Erythrocyte Sialic Acid Research Articles

Decreased erythrocyte binding of Siglec-9 increases neutrophil activation in sickle cell disease

Oxidative stress and inflammation promote vaso-occlusion in sickle cell disease (SCD). CD33-related Sialic acid-binding immunoglobulin-type lectins (CD33rSiglecs) are cell surface proteins that recognize sialic acids inhibit innate immune cell functions. We have shown that Siglec-9 on human neutrophils interact with erythrocyte sialic acids (prominently glycophorin-A (GYPA) to suppress neutrophil reactive oxygen species (ROS). We hypothesized that altered sickle erythrocyte membrane sialic acid leads to decreased Siglec-9 binding capability, and thus a decreased neutrophil oxidative burst. SS erythrocytes express significantly more sialic acid than AA erythrocytes (p = 0.02). SS erythrocytes displayed significantly less Siglec-9-Fc binding 39% ± 11 (mean ± SEM) compared to AA erythrocytes 78% ± 5 (p = 0.009). Treatment of AA erythrocytes with sialidase to remove sialic acid decreased binding to 3% ± 7.9 (p ≤ 0.001). When freshly isolated neutrophils were incubated with AA erythrocytes, neutrophils achieved 16% ± 6 of the oxidative burst exhibited by a stimulated neutrophil without erythrocytes. In contrast, neutrophils incubated with SS erythrocytes achieved 47% ± 6 of the oxidative burst (AA versus SS, p = 0.03). Stimulated neutrophils incubated with AA erythrocytes showed minimal NET formation while with SS erythrocytes NETs increased. SS erythrocytes are deficient in binding to neutrophil Siglec-9 which may contribute to the increased oxidative stress in SCD.

Decreased Erythrocyte Binding Capability for Neutrophil Siglec-9 Is a Source of Oxidative Stress in Sickle Cell Disease

IntroductionOxidative stress and inflammation promote hemolysis and vaso-occlusion in sickle cell disease (SCD). Erythrocytes can play a pro-oxidative and anti-oxidative role in disease-associated inflammation through iron-driven free radical production and endogenous anti-oxidants. In SCD, HbS accelerated auto-oxidation, iron de-compartmentalization, and inflammatory cell-derived oxidants drive this oxidative stress. CD33-related Sialic acid-binding immunoglobulin-type lectins (CD33rSiglecs) are cell surface proteins that recognize sialic acids in “Self-Associated Molecular Patterns” (SAMPs) and typically inhibit innate immune cell functions via cytosolic signaling. Recent studies have shown that Siglec-9 on human neutrophils in circulating blood interact with erythrocyte sialic acids (prominently glycophorin-A (GYPA) to suppress neutrophil reactivity, including reactive oxygen species (ROS) production. Modification of erythrocyte membrane sialic acids interferes with their ability to inhibit neutrophil activation and oxidative burst. As erythrocytes age and undergo cellular damage there is a loss of membrane bound sialoglycoproteins. Several studies have indicated that this reduction in the sialome is accelerated on the sickle erythrocyte. We hypothesize that altered sickle erythrocyte membrane sialic acid leads to decreased Siglec-9 binding capability, and that decreased binding of neutrophil Siglec-9 to sickle erythrocyte sialic acid enhances neutrophil activation and oxidative burst.Methods & ResultsBinding of recombinant Siglec-9-Fc protein to AA (n=9) and SS erythrocytes (n=7) was measured using flow cytometry. SS erythrocytes displayed significantly less Siglec-9-Fc binding 45% ± 11.9 (mean ± SEM) compared to AA erythrocytes 82% ± 5.4 (p=.03). Treatment of AA erythrocytes with neuraminidase to remove sialic acid decreased binding to 4% ± 7.9.Neutrophil ROS production was measured by flow cytometry using dihydrorhodamine 123 (DHR123). Neutrophils were purified from AA donors (n=5) and SS (n=11) or AA erythrocytes (n=5) were added to the neutrophils at a ratio of 50 erythrocytes to 1 neutrophil. Oxidative burst was stimulated using phorbol 12-myristate 13-acetate (PMA). AA erythrocytes decreased PMA-stimulated neutrophil ROS production by 84% ± 6.7. In contrast, SS erythrocytes decreased PMA-stimulated neutrophil ROS production by 53% ± 6.8 (p=0.03,).Recent studies have shown that neutrophil extracellular trap (NET) formation is pathogenic in SCD. We added AA and SS erythrocytes (50:1) to neutrophils stimulated with PMA and NET formation was assessed using Syto Orange, Syto Green and confocal microscopy. PMA-stimulated neutrophils incubated with AA erythrocytes showed minimal NET formation. In contrast, AA erythrocytes treated with neuraminidase to remove sialic acid had increased NET formation. PMA-stimulated neutrophils incubated with SS erythrocytes showed increased NET formation.ConclusionsA constant disease state of oxidative stress and inflammation underlies SCD pathophysiology. These data demonstrate that SS erythrocytes with decreased membrane sialic acid are deficient in binding to neutrophil Siglec-9. The decreased binding of SS erythrocytes to neutrophil Siglec-9 diminishes the ability of SS erythrocytes to properly modulate neutrophil activation, which may contribute to the oxidative stress and increased state of basal inflammation inherent to SCD. The overall reduction in number of RBCs in SS may also be a factor? [Display omitted] DisclosuresBelcher:CSL Behring: Research Funding. Vercellotti:CSL Behring: Research Funding.

Perturbations of erythrocyte membrane integrity by subchronic low-level lead exposure in New Zealand white rabbits

Lead (Pb) poisoning has been recognised as a clinical condition since ancient times. The anaemia frequently observed in Pb poisoning is thought to result from the shortening of erythrocytes, life span and inhibition of key enzymes of haem synthesis [viz. delta aminolevulinic acid dehydratase (ALAD), pyrimidine 5′-nucleotidase type 1 (P5N-1) and ferrochelatase]. However, the exact mechanism by which Pb shortens erythrocyte life span is still debatable. In the present study, the effect of ingested Pb on erythrocyte membrane sialic acid content and osmotic fragility of New Zealand White (NZW) rabbits was studied. Indices of Pb exposure, RBC counts, haemoglobin concentration (Hb), haematocrit (PCV), mean corpuscular volume (MCV), mean corpuscular haemoglobin (MCH) and mean corpuscular haemoglobin concentration (MCHC) of rabbits were investigated. Exposure to Pb significantly decreased (p < 0.05) RBC counts, Hb levels, MCV and MCH. Similarly, exposure to Pb significantly decreased erythrocyte sialic acid content and increased the erythrocyte osmotic fragility of Pb-treated animals. It is speculated that decreases in membrane sialic acid content due to subchronic low-level Pb exposure shortens red cell life span as observed by increases in osmotic fragility.

Erythrocyte sialic acid and filterability in type 2 diabetic patients

Erythrocyte sialic acid and filterability in type 2 diabetic patients

Erythrocyte Sialoglycoproteins Engage Siglec-9 to Maintain Neutrophil Quiescence in the Bloodstream

Neutrophils remain functionally quiescent in the bloodstream of healthy humans. Characterized by a short lifespan (for which estimates vary widely), they exit the bloodstream to mediate various functions such as pathogen elimination or wound healing--or undergo spontaneous apoptosis and macrophage-mediated clearance in the absence of an inflammatory stimulus. Understanding of neutrophil quiescence, activation and lifespan is relevant to many clinical situations such as infection, sepsis, reperfusion injury, granulocyte transfusions, and other neutrophil-mediated inflammatory pathologies.It has long been known that neutrophils purified from blood tend to be short-lived and show signs of activation, but this technical challenge has been assumed to be a reflection of their natural biology in vivo. We found that when purified from whole blood, neutrophils indeed rapidly express activation markers (low L-selectin; high CD11b) and progress to apoptosis. However, using a novel method to label neutrophils in situ followed by flow cytometry, we found that these activation events occurred at a much slower rate in neutrophils that remained in whole blood. This finding led us to hypothesize that during separation of neutrophils from other blood components, one may be removing an inhibitor that normally maintains neutrophil quiescence in the bloodstream.Co-incubation studies showed that erythrocytes are the primary blood component that dampens neutrophil activation, including chemotaxis, generation of reactive oxygen species, and release of neutrophil extracellular traps. The duration of neutrophil viability was also lengthened upon re-incubation with erythrocytes. We found that this maintenance of functional quiescence is mediated largely by erythrocyte surface sialoglycoproteins (particularly glycophorin A, GPA), which engage neutrophil Siglec-9, a sialic acid-binding receptor that is known to dampen innate immune cell activation via cytosolic inhibitory motifs. Modification of erythrocyte sialic acid side chains using a newly developed gentle method eliminated Siglec-9 binding, and allowed neutrophil activation as measured by reactive oxygen species production.We next sought evidence that this interaction occurs in vivoby studying freshly collected whole blood. Smears made from fresh whole blood showed a high degree of Siglec-9 clustering on neutrophils, which was evident at points of contact with GPA on erythrocytes. This clustering was markedly reduced when smears were made from buffy coat preparations, which involves initial physical separation of neutrophils from erythrocytes during centrifugation.Taken together, this data indicates that erythrocyte sialic acids have an unexpected function as carriers of "self-associated molecular patterns" (SAMPs), regulating innate immunity and maintaining neutrophil quiescence in the bloodstream, apparently by tonic engagement of inhibitory Siglec-9. Notably, this SAMP effect blunts but does not completely inhibit bacterial killing by neutrophils in whole blood, and yet presumably helps to limit unwanted neutrophil inflammatory activation in the bloodstream. Our findings are relevant to many physiological, pathological and clinical situations involving neutrophil biology, and may be useful in reevaluation of prior studies of activation, function and reinfusion-based kinetics that were performed using neutrophils isolated away from whole blood. DisclosuresNo relevant conflicts of interest to declare.

Effects of Vitamins C and E on Erythrocyte Osmotic Fragility, Serum Malondialdehyde concentrations and Surface Erythrocyte Sialic Acid in Rams following Road Transportation

This study was designed to investigate the effect of administration of combination of vitamin C + E and vitamin C alone on erythrocyte osmotic fragility (EOF), serum malondialdehde (MDA) and surface sialic acid (SSA) concentration in road transported rams. Twenty one (21) rams were used for the study. On the day of transportation, 7 rams (Group 1) were orally and individually administered with vitamin C (Juhel® Nigeria Ltd.) at the dose of 250 mg/kg dissolved in 10 ml of water and also vitamin E (100 mg DL-α-tocopherol) (Patterson Zoochonist Ltd. Nigeria) at the dose of 75 mg/kg, per os while another 7 rams (Group 2) was administered orally and individually with vitamin C (250 mg/kg) only. The 3rd group (7 rams) was the control, and they were administered orally and individually with only 10 ml of sterile water. The results indicated that the percentage haemolysis after 8 hours (end of the journey) increased in the entire treatment group which was significantly (P 0.05) different. There was progressive decline in the mean SSA concentrations in the control group with a value of 6.00 ± mg/ml at the end of the journey (8 h) which was not significantly (P > 0.05) different but had higher decrease of 17.43 % as against 4.52 % and 5.56 % in group 1 and 2 respectively. In conclusion the findings in this study have provided an insight into the role of erythrocyte SSA and ROS in explaining haemolysis associated with road transported ram and the possible ameliorative roles of Vitamins C and E on haemolysis

Erythrocyte sialic acid content during aging in humans: correlation with markers of oxidative stress.

Sialic acids are substituted neuraminic acid derivatives which are typically found at the outermost end of glycan chains on the membrane in all cell types. The role of erythrocyte membrane sialic acids during aging has been established however the relationship between sialic acid and oxidative stress is not fully understood. The present work was undertaken to analyze the relationship between erythrocyte membrane sialic acid with its plasma level, membrane and plasma lipid hydroperoxide levels and plasma total antioxidant capacity. Results show that sialic acid content decreases significantly (P < 0.001) in RBC membrane (r = −0.901) and increases in plasma (r = 0.860) as a function of age in humans. Lipid peroxidation measured in the form of hydroperoxides increases significantly (P < 0.001) in plasma (r = 0.830) and RBC membranes (r = 0.875) with age in humans. The Trolox Equivalent Total Antioxidant Capacity (TETAC) of plasma was found to be significantly decreased (P < 0.001, r = −0.844). We observe significant correlations between decrease of erythrocyte membrane sialic acid and plasma lipid hydroperoxide and TETAC. Based on the observed correlations, we hypothesize that increase in oxidative stress during aging may influence the sialic acid decomposition from membrane thereby altering the membrane configuration affecting many enzymatic and transporter activities. Considering the importance of plasma sialic acid as a diagnostic parameter, it is important to establish age-dependent reference.

Erythrocyte Sialic Acid Content during Aging in Humans: Correlation with Markers of Oxidative Stress

Sialic acids are substituted neuraminic acid derivatives which are typically found at the outermost end of glycan chains on the membrane in all cell types. The role of erythrocyte membrane sialic acids during aging has been established however the relationship between sialic acid and oxidative stress is not fully understood. The present work was undertaken to analyze the relationship between erythrocyte membrane sialic acid with its plasma level, membrane and plasma lipid hydroperoxide levels and plasma total antioxidant capacity. Results show that sialic acid content decreases significantly (P&lt; 0.001) in RBC membrane (r= −0.901) and increases in plasma (r= 0.860) as a function of age in humans. Lipid peroxidation measured in the form of hydroperoxides increases significantly (P&lt; 0.001) in plasma (r= 0.830) and RBC membranes (r= 0.875) with age in humans. The Trolox Equivalent Total Antioxidant Capacity (TETAC) of plasma was found to be significantly decreased (P&lt; 0.001,r= −0.844). We observe significant correlations between decrease of erythrocyte membrane sialic acid and plasma lipid hydroperoxide and TETAC. Based on the observed correlations, we hypothesize that increase in oxidative stress during aging may influence the sialic acid decomposition from membrane thereby altering the membrane configuration affecting many enzymatic and transporter activities. Considering the importance of plasma sialic acid as a diagnostic parameter, it is important to establish age-dependent reference.

Association between plasma glucose and glycoproteins in alcoholic smokers compared to alcoholics and teetotalers

Background and aim Diabetes mellitus is a metabolic disorder. However, the association between alcohol intake, cigarette smoking and risk of diabetes remains unclear. Present study was aimed to evaluate the association between blood glucose and glycoproteins in alcoholics and alcoholic smokers. Methods Human male volunteers were categorized into three groups viz., teetotalers who were non-alcoholics and non-smokers, alcoholics who consumed 70–120 g alcoholic beverage/day, and alcoholic smokers who use more than 12 cigarettes and 70–120 g alcoholic beverage/day. Results The results of the present study showed elevated levels of glucose, lipid peroxidation (LPO), protein carbonyls and altered glycoproteins in plasma, erythrocyte and platelet in alcoholics and alcoholic smokers when compared to teetotalers. Alcoholics and alcoholic smokers plasma glucose was positively correlated with plasma sialic acid ( r = 0.371; r = 0.528), hexose ( r = 0.213; r = 0.265) and hexosamine ( r = 0.314; r = 0.416), negatively correlated with plasma fucose ( r = −0.292; r = −0.348) and α-acid glycoprotein ( r = −0.194; r = −0.361) respectively. Further, alcoholics and alcoholic smokers plasma glucose was negatively correlated with erythrocyte sialic acid ( r = −0.392; r = −0.418), hexose ( r = −0.182; r = −0.268), hexosamine ( r = −0.381; r = −0.264) and fucose ( r = −0.216; r = −0.294) as well platelet sialic acid ( r = −0.17; r = −0.218), and fucose ( r = −0.247; r = −0.336) respectively. Conclusion Alterations in glycoproteins, glucose and increased oxidative stress in alcoholics and alcoholic smokers were observed compared to teetotalers.

Epigenetic control of the variable expression of aPlasmodium falciparumreceptor protein for erythrocyte invasion

Plasmodium falciparum can invade erythrocytes by redundant receptors, some of which have variable expression. A P. falciparum clone Dd2 requiring erythrocyte sialic acid for invasion can be switched to a sialic acid-independent progeny clone Dd2NM by growing the Dd2 clone with neuraminidase-treated erythrocytes. The RH4 gene is transcriptionally up-regulated in Dd2NM compared to Dd2, despite the absence of DNA changes in and around the gene. We determined the epigenetic modifications around the transcription start site (TSS) at the time of expression of RH4 in Dd2NM (44 h) and at an earlier time when RH4 is not expressed (24 h). At 44 h, the occupancy of the +1 nucleosome site downstream of the TSS of the active RH4 gene in Dd2NM was markedly reduced compared to Dd2; no difference was observed at 24 h. At 44 h, histone modifications associated with up-regulation were positively correlated to the active RH4 gene of Dd2NM compared to Dd2; no differences were observed at 24 h. Histone H3K9 trimethylation (a marker for silencing) was higher in Dd2 than Dd2NM along the 5'-UTRs of the RH4 gene at both 44 and 24 h. Our data indicate that the failure of Dd2 to express the sialic acid-independent invasion receptor gene RH4 is associated with the epigenetic silencing mark H3K9 trimethylation present throughout the cycle.

Semen sialic acid surge and modulation of α‐L‐fucosidase activity: possible link to loss in reproductive capacity during trypanosomiasis

The profiles of semen sialic acid and the enzyme alpha-L-fucosidase were studied in rams undergoing chronic infection by Trypanosoma congolense. Our data showed a significant surge in the level of sialic acid with parasitaemia. The pattern followed a polynomial function we had reported for erythrocyte sialic acid in mice undergoing acute infection by T. congolense. The activity of the enzyme alpha-fucosidase decreased progressively with approximately 60% decrease at the end of the 14 weeks of infection. Representative semen samples from the control and infected rams were subjected to kinetic characterization. While the uninfected semen sample showed two active pH peaks at 4.5-5.5 and at 6.8-7.2, respectively, there was an apparent shift to only a single pH optimum at 4.5-5.5 for the pathological semen. The fucosidases from both sources were optimally active at 35 degrees C albeit with contrasting activation energies (E(a)) with values 20.58 and 35 kJ/mol for the control and infected semen, respectively. Kinetic studies using methylumbelliferyl-beta-fucoside (4MU-Fuc) as substrate gave K(M) and V(max) values of 3.25 microM and 14.6 micromol. min(-1) mg(-1), respectively for the control semen. The values for the infected semen were 18.25 microM and 10.5 micromol. min(-1) mg(-1), respectively. The significance of these results is discussed as they relate to loss in reproductive capacity in trypanosomoses.

Involvement of a Host Erythrocyte Sialic Acid Content in Babesia bovis Infection

Host sialic acid (SA) has recently been suggested to play an important role in erythrocyte (RBC) infection by Babesia spp. The present study attempted to further determine the specific type of SAs important in the RBC invasion. Bovine RBC was found to bear abundant alpha2-3-linked SA residues but not alpha2-6-linked SA in nature, confirmed by flow cytometric analysis of the neuraminidase (Nm)-treated RBCs. Lectin-blot analyses revealed the removal of alpha2-3-linked SAs from the 97-, 33-, and 31-kDa bands by the Nm treatment. Addition of the Nm-treated RBCs into an in vitro culture of B. bovis resulted in a decreased population of the parasitized RBCs. The thin smear samples from the cultures were then observed under a confocal laser scanning microscope after staining with the alpha2-3-linked SA-specific lectin: a selective invasion of B. bovis was found only in the intact RBCs bearing the SAs, but not in the desialylated RBCs. Furthermore, a significant reduction of the parasitized RBCs was also observed in the culture supplemented with exogenous 3'-sialyllactose containing the alpha2-3-linked SAs. However, the complete inhibition of parasite proliferation was not achieved in the culture. These findings indicate that while the alpha2-3-linked SA-dependent pathway is needed for highly efficient invasion of host RBCs by B. bovis, there might also be other potential alternative pathways.

Comparative studies on erythrocyte sialic acid levels in apparently healthy indigenous Nigerian poultry species

The erythrocyte surface sialic acid levels of 200 apparently healthy indigenous Nigerian poultry species (pigeons, guinea fowls, ducks and chickens, n = 50 for each species) presented for slaughter at a poultry abattoir in Zaria, Nigeria was determined. Other parameters determined were packed cell volume (PCV) and plasma total protein (TP) concentration. The mean erythrocyte surface sialic acid concentration of pigeons, guinea fowls, ducks and chickens were 7.88 +/- 2.51, 14.6 +/- 2.51, 17.6 +/- 2.51 and 14.2 +/- 2.51 mg mg(-1) respectively. There was a statistically significant difference between the mean erythrocyte surface sialic acid concentration of all the species of poultry sampled (P < 0.05). The high erythrocyte surface sialic acid concentration in the indigenous Nigerian poultry species could be responsible for their resistance to infectious diseases, whose aetiologic agents produce neuraminidases. The mean PCV of the pigeons, guinea fowls, ducks and chickens were 46.22 +/- 6.91, 38.24 +/- 6.91, 36.50 +/- 6.91 and 36.46 +/- 6.91% respectively. The difference between the mean PCV values of pigeons on the one hand and guinea fowls, ducks and chickens on the other was statistically significant (P < 0.05). A negative correlation (r = -0.36, P < 0.05) between mean erythrocyte surface sialic acid concentrations and PCV was observed, i.e. the birds with the highest mean PCV values had the lowest levels of erythrocyte surface sialic acid. There was no correlation between TP concentration and either erythrocyte surface sialic acid concentration or PCV values. It is suggested, based on this study, that erythrocyte sialic acid types in these species should be determined, as the results may be vital in selective breeding.

Glycophorin A-mediated haemolysis of normal human erythrocytes: evidence for antigen aggregation in the pathogenesis of immune haemolysis.

The inexplicable severity of anti-Pr autoimmune haemolytic anaemia led us to test the hypothesis that the haemolysis was primarily due to a change in the function of glycophorin A, on which the Pr antigen is located. The lectins Maclura pomifera and wheat germ agglutinin that bind to glycophorin A induced the haemolysis of normal erythrocytes in vitro. Lectin binding led to an increase in erythrocyte membrane permeability to sodium and potassium, the former resulting in an influx of water and subsequent haemolysis. The response was glycophorin A specific as Concanavalin A, which binds to band 3, did not cause haemolysis and peanut agglutinin only did so after removal of erythrocyte sialic acid. The lectin-induced cation leak was not mediated by activation of cation channels as the inhibitors, tetrodotoxin, amiloride and 4,4' disothiocyanate stilbene 2,2'disulphonate, had no effect, suggesting that the haemolysis was due to exacerbation of the inherent cation permeability of the erythrocyte membrane. A human IgAK anti-Pr autoantibody and a mouse anti-human glycophorin A antibody increased erythrocyte permeability to sodium. The role of glycophorin A in stabilizing and, upon aggregation, destabilizing the phospholipid bilayer is discussed. Our findings may help explain the severity of anti-Pr autoimmune haemolytic anaemia and other pathophysiological changes in human erythrocytes.

Band 3 glycoprotein and glycophorin A from erythrocytes of children with congenital disorder of glycosylation type-Ia are underglycosylated.

Band 3 and PAS-1 (a dimer of glycophorin A) from erythrocyte membranes of three children with congenital disorder of glycosylation, type Ia (CDG-Ia), aged 1 month, 3 years and 10 years respectively, were examined by a new technique that allowed determination of carbohydrate molar composition of glycoproteins separated by sodium dodecyl sulfate polyacrylamide gel electrophoresis. In CDG children a single N-glycan of band 3 glycoprotein was hypoglycosylated and its mannose content was normal or elevated. Glycophorin A which is the major carrier of erythrocyte sialic acid, was deficient in N-acetylgalactosamine, and sialic acid residues. This finding indicated a partial unglycosylation of O-glycans in glycophorin A. In keeping with the results of PAS-1 analysis, total sialic acid in erythrocyte membranes from CDG children was reduced to 40-56% of normal values. A possible molecular mechanism of hypo- and unglycosylation of band 3 and glycophorin A, respectively, in CDG is discussed.

Erythrocyte disaggregation shear stress, sialic acid, and cell aging in humans.

Erythrocyte aggregation, which plays an important role in the physiological behavior of blood fluidity, was found to be enhanced in hypertension and hypercholesterolemia. While the role of macromolecule bridging force has been widely described, cellular factors related to membrane sialic acid content, which might contribute to the negative charge of cell surface causing the repulsion of erythrocytes, have been less studied. Cell age-dependent changes in membrane sialic acid content (in micromoles per gram of integral membrane protein) were investigated in 24 normotensive and 24 hypertensive matched subjects, each divided into 2 identical subgroups according to a cutoff of 6.2 mmol/L serum cholesterol. A progressive and significant (P<0.001) decrease in membrane sialic acid content associated with an increase (P<0.001) of disaggregation shear rate threshold (laser reflectometry in the presence of dextran) were observed with increased erythrocyte density (erythrocytes fractionated by density using ultracentrifugation) in both normotensive and hypertensive groups regardless of the cholesterol level. However, disaggregation shear rate threshold was significantly higher and sialic acid content was lower (P<0.001) in both hypertensive and normotensive subjects with hypercholesterolemia compared with either normotensive or hypertensive subjects with low cholesterol, respectively. A high membrane sialic acid content variance, beginning in the younger erythrocytes, was due mainly to triglyceride and LDL cholesterol levels (R2=0.49 for low, R2=0.43 for middle, and R2=0.54 for high densities, ie, young, mean, and senescent erythrocytes, respectively). We conclude that an early decrease in erythrocyte sialic acid content may influence the rheological properties of blood by increasing the adhesive energy of erythrocyte aggregates.

Influence of Sialic Acid on Erythrocyte Aggregation in Hypercholesterolemi

The respective role of adhesive forces induced by fibrinogen and repulsive forces induced by erythrocyte sialic acid content on erythrocyte aggregation, was investigated in hypercholesterolemic and control subjects. Aggregation index (AI) and disaggregation shear rate threshold (gamma t) were determined in the presence of either autologous plasma or dextran. Compared with controls, fibrinogen (p < 0.001) and aggregation parameters (AI p < 0.01; gamma t p < 0.01) were higher in hypercholesterolemics while erythrocyte sialic acid content (p < 0.001) was lower; in addition total serum sialic acid was increased (p < 0.01). The aggregation properties of erythrocytes, independent of plasma environment using dextran as a bridging macromolecule, showed an enhanced disaggregation shear rate threshold and an inverse relationship with erythrocyte sialic acid content. We conclude that decreased erythrocyte sialic acid content may intensify the effect of fibrinogen on aggregation and disaggregation of erythrocytes and participate in the development of atherothrombotic complications.

F166. Role of fibrinogen and erythrocyte sialic acid content on red blood cell aggregation in hypercholesterolemia

F166. Role of fibrinogen and erythrocyte sialic acid content on red blood cell aggregation in hypercholesterolemia

Biochemical Differences (O-Acetyl and Glycolyl Groups) in Erythrocyte Surface Sialic Acids of Trypanotolerant N'dama and Trypanosusceptible Zebu Cattle

Differences in the distribution and concentration of O-acetyl and glycolyl groups in erythrocyte sialic acids of trypanotolerant N'dama and trypanosusceptible Zebu cattle were investigated. Erythrocyte surface sialic acid concentrations were 25.4 +/- 5.5 mg per dl and 5.9 +/- 0.97 mg per dl in N'dama and Zebu animals, respectively. In N'damas, O-acetyl and glycolyl groups were present in concentrations of 16.4 +/- 4.3 mg per dl and 12.8 +/- 2.9 mg per dl, respectively, whereas the corresponding values in Zebus were 2.8 +/- 5.0 mg per dl and 7.6 +/- 1.7 mg per dl, respectively. The differences between N'dama and Zebu cattle in surface sialic acids and in O-acetyl and glycolyl groups were significant (P < 0.01 to < 0.001). N'dama erythrocyte sialic acids had more O-acetyl than glycolyl groups while those of Zebus had less O-acetyl than glycolyl groups. These findings may be relevant to the trypanotolerance of N'dama cattle.

Binding of Plasmodium falciparum 175-kilodalton erythrocyte binding antigen and invasion of murine erythrocytes requires N-acetylneuraminic acid but not its O-acetylated form

Sialic acid on human erythrocytes is involved in invasion by the human malaria parasite, Plasmodium falciparum. Mouse erythrocytes were used as a reagent to explore the question of whether erythrocyte sialic acid functions as a nonspecific negative charge or whether the sialic acid is a necessary structural part of the receptor for merozoites. Human erythrocytes contain N-acetylneuraminic acid (Neu5Ac), whereas mouse erythrocytes, which are also invaded by P. falciparum merozoites, contain 9- O-acetyl- N-acetylneuraminic acid (Neu5,9Ac 2) and N-glycoloylneuraminic acid (Neu5Gc), in addition to Neu5Ac. We compared the effects of sialidase and influenza C virus esterase treatments of mouse erythrocytes on invasion and the binding of a 175-kDa P. falciparum protein (EBA-175), a sialic acid-dependent malaria ligand implicated in the invasion process. Sialidase-treated mouse erythrocytes were refractory to invasion by P. falciparum merozoites and failed to bind EBA-175. Influenza C virus esterase, which converts Neu5,9Ac 2 to Neu5Ac, increased both invasion efficiency and EBA-175 binding to mouse erythrocytes. Thus, the parasite and EBA-175 discriminate between Neu5Ac and Neu5,9Ac 2, that is, the C-9 acetyl group interferes with EBA-175 binding and invasion by P.falciparum merozoites. This indicates that sialic acid is part of a receptor for invasion.