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PubMedDate not supplied

Anaphylatoxin C3a induces rapid protein phosphorylation in guinea pig platelets.

We investigated C3a signal transduction using guinea pig platelets. Anaphylatoxin C3a induced cytosolic calcium influx and turnover of inositol phospholipids and caused protein phosphorylation via specific C3a receptors on guinea pig platelets. Phosphorylation of 40 kDa and 20 kDa proteins was detected within 30 s after C3a stimulation. From phosphoamino acid analysis of both proteins, it was found that about 80% was serine and 20% was threonine. Phosphorylation was decreased by pretreatment of platelets with calcium blockers, diltiazem and TMB-8. A protein kinase C inhibitor, staurosporine, effectively blocked phosphorylation of both proteins, but H-7 did not. The effects of these inhibitors on platelet aggregation were correlated with their effects on protein phosphorylation. These results suggest that protein-serine/threonine phosphorylation is important for C3a signal transduction in guinea pig platelets and that the isoenzyme of protein kinase C or another kinase probably participates in such protein phosphorylation. C3a also caused protein-tyrosine phosphorylation of 56 kDa and 33-36 kDa proteins within 30 s. Staurosporine effectively blocked phosphorylation of these bands. It is suggested that tyrosine kinase also may be involved in C3a signalling in guinea pig platelets.

Animals↗
PubMed1987

Plasma levels of main granulocyte components in patients dialyzed with polycarbonate and cuprophan membranes.

Plasma levels of granulocyte lactoferrin, granulocyte myeloperoxidase and granulocyte elastase in complex with alpha 1-proteinase inhibitor (E-alpha 1PI) were investigated in regular hemodialysis patients dialyzed with hollow-fiber dialyzers made from polycarbonate (FD 100) or cuprophan (GFS 120 H). Plasma levels of all these main granulocyte components increased significantly during hemodialysis. E-alpha 1PI levels were significantly higher in patients dialyzed with the polycarbonate compared with the cuprophan membrane, whereas the increases of myeloperoxidase and lactoferrin were not different for the two dialyzers. On the other hand, plasma C3a levels were higher in patients dialyzed with the cuprophan compared with the polycarbonate dialyzer. Therefore, granulocyte activation during hemodialysis does not necessarily need complement activation.

Blood Proteins↗
PubMed1992

Effects of complement activation on platelet-activating factor and eicosanoid synthesis in rat mesangial cells.

The effects of in vitro complement activation and of isolated complement components (C3a, C5a, C3b) on the activity of the microsomal enzyme acetyl coenzyme A: 1-0-alkyl-glycero-3-phosphocholine acetyl transferase (AcTr) in cultured mesangial cells and on the synthesis of prostaglandin E2 (PGE2) were assessed. In vitro complement activation induced by the introduction of purified cobra venom factor (CVF) in culture media containing human serum enhanced mesangial cell PGE2 synthesis and had no effect on microsomal AcTr activity. When media containing C6-deficient serum were used, the stimulatory effect of CVF on PGE2 synthesis was abolished. Introduction of CVF in these media enhanced mesangial cell AcTr activity. These effects were partially reversed when the C6-deficient serum was supplemented with C7-deficient serum to allow formation of the C5b-9 complex. Isolated C3a, C5a, and C3b had opposite effects on PGE2 synthesis and on AcTr activity. Specifically, all components enhanced mesangial cell PGE2 synthesis. In contrast, AcTr activity was enhanced by C3a alone; C5a had no effect, whereas C3b had an inhibitory effect. The observations indicate that in response to complement activation and specific anaphylatoxin agonists, mesangial cell eicosanoid synthesis is not coupled with changes in the activity of AcTr and PAF synthesis.

Acetyltransferases↗
PubMed1996

Recurrent hematuria: a novel clinical presentation of hereditary complete complement C4 deficiency.

A 10-year-old boy suffered from recurrent attacks of fever, vomiting, and hematuria. During disease flares, circulating immune complexes were detected in the serum. Elevated levels of Bb, Ba, and C3a indicated complement activation through the alternative pathway. Complement C4 was undetectable. C4 phenotyping by agarose gel electrophoresis showed complete C4 deficiency. Restriction fragment length polymorphism (RFLP) studies showed a homozygous deletion of the C4B and 21-hydroxylase A genes. A mild mesangioproliferative glomerulonephritis with mesangial deposits of immunoglobulin (1g) G, IgM, IgA, Clq, C3, properdin, and terminal complement complex was probably caused by immune complex deposition and alternative complement pathway activation. Treatment with low-dose prednisolone substantially reduced the frequency of further episodes.

Biopsy↗
PubMed1992

[Reduction of neutrophil activation by prostaglandin E1 in open heart surgery].

This study was designed to demonstrate the effect of prostaglandin E1 (PGE1) on neutrophil activation in open heart surgery. Twenty adult patients undergoing cardiopulmonary bypass (CPB) for various cardiac operations were divided into 2 groups. PGE1 group consisted of 10 patients (7 males and 3 females) and the control group consisted of 10 patients (6 males and 4 females). In PGE1 group patients, 20-50 ng/kg/min of PGE1 was administered intravenously from the induction of anesthesia to the completion of CPB. Blood samples were taken before, during, after CPB, and in the morning of the first postoperative day. Differential counts of white blood cells, plasma neutrophil elastase (PNEL) activity, serum complements activity (C3a, CH50) and superoxide production of neutrophils were measured. Superoxide production by isolated neutrophils was evaluated utilizing luminol dependent chemiluminescence. After the initiation of CPB complements were activated markedly, and PNEL activity increased significantly in both groups. Although after CPB PNEL activity turned to decrease, it was still significantly higher on the first postoperative day than the preoperative value. There were no significant differences between two groups as for complements activation and PNEL activity. The total number of white blood cells unchanged during CPB and neutrophilia appeared after CPB, but no significant difference between two groups. Superoxide production of neutrophils relatively decreased during CPB and significantly increased after CPB in the control group. However, in PGE1 group superoxide production was reduced after CPB, especially on the first postoperative day. These results showed that PGE1 reduced neutrophil-mediated superoxide production in open heart surgery. In conclusions, PGE1 is useful agent to reduce the hazardous effects of neutrophils after CPB.

Adult↗
PubMed1998

Cloning and characterization of rat C3a receptor: differential expression of rat C3a and C5a receptors by LPS stimulation.

The anaphylatoxin C3a, generated during complement activation, is a factor known to mediate various inflammatory reactions. The human C3a receptor (C3aR) was recently cloned and identified to be a member of the G-protein-coupled receptor family. C3aR is characterized by seven transmembrane domains including a large second extracellular loop that appears to be a unique feature of this receptor. Here we report the isolation of the rat C3aR clone and confirm that the isolated cDNA coded for rat C3aR based on C3a binding analysis to stably transfected cells. Northern blot analysis of rat C3aR revealed expression in various tissues, similar to that of human C3aR but dissimilar to rat C5aR. We found that expression of rat C3aR in various tissues did not increase significantly after LPS injection, whereas rat C5aR expression is greatly increased. These results suggest that expression of C3aR and C5aR is independently regulated in rat cells and tissues.

Amino Acid Sequence↗
PubMed1999

Biocompatibility of a silicone-coated polypropylene hollow fiber oxygenator in an in vitro model.

BACKGROUND: A silicone-coated microporous hollow-fiber membrane oxygenator has been developed to prevent plasma leakage during long-term use. The objective of this study was to evaluate the biocompatibility of the oxygenator. METHODS: A silicone-coated oxygenator was compared with an uncoated oxygenator in an in vitro model of cardiopulmonary bypass. Simulated circulation was maintained for 6 h at 37 degrees C. RESULTS: Platelet counts decreased significantly (p < 0.05) and leukocyte counts tended to decline; however, the differences between groups were not significant. Concentrations of C3a increased significantly in both groups (p < 0.05), but levels were significantly less in the silicone-coated oxygenator (p = 0.008). In contrast, concentrations of C4a, beta-thromboglobulin, and granulocyte elastase increased significantly (p < 0.05), but the differences between groups were not significant. CONCLUSIONS: Silicone coating over a microporous hollow-fiber membrane may improve biocompatibility by reducing C3a activation.

Coated Materials, Biocompatible↗
PubMed1993

Increase in prostanoid formation in rat liver macrophages (Kupffer cells) by human anaphylatoxin C3a.

Human anaphylatoxin C3a increases glycogenolysis in perfused rat liver. This action is inhibited by prostanoid synthesis inhibitors and prostanoid antagonists. Because prostanoids but not anaphylatoxin C3a can increase glycogenolysis in hepatocytes, it has been proposed that prostanoid formation in nonparenchymal cells represents an important step in the C3a-dependent increase in hepatic glycogenolysis. This study shows that (a) human anaphylatoxin C3a (0.1 to 10 micrograms/ml) dose-dependently increased prostaglandin D2, thromboxane B2 and prostaglandin F2 alpha formation in rat liver macrophages (Kupffer cells); (b) the C3a-mediated increase in prostanoid formation was maximal after 2 min and showed tachyphylaxis; and (c) the C3a-elicited prostanoid formation could be inhibited specifically by preincubation of C3a with carboxypeptidase B to remove the essential C-terminal arginine or by preincubation of C3a with Fab fragments of a neutralizing monoclonal antibody. These data support the hypothesis that the C3a-dependent activation of hepatic glycogenolysis is mediated by way of a C3a-induced prostanoid production in Kupffer cells.

Animals↗
PubMed1988

Demonstration of a specific C3a receptor on guinea pig platelets.

Guinea pig platelets reportedly contain receptors specific for the anaphylatoxin C3a based on both ligand-binding studies and functional responses. A portion of the human 125I-C3a that binds to guinea pig platelets is competitively displaced by excess unlabeled C3a; however, the majority of ligand uptake was nonspecific. Uptake of 125I-C3a by guinea pig platelets is maximal in 1 min, and stimulation of guinea pig platelets by thrombin, ADP, or the Ca2+ ionophore A23187 showed little influence on binding of the ligand. Scatchard analysis indicated that approximately 1200 binding sites for C3a exist per cell with an estimated Kd of 8 x 10(-10) M. Human C3a des Arg also binds to guinea pig platelets, but Scatchard analysis indicated that no specific binding occurred. Because the ligand-binding studies were complicated by high levels of nonspecific uptake, we attempted to chemically cross-link the C3a molecule to a specific component on the platelet surface. Cross-linkage of 125I-C3a to guinea pig platelets with bis(sulfosuccinimidyl)suberate revealed radioactive complexes at 105,000 and 115,000 m.w. on SDS-PAGE gels by autoradiographic analysis. In the presence of excess unlabeled C3a, complex formation was inhibited. No cross-linkage could be demonstrated between the inactive 125I-C3a des Arg and the putative C3a-R on guinea pig platelets. Human C3a, but not C3a des Arg induces serotonin release and aggregation of the guinea pig platelets. Human C3a was unable to induce either serotonin release or promote aggregation of human platelets. Uptake of human 125I-C3a by human platelets was not saturable, and Scatchard analysis was inconclusive. Attempts to cross-link 125I-C3a to components on the surface of human platelets also failed to reveal a ligand-receptor complex. Therefore, we conclude that guinea pig platelets have specific surface receptors to C3a and that human platelets appear devoid of receptors to the anaphylatoxin.

Anaphylatoxins↗
PubMed1998

Murine macrophage behavior on peptide-grafted polyethyleneglycol-containing networks.

Polyethyleneglycol-based networks were employed as substrates to graft bioactive peptides to study macrophage interactions with materials. Our overall objective was to utilize biologically active factors to stimulate certain macrophage function on materials suitable for implantation in connective tissues. In this study, we sought to explore the bioactivity of several peptides derived from extracellular matrix adhesion proteins and macrophage-active proteins that are normally soluble. The candidate peptides examined corresponded to residues 63 to 77 of complement component C3a (C3a(63-77)), residues 178 to 207 of interleukin-1 beta (IL1beta(178-207)), residues 1615 to 1624 of fibronectin (FN(1615-1624)), endothelial-macrophage activating polypeptide II, complement component C5a inhibitory sequence, macrophage inhibitory peptide, and YRGDG; materials lacking peptides were used as negative controls. An established murine cell-line IC-21 was employed as a macrophage model, and human dermal fibroblasts were used for comparison. Our results showed that the substrates without grafted peptides were free from artifactual cell adhesion associated with the adsorption of serum or cellularly secreted proteins for long duration of culture. Of all grafted samples, IL1beta(178-207)- and C3a(63-77)-grafted surfaces supported higher adherent macrophage densities. C3a(63-77)- and FN(1615-1624)-grafted surfaces supported higher adherent fibroblast densities. From competitive inhibition studies, cell adhesion was determined to occur in a receptor-peptide specific manner. The presence of grafted YRGDG in addition to IL1beta(178-207), C3a(63-77), or FN(1615-1624) synergistically increased macrophage and fibroblast adhesion. Materials grafted with IL1beta(178-207) or C3a(63-77) co-grafted with or without YRGDG did not support the formation of multinucleated giant cells from the fusion of adherent macrophages in vitro.

Amino Acid Sequence↗
PubMed1987

Solution conformation of carboxy-terminal fragments of the third component of human complement C3: proton nuclear magnetic resonance study of C3a, des-Arg-C3a, and C3a Arg69.

A proton nuclear magnetic resonance (NMR) study is reported of the solution conformation of human C3a, that is released on activation of C3, the third component of complement. The intact C3a was used along with des-Arg-C3a, which is formed on cleavage of Arg-77 at the C terminal of C3a, and C3a Arg69, which is a 69-residue fragment produced on tryptic digestion of C3. A method of carboxypeptidase digestion/difference spectroscopy (Endo & Arata (1985) Biochemistry 24, 1561-1568) was extensively used for the spectral assignments of Ile-43, Ile-60, Leu-63, Tyr-15, and Tyr-59. On the basis of the results of nuclear Overhauser effect (NOE) measurements, we discuss the solution conformation of the C3a molecule. It has been concluded that removal of Arg-77, which is essential for expression of the biological activity of C3a, does not induce any significant change in the solution conformation of the C3a molecule. The C3a molecule is known to consist of a core region that comprises segment Tyr-15-Tyr-59. We conclude that in solution the C terminal segment sticks out of the core and takes on a helix-like conformation. Possible roles of the core region and the N terminal segment in maintaining the conformation of the C terminal segment are briefly discussed.

Amino Acid Sequence↗
PubMed1997

Synthetically modified cellulose: an alternative to synthetic membranes for use in haemodialysis?

Renal replacement therapy relies predominantly on the use of cellulose-based membranes. Such membranes have a biocompatibility profile which is inferior to membranes manufactured from synthetic polymers. Synthetically modified cellulose (SMC) is a new, low-flux haemodialysis membrane in which hydroxyl groups have been replaced with benzyl groups. The biocompatibility profile characterized by changes in white cell and platelet counts and the activation of complement components (C3a, C5a and C5b-9) have been studied in vivo and compared with those of cellulose acetate, unmodified cellulose (Cuprophan ) and low-flux polysulphone (Fresenius Polysulfone) in the same group of patients. For SMC, the white cell count at 15 min declined to 65.6% of pretreatment level, compared with 63.8% for the cellulose acetate, 79.6% for low-flux polysulphone and 28.1% for Cuprophan, thereafter returning to pretreatment levels. Both modified cellulose membranes were superior to unmodified cellulose (P = 0.001); the differences between the modified cellulose membranes were not significant statistically. The changes induced by all three cellulose-based membranes exceeded those for low-flux polysulphone (P = 0.001). Associated with the neutropenia was a reduction in platelet count, but this was independent of membrane type. The mean time-averaged concentrations of C3a(des Arg) over 150 min were 1168 ng ml(-1) (SMC), 1030 ng ml(-1) (cellulose acetate), 1297 ng ml(-1) (Cuprophan) and 790 ng ml(-1) (low-flux polysulphone). Equivalent values for C5a(des Arg) were 6.12 (SMC), 2.98 (cellulose acetate), 11.03 (Cuprophan) and 1.33 ng ml(-1) (low-flux polysulphone). C5b-9 values were 385 (SMC), 386 (cellulose acetate), 177 (Cuprophan) and 185 ng ml(-1) (low-flux polysulphone). For each of the complement components the differences between the membranes were significant [P = 0.0009 (C3a(des Arg)), P = 0.0001 (c5a(des Arg) and C5b-9)]. The levels of C5b-9 generated during dialysis also showed a significant positive correlation compared to C5a for all membranes considered as a single group (Pearson's correlation coefficient = 0.870, P = 0.0001). It is concluded that the modification of the cellobiosic unit is a promising approach to improve the biocompatibility profile of cellulose-based membranes. The two different methods of modification lead to similar improvements in biocompatibility compared with unmodified cellulose, but as yet do not match that of low-flux polysulphone.

Biocompatible Materials↗
PubMed1993

Expression of biologically active C3a as fusion proteins.

We selected three kinds of plasmids for expression of C3a as fusion proteins. The proteins were purified by affinity chromatography using the respective specific resins, and their activities were measured by guinea pig platelet aggregation. We showed that polyhistidine (polyHis)-C3a fusion protein was able to exhibit 30% of the activity of natural C3a. However, glutathione S-transferase (GST)-C3a fusion protein exhibited only 10% of such activity, and no activity was measured for maltose binding protein (MBP)-C3a fusion protein. The purified polyHis-C3a fusion protein was attached to the Ni-NTA agarose column in an attempt to isolate the C3a receptor from guinea pig platelets. The C3a binding protein isolated from digitonin-solubilized guinea pig platelet membrane was approximately 50 kDa on SDS-polyacrylamide gel. This is the first report of C3a fusion protein production with biological activity.

Adenosine Triphosphate↗
PubMed1997

Blood- and skin-derived monocytes/macrophages respond to C3a but not to C3a(desArg) with a transient release of calcium via a pertussis toxin-sensitive signal transduction pathway.

Controversial results have been published in the past regarding the functional reactivity of monocytes (Mo) and macrophages (M phi) to the anaphylatoxin C3a and its degradation product C3a(desArg). In this study we performed binding and calcium mobilization experiments with recombinant human C3a (rC3a) and rC3a(desArg). Blood Mo displayed non-inhibitable binding of FITC-labeled rC3a (rC3aFITC) but responded to rC3a with a transient release of the intracellular calcium concentration ([Ca2+]i), whereas rC3a(desArg) was completely inactive. In contrast, binding of rC3aFITC to eosinophilic granulocytes and the mast cell line HMC-1 which have been shown previously to express C3a binding sites could be blocked by a monoclonal anti-C3a antibody. The rC3a-induced [Ca2+]i release in blood Mo was pertussis toxin (PTX)-sensitive suggesting the involvement of G-proteins in the signal transduction pathway. Skin-derived Mo/M phi reacted similarly to blood Mo as no specific binding of rC3aFITC to these cells could be demonstrated, whereas an intracellular release of calcium ions in response to the anaphylatoxin was observed. Homologous desensitization to rC3a but not heterologous desensitization to rC5a was detected in further experiments. The functional effect of C3a, but not the unspecific binding of rC3aFITC to blood Mo and skin-derived Mo/M phi could be blocked by the monoclonal anti-C3a antibody. These results suggest the expression of the recently cloned G-protein-coupled receptor for C3a on human blood Mo and skin-derived Mo/M phi. However, the total number of specific C3a binding sites on these cells is distinctly lower as compared to eosinophilic granulocytes and cells of the mast cell line HMC-1. The small number of C3a receptors on Mo/M phi may be masked by a pronounced non-inhibitable binding of rC3aFITC. This binding, however, may contribute to the recently described biological effects of C3a(desArg) on Mo.

Arginine↗
PubMed1999

Complement and contact activation related to surfactant response in respiratory distress syndrome.

The activation of inflammation and coagulation cascades is part of the pathogenesis of adult respiratory distress syndrome (RDS). Previous studies have demonstrated contact activation in preterm infants with RDS, whereas no concordant results have been found with complement activation. In this study, both systems were investigated in preterm infants with severe RDS and related to surfactant response. Thirty preterm newborns with severe respiratory distress (FiO2 > 0.5), but with no evidence of infection or fetal acidosis, were studied. Eighteen healthy preterm newborns of similar gestational age and birth weight served as controls. The study group was divided into two subgroups, according to their response to a porcine natural surfactant 6 h after administration: responders (FiO2 reduction > 50%) and poor responders (FiO2 reduction < or = 50%). C1q, C4, factor B, C3a, C5a, complement, and C1-inhibitor activity, as well as factor XIIa, were determined in blood samples, drawn 24 h after birth. Except for C1-inhibitor concentration and C1-inhibitor activity, all parameters for infants with severe RDS were different from controls. Complement precursor proteins were lower, and activated split products of the complement and contact system were higher. Infants with a poor response after application of surfactant showed higher amounts of C3a, C5a, and factor XIIa but lower C1q and C4 levels compared with infants with a good response to surfactant. Activation of the complement and the contact system was demonstrated in all respiratory distress patients. This activation was more pronounced in poor responders to exogenous surfactant.

Complement Activation↗
PubMed1997

The human C3a receptor is expressed on neutrophils and monocytes, but not on B or T lymphocytes.

The pathophysiological relevance of the complement split product C3a as a proinflammatory mediator is still ill defined. The expression pattern of the human C3a receptor (C3aR) can provide important clues for the role of this anaphylatoxin in inflammation. There is strong evidence for C3aR expression on basophils, and eosinophils, but additionally, only on tumor cell lines of leukemic or hepatic origin. It is unclear whether neutrophils also express the C3aR, but need a costimulus provided by eosinophils for certain biological responses, or whether neutrophils lack the C3aR and respond to C3a via a secondary stimulus generated by eosinophils, i.e., by an indirect mode. In the present study, polyclonal antiserum raised against the second extracellular loop of the C3aR was used to characterize C3aR expression on peripheral blood leukocytes. For high degree purification of neutrophils, a negative selection method was established that decreased the contamination with CD9(bright+) eosinophils down to <0.2%. Flow cytometric analyses, functional assays, and binding assays on highly purified neutrophils confirmed C3aR expression and coupling. Monocytes were identified as an additional C3aR-positive cell population of the peripheral blood. The expression of the C3aR on eosinophils could be confirmed. In contrast, the receptor could not be detected on unchallenged B or T lymphocytes (or lymphocyte-derived Raji cells).

Animals↗