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PubMed1992

Cell-surface regulation of the human alternative pathway of complement. Sheep but not rabbit erythrocytes express factor I-dependent cofactor activity.

When incubated in normal human serum, rabbit erythrocytes are haemolysed as a result of activation of the alternative pathway of complement (APC), but sheep erythrocytes do not spontaneously activate the human APC under physiological conditions. The mechanism for this difference has been attributed to differences in the relative affinity of membrane-bound C3b for its natural ligands, factor B and factor H, that favour the formation and stability of the APC C3 convertase on rabbit erythrocytes and inhibit convertase activity on sheep erythrocytes. Previous studies have also suggested that factor I inactivated C3b on sheep erythrocytes more effectively than on rabbit erythrocytes. Further, sheep erythrocytes have recently been shown to have a membrane protein that associates non-covalently with cell-bound C3b, but rabbit erythrocytes lack a predominant C3b binding protein. Together, these results suggested the possibility that sheep but not rabbit erythrocytes have a membrane constituent with factor I cofactor activity. To investigate this hypothesis, rabbit and sheep erythrocytes bearing radiolabelled C3b were treated either with factor I or with factor I and factor H, and conversion to iC3b was assessed by autoradiography. Factor I caused a concentration-dependent conversion of C3b to iC3b on sheep erythrocytes; however, only trace amounts of C3b on rabbit erythrocytes were degraded even when high concentrations of factor I (83 micrograms/ml) were used. While C3b on rabbit erythrocytes was converted to iC3b by the combination of factor H and factor I, much less factor H was required for the same degree of conversion of C3b on sheep erythrocytes. Treatment of sheep erythrocytes with neuraminidase had no effect on either factor I-dependent cofactor activity or the capacity of factor H to serve as a factor I cofactor. Sheep erythrocytes did not manifest decay accelerating activity, however, suggesting that the factor I cofactor constituent is a functional analogue of the human membrane cofactor protein.

Animals↗

PubMed1983

Primate erythrocyte-immune complex-clearing mechanism.

Previous in vitro studies have shown that immune complexes (IC) that fix complement can bind to the C3b receptor on primate erythrocytes. The in vivo function of this erythrocyte receptor, however, is unknown. This study was undertaken to determine whether the binding of IC to erythrocytes in vivo might play a role in the removal of IC from the circulation. Baboons and rhesus monkeys were prepared with a catheter in the ascending aorta to infuse IC and in the abdominal aorta, renal, hepatic, and portal veins to monitor changes in binding and clearance of IC across kidney, liver, and spleen + gut, respectively. Autologous 51Cr-labeled erythrocytes were infused intravenously and allowed to equilibrate. Preformed IC (125I-labeled bovine serum albumin [BSA] rabbit anti-BSA) were then infused into the ascending aorta at a constant rate for 120 s. Blood samples were drawn at frequent intervals for 30 min from all catheters below the IC injection site. Each blood sample was then centrifuged on percoll to separate IC bound to erythrocytes from IC in plasma or bound to buffy coat cells. This resulted in an "erythrocyte fraction" beneath the percoll that contained the IC bound to erythrocytes, and a "plasma/buffy coat fraction" above the percoll that contained the IC in plasma and IC bound to buffy coat cells. Analysis of these data showed that the majority of the IC infused into the circulation rapidly became bound to erythrocytes. However, by 5 min after beginning the IC infusion, most of this IC load had been removed from the erythrocytes as they traversed the liver. In contrast, IC on erythrocytes did not deposit in kidney. The IC-bearing erythrocytes themselves were not trapped or detained by any organ. IC in the plasma/buffy coat fraction of blood were removed from the circulation but at a relatively low rate and almost entirely by the liver. These findings suggest that primate erythrocytes intercept large complement-fixing IC in the circulation causing the IC to adhere to the erythrocyte until th e IC-bearing erythrocyte traverses liver where the IC is deposited, and the erythrocyte is returned to the circulation. This primate erythrocyte-IC-clearing mechanism may be important in the protection against diseases mediated by deposition of circulating IC.

Animals↗

PubMed1991

[Membrane cholesterol and insulin receptor in erythrocytes].

The author studied influence of membrane cholesterol on insulin receptor behavior using human erythrocytes after modification of their membrane cholesterol contents. Insulin binding to the erythrocytes containing higher membrane cholesterol which were obtained from alcoholic liver cirrhosis (ALC) and familial lecithin: cholesterol acyl transferase (LCAT) deficiency patients was also examined. 1) Influence of modified membrane cholesterol contents on insulin binding to erythrocytes (in vitro experiments): The cholesterol content of human erythrocyte membranes was modified by incubating the cells with phosphatidylcholine/cholesterol and phosphatidylcholine vesicles. Then insulin binding to the erythrocytes was measured as a function of membrane cholesterol content. Low membrane cholesterol decreased insulin binding at 10 degrees C and 15 degrees C, but increased insulin binding at 25 degrees C and 37 degrees C. Scatchard plot analysis showed that low membrane cholesterol decreased a number of the receptor without alteration of affinity. Phosphatidylcholine spin probe showed a dual effect of cholesterol on membrane fluidity, fluidizing below but rigidifying membranes above 20 degrees C. It is suggested that membrane cholesterol affects insulin receptor behavior through alteration of membrane fluidity, depending on the phase state of the membranes. 2) Insulin binding and membrane lipid composition of erythrocytes from ALC and LCAT deficiency patients: The erythrocytes from both ALC and LCAT deficiency patients are known to have higher content of membrane cholesterol. Insulin binding and membrane lipid composition of erythrocytes in both diseases were measured to see the influence of cholesterol on insulin binding in sites. a) Erythrocytes from ALC patients: In the erythrocytes from ALC patients, cholesterol to phospholipid molar ratio was significantly increased as compared with normal subjects. In phospholipid acyl chains, contents of polyunsaturated fatty acid significantly decreased in ALC patients. Insulin binding to erythrocytes was significantly increased in the patients of ALC. Scatchard plot analysis (at 15 degrees C) showed significant increase of the receptor number in erythrocytes from the ALC patients as compared to the normal subjects, while the affinity did not differ between the groups. The increased cholesterol content in the erythrocyte membranes was suggested to be responsible for the increased receptor number and insulin binding to the erythrocytes from ALC patients. b) Erythrocytes from LCAT deficiency patients: Membrane cholesterol to phospholipid molar ratio was increased in two patients of LCAT deficiency studied. In phospholipid classes, increase of phosphatidylcholine and decreases of both phosphatidylethanolamine and sphingomyelin were observed in the erythrocytes from both patients.(ABSTRACT TRUNCATED AT 400 WORDS)

Adult↗

PubMed1990

[Preparation of partially fixed erythrocytes and its application for immunohematologic examination].

A suspension of washed human erythrocytes (2%) in PBS was mixed with an equal volume of 1 mM glutaraldehyde (GA) and allowed to stand at laboratory temperatures, followed by washing with normal saline. The agglutinability of the erythrocytes toward anti-A, anti-B, anti-M and anti-N reagents remained unchanged after GA treatment shorter than 20 minutes, and the agglutinability toward anti-C, anti-c, anti-D, anti-E, anti-e, anti-Lea, anti-Leb and anti-P1 did not decrease after treatment for 10 minutes. GA treatment for longer periods of time than the above caused a decrease of the reactivities. The agglutinability toward an anti-H (Ulex europaeus) and other lectins increased after 10 to 30 minutes of GA treatment and decreased after 40 minutes or more of exposure to GA. These results indicate that the immunologic agglutinability of erythrocytes were practically unchanged after a 10 minutes treatment with 1 mM GA (a mild fixation procedure hereafter called "partial fixation"). The properties of the partially fixed erythrocytes were closely similar to those of untreated erythrocytes as regards cell volume, membrane fragility in hypotonic solutions (measured by a modification of the Ribiere method), and membrane fragility under continuous shaking. The resistance of the partially fixed erythrocytes heating up to 50 degrees C was superior to that of the untreated erythrocytes. From these results, the deformability of the partially fixed erythrocytes was concluded to be similar to that of untreated erythrocytes. After storage for 6 months at 4 degrees C in Alsever solution containing adenine and inosine, the shapes of the untreated erythrocytes changed to "spherocytes" or "echinocytes", whereas the partially fixed cells retained the original discocyte shape. Blood grouping laboratory tests were performed with the partially fixed erythrocytes as indicators. Both anti-A and anti-B agglutinins in normal human sera could be detected without difficulty. Presence of irregular agglutinins, such as anti-H and anti-N, in healthy donors' sera could also be detected, as with the freshly obtained erythrocytes. The detection limits of an incomplete anti-D agglutinin were equal to those in the tests with untreated erythrocytes. The partially fixed erythrocyte (stored at 4 degrees C for 2.5 to 3 months) were used as indicators of ABO-blood grouping from blood stains, saliva stains and hairs by the agglutinin-inhibition test, absorption-elution test or mixed agglutination test. The results obtained were practically equal to those of the tests with freshly obtained erythrocytes, indicating the availabilities of the partially fixed erythrocytes.

Blood Grouping and Crossmatching↗

PubMed1984

Effects of diltiazem on the physicochemical properties of rat erythrocyte and liposome membrane: comparison with pentoxifylline and propranolol.

In vitro effects of the coronary vasodilator diltiazem on rat erythrocytes and liposomes were studied in comparison with propranolol and pentoxifylline. Diltiazem improved the deformability of rat erythrocytes, reduced the viscosity of rat erythrocyte suspensions, and protected the erythrocyte against hypotonic hemolysis at concentrations above 10(-4) M, 10(-5) M and 5 X 10(-7) M, respectively. Diltiazem at 5 X 10(-4) M also improved the impaired deformability of ATP-depleted erythrocytes, whereas it affected neither the adenine nucleotide level nor the phosphorylation of spectrin in the erythrocytes. Diltiazem enhanced the interaction of 1-anilino-naphthalene-8-sulfonate, a fluorescent probe, with erythrocyte ghosts at concentrations above 5 X 10(-6) M and, above 5 X 10(-5) M, inhibited the (Na+ + K+)-ATPase activity of the erythrocyte ghosts. Diltiazem reduced the microviscosity of both erythrocyte ghosts and liposomes prepared from rat erythrocyte lipids. Diltiazem induced aggregation of the liposomes prepared from rat erythrocyte lipids, phosphatidylserine or phosphatidylinositol, but it did not affect the liposomes prepared from a mixture of phosphatidylethanolamine and phosphatidylcholine (1:1). I-Diltiazem, a stereo-isomer of diltiazem, exhibited equipotent effects, compared with diltiazem, on these parameters. Propranolol showed similar properties, but pentoxifylline shared none of the above properties, except that it improved the deformability of erythrocytes. From these results, it is suggested that diltiazem may affect the erythrocyte membrane by interacting with acidic phospholipids and thus reduce the microviscosity of the membrane, improve erythrocyte deformability and protects the erythrocyte against hypotonic hemolysis.

Animals↗

PubMed2001

Direct measurement of erythrocyte deformability in diabetes mellitus with a transparent microchannel capillary model and high-speed video camera system.

To measure erythrocyte deformability in vitro, we made transparent microchannels on a crystal substrate as a capillary model. We observed axisymmetrically deformed erythrocytes and defined a deformation index directly from individual flowing erythrocytes. By appropriate choice of channel width and erythrocyte velocity, we could observe erythrocytes deforming to a parachute-like shape similar to that occurring in capillaries. The flowing erythrocytes magnified 200-fold through microscopy were recorded with an image-intensified high-speed video camera system. The sensitivity of deformability measurement was confirmed by comparing the deformation index in healthy controls with erythrocytes whose membranes were hardened by glutaraldehyde. We confirmed that the crystal microchannel system is a valuable tool for erythrocyte deformability measurement. Microangiopathy is a characteristic complication of diabetes mellitus. A decrease in erythrocyte deformability may be part of the cause of this complication. In order to identify the difference in erythrocyte deformability between control and diabetic erythrocytes, we measured erythrocyte deformability using transparent crystal microchannels and a high-speed video camera system. The deformability of diabetic erythrocytes was indeed measurably lower than that of erythrocytes in healthy controls. This result suggests that impaired deformability in diabetic erythrocytes can cause altered viscosity and increase the shear stress on the microvessel wall.

Adult↗

PubMed1996

Low erythrocyte Na/K-pump activity and number in northeast Thailand adults: evidence suggesting an acquired disorder.

Healthy northeastern Thais have a higher erythrocyte sodium concentration and a lower erythrocyte membrane Na,K-adenosine triphosphatase (ATPase) activity than central Thais. To elucidate whether the defect is hereditary or acquired, we studied plasma sodium and potassium and erythrocyte sodium, potassium, Na,K-ATPase activity, and ouabain-binding sites (OBS) in the following groups: healthy newborns of ethnic central Thais (group 1), healthy newborns of ethnic northeast Thais (group 2), healthy adults of central Thailand ethnicity who lived in the rural central region (group 3) or in Bangkok (group 4), healthy adults of northeast Thailand ethnicity who lived in the rural northeast region (group 5) or who migrated to work in Bangkok for at least 1 year (group 6). Erythrocyte Na was higher in group 2 than in group 1. Group 3 had lower erythrocyte Na,K-ATPase activity than group 4, and it was lower in group 5 than in group 6. Among all groups, group 5 had the highest erythrocyte Na (11.6 mmol/L,F < 0.0001) and the lowest Na,K-ATPase activity (63 mmol Pi/mg x h, F < 0.0001) and erythrocyte OBS (397 sites per cell, F < 0.05) than the other adult groups. There was a positive correlation between erythrocyte Na,K-ATPase and erythrocyte OBS (r = .416, P < .0001). Multiple regression analysis demonstrated a correlation between erythrocyte Na as a dependent variable and erythrocyte OBS, plasma potassium, erythrocyte potassium, and erythrocyte Na,K-ATPase (r = .517, P < .0001). The erythrocyte Na,K-ATPase/OBS ratio, an expression of Na,K-ATPase activity equalized for the number of Na,K-pump units, was lowest among rural adults of the central region (group 3) and the northeast region (group 5) (F < 0.0002). Our data suggest that rural dwellers in Thailand tend to have lower erythrocyte Na,K-ATPase activity than urban dwellers and that this is probably acquired after birth. It was more severe among those from the northeast versus the central region, and was less severe among those who migrated to an urban area. This defect in northeast rural dwellers was probably associated with low numbers of Na,K-pump units and a defect of the pump to express activity, whereas in central rural dwellers it was probably associated with the latter condition. We postulate that there might be circulating Na,K-pump inhibitors and metabolic disturbances that cause attenuation of Na,K-ATPase function and synthesis in the northeast Thailand rural population, and that such substances may have an environmental origin. There may be a relationship between these abnormalities and sudden unexpected deaths.

Adult↗

PubMed1986

Erythrocyte-neutrophil interactions: formation of leukotriene B4 by transcellular biosynthesis.

Studies on the mechanism of leukotriene B4 biosynthesis in suspensions composed of neutrophils plus erythrocytes indicate that human erythrocytes convert neutrophil-derived leukotriene A4 into leukotriene B4. Leukotriene B4 formation by neutrophils in the presence of erythrocytes exceeded that from corresponding suspensions of neutrophils alone. The increase was proportional to the erythrocyte content of the suspension. The erythrocyte-dependent increase in leukotriene B4 biosynthesis did not equal the arithmetic sum of calcium ionophore-dependent biosynthesis by neutrophils plus calcium ionophore-dependent biosynthesis by erythrocytes, since erythrocytes produced no leukotriene B4 upon incubation with ionophore A23187. Erythrocytes did not stimulate 5-lipoxygenase activity within neutrophils, since the erythrocyte effect was confined to enzymatic hydration: leukotriene B4 increased coincident with decreased formation of 5,12-dihydroxyicosatetraenoic acids derived from nonenzymatic hydration. Biosynthesis of leukotriene B4 within the erythrocyte, from neutrophil-derived leukotriene A4, was established by comparing the effect of normal erythrocytes with erythrocytes containing a leukotriene A4 hydrolase that was inactivated by the substrate. In the latter case, leukotriene B4 formation increased by only 30-40%; in the former case, it increased by 100-200%. Transcellular biosynthesis of leukotriene B4 from erythrocyte-neutrophil interactions explains the paradoxical presence of leukotriene A4 hydrolase within erythrocytes, a cell incapable of synthesizing leukotriene A4; affords a mechanism to overcome rate limitations or "suicide inactivation" of leukotriene A4 hydrolase in neutrophils; exploits a cryptic capacity within erythrocytes, provisionally dormant cells in terms of icosanoid biosynthesis; indicates that the biosynthetic capacity of cell combinations is not necessarily equivalent to the sum of their separate capacities.

Arachidonic Acids↗

PubMed2002

In vivo studies on mouse erythrocytes linked to transferrin.

Transferrin (Tf), a plasma protein with numerous, highly specific receptors in proliferating and differentiating cells was already discussed as a targeting ligand for drugs and liposomes in previous studies. In this paper, we deal with erythrocytes linked to Tf as possible physiological targeting carrier systems for delivering anticancer drugs. For that purpose we have used glutaraldehyde (0.1%) as a coupling agent between Tf and erythrocytes. The highest amount of Tf linked to erythrocytes turned out to be 0.76 +/- 0.13 microg Tf/10(6) cells, while reaching 65% of cell recovery. After 13 days, the Tf-erythrocytes hemolysis reached 50%, with transferrin still coupled to erythrocytes. The in vivo kinetic behaviour of intravenously injected 51Cr-Tf-erythrocytes showed a reduced half-life to hours as compared to days of controls. However, a considerable percentage of Tf-erythrocytes (close to 20%) remained circulating for a relatively long period (around 2 days), which made possible the specific targeting by these carrier systems. In vivo biodistribution studies indicated that 51Cr-Tf-erythrocytes rapidly accumulated in the different studied organs (liver, spleen, lungs, kidneys, femur-tibia, and heart), suggesting a selective removal of Tf-erythrocytes by the cells of the mononuclear phagocytic system present mainly in liver and spleen. On the other hand, Tf-erythrocytes showed a poor targeting of heart tissue, therefore a reduced cardiac toxicity should be expected after administration of erythrocyte-encapsulated drugs. The presence of Tf-erythrocytes in femur-tibia and spleen could be related to the Tf-specific binding to the hematopoietic cells containing Tf receptors. The final results of this study encourage additional research on Tf-erythrocyte to investigate the relationship between transferrin-mediated targeting by carrier erythrocytes and uptake of different erythrocyte-encapsulated drugs. Consequently, the current study showed possible use of these carriers as a potential therapeutic tool for drug targeting in animal models with alterations affecting mononuclear phagocytic system or carcinomas of various origins whose cells show elevated number of Tf receptors.

Animals↗

PubMed2000

Endotoxin promotes adhesion of human erythrocytes to human vascular endothelial cells under conditions of flow.

OBJECTIVE: To investigate the effects of endotoxin on adhesion of human red blood cells to human vascular endothelial cells under conditions of flow. DESIGN: Prospective, randomized, controlled in vitro study. SETTINGS: University-affiliated cell biology laboratory. SUBJECTS: Human erythrocytes and human vascular endothelial cells. INTERVENTIONS: Fresh human erythrocytes and human vascular endothelial cells grown as monolayers were incubated with either saline or endotoxin. After incubation, endothelial monolayers were superfused with erythrocytes, and the number of erythrocytes adhering to the endothelial monolayer was quantified. MEASUREMENTS AND MAIN RESULTS: Adhesion of erythrocytes to vascular endothelium was measured under conditions of continuous flow in different settings: a) exposure of both endothelial cells and erythrocytes to saline; b) incubation of both erythrocytes and endothelial cells with endotoxin; c) exposure of erythrocytes only to endotoxin; d) incubation of endothelial cells only to endotoxin; and e) both the endothelial cells and erythrocytes incubated with different concentrations of endotoxin. Erythrocyte adhesion in the saline control group was 71 +/- 8 cells/mm2. Incubation of both components with endotoxin increased the number of adhesive erythrocytes to 172 +/- 9 cells/mm2 (p < .05). When only the endothelial cells were treated with endotoxin, 142 +/- 8 cells/mm2 adhered to the endothelial monolayer, whereas the incubation of the erythrocytes only to endotoxin resulted in adhesion of 102 +/- 3 cells/mm2. Decreasing concentrations of endotoxin reduced adhesion from 172 +/- 9 cells/mm2 (endotoxin, 75 microg/mL) to 165 +/- 9 cells/mm2 (endotoxin, 25 microg/mL), 153 +/- 4 cells/mm2 (endotoxin, 1 microg/mL), and 146 +/- 6.1 cells/mm2 (endotoxin, 5 ng/mL). CONCLUSIONS: Exposure of human erythrocytes and human venous vascular endothelial cells to an inflammatory stimulus such as endotoxin promotes a dose-dependent adhesion of erythrocytes to endothelium in a dynamic environment. These adhesive erythrocyte-endothelium interactions can be produced by exposure of either red blood cells or endothelial cells to endotoxin, with a higher degree of adhesion after activation of the endothelial cell component.

Cell Adhesion↗

PubMedDate not supplied

[Arginase activity in plasma and erythrocytes in children with hematologic diseases].

INTRODUCTION: Arginase (EC 3.5.3.1) is one of the essential enzymes in the terminal stages of the urea cycle in the liver which participates in the elimination of ammonia from the human body [1, 7]. Except in liver tissue arginase is also present in many human tissues and in the circulating blood cells, especially in erythrocytes and leukocytes. Arginase splits arginase to urea and ornithine that serve for biosynthesis of amino acid proline, glutamic acid and biosynthesis of polyamines-spermine, spermidine and putrescine. Arginase activity is high during the mitotic cycle, with the function in phase S of the cell cycle. The aim of our study was to assess the arginase activity in the blood of children with some haematologic diseases. METHODS: We examined the arginase activity in blood plasma and erythrocytes of children who suffer from some haematological disorders (27 patients) and in healthy children (control group-15 subjects). The enzyme activity was measured with spectrophotometric method on the basis of the determination of the amount of liberated ornithine from substrate-arginine [3]. RESULTS: The obtained results suggest that arginase activity was much higher in the blood of ill children (Table 1 and Figure 1). In the control group of children (total 15) plasma arginase activity was in the range of 0 to 20 U/L x = 0.86 U/L), and enzyme activity in erythrocytes was 1.62-3.98 U/g Hb (x = 2.81 U/g Hb). Erythrocytes enzyme activity and plasma enzyme activity were in ranges of 4.03-5.26 U/L with the mean value of x = 4.56 U/L, and arginase activity in erythrocytes was in ranges of 9.38-14.16 U/g Hb, with mean value x = 11.34 U/g Hb, respectively. Arginase activity in erythrocytes was also significantly higher in children with non-spherocytic haemolytic anaemia (9 children) and was in ranges of 5.33-9.58 U/g Hb (x = 7.29 U/g Hb), with the relatively low values in plasma, 0-4.14 U/L (x = 1.75 U/L). In children with sideropenic anaemia (total number-11) arginase activity in erythrocytes was also very significantly increased with the range between 2.86 and 14.16 U/g Hb (x = 5.54 U/g Hb) while the plasma enzyme activity was relatively low, with the values in range of 0-4.98 U/L (x = 1.41 U/L); in myelodysplastic syndrome (4 pts) arginase activity in plasma was very low (0-0.71 U/L; x = 0.26 U/L) with higher values of arginase activity in erythrocytes (4.22-5.89 U/g Hb; x = 4.94 U/g Hb). DISCUSSION: We have concluded that the enzyme activity was the highest in erythrocyte haemolysates of patients with spherocytosis, non-sherocytic haemolytic anaemia; it was also high, but in a smaller degree, in erythrocytes of children with sideropenic anaemia and myelodysplastic syndrome; arginase activity in plasma of these children was higher in comparison with enzyme activity in plasma and erythrocytes of healthy children. Our results are in agreement with data from literature where it is stated that the younger erythrocytes have the highest arginase activity than the mature erythrocytes [4]. CONCLUSION: The measurement of arginase activity in plasma and erythrocytes is a good diagnostic indicator for the presence of young erythrocytes and reticulocytes in the circulating blood as is the good sign for the detection of haemolytic processes.

Anemia↗

PubMed1983

Deficiency of an erythrocyte membrane protein with complement regulatory activity in paroxysmal nocturnal hemoglobinuria.

Paroxysmal nocturnal hemoglobinuria (PNH) is an acquired hemolytic anemia in which the erythrocytes are abnormally sensitive to lysis by complement. A functional deficiency of membrane-associated complement regulators has been demonstrated on PNH erythrocytes. The two factor H-like proteins, the C3b receptor (CR1) and the decay-accelerating factor (DAF), were isolated from normal human erythrocytes, and specific antisera were prepared. Selective inhibition of the two proteins on normal erythrocytes by the antisera demonstrated (i) that the factor responsible for accelerated decay of erythrocyte-bound C3 convertase is DAF and (ii) that the cofactor required for inactivation of erythrocyte-bound C3b by factor I is CR1. PNH erythrocytes were deficient in both of these activities. Erythrocytes deficient in CR1, which were obtained from an apparently healthy individual, exhibited normal DAF activity but no factor I cofactor activity. These cells were not susceptible to complement-mediated lysis in acidified human serum, whereas PNH erythrocytes and Pronase-treated human erythrocytes (which lack DAF and CR1 activities) were lysed by this treatment. It is suggested that the protein primarily responsible for preventing complement activation on normal human erythrocytes is DAF. AMr 73,000 protein isolated from the normal erythrocyte membranes of one PNH patient by using anti-DAF IgG was largely absent from the abnormal erythrocytes of this individual, suggesting that PNH cells lack the DAF protein. CR1 antigen, however, was present on the abnormal PNH erythrocytes. The results suggest that the primary molecular defect underlying the clinical manifestations of PNH may be the lack of the membrane-associated DAF protein and that the abnormal cells may also exhibit impaired CR1 function.

Complement Activating Enzymes↗

PubMed1983

A monoclonal antibody to rhesus erythrocyte band 3 inhibits invasion by malaria (Plasmodium knowlesi) merozoites.

Receptors on erythrocytes and malaria parasites mediate specific attachment and junction formation between these cells that lead to invasion of the erythrocytes. We identified monoclonal antibody A9 and its subclone A9D3 that bound to rhesus erythrocytes and blocked invasion of the erythrocytes by Plasmodium knowlesi merozoites. The monoclonal antibodies did not block attachment, the initial step in invasion, although swelling and crenation of the erythrocyte, which normally occur after attachment, were rarely observed in the presence of antibody. The monoclonal antibody immunoprecipitated rhesus erythrocyte band 3. It bound to erythrocytes of another Old World monkey, the kra monkey, but not to erythrocytes of New World monkeys, chimpanzees, or man. Since the antibody did not bind to human erythrocytes, we could test for nonspecific toxicity to the parasite by studying the effect of the ascites and purified antibody on invasion of human erythrocytes. The antibody caused a minimal reduction in invasion of human erythrocytes, a reduction no greater than that seen with an unrelated monoclonal antibody. Further evidence that the inhibition was specific came from study of Fab fragments of A9D3. Column-purified Fab fragments reduced invasion of rhesus erythrocytes without affecting invasion of human erythrocytes. Fab fragments preabsorbed with rhesus erythrocytes did not inhibit invasion. From the above data, we conclude that band 3 is involved in a stage in the invasion process after initial recognition.

Absorption↗

PubMed2006

The influence of ferrylhemoglobin and methemoglobin on the human erythrocyte membrane.

The aim of the study was to examine and compare the effects of methemoglobin (metHb) and ferrylhemoglobin (ferrylHb) on the erythrocyte membrane. Kinetic studies of the decay of ferrylhemoglobin (*HbFe(IV)=O denotes ferryl derivative of hemoglobin present 5 min after initiation of the reaction of metHb with H(2)O(2); ferrylHb) showed that autoredecay of this derivative is slower than its decay in the presence of whole erythrocytes and erythrocyte membranes. It provides evidence for interactions between ferrylHb and the erythrocyte membrane. Both hemoglobin derivatives induced small changes in the structure and function of the erythrocyte membrane which were more pronounced for ferrylHb. The amount of ferrylHb bound to erythrocyte membranes increased with incubation time and, after 2 h, was twice that of membrane-bound metHb. The incubation of erythrocytes with metHb or ferrylHb did not influence osmotic fragility and did not initiate peroxidation of membrane lipids in whole erythrocytes as well as in isolated erythrocyte membranes. Membrane acetylcholinesterase activity increased by about 10% after treatment of whole erythrocytes with both metHb and ferrylHb. ESR spectra of membrane-bound maleimide spin label demonstrated minor changes in the conformation of label-binding proteins in ferrylHb-treated erythrocyte membranes. The fluidity of the membrane surface layer decreased slightly after incubation of erythrocytes and isolated erythrocyte membranes with ferrylHb and metHb. In whole erythrocytes, these changes were not stable and disappeared during longer incubation.

Acetylcholinesterase↗

PubMed1994

[Characterization of 24-hour survival rate and duration of survival of hydroxyethyl starch cryopreserved erythrocytes after autologous transfusion in the dog].

BACKGROUND: Cryopreservation of erythrocytes using hydroxyethyl starch (HES) as cryoprotecting additive could result in a nearly unlimited storage stability of preserved red cells. In addition, it would allow its immediate use for transfusion. In order to assess the therapeutic efficacy of erythrocytes cryopreserved with HES, their 24-hour post-transfusion survival and long-term survival was evaluated. MATERIALS AND METHODS: The experiments were carried out with dog erythrocytes as an animal model for human erythrocytes. To each of 6 German shepherd dogs a 15-ml sample of erythrocyte suspension, labeled with 51Cr (25 microCi) after thawing, was autologously injected. Caused by hemolysis 29% of the formerly cryopreserved erythrocytes have not been labeled. To each of 6 control animals 15 ml of a suspension of freshly drawn and 51Cr-labeled erythrocytes was injected. The 51Cr radioactivity in later taken blood samples was a measure for the number of injected erythrocytes having remained in the circulation until the moment of blood withdrawal. The effect of cryopreservation was assessed by comparison of the test group with the control group. RESULTS: In both groups 30% of the applied cells left the circulation within 30 min. This was effected by pharmacological enlargement of the dogs' spleen and not by hemolysis of the erythrocytes. After the first 24 h all of the cryopreserved labeled erythrocytes had survived to the same amount (> 95%) as the labeled fresh red cells. Between 12 h and 20 days after injection, in both groups the 51Cr activity decreased exponentially by 4.8 and 4.5%/d. This difference was not significant. The area under the curve amounted to 1253 and 1257% d, respectively. CONCLUSIONS: There exists a subpopulation of red cells that is destroyed by freezing stress. As a result the freed stroma would be a serious transfusion risk. All erythrocytes having survived the cryopreservation procedure resemble the fresh erythrocytes with regard to the in-vivo survival; their therapeutic efficacy is not impaired. In the context of in-vitro results with human erythrocytes it can be expected that at the present developmental state of the cryopreservation procedure at least 93% of the human erythrocytes cryopreserved with HES have a normal 24-hour and long-term post-transfusion survival.

Animals↗

PubMed1997

Binding of bilirubin to mammalian erythrocytes.

Binding of bilirubin to erythrocytes of various mammalian species in the presence of their respective plasma albumins was studied at pH 8.0, ionic strength 0.41 and at 37 degrees C. In human, buffalo, goat, and sheep, the amount of erythrocyte-bound bilirubin increased with the increase in both the bilirubin/albumin molar ratio (B/A) and the total bilirubin concentration. In all species, the binding patterns were qualitatively similar. However, at any given B/A, goat erythrocytes bound the highest amount of bilirubin, followed by buffalo and human erythrocytes; sheep erythrocytes bound the lowest amount of bilirubin. Increase in erythrocyte-bound bilirubin per unit increase in bilirubin concentration at a constant B/A as obtained from the values of the slope of the plot between erythrocyte-bound bilirubin and total bilirubin in the incubate, was found to be highest for goat erythrocytes, followed by buffalo, human, and sheep erythrocytes. At given bilirubin concentration, percentage fractional binding of bilirubin between any two B/As was found to be highest between 1.5 and 2.0, suggesting that a greater amount of bilirubin was transferred from plasma to cells between B/As 1.5 and 2.0. Percentage fractional binding of bilirubin was highest in goat erythrocytes, followed by buffalo, human, and sheep erythrocytes. These differences in the amount of erythrocyte-bound bilirubin were not due to the effect of various plasma albumins. These results suggest that the difference in bilirubin binding by different mammalian erythrocytes can be attributed to the difference in either the affinity of the erythrocyte receptors or their number or both.

Animals↗

PubMed1985

An antigen of Plasmodium yoelii that translocates into the mouse erythrocyte membrane upon entry into the host cell.

Monoclonal antibodies against the rodent malaria parasite, Plasmodium yoelii, have been prepared and characterized by indirect immunofluorescence on acetone-fixed infected mouse erythrocytes. The antibody of clone K2 reacted strongly with late trophozoites and schizonts, whereas it did so weakly and diffusely with ring forms and early trophozoites. Strong fluorescence was confined to granular structures in schizonts and merozoites. Parasites that invaded erythrocytes in vitro lost the strong fluorescence. Instead, immunofluorescence appeared in the membranes of erythrocytes infected in vitro with merozoites. Erythrocytes infected with more than one merozoite had intensified immunofluorescence in their membranes. Staining of the invaded erythrocytes with 4',6-diamidino-2-phenylindole (DAPI) hydrochloride demonstrated that membranes of all the invaded erythrocytes acquired the P. yoelii antigen. These results suggest that the P. yoelii antigen in merozoites is translocated into erythrocyte membranes upon entry into the host cell. Immunofluorescence continued to appear in membranes of infected erythrocytes throughout the intra-erythrocytic parasite growth. Staining of unfixed infected erythrocytes with the K2 antibody failed to detect the parasite antigen. In contrast, immunofluorescence was present in unfixed membranes of erythrocyte ghosts, which had been spontaneously formed after rupture of schizont-infected erythrocytes by merozoite release. No immunofluorescence appeared in either acetone-fixed or unfixed ghosts of normal erythrocytes. These results suggest the antigenic determinant of the P. yoelii antigen is exposed at the cytoplasmic surface of the infected erythrocyte membrane. Immunoprecipitation has revealed that the K2 antibody recognizes a 160 X 10(3) Mr P. yoelii antigen.

Animals↗

PubMed1994

A simple method for the assessment of macrophage scavenger receptor-ligand interaction: adherence of erythrocytes coated with oxidized low density lipoprotein and modified albumin to macrophages.

A simple method is described for the assessment of the binding of macrophage scavenger receptor and its ligands such as oxidized low density lipoprotein (ox-LDL) and maleylated bovine serum albumin (m-BSA). In this method, the binding of ox-LDL or m-BSA to macrophages is observed as the adherence of erythrocytes precoated with ox-LDL or m-BSA under phase-contrast microscopy. Erythrocytes were coated with native LDL or ox-LDL simply by incubating mouse erythrocytes with LDL. The ox-LDL-coated erythrocytes attached to the monolayer of mouse peritoneal macrophages without phagocytosis at 37 degrees C, while native LDL-coated erythrocytes did not. The extent of the adherence of the ox-LDL-coated erythrocytes to the macrophages was conveniently expressed as the proportion of the macrophages binding the coated erythrocytes. An optimal concentration of ox-LDL for erythrocyte coating giving maximum erythrocyte adherence to macrophages was 10 micrograms/ml as protein at an erythrocyte concentration of 2%. The binding of the ox-LDL-coated erythrocytes to the macrophages could be inhibited by ligands for scavenger receptor, and reflected the extent of LDL oxidation. Mouse erythrocytes were successfully coated with m-BSA using chromium(III) ion as adsorbent. The m-BSA-coated erythrocytes attached to the macrophages, while native BSA-coated erythrocytes did not. The adherence was inhibitable with ligands for scavenger receptor, and reflected the extent of maleylation. The method would be useful particularly for measurement of the ligand-binding activity of the receptor, and the receptor-binding activity of the ligands.

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