Formation Of Stomatocytes Research Articles

Hemolysis Pathways during Storage of Erythrocytes and Inter-Donor Variability in Erythrocyte Morphology

Background: Red blood cells (RBCs) stored for transfusions can lyse over the course of the storage period. The lysis is traditionally assumed to occur via the formation of spiculated echinocyte forms, so that cells that appear smoother are assumed to have better storage quality. We investigate this hypothesis by comparing the morphological distribution to the hemolysis for samples from different donors. Methods: Red cell concentrates were obtained from a regional blood bank quality control laboratory. Out of 636 units processed by the laboratory, we obtained 26 high hemolysis units and 24 low hemolysis units for assessment of RBC morphology. The association between the morphology and the hemolysis was tested with the Wilcoxon-Mann-Whitney U test. Results: Samples with high stomatocyte counts (p = 0.0012) were associated with increased hemolysis, implying that cells can lyse via the formation of stomatocytes. Conclusion: RBCs can lyse without significant echinocyte formation. Lower degrees of spiculation are not a good indicator of low hemolysis when RBCs from different donors are compared.

Motion Control of Polymeric Nanomotors Based on Host–Guest Interactions

AbstractControlling the motion of artificial self‐propelled micro‐ and nanomotors independent of the fuel concentration is still a great challenge. Here we describe the first report of speed manipulation of supramolecular nanomotors via blue light‐responsive valves, which can regulate the access of hydrogen peroxide fuel into the motors. Light‐sensitive polymeric nanomotors are built up via the self‐assembly of functional block copolymers, followed by bowl‐shaped stomatocyte formation and incorporation of platinum nanoparticles. Subsequent addition of β‐cyclodextrin (β‐CD) leads to the formation of inclusion complexes with the trans‐isomers of the azobenzene derivatives grafted from the surfaces of the stomatocytes. β‐CDs attachment decreases the diffusion rate of hydrogen peroxide into the cavities of the motors because of partly blocking of the openings of the stomatocyte. This results in a lowering of the speed of the nanomotors. Upon blue light irradiation, the trans‐azobenzene moieties isomerize to the cis‐form, which lead to the detachment of the β‐CDs due to their inability to form complexes with the cis‐isomer. As a result, the speed of the nanomotors increases accordingly. Such a conformational change provides us with the unique possibility to control the speed of the supramolecular nanomotor via light‐responsive host–guest complexation. We envision that such artificial responsive nano‐systems with controlled motion could have potential applications in drug delivery.

Motion Control of Polymeric Nanomotors Based on Host-Guest Interactions.

Controlling the motion of artificial self-propelled micro- and nanomotors independent of the fuel concentration is still a great challenge. Here we describe the first report of speed manipulation of supramolecular nanomotors via blue light-responsive valves, which can regulate the access of hydrogen peroxide fuel into the motors. Light-sensitive polymeric nanomotors are built up via the self-assembly of functional block copolymers, followed by bowl-shaped stomatocyte formation and incorporation of platinum nanoparticles. Subsequent addition of β-cyclodextrin (β-CD) leads to the formation of inclusion complexes with the trans-isomers of the azobenzene derivatives grafted from the surfaces of the stomatocytes. β-CDs attachment decreases the diffusion rate of hydrogen peroxide into the cavities of the motors because of partly blocking of the openings of the stomatocyte. This results in a lowering of the speed of the nanomotors. Upon blue light irradiation, the trans-azobenzene moieties isomerize to the cis-form, which lead to the detachment of the β-CDs due to their inability to form complexes with the cis-isomer. As a result, the speed of the nanomotors increases accordingly. Such a conformational change provides us with the unique possibility to control the speed of the supramolecular nanomotor via light-responsive host-guest complexation. We envision that such artificial responsive nano-systems with controlled motion could have potential applications in drug delivery.

The acetylcholinesterase (AChE) inhibitor and anti-Alzheimer drug donepezil interacts with human erythrocytes

Donepezil is used to treat symptomatically the Alzheimer's disease (AD). This drug is a specific inhibitor of the enzyme acetylcholinesterase (AChE), whose main physiological function is to hydrolyze the neurotransmitter acetylcholine. The main objective of this work was to study the effect of donepezil on human erythrocytes as AChE is present in its membrane. For this purpose, human erythrocytes and molecular model of its membrane built-up of dimyristoylphosphatidylcholine (DMPC) and dimyristoylphosphatidylethanolamine (DMPE) were used. The latter correspond to classes of phospholipids present in the outer and inner monolayers of the human erythrocyte membrane, respectively. Our experimental evidences obtained from X-ray diffraction and differential scanning calorimetry (DSC) analysis indicated that donepezil was capable of interacting with both phospholipids. Fluorescence spectroscopy results showed a moderate increase in the fluidity of the hydrophobic tails of DMPC and isolated unsealed human erythrocyte membranes (IUM). On the other hand, results by scanning electron microscopy (SEM) and optical defocusing microscopy (DM) showed that the drug changed the normal biconcave shape of the erythrocytes inducing the formation of stomatocytes (cup-shaped cells). This effect was explained by the incorporation of donepezil molecules into the erythrocyte membrane and interactions with AChE.

The Blood Bag Plasticizer Di-2-Ethylhexylphthalate Causes Red Blood Cells to Form Stomatocytes, Possibly by Inducing Lipid Flip-Flop

Background: During storage of red blood cell (RBC) concentrates, the plasticizer di-2-ethylhexylphthalate (DEHP) that keeps the blood bags soft leaches out and can be taken up by the RBCs. DEHP is known to be beneficial for the RBC storage quality, but the molecular mechanisms of the action are unknown. Methods: Aqueous suspensions of DEHP were added to RBCs in buffer. The morphological effects were observed on RBCs from 5 donors. Flow cytometry with annexin A5 binding was used to measure the exposed phosphatidylserine. Results: DEHP induced the formation of stomatocytes at concentrations as low as ng/ml, provided that the cell suspension was also sufficiently dilute. Some spherocytes, which were susceptible to lysis, were also formed; after lysis, RBC ghosts were seen to continue the transition to the cup-shaped stomatocyte form. Incubation with DEHP increased the exposed phosphatidylserine, an effect that was also observed in the presence of vanadate, which inhibits the ATP-dependent translocases that maintain the membrane's lipid asymmetry. Conclusions: DEHP can have an active effect on RBC shape, instead of just preventing the storage-related shape changes. The effect appears to be mediated by increased flip-flop of lipids between the leaflets of the RBC membrane.

Morphology Under Control: Engineering Biodegradable Stomatocytes.

Biodegradable nanoarchitectures, with well-defined morphological features, are of great importance for nanomedical research; however, understanding (and thereby engineering) their formation is a substantial challenge. Herein, we uncover the supramolecular potential of PEG–PDLLA copolymers by exploring the physicochemical determinants that result in the transformation of spherical polymersomes into stomatocytes. To this end, we have engineered blended polymersomes (comprising copolymers with varying lengths of PEG), which undergo solvent-dependent reorganization inducing negative spontaneous membrane curvature. Under conditions of anisotropic solvent composition across the PDLLA membrane, facilitated by the dialysis methodology, we demonstrate osmotically induced stomatocyte formation as a consequence of changes in PEG solvation, inducing negative spontaneous membrane curvature. Controlled formation of unprecedented, biodegradable stomatocytes represents the unification of supramolecular engineering with the theoretical understanding of shape transformation phenomena.

In vitro effects of the anti-Alzheimer drug memantine on the human erythrocyte membrane and molecular models

Memantine is a NMDA antagonist receptor clinically used for treating Alzheimer's disease. NMDA receptors are present in the human neurons and erythrocyte membranes. The aim of the present study was to investigate the effects of memantine on human erythrocytes. With this purpose, the drug was developed to in vitro interact with human red cells and bilayers built-up of dimyristoylphosphatidylcholine (DMPC) and dimyristoylphosphatidylethanolamine (DMPE). The latter represent lipids respectively present in both outer and inner monolayers of the red cell membrane. Results obtained by scanning electron microscopy (SEM) showed that memantine changed the normal biconcave shape of red cells to cup-shaped stomatocytes. According to the bilayer-couple hypothesis the drug intercalated into the inner monolayer of the erythrocyte membrane. Experimental results obtained by X-ray diffraction on multibilayers of DMPC and DMPE, and by differential scanning calorimetry on multilamellar vesicles indicated that memantine preferentially interacted with DMPC in a concentration-dependent manner. Thus, it can be concluded that in the low therapeutic plasma concentration of circa 1 μM memantine is located in NMDA receptor channel without affecting the erythrocyte shape. However, at higher concentrations, once the receptors became saturated excess of memantine molecules (20 μM) would interact with phosphoinositide lipids present in the inner monolayer of the erythrocyte membrane inducing the formation of stomatocytes. However, 40–50 μM memantine was required to interact with isolated phosphatidylcholine bilayers.

Breaking of the phosphodiester bond: a key factor that induces hemolysis.

In-depth understanding the toxicity of nanomaterials in red blood cells (RBCs) is of great interest, because of the importance of RBCs in transporting oxygen in blood circulation. Although the toxic effects of nanoparticles in RBCs have been revealed, the conclusions from the literature are conflicting, and in particular, the toxic mechanism is still at the infant stage. Herein, we investigated the size-dependent toxicity of well-known CdTe semiconductor quantum dots (QDs) and revealed the exact toxic mechanism at the molecular level by confocal microscopy and Fourier transform infrared (FT-IR) spectroscopy techniques. We found that smaller mercaptosuccinic acid-capped CdTe QDs (MSA-QDs) with the green-emitting color could cause hemagglutination whereas the middle-size yellow-emitting MSA-QDs induced the formation of stomatocytes and echinocytes and the bigger size red-emitting MSA-QDs induced heavy hemolysis and the formation of lots of ghost cells. The FT-IR data proved that all the MSA-QDs were likely to bond to the RBCs membranes and caused the structural changes of lipid and protein in RBCs. But only the red-emitting MSA-QDs caused the breakage of the phosphodiester bond, which might cause the heavy hemolysis. To some extent, this is the first example that reveals the hemolysis mechanism at the molecular level.

Anemia, Shortened Erythrocyte Lifespan and Stomatocytosis In a Flippase Mutant Mouse Strain

BackgroundMammalian erythrocytes are anucleate biconcave discs with marked flexibility and deformability, features necessary for efficient function. The cell membrane consists of a lipid bilayer containing proteins and an asymmetric distribution of phospholipids (PL). Phosphatidylcholine and sphingomyelin are predominantly concentrated in the outer layer and phosphatidylserine (PS) and phosphatidylethanolamine are mainly confined to the inner layer of the erythrocyte membrane. Maintenance of PL asymmetry is essential for erythrocyte survival and function as increased externalization of PS results in adherence of erythrocytes to vascular endothelium, activation of plasma blood clotting factors and premature removal from the circulation. Flippases, floppases and scramblases are the enzymes responsible for the establishment and maintenance of PL distribution.A mouse deficient in the flippase ATP11C was generated through ENU mutagenesis and was found to have reduced hemoglobin in comparison to wild type (WT) littermates. This study was performed to characterize the etiology of anemia in these mice. ResultsATP11C-deficient mice had significant reductions in erythrocyte numbers and hematocrit compared to WT but higher MCH and MCV. Reticulocyte numbers were comparable to WT as were serum iron parameters. Bone marrow and splenic erythropoiesis was normal in ATP11C mutant mice, however, erythrocyte lifespan was reduced by 35%. A marked increase in the frequency of PS+ erythrocytes was demonstrated in ATP11C mutant mice and was shown to increase with erythrocyte age. Bone marrow and splenic erythroblasts from ATP11C-deficient animals displayed a lower rate of PS translocation in vitro compared to WT confirming defective flippase activity.Erythrocytes and late-stage splenic erythroblasts in ATP11C mutants were significantly larger than WT based on flow cytometry. SEM revealed the majority of mutant erythrocytes displayed stomatocyte-like morphology, as confirmed on peripheral blood smears. The osmotic fragility of the ATP11C-deficient erythrocytes was comparable to WT erythrocytes as was Na+ and K+ homeostasis. DiscussionThese studies reveal the important role of flippases in maintaining normal erythrocyte function through the maintenance of the asymmetric distribution of PS within the membrane. Perturbation of this process, as seen in the ATP11C-deficient mice, results in (i) increased exposure of PS on the erythrocyte outer membrane, (ii) increased size of late-stage erythroblasts and erythrocytes with stomatocyte formation (iii) reduced erythrocyte lifespan and (iv) normochromic anemia. This study, therefore, reveals a new mechanism for stomatocytosis and raises the question of whether mutations in ATP11C may serve as a previously unclassified cause of anemia in humans. Disclosures:No relevant conflicts of interest to declare.

Entrapment of Metal Nanoparticles in Polymer Stomatocytes

Polymersomes assembled from amphiphilic block copolymers containing a glassy hydrophobic segment can be further re-engineered to perform a controlled shape transformation from a thermodynamically stable spherical morphology to a kinetically trapped stomatocyte structure. The stable bowl-shape stomatocyte morphology is ideal for the specific physical entrapment of nanoparticles for potential use in heterogeneous catalysis and drug delivery. Herein we report two approaches to obtain a selective and controlled entrapment of platinum nanoparticles (PtNP) of different sizes and shapes inside the stomatocyte structure. In the first approach, the stomach of the stomatocytes is used to template the growth of the PtNP by controlling and confining the nucleation points inside the cavity. In the second method, preformed nanoparticles are engulfed during the stomatocyte formation process. Synergistically, the reverse effect is observed, that is, differently shaped nanoparticles were shown to exhibit a templating effect on the stomach formation of the stomatocytes.

Cambios de la morfología de los eritrocitos inducidos por clorpromazina e imipramina

Chlorpromazine (CPZ), a phenothiazine derivative, is a potent antipsychotic agent and imipramine (IP) is a widely used tricyclic antidepressant. The interaction between these molecules and erythrocyte membranes is of particular interest considering the role of these cells in the transport and release of these drugs at the central nervous system. In the present paper, we intend to study the effects of IP on erythrocyte membranes and to compare these effects with those of CPZ. Erythrocytes from adult Sprague-Dawley rats were incubated separately with different concentrations of IP or CPZ for lh at room temperature, fixed and stained by Giemsa. Changes in erythrocyte morphology were quantified by an image analysis system. The interaction of both drugs, CPZ and IP, with the erythrocyte membrane causes similar changes in cell shape. Increasing concentrations of both drugs induces the formation of stomatocytes, spherostomatocytes and spherocytes, because of an irreversible loss of area and volume, probably due to endovesiculation. Our results also show that the CPZ is more potent than IP.

A study of the perturbation effects of the local anesthetic procaine on human erythrocyte and model membranes and of modifications of the sodium transport in toad skin

The interaction of the local anesthetic procaine with human erythrocytes, isolated unsealed human erythrocyte membranes (IUM), isolated toad skins, and molecular models is described. The latter consisted of phospholipid multilayers built-up of dimyristoylphosphatidylcholine (DMPC) and of dimyristoylphosphatidylethanolamine (DMPE), representatives of phospholipid classes located in the outer and inner monolayers of the human erythrocyte membrane, respectively. Optical and scanning electron microscopy of human erythrocytes revealed that procaine induced the formation of stomatocytes. Experiments performed on IUM at 37 °C by fluorescence spectroscopy showed that procaine interacted with the phospholipid bilayer polar groups but not with the hydrophobic acyl chains. X-ray diffraction indicated that procaine perturbed DMPC structure to a higher extent when compared with DMPE, its polar head region being more affected. Electrophysiological measurements disclosed a significant decrease in the potential difference (PD) and in the short-circuit current (Isc) after the application of procaine to isolated toad skin, reflecting inhibition of active ion transport.

Erythrocyte membrane modifying agents and the inhibition of Plasmodium falciparum growth: structure–activity relationships for betulinic acid analogues

The natural triterpene betulinic acid and its analogues (betulinic aldehyde, lupeol, betulin, methyl betulinate and betulinic acid amide) caused concentration-dependent alterations of erythrocyte membrane shape towards stomatocytes or echinocytes according to their hydrogen bonding properties. Thus, the analogues with a functional group having a capacity of donating a hydrogen bond (COOH, CH2OH, CONH2) caused formation of echinocytes, whereas those lacking this ability (CH3, CHO, COOCH3) induced formation of stomatocytes. Both kinds of erythrocyte alterations were prohibitive with respect to Plasmodium falciparum invasion and growth; all compounds were inhibitory with IC50 values in the range 7–28 μM, and the growth inhibition correlated well with the extent of membrane curvature changes assessed by transmission electron microscopy. Erythrocytes pre-loaded with betulinic acid or its analogues and extensively washed in order to remove excess of the chemicals could not serve as hosts for P. falciparum parasites. Betulinic acid and congeners can be responsible for in vitro antiplasmodial activity of plant extracts, as shown for Zataria multiflora Boiss. (Labiatae) and Zizyphus vulgaris Lam. (Rhamnaceae). The activity is evidently due to the incorporation of the compounds into the lipid bilayer of erythrocytes, and may be caused by modifications of cholesterol-rich membrane rafts, recently shown to play an important role in parasite vacuolization. The established link between erythrocyte membrane modifications and antiplasmodial activity may provide a novel target for potential antimalarial drugs.

In vitro Plasmodium falciparum drug sensitivity assay: inhibition of parasite growth by incorporation of stomatocytogenic amphiphiles into the erythrocyte membrane.

Lupeol, which shows in vitro inhibitory activity against Plasmodium falciparum 3D7 strain with a 50% inhibitory concentration (IC50) of 27.7 +/- 0.5 microM, was shown to cause a transformation of the human erythrocyte shape toward that of stomatocytes. Good correlation between the IC50 value and the membrane curvature changes caused by lupeol was observed. Preincubation of erythrocytes with lupeol, followed by extensive washing, made the cells unsuitable for parasite growth, suggesting that the compound incorporates into erythrocyte membrane irreversibly. On the other hand, lupeol-treated parasite culture continued to grow well in untreated erythrocytes. Thus, the antiplasmodial activity of lupeol appears to be indirect, being due to stomatocytic transformation of the host cell membrane and not to toxic effects via action on a drug target within the parasite. A number of amphiphiles that cause stomatocyte formation, but not those causing echinocyte formation, were shown to inhibit growth of the parasites, apparently via a mechanism similar to that of lupeol. Since antiplasmodial agents that inhibit parasite growth through erythrocyte membrane modifications must be regarded as unsuitable as leads for development of new antimalarial drugs, care must be exercised in the interpretation of results of screening of plant extracts and natural product libraries by an in vitro Plasmodium toxicity assay.

Passive transmembrane redistributions of phospholipids as a determinant of erythrocyte shape change. Studies on electroporated cells.

Echinocytes formed from discocytic erythrocytes by electric field pulses at 0 degree C return to the discoytic shape upon incubation at 37 degrees C and subsequently turn into stomatocytes. Active and passive components of phospholipid translocation are involved in this shape recovery. Following low-field-strength pulses (5 kV cm-1), shape recovery is fully suppressed by ATPase inhibitors, such as vanadate. When vanadate is only added after stomatocyte formation has been completed, the cells return to the stage of echinocytosis prevailing before recovery. At higher field strength (7 kV cm-1) and in particular after repetitive field pulses, the subsequent incubation at 37 degrees C results in partial shape recovery even in the presence of vanadate. On the basis of the enhanced passive transmembrane mobilities of phospholipid probes observed previously following electroporation, the shape changes in the presence of vanadate are proposed to be due to a passive net movement of phospholipids from the outer to the inner membrane leaflet, as a consequence of the different mobilities of the various membrane phospholipids. Repetitive pulses at higher field strengths lead to a progressively more discocytic stationary shape during subsequent resealing. This phenomenon is explained by the progressively increased transbilayer mobility of the normally almost immobile phospholipid sphingomyelin and a consecutive progressive symmetrization of all membrane phospholipids.

Electric field pulses induce reversible shape transformation of human erythrocytes.

Electric field pulses > 2-3 kV cm-1, long known to induce membrane poration and fusion of erythrocytes as well as to enhance the transbilayer mobility of phospholipids and to perturb aminophospholipid asymmetry, are shown to induce, at 0 degree C, transformation of the discocytic cells into echinocytes and spheroechinocytes. The extent of transformation increases with strength, duration and number of pulses. Its time course is biphasic, a major rapid phase (t/2 approximately 5 s) being followed by a minor one, lasting for 2-3 h. Shape transformation goes along with the exofacial exposure of phosphatidylserine (PS), detected by FITC-annexin V binding and quantified by a calibration curve established via externally inserted dilauroylphosphatidylserine. Incubation of these echinocytes at 37 degrees C leads to a rapid recovery of the discocytic shape followed by slower formation of stomatocytes. Shape recovery is temperature dependent (Ea approximately 100 kJ/mol), and can be impaired by depletion of ATP or Mg++ and by addition of vanadate or fluoride. Shape recovery and stomatocyte formation go along with a rapid loss of annexin binding in about 45% of the cells while the rest maintains its binding capacity. In the presence of vanadate, annexin binding increases in all cells. The results are discussed in the light of the bilayer couple concept of erythrocyte shape and the enhanced transverse mobility of phospholipids. Echinocyte formation is most likely caused by the reorientation of endofacial aminophospholipids to the outer leaflet of the bilayer. Shape recovery and stomatocyte formation probably result from a continuous reinternalization of PS via the ATP dependent aminophospholipid translocase, but may also be supported by downhill movement of PC to the inner leaflet and by other yet unidentified processes.

F145. Oxidative damage of cytoskeletal proteins of erythrocytes and the rheological and microcirculatory impairment

12-O-Tetradecanoylphorbol-13-acetate (TPA) (100 nM) when incubated with human erythrocytes under conditions of ATP depletion, delayed the onset of the morphological transition from discocytes to echinocytes so that at 2 h, when control incubations were estimated to contain 65% echinocytes, those treated with TPA contained 23% echinocytes. TPA did not alter the subsequent rate of the transition which was complete by 3 h in control cells and 5 h in TPA-treated cells. Addition of 100 nM TPA to ATP-depleted erythrocytes at 2.5 h (>80% echinocytes) for 0.5 h at 37 °C resulted in 17% reversal to a discocyte morphology, but as the time of incubation under conditions of ATP depletion was extended, the level of the reversal fell. TPA had no significant effect on the fall in ATP concentrations over the time course of the experiments (5 h). Preincubation of discocytes with TPA for 10 min also prevented, by approx. 50%, the echinocytosis induced by the calcium (0.2 mM) loading of discocytes using 5 μM A23187. TPA was unable to reverse the echinocyte morphology of calcium-loaded cells back to discocytes. The less potent tumour promotor 4-phorbol-12,13-didecanoate had no effect on this discocyte-echinocyte transition. Incubation of discocytes with the diacylglycerol 1-oleoyl-2-acetylglycerol (OAG) (1–10 μM) had complex effects on morphology, and the ATP-induced morphological transition, ranging from stomatocyte formation to echinocyte formation, depending upon the concentration of the agent and the time of incubation.

Changes in the ultrastructure of human erythrocytes and in their content of free fatty acids during incubation with hydroperoxide and calcium ionsin vitro

It is shown that incubation of a suspension of human erythrocytes with H2O2 and Ca2+ mainly results in echinocytic transformation and hemolysis; incubation with H2O2 in the absence of Ca2+ is attended by polymorphous changes in erythrocytes: discocyte swelling, formation of stomatocytes and echinocytes and their hemolysis. The level of free fatty acids in human erythrocytes increases for incubation with Ca2+ and calcimycin under anaerobic conditions and drops for H2O2-induced activation of lipid peroxidation.

The influence of chlorpromazine on the potential-induced shape change of human erythrocyte

The effect of chlorpromazine (CPZ) on the shape of human erythrocytes with different values of transmembrane potential (TMP) was investigated. The shape of red blood cells with negative values of the TMP remained unchanged after the formation of stomatocytes by chlorpromazine, while cells with positive TMP showed a characteristic time course of shape change during the incubation with CPZ. Experiments with vanadate show that this might be due to a difference in the activity of the phospholipid-translocase at different values of TMP.

Modulation of erythrocyte vesiculation by amphiphilic drugs

Release of acetylcholinesterase-containing vesicles from human erythrocyte membranes induced by dimyristoylphosphatidylcholine (DMPC) was inhibited by exposure of red cells to cationic amphiphilic drugs like tetracaine, chlorpromazine and primaquine which all are known to induce stomatocyte formation. On the other hand, the process was facilitated when red cells were exposed to crenators like the anionic drugs indomethacine and phenylbutazone or when DMPC was added to calcium-loaded red cells. The results suggest that agents which are known to modulate red cell shape do also influence the vesiculation behavior of the cells.