in a highly purified form. The yield of properdin was 5.9 mg from 3 liters of plasma, amounting to a 28% recovery. The crucial step in the purification was the application of affinity chromatography using C3b-ester-Sepharose.

Properdin binds to sulfatide [Gal(3-SO4)beta 1-1 Cer] and has a sequence homology with other proteins that bind sulfated glycoconjugates.

Properdin, which stabilizes the C3 convertase during the activation of the alternate complement pathway, contains amino acid sequence homologies with several proteins that bind sulfated glycoconjugates, including the adhesive protein thrombospondin and the leech salivary protein antistasin. This homology is based around the sequence Cys-Ser-Val-Thr-Cys-Gly-X-Gly-X-X-X-Arg-X-Arg. To determine if these homologous amino acid sequences are sulfated glycoconjugate-binding domains, purified native properdin, as well as activated properdin (a high molecular weight form of properdin), were examined for binding to various lipids in solid phase radioimmunoassays. Of the lipids tested, both native and activated properdin bind with high affinity only to sulfatide [Gal(3-SO4)beta 1-1 Cer], but not to comparable levels of cholesterol-3-SO4, or several neutral glycolipids, gangliosides, and phospholipids. Sulfatide binding by both forms of properdin is inhibited by dextran sulfate (Mr = 500,000) or fucoidan, whereas only the activated form is inhibited by dextran sulfate (Mr = 5,000) or heparin. Comparable levels of chondroitin sulfates A, B, and C, keratan sulfate, dextran (Mr = 90,000), or hyaluronic acid do not inhibit binding. Taken together, these data suggest that properdin, like antistasin and thrombospondin, binds sulfated glycoconjugates and supports the conclusion that the homologous sequences are sulfated glycoconjugate-binding domains.

Biosynthesis of properdin.

Properdin (P) is synthesized by the human promyelocytic cell line, HL-60, after differentiation with DMSO. The secreted P was physiochemically indistinguishable from purified plasma P. It was polymerized and able to bind to C3IBb-Sepharose but not to C3i-Sepharose. No extracellular precursor was present. The intracellular form, detected between 1 and 4 h after labeling, was similar but had a slightly lower Mr. It also bound reversibly to C3iBb-Sepharose, and polymers could be demonstrated by cross-linking. Pulse-chase experiments suggested the existence of an earlier, but undetectable, intracellular precursor(s). This form could not be immunoprecipitated even when harsh solubilization conditions and/or antibodies against reduced and denatured P were utilized. Studies with endoglycosidases F and H and tunicamycin indicated that the detectable intracellular precursor contains high mannose N-linked carbohydrate that is processed to the complex form before secretion. The sugars are not required for polymerization, secretion, or functional activity, or responsible for the electrophoretic heterogeneity. Polymerization of P is therefore an early intracellular event, perhaps carefully controlled to prevent anomalous aggregation.

Analysis of the natural polymeric forms of human properdin and their functions in complement activation.

Many of the anomalies observed in studies or properdin may be explained on the basis of its ability to form a series of multi-subunit polymers and by differences in the functions of these forms of properdin. Dimers (P2), trimers (P3), tetramers (P4), and higher Mr polymers (Pn) of the 46,000-Da subunit were separated by gel filtration or by cation exchange chromatography of purified properdin. The specific activity of each form was measured in two assays. The native properdin activity of P4 was 10 times that of P2 (on a molar basis) with the order: P4 greater than P3 greater than P2 greater than Pn. During C activation P4 was found to be consumed first, P3 second, and P2 last, consistent with their measured specific activities. Assays for activated properdin showed that only Pn caused fluid phase C consumption when incubated in serum at normal concentrations. Pn accumulated during long term storage of purified P and freezing rapidly converted the smaller oligomers to Pn. The isolated oligomers were extremely stable, but did redistribute after denaturation-renaturation cycles by using low pH or guanidine. Renaturation after exposure of any species to denaturing conditions yielded mixtures of 20:54:26 (P4:P3:P2). This distribution was almost identical to that found in fresh normal human serum or plasma, suggesting that a distinct distribution of oligomers exists in blood that provides the C system with an apparently advantageous range of specific activities.

Resolution and analysis of 'native' and 'activated' properdin.

A rapid and reproducible procedure for the resolution of 'native' and 'activated' forms of properdin (a component of the alternative activation pathway of complement), by gel filtration on the polyvinyl matrix Fractogel TSK HW-55(S), is reported. This fractionation permitted effective screening of samples for conditions that cause activation. Only 'native' properdin was detected in serum, even after activation of the alternative pathway by yeast cell walls. Transformation of 'native' into 'activated' properdin in vitro was produced by freeze-thawing of the protein, but not upon binding to and dissociation from the C3 convertase, C3bBb. Electron microscopy showed that only the 'native' population contained the discrete cyclic structures described previously by Smith, Pangburn, Vogel & Müller-Eberhard [(1984) J. Biol. Chem. 259, 4582-4588]. 'Activated' properdin, which was eluted from the gel-filtration column close to the breakthrough peak, was mainly composed of large amorphous aggregates. We therefore conclude that properdin 'activation' is not a physiological event that occurs in serum on complement activation, but is an artifact of isolation. Fractionation of properdin on Fractogel TSK HW-55(S) has, however, enabled detailed analysis of functional heterogeneity within the 'native' population.

Isolation and characterization of rabbit properdin of the alternative complement pathway.

Rabbit factor P, which is involved in stabilizing the labile C3 convertase of the alternative complement pathway, was isolated from rabbit serum by a simple two-step procedure: polyethylene glycol precipitation and QAE-Sephadex A-50 column chromatography. The chemical properties, and functional activities of the rabbit P were very similar to those of human P, except that there were slight differences in the SDS-PAGE patterns of limited tryptic digests and in the amino terminal sequences. The molecular weights were estimated to be 58 000 for rabbit P and 59 000 for human P on SDS-PAGE under reducing conditions. The amino terminal 36 residues of rabbit P showed 78% homology to the equivalent region of human P.

Molecular architecture of human properdin, a positive regulator of the alternative pathway of complement.

The structure of the human properdin molecule was investigated by hydrodynamic, spectroscopic, and transmission electron microscope studies. Sucrose density gradient ultracentrifugation of purified, functionally active properdin showed a single component sedimenting at 5.5 S. Electron microscopic examination by two different methods, however, revealed polydispersity of the protein which consisted of cyclic dimers, trimers, tetramers, pentamers, and higher cyclic oligomers. Approximately 80% of the oligomers were dimers, trimers, and tetramers. Monomers could not be detected. These polymers could be partially separated by gel filtration on Sephacryl S-300 and all fractions were active in terms of binding to C3b. The specific activity increased with oligomer size. When reexamined after incubation at 37 degrees C for 4 h or at 4 degrees C for 2 weeks, the chromatographic behavior of the oligomers and their electron microscopic appearance were unchanged, suggesting that in vitro no rapid equilibration occurred. The protomer is clearly visualized within polymers as a flexible, rod-like structure 26.0 nm in length and 2.5 nm in diameter, with pronounced thickening at each end. The monomer is bivalent with respect to binding to other properdin monomers and the binding sites are localized to the ends of the structure. A model could be devised which is consistent with the distinct geometry of the intersubunit contacts observed in micrographs. The circular dichroism spectrum of properdin suggests the presence of little alpha helix or beta structure and shows positive ellipticity at 231 nm. In contrast to previous investigators, we conclude that isolated human properdin is polydisperse and consists of a set of cyclic polymers constructed from a single highly asymmetric and flexible protomer.

The rabbit properdin system: I. Identification of a new factor and purification of rabbit properdin.

Pure preparations of rabbit properdin were obtained from rabbit serum by ion-exchange chromatography. These preparations functioned as properdin when they were measured with the zymosan assay or with a serum reagent selectively depleted of properdin by a specific immunoabsorbent. Properdin in these preparations was in its activated state. A new serum factor was required to measure properdin activity when purified preparations of rabbit properdin were tested with the zymosan assay. This factor was designated as ZBP, or zymosan-binding protein. ZBP appeared to be distinct from known components of the alternative complement pathway and the classical complement system, and it did not appear to be an immunoglobulin.

Native and activated properdin: interconvertibility and identity of amino- and carboxy-terminal sequences.

The development of a two-step purification procedure of native properdin with good yield has allowed the physical and chemical comparison of native and activated properdin. The two forms of properdin have identical electrophoretic mobility, subunit size, and amino- as well as carboxyl-terminal amino acid sequences. The two forms of properdin can be interconverted by using mild denaturing agents, indicating that the change in biologic activity is conformational. Circular dichroism analysis of properdin reveals a significant variability in the tertiary structure. However, the differences are a result of the method of purification and do not correspond to the biologic activity of the protein, because the spectra of the interconverted forms of properdin do not change. This indicates that the conformational transition that causes biologic activity changes is small, relative to the conformational variations produced by other conditions that do not alter the biologic activity.

Assembly of the cytolytic alternative pathway of complement from 11 isolated plasma proteins.

The known cytolytic function of the alternative pathway in serum was quantitatively reproduced by combining 11 isolated plasma proteins at their respective serum concentrations. These proteins are: C3, Factor B, Factor D, C3b inactivator, beta1H, native properdin, C5, C6, C7, C8, and C9. In absence of activators of the alternative pathway, this mixture was stable at 37 degrees C as evidenced by lack of consumption of Factor B, C3, and C5. Upon addition of either rabbit erythrocytes or neuraminidase-treated sheep erythrocytes, cell lysis ensued and the extent of lysis was dependent on dose of the component mixture. The dose-response curves obtained with the isolated component mixture and with C4-depleted serum were virtually indistinguishable. Nonactivator erythrocytes (untreated sheep erythrocytes) were not lysed by the component mixture. Deletion of properdin resulted only in a twofold diminution of the hemolytic activity of the component mixture. No immunoglobulin requirement was apparent. These results indicate that the cytolytic systems studied are internally sufficient and capable of coupling the initiation and amplification sequence with the cytolytic membrane attack sequence.

Alternate pathway activation of complement in a Proteus mirabilis ulceration of the cornea.

A 63-year-old patient had an extensive infiltration, ulceration, and eventual perforation of the cornea caused by Proteus mirabilis. Histopathological examination of corneal tissue that was obtained at keratoplasty disclosed that the stroma was ulcerated with a diffuse infiltration of polymorphonuclear leukocytes. Immunopathological examination of the cornea revealed prominent and diffuse staining for properdin and C3 complement. There was an absence of staining for IgG, IgA, IgM, IgE, and C4 complement. These findings suggest that the alternate pathway of complement was activated in this ulcerated cornea and that immunopathological phenomena contribute to the polymorphonuclear infiltration found with Gram-negative ulcerations of the human cornea.

[Activation of the complement system in different forms of glomerulonephritis]. / Komplementaktivierung bei Glomerulonephritiden.

The complement system may be activated by two pathways, the classical and the alternate. To evaluate their respective participation in different forms of glomerulonephritis, the plasma values of C3, C4, C3PA, C1q and properdin were determined in 70 patients. In systemic lupus erythematosus (LED), acute poststreptococcal glomerulonephritis (AGN) and septicemia the classical pathway appears to be mainly involved, whereas the amplification loop and the alternate pathway seem to be of secondary importance. By contrast, in membranoproliferative glomerulonephritis (MPGN) the alternate pathway plays a major role. However, the present data suggest that activation of the classical pathway may often be involved as well. In minimal change glomerulonephritis no signs indicating involvement of the complement system were apparent. Follow-up observation demonstrated a correlation between decreases in plasma complement concentrations and the clinical severity of the primary disease in LED, AGN and septicemia, but not in MPGN.