Effects of Cholesterol on Membrane Stability of Human Erythrocytes

Abstract

Human erythrocytes contain abundant cholesterol as membrane lipids. Cholesterol contributes to the stability and function of the membrane. Membrane stability of the erythrocyte has been mainly examined under hypotonic conditions, but not under high hydrostatic pressure. So, the effect of cholesterol on the membrane stability of human erythrocyte was examined under a pressure of 200 MPa. As with hypotonic hemolysis, the pressure-induced hemolysis was enhanced by depletion of cholesterol from the intact erythrocyte membrane, whereas suppressed by cholesterol loading to the intact one. Enhancement of such hemolysis was associated with the suppression of fragmentation, whereas the hemolysis was suppressed by the facilitation of vesiculation. Cholesterol induced the tight linkage of the lipid bilayer with cytoskeleton. Taken together, these results suggest that the erythrocyte membrane stability is affected by such tight linkage by cholesterol.

INTRODUCTION

Human erythrocyte membrane is characterized by its stability and deformability.¹⁾ Human erythrocyte has a life-span of about 120 d and can squeeze through blood vessels of 3 µm in diameter by deforming biconcave shape of diameter 8 µm.²⁾ Membrane stability of erythrocytes is evaluated by examining the degree of hemolysis under stress conditions.³⁾ Here, we consider membrane damages induced by stress like the cell volume change. When the erythrocytes suspended in isotonic buffer are exposed to a high hydrostatic pressure (simply, called pressure), the cells are compressed so that the vesiculation, fragmentation, and hemolysis occur.^4–7) The proportion of vesicles, fragmented particles, and open ghosts is modulated by the properties of the erythrocyte membrane.^6,7) The pressure-induced hemolysis is dependent on the size of vesicles released from the membrane surface.^6,7) On the other hand, when the erythrocytes are suspended in hypotonic buffer, the cells are swollen by water influx and finally burst.⁸⁾ Here, such burst is called hypotonic hemolysis. Hypotonic hemolysis is readily induced in the erythrocyte with a decreased surface area-to-volume ratio.⁹⁾

Cholesterol is abundant molecules in membrane lipids of human erythrocytes.¹⁰⁾ Parts of these cholesterol molecules form the complex with sphingomyelin in the outer leaflet of the lipid bilayer.^11,12) Such complex is insoluble in non-ionic detergents and called as a raft.^11,12) The remaining cholesterol is mainly distributed in the inner leaflet.¹³⁾ The flip-flop rate of cholesterol is fast.¹⁴⁾ Up to date, it is well known that cholesterol plays an important role in the regulation of membrane fluidity and the maintenance of membrane stability.¹⁵⁾ However, the detailed mechanism concerning such actions of cholesterol is elusive. So, it is of interest to examine the effect of cholesterol on stress-induced hemolysis. In this article, we demonstrate that the pressure-induced hemolysis and hypotonic one are suppressed by cholesterol and that cholesterol strengthens the interaction of the bilayer with cytoskeleton, perhaps via its binding to transmembrane proteins.

MATERIALS AND METHODS

Materials

Cholesterol and methyl-β-cyclodextrin (CD) were purchased from Nacalai Tesque (Kyoto, Japan) and Sigma-Aldrich (U.S.A.), respectively. All other chemicals were of reagent grade.

Pressure Treatment of Human Erythrocytes

Human erythrocytes were obtained from the Fukuoka Red Cross Blood Center. The erythrocytes were washed three times with phosphate-buffered saline (PBS) (10 mM sodium phosphate, 150 mM NaCl, pH 7.4). To decrease the membrane cholesterol contents, the erythrocytes were incubated for 1 h at 37 °C at a 20% hematocrit in 0.5% CD-containing PBS, which was preincubated for 30 min at 37 °C, and washed with PBS.¹⁶⁾ For the loading of cholesterol, the erythrocytes were incubated for 1 h at 37 °C at a 20% hematocrit in PBS containing 0.5% CD-1.3 mM cholesterol, which was preincubated for 1–2 h at 37 °C. After the incubation, the erythrocytes were washed with PBS. For the pressure treatment, the erythrocytes in PBS were incubated for 30 min at 200 MPa and 37 °C, and decompressed up to atmospheric pressure. Such pressure-treated erythrocyte suspensions were used for measurements of hemolysis and flow cytometry. For the hemolysis, the suspensions were centrifuged for 1 min at 1000 × g and room temperature (approx. 25 °C), and the absorbance of hemoglobin in the supernatant was measured at 542 nm.⁴⁾ Moreover, the properties of particles produced from the erythrocytes by pressure were measured using an EPICS XL-MCL flow cytometer (Coulter, U.S.A.).⁷⁾ The morphology of pressure-treated erythrocytes was examined using a scanning electron microscope (SEM).¹⁷⁾ Pressure-treated erythrocytes were fixed for 2 h at room temperature using 1% glutaraldehyde. The fixed cells were dehydrated by ethylalcohol. The dehydrated samples were immersed in t-butylalcohol–ethylalcohol mixed solution (v/v = 1 : 1) for 30 min and then 100% t-butylalchohol for 30 min. The samples in t-butylalchohol were frozen at −5 °C and dried. The dried samples were coated with Pt. The cell morphology was observed using an SEM (model JSM-LV, JEOL).

For the hypotonic hemolysis, the erythrocytes were suspended in 10 mM sodium phosphate (pH 7.4) containing 54 mM NaCl, incubated for 10 min at 37 °C, and centrifuged for 1 min at 1000 × g. The degree of hemolysis was estimated, as mentioned above.

Analysis of Erythrocyte Membrane Lipids

Membrane lipids were extracted from erythrocytes by using a mixed solvent of chloroform and methanol (v/v = 2 : 1).¹⁸⁾ The cholesterol concentration was determined by the method of Zlatkis et al.¹⁹⁾ The total phospholipids were estimated by the method of Ames.²⁰⁾

Bilayer–Cytoskeleton Interaction

To extract the cytoskeletal proteins from the membrane, open ghosts, which were prepared from the erythrocytes using 5 mM sodium phosphate, pH 8 (5P8) at 0 °C, were suspended in 7 volume of 5P8 and incubated for 1 h at 37 °C.²¹⁾ After the incubation, ghost suspensions were centrifuged for 20 min at 20000 × g and 4 °C. The protein concentration in the supernatant was determined by the method of Lowry et al.²²⁾

RESULTS

Effects of Cholesterol on Membrane Stability of Human Erythrocytes

To examine effects of cholesterol on the membrane stability of human erythrocytes, the amount of membrane cholesterol was modulated by using a CD or a CD-cholesterol complex. The cholesterol content was evaluated for total membrane phospholipid one. Pressure (200 MPa)-induced hemolysis and hypotonic one¹⁵⁾ were enhanced upon depletion of cholesterol from intact erythrocyte membranes (cholesterol/phospholipid approx. 0.6 at molar ratio), whereas suppressed upon cholesterol loading (Fig. 1). Moreover, when the cholesterol-depleted erythrocyte membranes were reloaded with cholesterol to cholesterol/phospholipid approx. 1.6, the hemolysis at 200 MPa and hypotonic lysis were suppressed, as seen in cholesterol loading of intact erythrocyte membranes (Fig. 1). These results suggest that the membrane stability under pressure or hypotonic stress is dependent on cholesterol content.

Fig. 1. Effects of Cholesterol on Stress-Induced Hemolysis in Human Erythrocytes

Cholesterol contents in intact cell membranes were modulated using a CD or a CD-cholesterol complex (circle). Cholesterol-depleted cell membranes were reloaded using a CD-cholesterol complex (triangle). (A) 200 MPa-induced hemolysis, (B) Hypotonic hemolysis. Values are means ± standard deviation (S.D.) for three independent experiments.

The shape of 200 MPa-treated erythrocytes was examined using a SEM (Fig. 2). The shape of intact erythrocytes exposed to a pressure of 200 MPa changed from biconcave disk to spherical form, and many vesicles were observed on the surface of mother cells (Fig. 2A). Here, mother cells only were observed since other particles such as open ghosts and released vesicles were removed by centrifugation. In cholesterol-depleted erythrocytes, pressure-treated cells became spherical and vesicles were formed on the cell surface (Fig. 2B). In cholesterol-loaded erythrocytes, on the other hand, few vesicles were observed on the surface of pressure-treated cells (Fig. 2C).

Fig. 2. Scanning Electron Microscopy of Pressure-Treated Human Erythrocytes

Intact (none, A), cholesterol-depleted (−, B), and cholesterol-loaded (+, C) erythrocytes were exposed to a pressure of 200 MPa for 30 min at 37 °C.

When the erythrocyte suspensions exposed to a pressure of 200 MPa are examined by a flow cytometer, mother cells, fragmented particles, released vesicles, and open ghosts are observed in regions a, b, c, and d, respectively^7,23) (Fig. 3A). Here, the size of closed particles is as follows: mother cells > fragmented particles > vesicles.²³⁾ The increment of open ghosts indicates the enhancement of hemolysis.²³⁾ Upon facilitation of fragmentation and vesiculation, the hemolysis at 200 MPa is suppressed.^7,23) Moreover, the effect of fragmentation on the hemolysis at 200 MPa is more dominant than that of vesiculation.²³⁾ When cholesterol-depleted erythrocytes were exposed to a pressure of 200 MPa, particles in regions a and b deceased and particles in regions c and d increased (Fig. 3B). This indicates that the hemolysis enhances upon suppression of fragmented particles. In cholesterol-loaded erythrocytes, on the other hand, the vesiculation was facilitated so that pressure-induced hemolysis was significantly suppressed (Fig. 3C)

Fig. 3. Flow Cytometry of 200 MPa-Treated Erythrocyte Suspensions

Intact (A), cholesterol-depleted (B), and cholesterol-loaded (C) erythrocytes were exposed to a pressure of 200 MPa for 30 min at 37 °C. After the decompression, the erythrocyte suspensions were used for flow cytometric measurements. Values are means ± S.D. for three independent experiments.

Effects of Cholesterol on the Bilayer–Cytoskeleton Interaction

Membrane stability of human erythrocytes is affected by the bilayer–cytoskeleton interaction.^1–3) Parts of cytoskeletal proteins such as spectrin and actin are detached from the bilayer when ghosts are exposed to hypotonic buffer.²¹⁾ The amount of detached proteins reduces under tight linkage between bilayer and cytoskeleton.²¹⁾ So, the effect of cholesterol on such detachment was tested. The amount of detached proteins from the bilayer under hypotonic buffer significantly enhanced upon depletion of cholesterol from intact membrane, and reduced upon sterol loading (Fig. 4A). Thus, cytoskeletal proteins became refractory to such detachment under cholesterol loading (Fig. 4B). These results suggest that cholesterol strengthens the bilayer–cytoskeleton interaction.

Fig. 4. Effects of Cholesterol on Protein Detachment from the Membrane and Cholesterol Binding Amino Acid Sequences

(A) Ghosts were prepared from intact (none) erythrocytes, cholesterol-depleted (−) or cholesterol-loaded (+) ones. For detachment of cytoskeletal proteins, these ghosts were incubated in 5P8 for 30 min at 37 °C. (B) Relation between detached proteins and cholesterol contents. (C) Transmembrane segments (TM) of band 3,³²⁾ glycophorin A (Human #P02724), and glycophorin C (Human #P04921). Under lines of straight and wave present CARC and CRAC motifs, respectively. Values are means ± S.D. for three independent experiments. *^, ** p < 0.01 vs. None.

DISCUSSION

Under hypotonic conditions, the erythrocytes are swollen and hemolyzed.⁸⁾ The swollen cells are characterized by reduction of the surface area-to-volume ratio.⁹⁾ Upon compression at 200 MPa of erythrocytes under isotonic conditions, on the other hand, the cell volume decreases and the hemolysis occurs upon release of large vesicle.^6,7) For instance, hereditary spherocytic (HS) erythrocytes are characterized by a decreased surface area-to-volume ratio,^9,24) which is ascribed to an abnormal interaction between cytoskeleton and band 3.^9,24) Compared with normal erythrocytes, HS cells are more fragile under hypotonic buffer, but not under compression at 200 MPa.²⁵⁾ Moreover, the erythrocytes treated with 4,4′-diisothiocyanostilbene-2,2′-disulfonate (DIDS), an inhibitor of anion transporter band 3,²⁶⁾ exhibit a 10% increased cell volume due to water uptake.²⁷⁾ In DIDS-modified erythrocytes, the hypotonic hemolysis is enhanced, whereas the hemolysis at 200 MPa is suppressed.²⁶⁾ Interestingly, in cholesterol-depleted erythrocytes that exhibit a decreased surface area-to-volume ratio,^28,29) hypotonic hemolysis¹⁵⁾ and pressure-induced one are enhanced. Taken together, the hypotonic hemolysis of erythrocytes is enhanced upon reduction of the surface area-to-volume ratio, whereas pressure-induced hemolysis is independent. Furthermore, denatured spectrin in 49 °C-treated erythrocytes is tightly attached to the bilayer.⁶⁾ In these erythrocytes, 200 MPa-induced hemolysis and hypotonic one are suppressed.⁶⁾ Similar results are obtained in cholesterol-loaded erythrocytes (Fig. 1). These results suggest the modulation of the bilayer–cytoskeleton interaction by cholesterol.

Pressure-induced hemolysis is greatly suppressed by facilitation of vesiculation, as with 49 °C-heated erythrocytes⁶⁾ or ATP-depleted ones²³⁾ and also of fragmentation, as seen under hypertonic buffer of normal cells.²³⁾ The present work shows that the hemolysis at 200 MPa is suppressed by the facilitated vesiculation in cholesterol-loaded erythrocytes, whereas is enhanced by the suppressed fragmentation in cholesterol-depleted cells, as with ATP-rich ones.²³⁾ Why does cholesterol loading into the membrane facilitate the vesiculation under pressure? In 49 °C-treated erythrocytes and ATP-depleted ones, the bilayer interacts tightly with cytoskeleton so that the vesiculation under pressure is facilitated.^6,23) This work also shows the tight linkage of the bilayer with cytoskeleton by cholesterol. This tight linkage induces the facilitated vesiculation under pressure.²³⁾ Cholesterol is distributed in both leaflets of lipid bilayer. β-Hydroxyl group at C-3 of cholesterol is located in the interface of the bilayer, whereas its isooctyl chain is oriented in the hydrophobic region of the bilayer.³⁰⁾ Thus, it seems unlikely that cholesterol interacts directly with cytoskeletal proteins such as spectrin. Therefore, cholesterol may mediate indirectly the bilayer–cytoskeleton interaction.

Spectrin is mainly attached to the bilayer via linking proteins such as ankyrin and protein 4.1R.¹⁾ Ankyrin links spectrin to the transmembrane protein band 3, whereas protein 4.1R links spectrin to the transmembrane protein glycophorin C.¹⁾ Interestingly, cholesterol binds transmembrane proteins.³¹⁾ The cholesterol-binding motif is the following amino acid consensus sequences: L/V-X_(1–5)-Y/F-X_(1–5)-K/R (CRAC) and K/R-X_(1–5)-Y/F-X_(1–5)-L/V (CARC).³¹⁾ The both motifs are located on the same transmembrane segment in membrane receptors.³¹⁾ Similar motifs are observed in band 3 that comprises 14 transmembrane segments.³²⁾ Indeed, the interaction of cholesterol with band 3 is reported.^30,33) As shown in Fig. 4C, cholesterol binding motifs are located on the fifth transmembrane segment (TM 5) of band 3.³²⁾ Here, the CRAC (LIFIYETFS⁵³⁹K) motif is located in the outer leaflet of lipid bilayer and CARC in its inner leaflet. Moreover, the TM 6 or 7 of band 3 has CRAC or CARC motif, respectively.³²⁾ Importantly, the interaction of spectrin with the bilayer is modulated by the binding of DIDS to Lys-539, which is located at exofacial site on the TM 5.²⁶⁾ Thus, the TM 5 might be predominant than TMs 6 and 7 as a modulator of the bilayer–cytoskeleton interaction. Another transmembrane protein such as glycophrin A interacts with cytoskeletal proteins upon ligand binding.³⁴⁾ Binding motif for cholesterol is observed as a CRAC on the transmembrane segment of glycophorins A and C (Fig. 4C). Thus, it seems likely that the interaction of the bilayer with cytoskeleton is modulated by the binding of cholesterol to the transmembrane proteins such as band 3, glycophorins A, and C. So, we propose that the membrane stability of erythrocytes under pressure is mediated by the cholesterol–transmembrane protein interaction, which is followed by the bilayer–cytoskeleton interaction.

Roles of cholesterol in the membrane stability have been mainly explained by cholesterol–phospholipid interactions.^12,35) Cholesterol in lipid bilayer induces the ordering of hydrocarbon chains in phospholipids so that the liquid ordered phase is formed.¹⁵⁾ Such ordered phase is also induced by high pressure.³⁶⁾ Interestingly, giant liposomes composed of egg phosphatidylcholine (PC) only show a spherical form at a pressure of 285 MPa, but a budded form after decompression.³⁵⁾ However, cholesterol-containing egg PC liposomes remain spherical during such pressure treatment,³⁵⁾ suggesting the stabilization of the liposome structure under pressure by cholesterol. On the other hand, the compression at 200 MPa of cholesterol-loaded erythrocytes induces the vesiculation. This vesiculation is ascribed to the tight linkage of bilayer with cytoskeleton,²³⁾ via cholesterol–transmembrane protein interactions. Thus, we have demonstrated that the modulation of bilayer–cytoskeleton interaction by cholesterol plays an important role in the mechanical stability of human erythrocyte membrane. Further studies on cholesterol–phospholipid interactions under pressure using liposomes composed of erythrocyte membrane phospholipids promise to supplement our idea.

Acknowledgment

This work was supported in part by a Grant (No. 155003) from the Central Research Institute of Fukuoka University.

Conflict of Interest

The authors declare no conflict of interest.

REFERENCES

• 1) Mohandas N, Gallagher PG. Red cell membrane: past, present, and future. Blood, 112, 3939–3948 (2008).
• 2) Bull BS, Breton-Gorius J. Morphology of the erythron. Williams Hematology. (Beutler E, Lichtman MA, Coller BS, Kipps TJ eds.) 5th ed., McGraw-Hill, New York, pp. 349–363 (1995).
• 3) Kitajima H, Yamaguchi T, Kimoto E. Hemolysis of human erythrocytes under hydrostatic pressure is suppressed by cross-linking of membrane proteins. J. Biochem., 108, 1057–1062 (1990).
• 4) Yamaguchi T, Kawamura H, Kimoto E, Tanaka M. Effects of temperature and pH on hemoglobin release from hydrostatic pressure-treated erythrocytes. J. Biochem., 106, 1080–1085 (1989).
• 5) Yamaguchi T, Kajikawa T, Kimoto E. Vesiculation induced by hydrostatic pressure in human erythrocytes. J. Biochem., 110, 355–359 (1991).
• 6) Yamaguchi T, Miyamoto J, Terada S. Suppression of high-pressure-induced hemolysis of human erythrocytes by preincubation at 49˚C. J. Biochem., 130, 597–603 (2001).
• 7) Yamaguchi T, Satoh I, Ariyoshi N, Terada S. High-pressure-induced hemolysis in papain-digested human erythrocytes is suppressed by cross-linking of band 3 via anti-band 3 antibodies. J. Biochem., 137, 535–541 (2005).
• 8) Singh S, Ponnappan N, Verma A, Mittal A. Osmotic tolerance of avian erythrocytes to complete hemolysis in solute free water. Sci. Rep., 9, 7976 (2019).
• 9) King MJ, Zanella A. Hereditary red cell membrane disorders and laboratory diagnostic testing. Int. J. Lab. Hematol., 35, 237–243 (2013).
• 10) Koumanov KS, Tessier C, Momchilova AB, Rainteau D, Wolf C, Quinn PJ. Comparative lipid analysis and structure of detergent-resistant membrane raft fractions isolated from human and ruminant erythrocytes. Arch. Biochem. Biophys., 434, 150–158 (2005).
• 11) Bieberich E. Sphingolipids and lipid rafts: novel concepts and methods of analysis. Chem. Phys. Lipids, 216, 114–131 (2018).
• 12) Pike LJ. Lipid rafts: heterogeneity on the high seas. Biochem. J., 378, 281–292 (2004).
• 13) Courtney KC, Pezeshkian W, Raghupathy R, Zhang C, Darbyson A, Ipsen JH, Ford DA, Khandelia H, Presley JF, Zha X. C24 Sphingolipids govern the transbilayer asymmetry of cholesterol and lateral organization of model and live-cell plasma membranes. Cell Reports, 24, 1037–1049 (2018).
• 14) Brasaemle DL, Robertson AD, Attie AD. Transbilayer movement of cholesterol in the human erythrocyte membrane. J. Lipid Res., 29, 481–489 (1988).
• 15) Bernecker C, Köfeler H, Pabst G, Trötzmüller M, Kolb D, Strohmayer K, Trajanoski S, Holzapfel GA, Schlenke P, Dorn I. Cholesterol deficiency causes impaired osmotic stability of cultured red blood cells. Front. Physiol, 10, 1529 (2019).
• 16) Steck TL, Ye J, Lange Y. Probing red cell membrane cholesterol movement with cyclodextrin. Biophys. J., 83, 2118–2125 (2002).
• 17) Kiyoatake K, Nagadome S, Yamaguchi T. Membrane perturbations induced by the interactions of zinc ions with band 3 in human erythrocytes. Biochem. Biophys. Rep., 2, 63–68 (2015).
• 18) Bligh EG, Dyer WJ. A rapid method of total lipid extraction and purification. Can. J. Biochem. Physiol., 37, 911–917 (1959).
• 19) Zlatkis A, Zak B, Boyle AJ. A new method for the direct determination of serum cholesterol. J. Lab. Clin. Med., 41, 486–492 (1953).
• 20) Ames BN. Assay of inorganic phosphate, total phosphate and phosphatases. Methods Enzymol., 8, 115–118 (1966).
• 21) Yamaguchi T, Ozaki S, Shimomura T, Terada S. Membrane perturbations of erythrocyte ghosts by spectrin release. J. Biochem., 141, 747–754 (2007).
• 22) Lowry OH, Rosebrough NJ, Farr AL, Randall RJ. Protein measurement with the Folin phenol reagent. J. Biol. Chem., 193, 265–275 (1951).
• 23) Yamaguchi T, Fukuzaki S. ATP effects on response of human erythrocyte membrane to high pressure. Biophys. Physicobiol., 16, 158–166 (2019).
• 24) Da Costa L, Galimand J, Fenneteau O, Mohandas N. Hereditary spherocytosis, elliptocytosis, and other red cell membrane disorders. Blood Rev., 27, 167–178 (2013).
• 25) Yamaguchi T, Terada S, Ideguchi H. High-pressure-induced hemolysis of hereditary spherocytic erythrocytes is not suppressed by DIDS labeling. Jpn. J. Physiol., 47, 571–574 (1997).
• 26) Yamaguchi T, Matsumoto M, Kimoto E. Effects of anion transport inhibitors on hemolysis of human erythrocytes under hydrostatic pressure. J. Biochem., 118, 760–764 (1995).
• 27) Blank ME, Hoefner DM, Diedrich DF. Morphology and volume alterations of human erythrocytes caused by the anion transporter inhibitors, DIDS and p-azidobenzylphlorizin. Biochim. Biophys. Acta, 1192, 223–233 (1994).
• 28) Chabanel A, Flamm M, Sung KL, Lee MM, Schachter D, Chien S. Influence of cholesterol content on red cell membrane viscoelasticity and fluidity. Biophys. J., 44, 171–176 (1983).
• 29) Bernecker C, Köfeler H, Pabst G, Trötzmüller M, Kolb D, Strohmayer K, Trajanoski S, Holzapfel GA, Schlenke P, Dorn I. Cholesterol deficiency causes impaired osmotic stability of cultured red blood cells. Front. Physiol., 10, 1529 (2019).
• 30) Kalli AC, Reithmeier RAF. Interaction of the human erythrocyte Band 3 anion exchanger 1 (AE1, SLC4A1) with lipids and glycophorin A: molecular organization of the wright (Wr) blood group antigen. PLOS Comput. Biol., 14, e1006284 (2018).
• 31) Fantini J, Di Scala C, Evans LS, Williamson PT, Barrantes FJ. A mirror code for protein–cholesterol interactions in the two leaflets of biological membranes. Sci. Rep., 6, 21907 (2016).
• 32) Arakawa T, Kobayashi-Yurugi T, Alguel Y, Iwanari H, Hatae H, Iwata M, Abe Y, Hino T, Ikeda-Suno C, Kuma H, Kang D, Murata T, Hamakubo T, Cameron AD, Kobayashi T, Hamasaki N, Iwata S. Crystal structure of the anion exchanger domain of human erythrocyte band 3. Science, 350, 680–684 (2015).
• 33) Schubert D, Boss K. Band 3 protein–cholesterol interactions in erythrocyte membranes. Possible role in anion transport and dependency on membrane phospholipid. FEBS Lett., 150, 4–8 (1982).
• 34) Chasis JA, Mohandas N, Shohet SB. Erythrocyte membrane rigidity induced by glycophorin A–ligand interaction. Evidence for a ligand-induced association between glycophorin A and skeletal proteins. J. Clin. Invest., 75, 1919–1926 (1985).
• 35) Beney L, Perrier-Cornet JM, Hayert M, Gervais P. Shape modification of phospholipid vesicles induced by high pressure: influence of bilayer compressibility. Biophys. J., 72, 1258–1263 (1997).
• 36) Barriga HMG, Law RV, Seddon JM, Ces O, Brooks NJ. The effect of hydrostatic pressure on model membrane domain composition and lateral compressibility. Phys. Chem. Chem. Phys., 18, 149–155 (2016).