Squeezing for Life – Properties of Red Blood Cell Deformability

1The primary function of RBCs is to enable respiration in tissues by providing oxygen and removing carbon dioxide via gas exchange in the lungs. During a typical 120 days lifespan of a RBC, it circulates through arteries, veins and small capillaries traveling –in total– a distance of 500 km. RBC deformability, i.e., the ability of the RBC to change shape is essential for successful passage through these capillaries and splenic sinuses.

2Deformability of RBC depends on the (i) structural properties of the “horizontal” cytoskeletal components such as spectrin, (ii) vertical interaction of cytoskeleton and integral transmembrane complexes that is accomplished by ankyrin, 4.1 and 4.2 protein and the cytosolic domain of band 3 protein (also known as the anion exchanger 1 (AE1) or solute carrier family 4 member 1 (SLC4A1)) and (iii) resistance of the cytosolic pool (i.e., intracellular viscosity, RBC hydration state and surface-volume interaction).

3Deformability is also affected by metabolic processes controlling ATP levels and redox state. These factors control ion handling by pumps and passive transport pathways, proteolytic activity of Ca2+-dependent protease calpain, and mutations and structural integrity of each element of the membrane architecture. Failure to sustain deformability results in shortening of RBC life span and, when not compensated by de novo RBC production, in hemolytic anemia.

4Therefore, reliable estimation of RBC deformability and understanding of the processes in control of it are essential for evaluation of severity of patients’ state and choosing of the optimal therapeutic strategy. This particularly relates to the feasibility of splenectomy as an option to improve or worsen condition of patients with anemic state.

5In this review, we provide an overview of the current knowledge on the primary and secondary mechanisms involved in regulation of RBC deformability in hereditary hemolytic anemia. We discuss methodologies that are currently used to assess RBC deformability in the clinical and research laboratories. We link different processes, such as ion channel activity, intracellular energy metabolism and phosphorylation of membrane proteins to RBC deformability and illustrate how these processes are affected in various RBC pathologies, such as sickle cell disease, thalassemia, HS and metabolic defects of RBCs. Finally, we describe the influence of shedding of nano-sized membrane vesicles from the RBC, the oxygenation state of hemoglobin and adaptive responses (such as exercise and high-altitude) on RBC deformability. Increased shedding of RBC vesicles, for example, is a feature of various RBC pathologies and vesicles are increasingly being considered to be a novel biomarker of RBC disorders. They are considered to be involved in thrombosis and hemostasis and associated with reduced RBC deformability.

6Anemia is considered to be hemolytic when RBCs are prematurely cleared from the circulation. Hemolytic anemia can be further subdivided into intra- or extravascular hemolytic anemia, and the underlying cause can be either inherited or acquired. Intravascular hemolysis is, as the name suggests, lysis of RBC in the vasculature. The cause can be hereditary, as seen in sickle cell disease, but intravascular hemolysis can also be initiated by certain drugs, by mechanical stress (for example through shear forces generated by artificial heart valves), by cold-agglutination or as a result of exhaustive exercise. Intravascular hemolysis causes the release of hemoglobin into the plasma. Free hemoglobin is toxic and can lead to various clinical manifestations, such as hemoglobinuria, renal dysfunction, pulmonary hypertension and platelet activation.

7Extravascular hemolysis is directly related to reduced RBC deformability. RBCs with reduced deformability fail to pass the spleen, which acts as an RBC quality-control organ. The red pulp of the spleen contains narrow inter-endothelial slits. Failure to pass through these narrow slits leads to the uptake and breakdown of RBCs by macrophages. A number of hereditary RBC disorders result in reduced RBC deformability, which, as a consequence, leads to premature removal of RBCs in the spleen. Removal of RBCs by the spleen is, however, not only dependent on reduced deformability, but also occurs after recognition by macrophages. Senescent RBCs can be recognized and phagocytized by macrophages in the spleen upon binding of autologous antibodies to band 3, exposure of conformational altered CD47 or exposure of PS.

8Hereditary forms of hemolytic anemia can affect the RBC membrane (i.e., HS, elliptocytosis, and pyropoikilocytosis), its metabolism (i.e., enzymopathies), cell hemoglobin (i.e., sickle cell anemia, unstable hemoglobin variants), or cellular hydration (i.e., HS, hereditary xerocytosis or Gardos Channelopathy). While the primary genetic causes of these disorders are often well determined, less is known about the factors triggering the actual hemolysis. Striking examples are sickle cell disease, thalassemia, HS and the metabolic disorders of the RBC. All have well-known and well-studied primary genetic and molecular defects. However, little is known about the secondary mechanisms that may decrease RBC deformability and thereby contribute to the premature removal of these affected RBCs.

9For example, the homozygous single point mutation in the HBB gene, substituting glutamic acid for valine at position 6 leads to sickle cell disease. Although the discovery that these RBCs tend to sickle at low oxygen tension was already provided by,, it took decades to unravel the processes that contribute to sickle cell dehydration, decreased deformability and increased endothelial cell adhesion. We still do not understand how does a point mutation in hemoglobin beta chain cause these secondary pathological alterations in density and adhesiveness and how all these changes in turn impact the disease symptoms of the patient. Other examples are the enzymopathies, which are caused by mutations in, for example, PK or HK and lead to a shortage of metabolic energy inside the RBC. Again, although the primary effect on decreased levels of ATP in these diseases is well understood, secondary processes such as altered K+ fluxes and phosphorylation of intracellular proteins, need further elucidation.

10Moreover, for a substantial number of patients with hereditary hemolytic anemia – the primary causes of disease remain unknown, or were only identified very recently. For these patients, a comprehensive understanding of both primary and secondary defects of the affected RBCs is still lacking. This is illustrated by the recently discovered mutations in patients with hereditary xerocytosis, also known as dehydrated stomatocytosis. In these patients, stomatocytes are typically found in peripheral blood smears. However, the primary molecular cause for the anemia and morphological change of the RBC remained unknown for a long time, let alone that the secondary processes that are involved in the disease process were elucidated. Only recently, it was shown that hereditary xerocytosis was caused by mutations in genes for the mechanosensitive PIEZO1 channel. Very recently, a mutation in the calcium-activated potassium channel subfamily N member KCNN4 (encoded by the human KCa3.1 gene, and also known as the Gardos channel in RBCs) was shown to cause ‘Gardos channelopathy’ a disease resembling yet at some points different from hereditary xerocytosis. Only then, the molecular diagnosis of these diseases was established in these patient groups and new insight on RBC deformability and RBC ion homeostasis with respect to ion channel function were obtained. The secondary processes that contribute to RBC clearance in these diseases are now being explored.

11A currently well-established tool to probe RBC deformability is osmotic gradient ektacytometry, which is routinely used in the diagnosis of patients with hereditary hemolytic anemia. The technique is performed on a Laser-assisted Optical Rotational Cell Analyzer (Lorrca). It assesses RBC deformability, osmotic fragility and cellular hydration status (see Figure 1). In osmotic gradient ektacytometry, the maximum RBC deformability is represented by the maximum elongation index (EImax). The Omin represents the osmotic value (mOsmol/kg H2O) where the elongation index (EI) is minimal, corresponding to the 50% lysis point as determined by the classical osmotical fragility test. The hydration status (or intracellular viscosity) is represented by Ohyper (mOsmol/kg H2O). The value corresponds with the hypertonic osmolarity where the EI is 50% of EImax. The value of these three parameters as well as the shape of the curve is used in the diagnosis of various disorders of the RBC membrane and hydration station (Figure 1B).

12Ohyper correlates with the reciprocal function of the MCHC. In healthy individuals, during RBC aging increased MCHC is observed and this correlates with decreased RBC deformability. A similar observation has been made for RBCs from patients with sickle cell disease and HS. Also here, a decrease in RBC hydration (i.e., decreased Ohyper and increased MCHC) of patient’s cells is associated with a decrease in the maximum RBC deformability (EImax).

13Besides osmotic gradient ektacytometry, there are other methods to measure RBC deformability. A number of techniques that are also used in diagnostics measures RBC deformability as a function of shear stress, for example the RheoScan-D, Automated Rheoscope and Cell Analyser (ARCA) and the deformability measurement module on the Lorrca. The results of RheoScan-D and the deformability measurements (shear-stress module) on the Lorrca were observed to be comparable. However, RheoScan-D measures RBC deformability under microfluidic conditions after the application of shear stress, while shear-stress on RBCs in the deformability measurements on the Lorrca requires a larger set-up with a rotating cup. The ARCA measures RBC deformability with a microscope after the application of shear-stress, and therefore has the potential to measure the RBC deformability of subpopulations.

14Most direct measurements of single cells include: micropipette, atomic force spectroscopy and holographic optical tweezers (HOT). The mechanical probes and the optical approach are complementary since 30 pN is a lower force limit for the mechanical measurements and an upper limit for the HOT in the context of investigating RBCs. Currently microfluidic approaches becoming increasingly popular to investigate RBC deformability. While the latter method has the potential to become a routine diagnostic tool, the former single cell methods are mostly dedicated to basic research due to their instrumental complexity.

15A further very simple method to measure RBC deformability is the readout of RBC filterability, e.g., by measuring RBC passage through cellulose columns. Unfortunately there is currently no standardized column/readout system on the market that would allow comparison of filterability between diagnostic laboratories. In addition, RBC passage through cellulose columns or filters is highly subjected to changes in cellular volume (such as MCV), which may affect results. A summary of available techniques to measure RBC deformability is depicted in Table 1.

16Red blood cell deformability is highly influenced by RBC volume control and by ion content, both regulated by ion pumps, ion channels, symporters and antiporters (see Figure 2). When ion channels are open, ions move following their electrochemical gradients, while ion pumps can actively move these ions against the gradient. Symporters and antiporters may also create secondary ion gradients, but require the pre-existing gradients for at least one type of ions as a driving force to transport the other ion types against the gradient. Symporters transport two (e.g., K+-Cl- cotransporter) or more (e.g., Na+-K+-2Cl- cotransporter) ions in the same direction using driving force for one of them, while antiporters (e.g., Na+/H+ exchanger or anion exchanger) exchange two ions that move in the opposite direction. These transporters, pumps and channels are crucial in resisting/adapting to local osmotic changes and maintaining RBC volume. RBC volume changes result from differences in osmotic pressure, which results in water transport by aquaporins of which AQP1 and AQP3 are found in human RBCs. Cotransport of water is, however, also possible through the K+-Cl- cotransporter (KCC). Volume changes are also associated by in- and efflux of several amino acids and amino-acid derivates, such as taurine.

17As mentioned previously, MCHC is a parameter that reflects the RBC hydration state, and is dependent on the hemoglobin concentration in RBCs, RBC volume, RBC membrane loss and water content. Quick movements of ions and water result in acute changes in MCHC whereas long-term effects on MCHC are often associated with release of hemoglobin-free vesicles. RBC hydration may be quantified using the Ohyper value as obtained by osmotic gradient ektacytometry (see Figure 1 and see section “Techniques to Determine RBC Deformability”). Changes in RBC density detected using cell distribution within Percoll density gradients reflects both membrane loss and the changes in RBC surface-to-volume ratio and alterations in intracellular ion and water content. In-depth morphological analysis could be used as a complementary approach to discriminate between membrane and ion/water loss.

18In this section we shall address hereditary hemolytic anemias associated with the changes in the intracellular ion and secondary to the latter changes in water content. One of the players in this type of disorder is a mechano-sensitive PIEZO1 channel, a non-selective cation channel permeable for Ca2+ that in turn can activate the calcium activated K+ channel subfamily N member KCNN4 (or Gardos channel in RBCs). Thus, hydration state is largely dependent on the shear stress intensity and the hydration state of RBCs from splenectomized and non-splenectomized patients with the same mutation may vary substantially. Furthermore, hydration state is a function of RBC age, young RBCs being more hydrated than the senescent cells.

19A RBC disorder characterized by primary hydration changes is hereditary xerocytosis (HX), also known as dehydrated stomatocytosis. HX is a form of hereditary hemolytic anemia, and patients often suffer from iron-overload. In addition, in many RBC disorders splenectomy relieves symptoms and increases RBC survival, but in HX, splenectomy is contra-indicated as it drastically increases the risks for thrombotic events. HX was related to mutations in the mechanosensitive PIEZO1 channel. PIEZO1 mutations lead to stabilization of the “active” state or destabilization of the “inactive” state of the PIEZO1 protein. These alterations in channel function lead to increased intracellular levels of Ca2+ and subsequent KCNN4 (Gardos channel) activation. KCNN4 activation leads to efflux of K+ and dehydration. The relatively low intracellular levels of K+ could therefore serve as a biomarker for HX. Although PIEZO1 mutations leads to KCNN4 (or Gardos channel) activation, RBCs with mutations in KCNN4 (Gardos channelopathy) exhibit a different pathology. For example, HX caused by mutations in PIEZO1 lead to severe RBC dehydration, which is also shown by a left-shift of Ohyper in the osmotic gradient curve (see Figure 1B and see section “Techniques to Determine RBC Deformability”) while mutations in KCNN4 (or Gardos channel) may cause only slight alterations in the osmotic gradient curve. In addition, RBCs from patients with KCNN4 mutations were found to exhibit increased activity of Na-K-ATPase that is most likely triggered by the increase in the intracellular Na+ concentrations. The hyperactivated Na, K-pump compensates for the dissipation in Na/K gradients and pumps K+ back into the cells, but fails to restore normal K+ content completely. Currently, however, it is difficult to measure intracellular water content or RBC volume directly and accurately enough, which makes it challenging to discriminate between water loss and membrane loss.

20In addition to the protein defects, described above, that directly disturb the membrane and thereby the ability of the RBC to deform there are also secondary causes affecting membrane (protein) function and, thereby, RBC deformability. Examples of these are membrane protein phosphorylation, RBC density and RBC vesiculation.

21The RBC membrane is built from a basic triangular network of α- and β-spectrin molecules connected to band 3, ankyrin and protein 4.1. This network provides the RBC with a certain stability and simultaneously the ability to deform. HS (HS) is characterized by defects in proteins forming the cytoskeletal network or proteins forming ankyrin and junctional complexes. This usually concerns mutations in band 3, protein 4.2, α-spectrin, or ankyrin. These mutations lead to membrane instability, membrane loss through release of hemoglobin-free vesicles, with consequent decreased surface area-to-volume ratio, increase in MCHC and density and formation of spherocytes. Spherocytes show a characteristic loss of RBC deformability, leading to premature removal of these RBCs from the circulation. Hereditary elliptocytosis (HE) is a disorder characterized by disturbances of the horizontal cytoskeletal interactions and are usually caused by mutations in α-spectrin, β-spectrin and protein 4.1R. As the name suggests, the disturbed horizontal interactions lead to the formation of elliptocytes. The majority of HE patients are clinically asymptomatic, but few patients suffer from hemolysis, jaundice and splenomegaly.

22The triangular spectrin network, connected through ankyrin and protein 4.1R to band 3 supports membrane stability and contributes to RBC flexibility and deformability. Tyrosine phosphorylation of in the individual proteins regulates interaction forces between the elements of this network. Tyrosine phosphorylation is regulated by phosphotyrosine kinases and by phosphotyrosine phosphatases. Under normal conditions, protein regulatory tyrosine residues are only phosphorylated to a limited degree. In the RBC membrane, phosphorylation of protein 4.1R, for example, leads to a reduced membrane stability, and phosphorylation of ankyrin regulates its binding to band 3. The dissociation constant (Kd) between ankyrin and band 3 influences RBC deformability without a loss in elasticity, underlining that cytoskeletal protein phosphorylation has implications on RBC deformability.

23The effect of phosphorylation of band 3, the most abundant membrane protein, on RBC deformability is not entirely clear. The possible physiological relevance of band 3 protein Tyr phosphorylation state emerges from the fact that it is controlled by hormones such as insulin and that it is altered in patients with hemoglobinopathies. showed that dephosphorylation of band 3 Tyr residues does not affect RBC deformability. It, therefore, appears that phosphorylation of band 3 does not affect RBC deformability directly, but may alter band 3 protein aggregation and its interaction with extracellular matrix components.

24showed that high concentrations of fibrinogen in combination with dephosphorylated band 3, leads to mildly increased RBC deformability at low shear stress. In contrast to these findings, report decreased RBC deformability in diabetic patients with hyperfibrinogenemia in absence of dephosphorylating agents. The decreased RBC deformability in the absence of dephosphorylating agents seems to underline that phosphorylation is not involved. However, insulin is implicated in the activation of tyrosine kinases that in turn phosphorylate band 3. In diabetes types I and II, glucose and insulin homeostasis is disturbed leading to increased protein tyrosine phosphatase activities and aberrant band 3 phosphorylation patterns, potentially leading to reduced RBC deformability and increased RBC turnover. This same principle may also play a role in patients with sepsis, where investigators have shown that tyrosine phosphorylation of band 3 was increased in septic mice and that this increase was accompanied by reduced RBC deformability.

25Band 3 phosphorylation was found to be increased by a rise in intracellular Ca2+-concentrations due to dissociation of phosphotyrosine phosphatase from band 3. Interestingly, sickle cell disease is characterized by an increase in both intracellular Ca2+ concentration and tyrosine phosphorylation of band 3, which potentially could thus influence RBC membrane stability. The importance of band 3 phosphorylation for RBC shape and deformability is highlighted by a number of in vitro experiments. For example, morphological changes of RBCs were observed after Tyr-phosphorylation of band 3 mediated by pervanadate. In contrast, Ser/Thr-phosphorylation of spectrin, which was selectively induced by okadaic acid did not result in morphological changes. Unfortunately, the deformability of RBCs after pervanadate and okadaic treatment was not measured in these studies, but it seems reasonable to assume that RBC deformability is affected since both membrane protein phosphorylation and RBC morphology have been associated with altered RBC deformability.

26Although reduced RBC deformability in HS mainly results from cytoskeletal disturbances, spherocytes experience dehydration and increased leakage of K+. To compensate for this increased K+ leakage, spherocytes are also found to have increased activity of the Na-K pump and the NKCC1 (also known as the Na-K-2Cl cotransporter, coded by gene SLC12A2). RBCs of transgenic mice deficient for ankyrin or spectrin show increased activity of the Na-K pump, but normal activities of the NKCC1 and K+-Cl- cotransporters. This contrasts with protein 4.2-deficient mice, where Na-K pump activity is normal, while activities of NKCC1 and K+-Cl- cotransporters are increased. These differences in pump activities between protein 4.2 deficient and spectrin- or ankyrin-deficient mice support the concept that membrane proteins interact with ion channels of the RBC.

27The RBC dehydration in HS also involves increased intracellular Ca2+ levels. Increased intracellular Ca2+ correlates with decreased RBC deformability and RBC membrane stiffening. For example, after in vitro ATP-depletion of HS RBCs, Ca2+ influx is increased compared to healthy RBCs. The detailed mechanisms beyond this Ca2+ influx have up to now not been identified and may include PIEZO1 or NMDA receptors both of which are activated by shear stress in “stiff” less deformable cells.

28In protein 4.2-/- mice, displaying the HS phenotype, KCNN4 (or known as Gardos channels in RBCs) were noted to be functionally up-regulated, or at least more KCNN4 activity was measured. This can be considered as a protective mechanism that leads to an outward flux of K+ and water from RBCs. This mechanism may compensate for the decreased surface-to-area ratio. Indeed, increased lysis was observed when these RBCs were exposed to KCNN4 (or Gardos-channel) inhibitors. These findings indicate that KCNN4 is activated in HS in order to facilitate RBC dehydration, which maintains hemoglobin in RBCs at the expense of water. Recent research confirms the finding that RBC dehydration and formation of denser RBCs may protect the RBC in HS from lysis and premature uptake. We observed that mild HS is accompanied with prolonged RBC life-span, relatively mild reductions in RBC deformability and denser RBCs when compared with RBCs from patients with moderate/severe HS. Most likely, the mild reduction in RBC deformability in patients with mild HS avoids premature splenic uptake and thus may provide the HS RBC time to lose membrane and to become dense.

29Mature RBCs of healthy individuals lack mitochondria and are therefore entirely dependent on the glycolytic pathway for the production of energy in the form of ATP. In the glycolytic pathway, glucose is converted to lactate by several enzymatic steps (Figure 3). Key regulatory enzymes in the glycolytic pathway are HK, phosphofructokinase and PK. Hereditary metabolic disorders, or enzymopathies, are disorders impairing cellular energy production and balance, in particular ATP production. ATP is essential in the regulation of RBC deformability, viability, regulatory cascades involving phosphorylation and activity of ion transport involving Na, K-ATPase, and Ca ATPase.

30Adenosine triphosphate plays a key role in maintenance of ion gradients and should impact cytoskeletal structure and RBC shape. However, glycolytic enzymopathies usually do not show altered RBC morphology. For reasons that are not yet well understood, metabolically defective RBCs are prematurely removed by the spleen, causing chronic Hereditary Non-Spherocytic Hemolytic Anemia (HNSHA).

31Several observations point to mechanisms that may be involved in the premature clearance. RBCs from a patient with pyruvate kinase deficiency (PKD) were found to leak K+ more rapidly and were found to consume ATP at an accelerated rate. This most likely contributes to the decreased RBC viability in this disease and could result in reduced RBC deformability. A possible explanation for increased clearance of RBCs with metabolic defects is the imbalance in Ca2+ uptake and extrusion. In agreement with the hypothesis that ATP deprivation will cause Ca2+ overload due to the inability to actively extrude Ca2+ from the cells by the Ca2+ pumps, decreased ATP levels in RBCs with metabolic defects were associated with abnormally high intracellular Ca2+ and exposure of PS, an important cellular ‘eat-me’ signal. Alternatively, defective metabolism may impact the redox state of RBCs making them more susceptible to oxidative stress.

32Despite the intuitive feeling that intracellular ATP levels should correlate with cellular deformability, evidence for this is so far inconclusive. This may be related to the techniques used. For example, research performed by did not show any correlation between intracellular ATP and deformability in RBC-concentrates intended for transfusion when assessing deformability by increasing shear stress. On the other hand, studies using viscosity and filterability assays did find correlations between intracellular ATP and deformability and showed that deformability is indeed dependent on intracellular ATP levels. Atomic force microscopy studies also revealed increased membrane stiffness after ATP depletion due to reduced spectrin phosphorylation. We hypothesize that the different results from these experiments are possibly caused by the fact that ATP is not required for RBCs to elongate after the application of external forces, such as shear stress but ATP might be essential in the process of RBC deformation toward different shapes, as seen in filterability measurements.

33Cyclic AMP (cAMP) is a second messenger and is derived from ATP. In nucleated human cells, cyclic AMP (cAMP) is synthesized from ATP by adenylate cyclase (AC) upon stimulation by G-protein coupled receptors (GPCR) and there is evidence that this process also occurs in human RBCs (see Figure 4). Examples of GPCRs on RBCs are the erythrocyte β2-adrenergic receptor, the lysophosphatidic acid (LPA) receptor and the purinergic (P2Y) receptor. The activation of the erythrocyte β2-adrenergic receptor by catecholamines such as epinephrine (or adrenaline) affects RBC deformability. In vitro experiments with increased concentration of adrenaline, lead to increased membrane fluctuations and increased RBC filterability.

34Phosphodiesterases tightly regulate levels of cAMP in RBCs. PDEs catabolize the phosphodiesters in cyclic nucleotides, such as cAMP to AMP or cGMP to GMP. By inhibiting these PDEs, cyclic nucleotides remain intact and active. The reports of cAMP effects on RBC deformability are, however, ambivalent. Recently it was found that sildenafil, which is a PDE inhibitor and well-known for its function in the treatment of erectile dysfunction and pulmonary hypertension, has beneficial effects on RBC deformability in sickle cell disease at low concentrations in vitro. Increase in nitric monoxide bioavailability was suggested to improve RBC deformability. However, increased concentrations of sildenafil impaired sickle RBC filterability. Typically, inhibition of cGMP-specific PDE type 5 (PDE-5) by sildenafil reduces the degradation of the second messenger cGMP to GMP and increases smooth muscles relaxation. In RBCs, this PDE-5 inhibition increases not only intracellular levels of cGMP but also preserves intracellular levels of cAMP via the inhibitor activity of cGMP on PDE-3 (see Figure 5). The effect of increased cAMP levels was also studied using a spleen-like microfiltration system. Increased retention in the microfiltration system, reflecting decreased deformability, was observed after incubation of RBCs with sildenafil. Moreover, the production of cGMP from GTP is synthesized by guanylate cyclase. Guanylate cyclase is activated by nitric oxide (NO) and the addition of sodium nitroprusside to RBCs as an artificial nitric oxide donor does prevent Ca2+ influx and Ca2+-mediated stiffness of RBCs.

35In addition, the effect of cAMP on the release of ATP is still under debate. A role for cAMP in the release of ATP to its extracellular environment via PKA was proposed (see Figure 4). Others, however, have attributed the release of ATP solely to hemolysis of the RBC or to release via transporters such as Pannexin 1 or PIEZO1.

36Recently AMPK was also found to be present in RBCs. The exact function of AMPK in RBCs is unknown, however, in other cells, AMPK is essential to balance ATP consumption and ATP production. In muscles and fat AMPK regulates glucose uptake. Likely, AMPK also regulates these processes in RBCs, as RBCs from AMPK-/- mice were less viable after in vitro glucose depletion. Interestingly, these AMPK-/- mice had increased reticulocytes count, decreased RBC deformability, increased osmotic fragility, increased spleen size and decreased RBC lifespan. This indicates a significant contribution of AMPK in maintaining RBC function and viability. AMPK is an energy-sensing enzyme, which is activated by low AMP: ATP ratios and inhibited by high AMP: ATP ratios. Under low ATP concentrations, AMP and ADP can bind to AMPK and change its conformation, thereby preventing the protein from activation by phosphorylation. Chemical agents that increase intracellular levels of cAMP are forskolin and isobutyl-methyl-xanthine (IBMX). Forskolin activates AC and thus leads to increased formation cAMP from ATP, while IBMX inhibits PDEs and maintains cAMP levels. Both forskolin and IBMX lead to AMPK inhibition, which reveals a molecular interaction between cAMP and AMPK activity. This interaction could play a role in altered deformability in patients suffering from glycolytic enzymopathies. The low RBC ATP concentrations in these patients would lead to low levels of cAMP and increased intracellular levels of non-phosphorylated (inactive) AMPK.

37Phosphorylation of AMPK may also be involved in G6PD-deficiency. G6PD deficiency is the most common RBC enzyme deficiency with over 400 million affected people. Deficiency of this enzyme is associated with decreased ability of the RBC to withstand oxidative stress. Exposure of normal and G6PD-deficient RBCs to the oxidant diamide leads to a decrease in RBC deformability of the G6PD-deficient RBCs due to the inability of the RBC to maintain the intracellular glutathione-pool. Depletion of glutathione also leads to activation of AMPK by phosphorylation in G6PD-deficient RBCs. The activation of AMPK in G6PD-deficient RBCs after exposure to oxidative stress is most likely a consequence of ATP-consuming compensation mechanisms after excessive glutathione loss.

38Protein kinase C alpha (PKCα) possibly also regulates RBC deformability. PKCα is involved in the phosphorylation of proteins in the RBC, such as adducin, protein 4.1R and protein 4.9. Moreover, PKCα is involved in stimulation of glucose uptake by phosphorylation of the glucose transporter and participates in the activity of calcium channels. Consequently, phosphorylation and dephosphorylation of these proteins can be altered upon depletion of glucose. Glucose depletion was found to activate PKCα and this is accompanied by an increase in intracellular Ca2+ and, consequently, exposure of the membrane phospholipid PS. The phosphorylation of adducin and protein 4.1R by PKCα could change RBC behavior and could influence RBC deformability in a way similar to what is seen, for example, after phosphorylation of band 3. Interestingly, homology analysis between murine, rat and human showed a conserved region in both liver- and RBC-specific PK for a PKCα binding site. PKCα regulates RBC membrane stability and this conserved region for PK indicates a possible role for PK in this process. In addition, PK converts phosphoenolpyruvate (PEP) to pyruvate (Figure 3) and previously it has been recognized that phosphorylation of PK by cAMP-dependent protein kinases leads to a reduced affinity of PEP for PK, which leads to a reduced production of ATP. The decreased cAMP and ATP production in metabolic disorders of the RBC, such as PK-deficiency, could subsequently result in disturbed RBC deformability.

39It has been suggested that pools of ATP are intracellular enclosed by the membrane proteins ankyrin, β-spectrin, band 3, and GAPDH, serving as substrates for both the Na+, K+-, and Ca2+ -pumps. This indicates an interplay between membrane proteins, energy stores and ion channel function of the RBC. Interestingly, the chemotherapeutic agent 5-FU was shown to induce changes in RBC rigidity, morphology and ion balance, most likely by altering ATP levels in the RBC. 5-FU is a pyrimidine antagonist and since purine and pyrimidines play key roles in cellular metabolism and energy homeostasis. 5-FU could, therefore, influence RBC deformability. The activating and feedback mechanisms of purines and pyrimidines on glycolytic enzymes have previously been described by and. For example, in bovine RBCs incubated with glucose, adenosine stimulated ATP production compared to incubation with glucose alone. Most likely, adenine (i.e., the nucleobase of adenosine) stimulates HK activity. The effect of pyrimidines in RBCs has also been studied in two patients with a pyrimidine 5′-nucleotidase (P5N) deficiency, which lack this enzyme involved in clearance of pyrimidines from the RBC. P5N deficiency increases intracellular concentrations of pyrimidine nucleotides, which eventually leads to hemolytic anemia. P5N-deficient patients were also found to have increased levels of reduced glutathione. At the same time, pyrimidine 5′-nucleotides (such as cytidine mono-, di-, and triphosphate (CMP, CDP, CTP) or uridine mono-, di-, and triphosphate (UMP, UDP, UTP)) decreased the activity of glucose-6-phosphate dehydrogenase (G6PD, Figure 3). As mentioned earlier G6PD activity is crucial in the anti-oxidative defense of RBCs. Indeed, RBCs from P5N-deficient patients were found to be more susceptible to oxidative stress, as reflected by increased formation of Heinz bodies, even despite increased reduced glutathione concentrations. Besides their role as a morphological marker of oxidative stress, Heinz bodies can lead to a decreased deformability by themselves.

40Hemoglobin is the main component of the RBC and responsible for the delivery and removal of oxygen and carbon dioxide to and from the tissues, respectively. Intracellular hemoglobin concentrations and its state (polymerization, crystallization, degradation, and oxidation) also defines cytosolic viscosity making up 19.9–22.3 mmol/L in cells of healthy humans. Hemoglobin is composed of two α- and β-hemoglobin molecules together composing a heterotetramer. Disorders of hemoglobin can be subdivided into hemoglobinopathies (e.g., sickle cell anemia) and thalassemias (α- and β-thalassemia).

41Sickle cell anemia is caused by a single point mutation in the HBB gene at position 6 substituting glutamic acid to valine (HbS). This substitution causes formation of HbS polymers of deoxygenated hemoglobin. This transition is usually rapidly reversed upon reoxygenation, but induces progressive damage of membrane driving dehydration to the extreme to the state when HbS polymers do not dissociate as its concentration exceeds it solubility threshold. Destabilization of the membrane and decrease in deformability leads to intravascular hemolysis and to vaso-occlusive events. Sickle RBCs are poorly deformable and this is, in part, due to changes in hydration status of the RBC.

42In α- and β-thalassemia, α- and β-hemoglobin chains are affected, respectively, which leads to an imbalance in the synthesis of globin chains and to an inability to form sufficient quantities of hemoglobin heterotetramers. The imbalanced synthesis in thalassemia leads to the formation of hemoglobin precipitates, so-called Heinz bodies. RBCs in α-thalassemia show increased hydration, whereas the hydration of RBCs in β-thalassemia is either decreased or increased. Both Heinz bodies and the altered hydration state of the RBC are known to impair RBC deformability.

43The main function of RBCs is to transport oxygen to the tissues in the human body. The oxygenation state of hemoglobin is known to influence various processes of the RBC. Several ion transporters are oxygen sensitive, such as K+-Cl- cotransporter (KCC1) and the NKCC1 (Na-K-2Cl cotransporter). The function of band 3 seems, however, unaltered upon deoxygenation and oxygenation. On the other hand, hemoglobin binds the cytosolic domain of band 3 and this binding is regulated by pH. Deoxygenation reduces the binding of ankyrin to band 3 and dissociation leads to more freely diffusible band 3. In addition, the binding between ankyrin and band 3 is also regulated by 2,3-disphosphoglycerate (2,3-DPG). 2,3-DPG promotes the dissociation of oxygen from hemoglobin and promotes the release of oxygen from the RBC to the tissues. In the oxygenated state 2,3-DPG is not bound to hemoglobin and leads to less deformable and more fragile RBCs, while in the deoxygenated state 2,3-DPG is bound to hemoglobin and leads to an increase in RBC deformability and less fragile RBCs. The exact role of the reduced binding between ankyrin and band 3 after deoxygenation and binding of 2,3-DPG to hemoglobin is currently unknown, although it is hypothesized that a mild membrane weakening is beneficial in deoxygenated capillaries to facilitate smooth traveling of the RBC.

44The RBC dehydration in sickle cell anemia is caused by facilitated K+ loss through hyperactivated KCNN4 (also known as the Gardos Channel in RBCs) and K+-Cl- cotransporter that is not compensated by uptake of equal amounts of Na+. Regardless of the ion transport pathway involved, RBC dehydration raises the HbS concentration, thereby affecting the equilibrium of HbS-polymerization and depolymerization in favor of the polymerized version. Na/K/ATPase was found to be more active in RBCs from sickle cell disease patients and can contribute to RBC dehydration. Dehydration of RBCs in sickle cell disease contributes significantly to the decreased deformability since dehydration promotes the probability of polymerization of HbS by 20–40-fold and result in prolonged polymerization of hemoglobin and thus prolonged sickling of RBCs. Sickled RBCs are far less deformable.

45Two mechanisms involved in abnormally high K+ loss are presented in Figure 6. The first mechanism of K+ loss involves the ubiquitously expressed K-Cl co-transporter (KCC), of which the isoforms KCC1, KCC3, and KCC4 are expressed in human RBCs.

46KCC1, is most often studied in RBCs and referred to as KCC. KCC in RBCs are known to be regulated by intracellular pH, Mg2+ concentrations as well as volume and oxygenation states of hemoglobin. Under physiological conditions, KCC activation restores cell volume after swelling, but KCC activation may also respond to the changes in the RBC redox state. In healthy RBCs, KCC is oxygen sensitive but only activated at high oxygen tension (pO2). At low oxygen tension, KCC becomes sensitive to other stimuli. In sickle cell disease, in contrast, KCC is also activated at low oxygen tension, most likely due to increased phosphorylation of KCC in sickle RBCs. In addition, KCC activity is intrinsically higher, even in older sickle RBCs.

47The loss of deformability caused by the formation of HbS-polymers at low oxygen tensions is enhanced by the KCC activation and leads to additional cell shrinkage. The clinical relevance of KCC activity is emphasized by observations in individuals with mild sickle cell disease. In these patients, an increased K-Cl cotransport activity is associated with increased likelihood of hospitalization, because of acute vaso-occlusive problems caused by less deformable dehydrated RBCs.

48One more trigger of K+ leak from RBCs is the increase in the intracellular Ca2+ levels and the activation of Ca2+-sensitive K+ channels (KCNN4 or known as the Gardos channel in RBCs). These channels driving K+ loss and dehydration are hyperactivated in RBCs of sickle cell disease patients secondary to the high intracellular Ca2+ levels (see Figure 6). KCNN4 is expressed in various cell types and are inhibited by imidazole antimycotics, such as clotrimazole. In sickle cell disease, KCNN4 (or known as the Gardos channel in RBCs) is activated by two pathways. Firstly, KCNN4 can be activated by a signaling cascade initially triggered by several cytokines, such as PAF, interleukin-10 (IL-10) and endothelin 1 (ET-1) also involving activation of PKCα. In addition, PAF and ET-1 concentrations are increased in plasma from patients with sickle cell disease and are assumed to contribute to the adhesion of sickle RBCs to endothelium that is responsible for the vaso-occlusive events. Also, these cytokines generate denser RBCs through RBC dehydration after an oxygenation/deoxygenation cycle. Secondly, sickle cell disease patients have increased expression of the NMDA-receptor on their RBC membranes, causing pathological influx of Ca2+ after stimulation with receptor agonists (such as glycine and homocysteic acid) which could contribute to dehydration mediated by KCNN4. Currently, a clinical trial using memantine as a NMDA-receptor antagonist is being tested in sickle cell disease patients. In addition, the KCNN4 (or Gardos channel) blocker senicapoc (ICA-17043) has recently been tested in sickle cell disease. Senicapoc increased hemoglobin concentrations and hematocrit, and improved RBC hydration. Despite these encouraging findings, this study has been terminated prematurely because senicapoc did not meet its primary efficacy endpoint, defined as a decrease in painful crises. From the clinical results obtained with senicapoc it can be hypothesized that the RBC dehydration and changes in RBC hydration after treatment with senicapoc are not causally related to the vaso-occlusive events which are often observed in sickle cell anemia.

49Another pathway that increases intracellular Ca2+ levels in sickle cell disease is the mechanosensitive PIEZO1 channel (also described at section “Primary Changes of RBC Hydration”). PIEZO1 channels are widely expressed in vertebrates across various cell types and in RBC these channels are responsible for regulation of volume homeostasis upon mechanical signals. Sickle RBCs show increased permeability for Ca2+ and Mg2+ upon deoxygenation. This increased permeability results in an increased deoxygenation-induced cation conductance in sickle cell disease, which can be entirely blocked by GsMTx4. The gating modifier GsMTx4 blocks the mechanically sensitive part of PIEZO1 that supports a closed state of this ion channel, preventing Ca2+ influx.

50Three increasingly severe phenotypes can be distinguished in β-thalassemia, i.e., β-thalassemia minor, intermedia and major. Intracellular concentrations of Na+ in RBCs from patients with β-thalassemia minor and β-thalassemia major are comparable with those in RBCs from healthy adults, but K+ levels are slightly increased. Upon in vitro incubation at 37°C, RBCs from patients with β-thalassemia major show increased leakage of K+ and elevation of intracellular Na+ levels when compared with healthy RBCs. In RBCs from β-thalassemia minor patients, leakage of K+ and intracellular Na+ levels are comparable to the healthy RBCs after in vitro incubation at 37°C.

51Leakage of K+ from RBCs in thalassemia is possibly linked to the precipitation of excess α- or β-globin chains inside the RBCs, which is a characteristic feature of this disease. The precipitated globin chains in RBCs can lead to oxidative stress and can affect transport of Na+ and K+ in thalassemia RBCs. In both β-thalassemia and α-thalassemia, RBCs show increased efflux of K+. Moreover, the amount of hemoglobin aggregates in RBCs is correlated with K+ efflux. The presence of these aggregates reduces RBC deformability thereby revealing correlation between K+ fluxes, Heinz body formation, and reduced RBC deformability. The precipitation of globin chains and leakage of K+ from RBCs in thalassemia is also linked, or may even be augmented, by overload of Ca2+.

52Another important factor in K+ loss from RBCs in β-thalassemia involves the K+-Cl- cotransporter, which can be affected by oxidative stress. This K+ loss by the K+-Cl- cotransporter is inhibited by increased intracellular concentrations of Mg2+. In a trial with β-thalassemia intermedia patients, dietary Mg2+ supplementation did lead to increased intracellular concentrations of Mg2+ and to a reduction in the activities for the K+-Cl- cotransporter and Na-K pump (Na/K/ATPase). However, Mg2+ did not influence the activities of NKCC1 (also known as the Na-K-Cl cotransporter). Although dietary Mg2+ supplementation in β-thalassemia patients does not influence the hemoglobin concentration in blood, it does lead to a significant decrease in the absolute reticulocyte number. This is possibly due to improved RBC survival time.

53Little research has been performed on KCNN4 (or also known as the Gardos channel in RBCs) in thalassemia. An exception is the work on a mouse model with a homozygous deletion in the β-globin chain. These mice were treated with clotrimazole, an antifungal imidazole derivate with KCNN4 blocking properties. During treatment with clotrimazole, hemoglobin concentrations in blood remained constant. In addition, it caused a decrease in MCHC whereas hematocrit and intracellular K+ levels increased indicating that hydration state of the RBC was restored. Combination of clotrimazole with erythropoietin administration did increase hemoglobin levels in these mice to a higher extent than erythropoietin alone, possibly by promoting proliferation and differentiation during erythropoiesis.

54Intracellular levels of Ca2+ tightly regulate RBC vesiculation and elevated levels of intracellular Ca2+ are known to induce PS exposure and RBC vesiculation (Figure 7).

55The RBC-vesiculation is a physiological process and leads to reduced RBC deformability. During its 120-day life span, RBC lose membrane surface and hemoglobin content through vesiculation a process in particular prominent in the youngest RBCs. As membrane is shed, this leads to a decreased surface-to-volume ratio in the RBC. During this RBC aging, hemoglobin-free vesicles are shed which leads to RBC dehydration and older RBCs with increased MCHC. Altogether, this leads to a reduction in membrane elasticity and RBC deformability. This implies that there is an important role for vesiculation in RBC deformability.

56While RBC-vesiculation is considered a physiological process, RBC vesiculation is increased in various forms of hereditary hemolytic anemia, such as HS and sickle cell disease. In HS, differences in deformability between spectrin/ankyrin-deficient and band 3-deficient RBCs were observed after splenectomy. While splenectomy prevented premature removal of young RBCs from the circulation in both groups, the loss of deformability during RBC-aging was delayed after splenectomy in spectrin/ankyrin-deficient RBCs. This contrasts with band 3-deficient RBCs, where splenectomy did not lead to a delay in the deformability decrease during RBC-aging. This could be explained by the fact that spectrin/ankyrin-deficient RBCs are more prone to shed band 3-containing vesicles. Clustered band 3 is known to induce binding of autologous IgG, which facilitates removal of RBC by macrophages. Shedding of band 3-containing vesicles from spectrin/ankyrin-deficient RBCs, therefore, would avoid IgG-opsonisation and clearance of the RBC.

57Red blood cell vesiculation is suggested as a mechanism to protect the cell from removal from the circulation. By vesiculation, the RBC can shed “eat-me” signals such as PS and specific band 3 cleavage products that react with senescent antigens. By shedding these “eat-me” signals, the RBC may escape clearance until the reduction in RBC deformability causes trapping in the spleen. In addition, the lipid bilayer of the RBC is a complex system with various microdomains. These microdomains of the RBC, or so called ‘lipid rafts’ are specific membrane parts with high concentrations of cholesterol, sphingomyelin and gangliosides. These specific lipid rafts have a high abundance of the membrane proteins stomatin, flotillin-1, flottilin-2 and band 3. RBC vesicles shed during storage are enriched in the lipid raft marker stomatin and indicates that specific lipid domains are shed during RBC vesiculation.

58The relationship between vesiculation and deformability is nicely illustrated by an experiment where chlorpromazine is added to RBCs to stabilize the RBC membrane. During overnight incubation with chlorpromazine at 37°C in a glucose-free buffer, vesiculation is inhibited and RBC deformability is maintained. Without chlorpromazine strong vesiculation of RBCs under these conditions occur as PS is exposed on the RBC surface. The activity of chlorpromazine may be explained by the amphiphilic properties of the molecule directly affecting the RBC membrane.

59High-throughput screening with chemical compound libraries also revealed that RBC-vesiculation can be driven by drugs and chemical compounds. For example, vesiculation of RBCs is driven by certain kinase pathways, including Jak-STAT and protein kinase B. Increased RBC vesiculation is also observed after addition of paclitaxel to blood, probably through its formulation excipient containing Cremophor. Not only RBC vesiculation is influenced by the vehicle Cremophor, but the compound also increases whole blood viscosity and transforms RBC to stomatocytes.

60Exercise leads to increased heart rate as a response to provide tissues with sufficient amount of oxygen. Exercise is accompanied with several stress factors that may affect RBC deformability, such as shear stress, hyperthermia, and glucose consumption. These stress factors can lead to mechanical rupture, stimulated erythropoiesis and can decrease the average RBC age in athletes.

61In a study with 24 trained cyclists, RBC deformability was decreased directly after exercise. The biology beyond the decrease in RBC deformability is unknown, although increased blood flow may be involved. The change in deformability after exercise can be the results of cytoskeletal changes, such as membrane loss. In addition, decreased levels of haptoglobin and increased levels of bilirubin were observed after marathon races. On the other hand, RBC deformability seems to be generally increased in well-trained athletes. A study comparing RBC membrane fluidity with the RBC membrane composition in controls and runners observed increased RBC membrane fluidity in endurance runners and sprinters. Although the intake of nutrients was comparable between the running groups and the control group, endurance running was accompanied with reduced concentrations of saturated fatty acids in RBC membranes. These results indicate that the RBC can adapt upon exposure to exercise and that these adapted RBCs facilitate exercise and proper delivery of oxygen to the tissues. Interestingly, observed a higher maximal RBC deformability in elite cyclists when compared with sedentary healthy controls. Also, RBC populations with lower RBC densities were observed in the elite cyclists, which could indicate increased RBC turnover. A possible explanation for the increased RBC deformability in elite cyclist could be that extreme exercise leads to membrane loss and early RBC uptake, which is likely to be compensated with relatively young and good deformable RBCs. Whether increased RBC deformability would lead to better sports performances is unknown.

62Autologous erythropoietin production is stimulated upon exposure to high altitudes. Acute exposure to high altitude, however, does not affect RBC deformability and RBC rheology. The effects of chronic exposure to high altitude seems largely unexplored. RBC deformability related processes were investigated in RBC concentrates obtained from Tibetans living at high altitude and from Tibetans at living at lowland. RBC viscosity was increased, and RBCs were more osmotic fragile in Tibetians living at low altitude when compared to RBC concentrates obtained from their lowland residents. The increased viscosity can, however, be affected by increased hemoglobin levels and the decreased osmotic fragility can be affected by the decreased MCV values in Tibetans living at high altitude. The effects of exercise under hypoxia on RBC properties were investigated by. Exercise under hypoxic conditions was found to decrease KCNN4 (or Gardos-channel) modulated deformability and KCNN4 modulated volume and down-regulated the senescence markers CD47 and CD147. These results indicate RBC dysfunction after exercise under hypoxia. The dysfunction of RBCs under hypoxic conditions in combination with the erythropoietin-driven increase in RBC production leads to RBCs with relative low densities, possibly caused by both increased RBC turnover and increased RBC production.

63Deformability is an important parameter that regulates RBC rheology, its longevity, and efficacy of O2 transport. Altered deformability is a characteristic feature of multiple forms of hereditary hemolytic anemias and is likely related to the severity of the disease. Factors regulating deformability at the cellular level are dehydration, membrane protein phosphorylation, cytoskeletal integrity, metabolism and the integrity of hemoglobin. Interaction of these factors makes RBCs more or less deformable. Measuring RBC deformability in research and diagnostic laboratories can be challenging as there are many techniques available with all their specific advantages and disadvantages.

64Measuring RBC deformability is important from both a diagnostic and research point of view. RBC deformability (i) could provide information about the patient’s disease and clinical severity, and (ii) could be a target for pharmacological intervention or predict the toxicity of drugs for patients with hemolytic anemias.

65Reduced RBC deformability leads to an inability of the RBC to pass the splenic circulation and leads to premature removal of RBCs from the blood. Altered RBC deformability can be attributed to primary and secondary changes of RBC deformability. Primary changes of RBC deformability are directly related to the disease, such as the membrane weakening in HS or the formation of poorly deformable sickle cells in sickle cell disease. Secondary changes of RBC deformability, such as altered ion fluxes, aberrant membrane protein phosphorylation or RBC vesiculation, are not directly related to the cause of disease. Thus, both primary and secondary causes of RBC deformability can contribute to premature uptake of RBCs in the spleen. In this review and in Table 2, we have summarized the current knowledge on primary and secondary mechanisms of RBC deformability in sickle cell anemia, thalassemia, HS, hereditary xerocytosis, and a number of metabolic disorders of the RBC. We have addressed and discussed the effects of ion regulation, ion channels and RBC (de)hydration on RBC deformability. In addition, we discuss the role and current knowledge of the adaptive responses on RBC deformability, intracellular energy-sensing molecules, membrane protein phosphorylation, hemoglobin deoxygenation and RBC vesiculation on RBC deformability. Knowledge about these processes in the RBC will lead to better understanding of the secondary processes involved in premature removal, and could lead to the discovery of new targets for pharmacological treatment. Furthermore, we postulate that a number of currently undiagnosed, patients with hereditary hemolytic anemia may have (genetic) defects in the here discussed secondary pathways or secondary processes that regulate RBC deformability. This review helps to understand the molecular mechanisms that maintain RBC deformability in healthy and diseased individuals, and enumerates the molecular mechanisms that are altered in RBC disorders leading to hereditary hemolytic anemia.

66RvW, RS, and WvS encouraged RH to investigate the properties of RBC deformability. RH and RvW wrote the manuscript with support from RS, AB, LK, and WvS.

67The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.