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PubMed2001

Human serum-induced expression of E-selectin on porcine aortic endothelial cells in vitro is totally complement mediated.

BACKGROUND: Whereas complement is a key mediator of hyperacute xenograft rejection, its role in acute vascular rejection (AVR) is a matter of controversy. AVR is associated with de novo synthesis of endothelial cell-derived inflammatory mediators, including the leukocyte-recruiting adhesion molecule E-selectin. Here we investigate the role and mechanism of complement in human serum-induced porcine endothelial cell activation. METHODS: An in vitro xenotransplantation method was designed using porcine aortic endothelial cells stimulated with human serum in microculture wells. E-selectin expression was measured by cell-enzyme immunoassay. Complement inhibitors acting at different levels in the cascade were investigated for their effect on E-selectin expression. RESULTS: E-selectin was strongly induced by normal human serum but not by heat-inactivated serum. Compstatin, a synthetic C3 inhibitor, markedly reduced human serum-induced E-selectin expression. Purified C1-inhibitor suppressed E-selectin induction completely, indicating activation through the classical or lectin pathway. Furthermore, a monoclonal antibody (mAb) that inhibits cleavage of C5 or another mAb that blocks the function of C7, completely inhibited the expression of serum-induced E-selectin, consistent with the terminal C5b-9 complement complex being the mediator of the endothelial cell activation. Inhibition of the alternative pathway using a novel antifactor D mAb did not reduce E-selectin expression. CONCLUSION: Human serum-induced expression of porcine E-selectin is totally complement dependent, induced by a C1-inhibitor regulated pathway and mediated through the terminal complement complex. The data may have implications for therapeutic strategies, particularly of C1-inhibitor and anti-C5 mAb, to protect against endothelial cell activation and subsequent AVR of porcine xenografts.

Animals↗

PubMed1991

Ultrastructural and biochemical observations on antineutrophil antibody- and complement-induced immuno-injury to equine neutrophils.

Antibody-induced damage to neutrophils was studied to elucidate processes associated with destruction of neutrophils in immune-mediated neutropenias. Cytomorphological changes and release of certain cellular constituents were determined for neutrophils treated with an antineutrophil serum in the presence or absence of rabbit complement. Neutrophils exposed to the antineutrophil serum alone showed endocytotic vacuoles and degranulation. In contrast, neutrophils exposed to the antineutrophil serum and complement showed marked morphologic changes. The plasma membrane developed numerous vesicles, villous processes and minute areas of bilayer discontinuity. Highly damaged cells exhibited cellular and nuclear swellings, disruption of cytoplasmic integrity and disordered distribution of lysosomal granules. Cytoplasmic constituents (K+ and lactate dehydrogenase) were released extracellularly from neutrophils exposed to the antineutrophil serum with or without complement. Cytological changes induced by the antineutrophil serum and complement were analogous to those reported for leucocytes exposed to the activated complement components C5b-9 (the membrane attack complex) and bacterial toxins. It was concluded that the cytological abnormalities observed were most probably associated with immune-mediated damage to the cell membrane, leading to leakage of cytoplasmic constituents like K+, colloidal osmotic swelling, and disruption of the cytoskeletal system.

Alkaline Phosphatase↗

PubMed2004

A role for mannose-binding lectin dysfunction in generation of autoantibodies in systemic lupus erythematosus.

OBJECTIVE: To investigate the possible association of the mannose binding lectin (MBL) pathway of complement activation with different disease parameters and disease activity in patients with systemic lupus erythematosus (SLE). METHODS: MBL genotype, MBL serum concentration, MBL complex activity and MBL pathway activity were assessed in 53 patients. The activity of the MBL-MASP complex was assessed on the basis of its ability to activate exogenous C4. For MBL pathway activity the formation of the terminal complex of complement activation (C5b-9) was measured. Results were analysed in relation to clinical variables and autoantibody profiles in these patients. RESULTS: MBL complex activity and MBL pathway activity were both reduced in patients carrying MBL variant alleles. Anticardiolipin and anti-C1q autoantibodies were observed significantly more frequently in patients with MBL variant alleles. Furthermore, the presence of these autoantibodies was associated with a decreased MBL concentration and function. In contrast, anti-MBL autoantibodies were not found in patients with MBL variant alleles, possibly related to impaired binding of variant MBL to apoptotic material. CONCLUSION: In patients with SLE, a reduced functional activity of the MBL pathway of complement, in relation to expression of MBL variant alleles, is associated with increased levels of autoantibodies against cardiolipin and C1q, but not against MBL. We hypothesize that an enhanced production of autoantibodies may be related to disturbed clearance of apoptotic material due to impaired MBL function.

Adult↗

PubMed2002

Role of the human C8 subunits in complement-mediated bacterial killing: evidence that C8 gamma is not essential.

Human C8 is one of five complement components (C5b, C6, C7, C8 and C9) that interact to form the cytolytic membrane attack complex (MAC) on bacterial cell membranes. It is an oligomeric protein composed of a disulfide-linked C8 alpha-gamma heterodimer and a non-covalently associated C8 beta chain. Previous studies revealed that C8 alpha and C8 beta have distinct roles in the formation of the MAC on simple cells such as erythrocytes and that both subunits are essential for cell lysis. These studies also determined that C8 gamma is not required for expression of MAC hemolytic activity. To determine if these conclusions are applicable to more biologically relevant systems, the C8 subunits were examined for their ability to support complement-mediated killing of Gram-negative bacteria. Results indicate: (1) C8 alpha-gamma, C8 alpha, C8 beta and C8 gamma have no independent bactericidal activity; (2) bacterial killing requires C8 beta and either C8 alpha-gamma or C8 alpha; (3) C8 alpha is an effective substitute for C8 alpha-gamma in bacterial killing; and (4) C8 gamma enhances, but is not required for C8 bactericidal activity. Together, these data suggest that C8 alpha and C8 beta have correspondingly similar roles in MAC-mediated lysis of erythrocytes and bacterial killing. Furthermore, they provide the first direct evidence that C8 gamma is not required for complement-mediated killing of Gram-negative bacteria.

Blood Bactericidal Activity↗

PubMed2004

The study of mitoxantrone as a potential immunosuppressor in transgenic pig renal xenotransplantation in baboons: comparison with cyclophosphamide.

Mounting evidence suggests that delayed xenograft rejection (DXR) of discordant xenografts has a strong humoral component. To explore the possibility of targeting this humoral response more efficiently, we performed a preliminary study in baboons immunized against pig blood cells using the immunosuppressor mitoxantrone (Mx). The results from this study showed that, in comparison with cyclophosphamide (CyP), Mx induced a long-lasting depletion of circulating B cells within 6 days of its administration and delayed secondary anti-Gal antibody (Ab) responses to pig blood cell immunizations. Given these results, we next evaluated Mx in an in vivo model of pig to baboon renal xenotransplantation. We performed a series of renal xenotransplantations in baboons using human CD55-CD59 transgenic donor pigs. In the first group of baboons (Mx group; n = 4) Mx was administered 6 days prior to the day of transplantation, the objective being to perform the xenotransplantation in a context where the recipient would have few remaining circulating B cells and thus have an impaired capacity to mount an Ab response to the xenograft. We compared this group to a second group of baboons treated with CyP starting 1 day prior to transplantation (CyP group; n = 2). All baboons receiving Mx or CyP received an additional immunosuppression of cyclosporin A, mycophenolate mofetil and steroids. No hyperacute rejection was observed in either group but all xenografts underwent DXR. Mx did not show superiority to CyP in terms of graft survival with a mean survival time of 8 +/- 2 days compared with 9 days for both CyP-treated baboons. Neither CyP nor Mx decreased serum levels of pre-existing anti-Gal Abs but levels of these Abs decreased dramatically within 1 day of transplantation, likely reflecting their immediate trapping within the xenograft. Interestingly however, in contrast to CyP, Mx inhibited the return of anti-Gal immunoglobulin M (IgM) to the circulation, even at the time of rejection. Nevertheless, strong intragraft deposits of IgM, IgG and the activated complement complex C5b-9 were observed in biopsies at rejection. Furthermore, despite the expected profound depletion of circulating B cells by Mx within 6 days of its administration, biopsies from both groups at rejection displayed a mild B cell infiltrate accompanied by a strong macrophage and intermediate T-cell infiltration, the latter tending to be more abundant in Mx-treated animals. Our data show that in this particular model of pig to baboon xenotransplantation and at the dose used, Mx was not superior to CyP in conferring protection against rejection, despite its capacity to profoundly deplete circulating B cells and to inhibit anti-Gal Ab responses to xenografts. DXR was thus possible without the return of anti-Gal Abs and may have been mediated by the early fixation of pre-existing Abs with secondary complement activation. However, although Mx was not more efficient than CyP in controlling DXR, its capacity to deplete B cells and delay Ab recovery may be beneficial in the context of Gal knockout organ transplantation where the induced Ab response is likely to take precedence over the preformed response.

Animals↗

PubMed2006

Antagonism of PDGF-D by human antibody CR002 prevents renal scarring in experimental glomerulonephritis.

Glomerular mesangial cell proliferation and/or matrix accumulation characterizes many progressive renal diseases. PDGF-D was identified recently as a novel mediator of mesangial cell proliferation in vitro and in vivo. This study investigated the long-term consequences of PDGF-D inhibition in vivo. Rats with progressive mesangioproliferative glomerulonephritis (uninephrectomy plus anti-Thy-1.1 antibody) received the PDGF-D-neutralizing, fully human mAb CR002 on days 3, 10, and 17 after disease induction. Glomerular mesangioproliferative changes on day 10 were significantly reduced by anti-PDGF-D treatment as compared with control antibody. Eight weeks after disease induction, anti-PDGF-D therapy significantly ameliorated focal segmental glomerulosclerosis, podocyte damage (de novo desmin expression), tubulointerstitial damage, and fibrosis as well as the accumulation of renal interstitial matrix including type III collagen and fibronectin. Treatment with anti-PDGF-D also reduced the cortical infiltration of monocytes/macrophages on day 56, possibly related to lower renal cortical complement activation (C5b-9 deposition) and/or reduced epithelial-to-mesenchymal transition (preserved cortical expression of E-cadherin and reduced expression of vimentin and alpha-smooth muscle actin). In conclusion, these data provide evidence for a causal role of PDGF-D in the pathogenesis of renal scarring and point to a new therapeutic approach to progressive mesangioproliferative renal disease.

Animals↗

PubMed1999

C6 produced by macrophages contributes to cardiac allograft rejection.

The terminal components of complement C5b-C9 can cause significant injury to cardiac allografts. Using C6-deficient rats, we have found that the rejection of major histocompatibility (MHC) class I-incompatible PVG.R8 (RT1.A(a)B(u)) cardiac allografts by PVG.1U (RT1.A(u)B(u)) recipients is particularly dependent on C6. This model was selected to determine whether tissue injury results from C6 produced by macrophages, which are a conspicuous component of infiltrates in rejecting transplants. We demonstrated that high levels of C6 mRNA are expressed in isolated populations of macrophages. The relevance of macrophage-produced C6 to cardiac allograft injury was investigated by transplanting hearts from PVG. R8 (C6-) donors to PVG.1U (C6-) rats which had been reconstituted with bone marrow from PVG.1U (C6+) rats as the sole source of C6. Hearts grafted to hosts after C6 reconstitution by bone marrow transplantation underwent rejection characterized by deposition of IgG and complement on the vascular endothelium together with extensive intravascular aggregates of P-selectin-positive platelets. At the time of acute rejection, the cardiac allografts contained extensive perivascular and interstitial macrophage infiltrates. RT-PCR and in situ hybridization demonstrated high levels of C6 mRNA in the macrophage-laden transplants. C6 protein levels were also increased in the circulation during rejection. To determine the relative contribution to cardiac allograft rejection of the low levels of circulating C6 produced systemically by macrophages, C6 containing serum was passively transferred to PVG.1U (C6-) recipients of PVG.R8 (C6-) hearts. This reconstituted the C6 levels to about 3 to 6% of normal values, but failed to induce allograft rejection. In control PVG.1U (C6-) recipients that were reconstituted with bone marrow from PVG.1U (C6-) donors, C6 levels remained undetectable and PVG.R8 cardiac allografts were not rejected. These results indicate that C6 produced by macrophages can cause significant tissue damage.

Animals↗

PubMed1997

Effects of cardiopulmonary bypass on neonatal and paediatric inflammatory profiles.

OBJECTIVE: Cardiopulmonary bypass (CPB) causes significant morbidity in paediatric patients, yet the mechanisms involved in the related inflammatory processes (resulting in capillary leak and edema) are poorly understood. Moreover, earlier palliative and corrective intervention in neonates and infants has provided the cohorts of patients about whom little is known of their proinflammatory response. METHODS: In the present two group study, 14 neonates (age 1-28 days, 2.5-4.5 kg) and 13 infants (2-12 months, 3-7 kg), undergoing CPB for congenital heart disease were consecutively recruited. The two cohorts were well matched in terms of CPB and aortic cross-clamp times (P > 0.1). Blood samples were collected on induction of anaesthesia, 5 min following onset of CPB, at the end of CPB, and 30 min, 2 and 24 h post-protamine (PP) administration. Plasma concentration of cytokines interleukin-6 (IL-6) and interleukin-8 (IL-8), terminal complement complex (C5b-9) neutrophil counts and leucocyte elastase were measured. RESULTS: Plasma levels of all inflammatory markers significantly increased in both groups during and following CPB as compared to baseline. During and following CPB the change in IL-8 level was more pronounced in neonates (peak 30 min PP, median(range): 1062 (182-3872) pg/ml) than in infants 568 (172-1368) pg/ml), P = 0.01. Changes in IL-6 level were indistinguishable between groups intraoperatively, but remained significantly higher at 24 h in neonates (P = 0.02). Peri and postoperative levels of C5b-9 were significantly higher in infants than in neonates (peak 30 min PP, median (range): 984 (118-1142) ng/ml vs 458 (22 1340) ng/ml in neonates respectively, P = 0.01) but were similar at 24 h. Despite this, leucocyte elastase profiles did not differ significantly between the respective cohorts. CONCLUSION: These results indicate that there may be differences between neonates and infants with regard to the inflammatory response to CPB and neonatal patients merit further investigation in order to elucidate whether the pathophysiology of their CPB related inflammatory response and its clinical sequelae differs from their older counterparts.

Cardiopulmonary Bypass↗

PubMed2000

Biocompatibility of silicone-coated oxygenator in cardiopulmonary bypass.

BACKGROUND: This study was designed to analyze the biocompatibility of silicone-coated oxygenators using inflammatory response as the outcome measure, and to investigate whether the silicone-coated oxygenators perform better in terms of postoperative organ dysfunction. METHODS: The 32 patients who underwent cardiopulmonary bypass (CPB) were divided into 3 groups: group A (n = 10), heparin-coated circuit with silicone-coated oxygenator; group B (n = 11), whole heparin-coated circuit; and group C (n = 11), whole untreated circuit. The plasma concentrations of the proinflammatory markers, made of inflammatory cytokines (tumor necrosis factor-alpha, interleukin-1beta, interleukin-6, interleukin-8), terminal complement complex (C5b-9), and polymorphonuclear elastase (PMN-E), were measured by enzyme-linked immunosorbant assay. RESULTS: All proinflammatory markers were significantly lower in groups A and B than in group C, especially C5b-9 and PMN-E concentrations, which were significantly lower in group A than in group B. The alveolar-arterial oxygen gradients (A-aDO2) and the respiratory index were significantly better in group A than in group C. In group B, however, only the A-aDO2 was significantly better than in group C. The duration of intubation and the length of stay in the intensive care unit stay were significantly shorter in groups A and B than in group C. CONCLUSIONS: Silicone-coated oxygenators are biocompatible and prevent postoperative organ dysfunction.

Anticoagulants↗

PubMed1997

A flow cytometric immunoassay to quantify adsorption of complement activation products (iC3b, C3d, SC5b-9) on artificial surfaces.

Crosslinked agarose microspheres and various polystyrene microspheres were analyzed for complement components after incubation with serum at 37 degrees C for times up to 2 h. Quantification involved direct flow cytometric analysis of the beads after the bound complement proteins were indirectly fluorescently tagged by use of a monoclonal antibody against a complement protein: C5b-9, iC3b, C3d, C4d, Bb, C3a, and C1q. Calibration with fluorescein microbead standards demonstrated that the membrane attack complex (SC5b-9) was surface bound on all surfaces and that the surface concentration gradually increased to levels as high as 0.5 micrograms/cm2. Further, the surface bound represented a substantial percentage of the total generated. The iC3b level on polystyrene beads rapidly reached 0.09 micrograms/cm2 and the C3d levels were an order of magnitude less. On agarose beads the iC3b levels continually rose to 0.17 micrograms/cm2 and, as before, the C3d levels were substantially lower. The surface concentration of C4d and Bb on both surfaces were significant but less than 1.0 ng/cm2. There was minimal evidence of C3a and C1q adsorption for any surface. Use of amino-polystyrene beads moderately reduced the level of bound iC3b, C3d, and SC5b-9, whereas carboxylated beads reduced the levels by almost a factor of two. The appreciable amounts of iC3b and SC5b-9 consistently noted on the artificial surfaces tested in this paper suggests that for these two activation products in vitro analysis of material induced complement activation should also include surface analysis.

Adsorption↗

PubMed1987

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.

Binding Sites↗

PubMed1989

Assembly of the platelet prothrombinase complex is linked to vesiculation of the platelet plasma membrane. Studies in Scott syndrome: an isolated defect in platelet procoagulant activity.

Activation of human platelets by complement proteins C5b-9 is accompanied by the release of small plasma membrane vesicles (microparticles) that are highly enriched in binding sites for coagulation factor Va and exhibit prothrombinase activity. We have now examined whether assembly of the prothrombinase enzyme complex (factors VaXa) is directly linked to the process of microparticle formation. Gel-filtered platelets were incubated without stirring with various agonists at 37 degrees C, and the functional expression of cell surface receptors on platelets and on shed microparticles was analyzed using specific monoclonal antibodies and fluorescence-gated flow cytometry. In addition to the C5b-9 proteins, thrombin, collagen, and the calcium ionophore A23187 were each found to induce formation of platelet microparticles that incorporated plasma membrane glycoproteins GP Ib, IIb, and IIIa. These microparticles were enriched in binding sites for factor Va, and their formation paralleled the expression of catalytic surface for the prothrombinase enzyme complex. Little or no microparticle release or prothrombinase activity were observed when platelets were stimulated with epinephrine and ADP, despite exposure of platelet fibrinogen receptors by these agonists. When platelets were exposed to thrombin plus collagen, the shed microparticles contained activated GP IIb-IIIa complexes that bound fibrinogen. By contrast, GP IIb-IIIa incorporated into C5b-9 induced microparticles did not express fibrinogen receptor function. Platelets from a patient with an isolated defect in inducible procoagulant activity (Scott syndrome) were found to be markedly impaired in their capacity to generate microparticles in response to all platelet activators, and this was accompanied by a comparable decrease in the number and function of inducible factor Va receptors. Taken together, these data indicate that the exposure of the platelet factor Va receptor is directly coupled to plasma membrane vesiculation and that this event can be dissociated from other activation-dependent platelet responses. Since a catalytic membrane surface is required for optimal thrombin generation, platelet microparticle formation may play a role in the normal hemostatic response to vascular injury.

Antibodies, Monoclonal↗

PubMed1991

Clusterin (complement lysis inhibitor) forms a high density lipoprotein complex with apolipoprotein A-I in human plasma.

Clusterin/human complement lysis inhibitor (CLI) is incorporated stoichiometrically into the soluble terminal complement complex and inhibits the cytolytic reaction of purified complement components C5b-9 in vitro. Using an anti-clusterin affinity column, we found that an additional protein component with a molecular mass of 28-kDa co-purifies with clusterin from human plasma. We show by immunoblotting and amino acid sequencing that this component is apolipoprotein A-I (apoA-I). By using physiological salt buffers containing 0.5% Triton X-100, apoA-I is completely dissociated from clusterin bound to the antibody column. Free clusterin immobilized on the antibody-Sepharose selectively retains apoA-I from total human plasma. Delipidated apoA-I and to a lesser extent ultracentrifugation-purified high density lipoproteins (HDL) adsorbed to nitrocellulose also have a binding affinity for purified clusterin devoid of apoA-I. The isolated apoA-I-clusterin complex contains approximately 22% (w/w) lipids which are composed of 54% (mole/mol) total cholesterol (molar ratio of unesterified/esterified cholesterol, 0.58), 42% phospholipids, and 4% triglycerides. In agreement with the low lipid content, apoA-I-clusterin complexes are detected only in trace amounts in HDL fractions prepared by density ultracentrifugation. In free flow isotachophoresis, the purified apoA-I-clusterin complex has the same mobility as the native clusterin complex in human plasma and is found in the slow-migrating HDL fraction of fasting plasma. Our data indicate that clusterin circulates in plasma as a HDL complex, which may serve not only as an inhibitor of the lytic terminal complement cascade, but also as a regulator of lipid transport and local lipid redistribution.

Apolipoprotein A-I↗

PubMed1983

Interaction of human beta-endorphin with nonopiate binding sites on the terminal SC5b-9 complex of human complement. Significance of COOH-terminal beta H-endorphin fragments.

We have characterized the binding of 125I-labeled human beta-endorphin (125I-beta H-endorphin) to sites present on the terminal fluid-phase complex of human complement, consisting of complement components C5b, C6, C7, C8, C9, and the S-protein (SC5b-9 complex). Specific binding exhibited saturability, reversibility, structural specificity, temperature dependence, and absence of negative cooperative effects. Binding was maximal at 4 degrees C and pH 7.0; it was diminished by monovalent and divalent cations as well as by increasing concentrations of urea and Triton X-100 and apparently required intact disulfide groups. Binding was not inhibited by a number of opioid peptides sharing common sequences with the NH2 terminus of beta H-endorphin. In contrast, binding was inhibited by beta H-endorphin, N-acetyl-beta H-endorphin, and a series of COOH-terminal beta H-endorphin fragments, where of the COOH-terminal dipeptide Gly-Glu represented the minimal effective structure. Stepwise extension towards the NH2 terminus led to an increased binding affinity of the respective fragment. Computer resolution of competition curves yielded one binding component for several shorter COOH-terminal beta H-endorphin fragments and for beta H-endorphin (1-5) + (16-31), whereas two distinct binding components were obtained when beta H-endorphin (27-31), beta H-endorphin (6-31), N-acetyl-beta H-endorphin or beta H-endorphin were used as inhibitors. This study presents detailed data on the binding of COOH-terminal beta H-endorphin fragments to specific nonopiate binding sites present on the terminal SC5b-9 complex of human complement. We suggest that through this interaction, beta H-endorphin may modulate certain functions within the immune system.

Binding Sites↗

PubMed1993

Contribution of platelet microparticle formation and granule secretion to the transmembrane migration of phosphatidylserine.

Activation of human platelets by complement proteins, C5b-9, thrombin plus collagen, or a Ca2+ ionophore results in surface exposure of phosphatidylserine (PS), accompanied by the expression of membrane catalytic activity for the tenase (VIIaIXa) and prothrombinase (VaXa) coagulation enzyme complexes. The mechanism underlying this surface exposure of PS upon platelet activation remains unresolved. Using fluorescent derivatives of PS (NBD-PS), we have investigated how the transmembrane migration of PS is related to microvesiculation of the platelet plasma membrane and to fusion of storage granules with the plasma membrane. Gel-filtered platelets were incubated with NBD-PS, allowing 90 +/- 10% of the incorporated NBD-PS to accumulate into the inner leaflet of the plasma membrane. Migration of NBD-PS from the inner leaflet to the plasma membrane surface was monitored by time-based flow cytometry, and correlated with the appearance of platelet microparticles and alpha-granule secretion. Platelet activation by C5b-9 or the Ca2+ ionophore, A23187, increased surface exposure of NBD-PS, due to acceleration of the apparent rate of migration from inner to outer plasma membrane leaflets. The onset of this accelerated migration of NBD-PS to the surface coincided with the onset of plasma membrane vesiculation, and the NBD-PS that partitioned into the membrane of the shed microparticle was also rapidly exposed to the surface (t1/2 < 2 min). In addition to a temporal correlation, microparticle formation and the surface exposure of inner leaflet NBD-PS showed a similar requirement for Ca2+. These results demonstrate that agonist-induced microvesiculation of the platelet plasma membrane is accompanied by accelerated migration of a PS analogue from the inner leaflet to the surface of the shed microparticle membrane, suggesting the mechanism by which induction of platelet microparticle formation exposes catalytic surface for tenase and prothrombinase assembly.

Alkaloids↗

PubMed1997

Proinflammatory cytokine release during pediatric cardiopulmonary bypass: influence of centrifugal and roller pumps.

OBJECTIVE: It has been proposed that nonocclusive centrifugal pumps may elicit less blood cell trauma and hence a reduced inflammatory response than standard roller pumps. However, there have been no reports describing the impact of such pumps on proinflammatory cytokine release in pediatric cohorts. DESIGN: A prospective randomized study was undertaken. SETTING: A regional cardiothoracic center of a university hospital. PARTICIPANTS: Thirty-four pediatric patients undergoing cardiopulmonary bypass (CPB) for the correction of complex congenital heart defects were recruited. INTERVENTIONS: Either standard twin roller (n = 17), or centrifugal vortex (Biopump, Medtronic Biomedicus Inc, MN) (n = 17) blood pumping. MEASUREMENTS AND MAIN RESULTS: Venous blood was drawn (1) on induction of anesthesia, (2) 5 minutes on bypass, (3) end of CPB, (4) 30 minutes post-protamine, (5) 2 hours and (6) 24 hours postoperation. Neutrophil count, level of plasma leukocyte elastase, terminal complement complex (C5b-9); interleukin-6 (IL-6) and interleukin-8 (IL-8) were increased during and after CPB compared with the postinduction baseline. C5b-9 levels in both groups peaked at the end of CPB before returning to baseline at 24 hours: (median [range]), 564 (16 to 1,136) ng/mL in centrifugal group versus 508 (0 to 1,128) ng/mL in the roller group. IL-6 in both groups reached its peak level at 2 hours postprotamine (208 [98 to 411] pg/mL in centrifugal versus 205 [60-327] pg/mL in the roller group), before coming back to baseline at 24 hours. Plasma leukocyte elastase and IL-8 reached their maximum level 15 minutes after protamine administration: 215 (64 to 375) pg/mL in centrifugal versus 235 (87 to 410) pg/mL in roller group; and 700 (90 to 5,925) ng/mL versus 362 (120 to 3,400) ng/mL, respectively. CONCLUSIONS: The current study confirms the proinflammatory nature of pediatric CPB surgery, but failed to show a significant advantage of centrifugal pumping over roller perfusion in terms of the inflammatory response.

Cardiopulmonary Bypass↗

PubMed2002

Interaction between the C8 alpha-gamma and C8 beta subunits of human complement C8: role of the C8 beta N-terminal thrombospondin type 1 module and membrane attack complex/perforin domain.

Human C8 is one of five complement components (C5b, C6, C7, C8, and C9) that interact to form the cytolytic membrane attack complex (MAC). It is an oligomeric protein composed of a disulfide-linked C8alpha-gamma heterodimer and a noncovalently associated C8beta chain. C8alpha and C8beta are homologous; both contain an N-terminal thrombospondin type 1 (TSP1) module, a low-density lipoprotein receptor class A (LDLRA) module, an extended central segment referred to as the membrane attack/perforin (MACPF) domain, an epidermal growth factor (EGF) module, and a second TSP1 module at the C-terminus. In this study, the segment of C8beta that confers binding specificity toward C8alpha-gamma was identified using recombinant C8beta constructs in which the N- and/or C-terminal modules were deleted or exchanged with those from C8alpha. Constructs were tested for their ability to bind C8alpha-gamma in solution and express C8 hemolytic activity. Binding to C8alpha-gamma was found to be dependent on the TSP1 + LDLRA + MACPF segment of C8beta. Within this segment, the TSP1 module and MACPF domain are principally involved and act cooperatively to mediate binding. Results from activity assays suggest that residues within this segment also mediate binding and incorporation of C8 into the MAC.

Amino Acid Motifs↗

PubMed1995

Genomic organization of human complement protein C8 alpha and further examination of its linkage to C8 beta.

Human C8 is one of five complement components (C5b, C6, C7, C8, C9) that interact to form the cytolytic C5b-9 complex on target membranes. It is composed of three nonidentical subunits (C8 alpha, C8 beta, C8 gamma) encoded by separate genes. C8 alpha and C8 beta are linked on chromosome 1p32, whereas C8 gamma is located on 9q22.3-q32. In this study, overlapping genomic clones were isolated and used to decipher the organization of the human C8 alpha gene. The gene contains at least 11 exons spanning approximately 70 kb of DNA. When compared to C6, C8 beta and C9, there is a remarkable similarity in genomic organization, consistent with amino acid sequence comparisons that suggest these proteins are ancestrally related. Regions of each protein that are structurally similar are encoded in exons of correspondingly similar lengths with highly conserved boundaries and phases. Availability of genomic sequence also facilitated a more detailed analysis of C8 alpha and C8 beta linkage. Based on analysis of genomic digests with cDNA probes, the loci were previously reported to be physically linked (< 2.5 kb) and in a 5' alpha-beta 3' orientation. In the present study, results obtained using exon-specific probes indicate the loci are not as closely linked as initially believed. Furthermore, they suggest that cDNA probes used earlier yielded misleading information because they encode exons that are distributed across large segments of genomic DNA.

Amino Acid Sequence↗