Iron mobilization from hepatocyte monolayer cultures by chelators: the importance of membrane permeability and the iron-binding constant

A series of bidentate hydroxypyridinone iron chelators that have therapeutic potential as oral iron chelators, have been studied systematically to determine which properties are the most critical for the mobilization of hepatocyte iron. The relationship between lipid solubility of the free and complexed forms of each chelator and hepatocyte iron release has been investigated as well as the contribution of the binding constant for iron (III). Hydroxypyridin-4- ones that were approximately equally soluble in lipid and aqueous phases were the most active compounds, the partition coefficient of the free chelator appearing to be more critical in determining iron release than that of the iron-complexed form. Highly hydrophilic chelators did not mobilize intracellular iron pools, whereas highly lipophilic compounds were toxic to hepatocytes. The contribution of the binding constant for iron (III) to cellular iron release was assessed by comparing hydroxypyridin-4-ones (log beta 3 = 36) and hydroxypyridin-2- ones (log beta 3 = 32), which possess similar partition coefficients. The results show that the binding for iron (III) is particularly important at low concentrations of chelator (less than 100 mumol/L) and that at higher concentrations (greater than 500 mumol/L) iron mobilization is limited by the available chelatable pool. Measurement of iron release with other chelators confirms the importance of both the lipid solubilities and iron (III)-binding constants to iron mobilization. The most active hydroxypyridin-4-ones released more hepatocyte iron than did deferoxamine when compared at equimolar concentrations. The results suggest that the ability of an iron chelator to enter the cell is crucial for effective iron mobilization and that once within the cell the binding constant of the chelator for iron (III) becomes a dominant factor.     

Deferiprone (L1) chelates pathologic iron deposits from membranes of intact thalassemic and sickle red blood cells both in vitro and in vivo

Red blood cell (RBC) membranes from patients with the thalassemic and sickle hemoglobinopathies carry abnormal deposits of iron presumed to mediate a variety of oxidative-induced membrane dysfunctions. We hypothesized that the oral iron chelator deferiprone (L1), which has an enhanced capacity to permeate cell membranes, might be useful in chelating these pathologic iron deposits from intact RBCs. We tested this hypothesis in vitro by incubating L1 with RBCs from 15 patients with thalassemia intermedia and 6 patients with sickle cell anemia. We found that removal of RBC membrane free iron by L1 increased both as a function of time of incubation and L1 concentration. Thus, increasing the time of incubation of thalassemic RBCs with 0.5 mmol/L L1 from 0.5 to 6 hours, enhanced removal of their membrane free iron from 18% +/- 9% to 96% +/- 4%. Dose-response studies showed that incubating thalassemic RBC for 2 hours with L1 concentrations ranging from 0.125 to 0.5 mmol/L resulted in removal of membrane free iron from 28% +/- 15% to 68% +/- 11%. Parallel studies with sickle RBCs showed a similar pattern in time and dose responses. Deferoxamine (DFO), on the other hand, was ineffective in chelating membrane free iron from either thalassemic or sickle RBCs regardless of dose (maximum, 0.333 mmol/L) or time of incubation (maximum, 24 hours). In vivo efficacy of L1 was shown in six thalassemic patients whose RBC membrane free iron decreased by 50% +/- 29% following a 2-week course of L1 at a daily dose of 25 mg/kg. As the dose of L1 was increased to 50 mg/kg/d (n = 5), and then to 75 mg/kg/d (n = 4), 67% +/- 14% and 79% +/- 11%, respectively, of their RBC membrane free iron was removed. L1 therapy-- both in vitro and in vivo--also significantly attenuated the malondialdehyde response of thalassemic RBC membranes to in vitro stimulation with peroxide. Remarkably, the heme content of RBC membranes from L1-treated thalassemic patients decreased by 28% +/- 10% during the 3-month study period. These results indicate that L1 can remove pathologic deposits of chelatable iron from thalassemic and sickle RBC membranes, a therapeutic potential not shared by DFO. Furthermore, membrane defects possibly mediated by catalytic iron, such as lipid peroxidation and hemichrome formation, may also be alleviated, at least in part, by L1.     

Iron mobilization from myocardial cells by 3-hydroxypyridin-4-one chelators: studies in rat heart cells in culture

The ability of 3-hydroxypyridin-4-ones (CP), a family of bidentate orally effective iron chelators, to remove iron and to prevent iron-induced lipid peroxidation was studied in beating rat myocardial cells in culture. The iron (III) binding constant (log beta 3) of all CP compounds is 36, but their lipophilicity may be modified by altering the length of the R2 substituent on the ring nitrogen. There was a direct relation between lipid solubility and chelating efficiency. Although at high concentrations all CP compounds were more effective in iron mobilization than deferoxamine, the opposite was true for low concentrations. Further studies with 1,2-diethyl-3-hydroxypyridin-4-one (CP94), the most effective CP compound, have shown that iron mobilization is completed within 6 hours, that effective mobilization requires a drug: iron molar ratio exceeding 3:1 permitting the formation of a hexadentate complex, and that the beneficial effects of iron mobilization are manifested in a marked reduction in membrane lipid peroxidation as indicated by cellular malonaldehyde content. Our study represents the first demonstration of a direct interaction between myocardial cells and an orally effective iron chelator, and underlines the need for high molar concentrations for achieving an optimal therapeutic effect.     

Selection of a new generation of orally active α-ketohydroxypyridine iron chelators intended for use in the treatment of iron overload

The prospect of selecting oral α-ketohydroxypyridine chelators intended for clinical use in iron overload has been examined using several animal models of efficacy and toxicity. Studies using iron dextran-loaded mice labelled with ⁵⁹Fe have shown that only the 1-substituted methyl, ethyl, (n)propyl, allyl, cyclopropyl, 2′-methoxyethyl, 3′-ethoxypropyl, or 2-methyl- or 2-ethyl- 3-hydroxypyrid-4-one chelators were orally effective in increasing iron (⁵⁹Fe) excretion by comparison to intraperitoneally administered desferrioxamine at the same dose (250 mg/kg). In contrast, chelators containing -H, mono- or dihydroxyalkyl and diethoxyethyl 1-substituents caused very little or no increase in iron (⁵⁹Fe) excretion by the oral or intraperitoneal routes. In vitro studies using ferritin and haemosiderin have shown that equivalent iron release took place with both groups of chelators irrespective of their in vivo effects. In most cases there was no correlation between the n-octanol/water partition coefficient (K_par) and iron removal efficacy but positive correlation between the lipophilicity and acute or subacute toxicity of these chelators in rats. The most toxic chelator in the chronic toxicity studies in rats was the lipophilic 1, 2-diethyl-3-hydroxypyrid-4-one (EL1NEt). The most effective chelator in increasing iron excretion in mice and rabbits was 1-allyl-2-methyl-3-hydroxypyrid-4-one (L1NAll), and the chelator with the highest safety margin in mice and rats was 1, 2-dimethyl-3-hydroxypyrid-4-one (L1). Overall the oral effectiveness in increasing iron excretion by these chelators in animals does not appear to be related to their lipophilicity or their ability to mobilise polynuclear iron in vitro but rather to other properties possibly related to their rate of biotransformation and excretion. © 1993 Wiley-Liss, Inc.     

Iron chelators for the treatment of iron overload disease: relationship between structure,redox activity,and toxicity

The success of the iron (Fe) chelator desferrioxamine (DFO) in the treatment of beta-thalassemia is limited by its lack of bioavailability. The design and characterization of synthetic alternatives to DFO has attracted much scientific interest and has led to the discovery of orally active chelators that can remove pathological Fe deposits. However, chelators that access intracellular Fe pools can be toxic by either inhibiting Fe-containing enzymes or promoting Fe-mediated free radical damage. Interestingly, toxicity does not necessarily correlate with Fe-binding affinity or with chelation efficacy, suggesting that other factors may promote the cytopathic effects of chelators. In this review, we discuss the interactions of chelators and their Fe complexes with biomolecules that can lead to toxicity and tissue damage.     

Variable inhibitory effects on the formation of dinitrosyl iron complexes by deferoxamine and salicylaldehyde isonicotinoyl hydrazone in K562 cells

A prerequisite of dinitrosyl iron complexes (DNIC) formation is the presence of nitric oxide (NO), iron (Fe) and thiol/imidazole groups. The aim of this study was to investigate the influence of Fe chelators on the formation of DNIC in erythroid K562 cells. The cells were treated with lipophilic salicylaldehyde isonicotinoyl hydrazone (SIH) (0.1 mM) and hydrophilic deferoxamine mesylate (DFO) (1 mM), a membrane permeable and non permeable Fe chelator, respectively. Dinitrosyl Fe complexes were generated by addition of 0.07 mM diethylamine NO. The DNIC formation was recorded using electron paramagnetic resonance (EPR). Both chelators inhibited DNIC formation up to 50% after 6 hours of treatment. Taken together, our data suggest that an intracellular low molecular weight labile Fe pool (LIP) and protein-bound Fe participate in DNIC formation in K562 cells to a similar extent.     

Variable Inhibitory Effects on the Formation of Dinitrosyl Iron Complexes by Deferoxamine and Salicylaldehyde Isonicotinoyl Hydrazone in K562 Cells

A prerequisite of dinitrosyl iron complexes (DNIC) formation is the presence of nitric oxide (NO), iron (Fe) and thiol/imidazole groups. The aim of this study was to investigate the influence of Fe chelators on the formation of DNIC in erythroid K562 cells. The cells were treated with lipophilic salicylaldehyde isonicotinoyl hydrazone (SIH) (0.1 mM) and hydrophilic deferoxamine mesylate (DFO) (1 mM), a membrane permeable and non permeable Fe chelator, respectively. Dinitrosyl Fe complexes were generated by addition of 0.07 mM diethylamine NO. The DNIC formation was recorded using electron paramagnetic resonance (EPR). Both chelators inhibited DNIC formation up to 50% after 6 hours of treatment. Taken together, our data suggest that an intracellular low molecular weight labile Fe pool (LIP) and protein-bound Fe participate in DNIC formation in K562 cells to a similar extent.     

Hydroxyl radical formation by sickle erythrocyte membranes: role of pathologic iron deposits and cytoplasmic reducing agents.

Sickle erythrocyte (RBC) membranes were previously shown to manifest increased Fenton activity (iron-dependent, peroxide-driven formation of hydroxyl radical [.OH]) compared with normal RBC membranes, but the nature of the catalytic iron was not defined. We now find that sickle membranes exposed to superoxide (.O2-) and hydrogen peroxide (H2O2) have three distinct iron compartments able to act as Fenton catalysts: preexisting free iron, free iron released during oxidant stress, and a component that cannot be chelated with deferoxamine (DF). In a model system, addition of iron compounds to normal ghosts showed that free heme, hemoglobin, Fe/adenosine diphosphate (ADP), and ferritin all catalyze .OH production; concurrent inhibition studies using DF documented that the unchelatable Fenton component is free heme or hemoglobin. During exposure to peroxide only, the iron in sickle membranes was unable to act as a Fenton catalyst without addition of a reducing agent. At physiologic concentrations, both ascorbate and glutathione restored Fenton activity. Lipid peroxidation studies showed that at physiologic levels ascorbate acts primarily as an antioxidant; however, as pharmacologic levels are reached, its pro-oxidant effects predominate. This study elucidates the catalytic ability of the iron compartments in the sickle cell membrane, the importance of which relates to the potential role of .OH in membrane damage. It also illustrates the potential participation of cytoplasmic reducing agents in this process, which may be especially relevant in the context of administration of supraphysiologic doses of ascorbate to sickle cell patients.     

Fluorescence measurements of the labile iron pool of sickle erythrocytes

Sickle erythrocytes have increased ferritin and increased molecular iron on the inner membrane leaflet, and we postulated that cytosolic labile iron is also elevated. We used the fluorescent metallosensor, calcein, and a permeant Fe2+ chelator to estimate labile cytoslic Fe2+, and calcein plus an Fe3+ chelator to estimate total cytosolic labile iron (Fe2+ + Fe3+). We measured membrane nonheme iron by its reactivity with ferrozine. As estimated by calcein and Fe2+ chelator, the mean +/- SD labile Fe2+ concentration was significantly lower in hemoglobin (Hb) SS (n = 29) than hemoglobin AA (n = 17) erythrocytes (0.56 +/- 0.35 microM versus 1.25 +/- 0.65 microM; P <.001). In contrast, as estimated by calcein and Fe3+ chelator, total erythrocyte labile iron was similar in hemoglobin SS (n = 12) and hemoglobin AA (n = 10) participants (1.75 +/- 0.41 microM versus 2.14 +/- 0.93 microM; P =.2). Mean membrane nonheme iron levels were higher in hemoglobin SS cells than hemoglobin AA cells (0.0016 x 10-4 versus 0.0004 x 10-4 fmol/cell; P =.01), but much lower than the mean amounts of total labile iron (1.6-1.8 x 10-4 fmol/cell) or hemoglobin iron (18 000-19 000 x 10-4 fmol/cell). Both membrane iron and total labile iron were much less than the mean amount of iron potentially present in erythrocyte ferritin as calculated from results of other investigators (15 x 10-4 versus 34 x 10-4 fmol/cell in HbAA versus HbSS erythrocytes). We conclude that cytosolic labile iron is not elevated in hemoglobin SS erythrocytes and that elemental membrane iron is present in only trace amounts.     

Catalysis of soluble hemoglobin oxidation by free iron on sickle red cell membranes

Abnormal deposition of hemichrome on the inner aspect of the sickle red cell membrane promotes premature cell demise. The steps proximate to hemichrome formation in these cells are poorly understood. To test the hypothesis that the pathologic deposits of free ferric iron located on the inner aspect of sickle cell membranes would be redox active and promote oxidation of soluble oxyhemoglobin, we incubated native versus iron-stripped sickle or normal ghost membranes with oxyhemoglobin S. We found that sickle membranes exerted an exaggerated effect on methemoglobin formation in solution, an effect completely accounted for by their abnormal content of free iron. This ability of sickle membranes to promote hemoglobin oxidation was not diminished by catalase or by presence of a high-affinity, iron-inactivating chelator that is unable to remove membrane iron. Examination of those membranes likewise revealed that their free iron content promoted deposition of additional heme-protein. These results establish that the potential redox couple formed by membrane-associated ferric iron and cytoplasmic oxyhemoglobin is promotive of hemoglobin oxidation and deposition of hemichrome on the membrane. This predicts that removal of pathologic membrane iron might help prevent the detrimental formation of methemoglobin and hemichrome in vivo, insofar as this is accelerated by transition metal.     

Determinants of red cell survival and erythropoietic activity in patients with sickle cell anemia in the steady state

We have studied 26 patients with sickle cell anemia to determine the factors that affect red blood cell (RBC) survival and other parameters of erythropoietic activity in the steady state. Determinants of erythropoietic activity included RBC survival by the 51Cr method, RBC production/destruction rate, alpha genotype, beta(s) haplotype, plasma 59Fe clearance, plasma iron turnover, erythron transferrin uptake), RBC Fe utilization, reticulocyte count, and erythropoietin levels. The alpha genotype was the most significant determinant of RBC survival followed, to a lesser extent, by the beta(s) haplotype. Hb F showed no correlation with RBC survival due to patient selection bias - the patients studied had comparable Hb F levels to start with. Other determinants of erythropoietic activity (hemoglobin level, mean corpuscular volume, reticulocyte count, RBC mass, RBC production/destruction rate, and erythropoietin level) were most likely secondary determinants associated with the alpha genotype, and not independent determinants in themselves. The data suggest that the alpha genotype and, and to a lesser extent, the beta(s) haplotype, might be determinants of the severity of the anemia of sickle cell disease, and should be considered in genetic counseling and patient selection for aggressive therapeutic interventions.     

Nonheme iron in sickle erythrocyte membranes: association with phospholipids and potential role in lipid peroxidation

Previous studies documented the abnormal association of heme and heme proteins with the sickle RBC membrane. We have now examined RBC ghosts and inside-out membranes (IOM) for the presence of nonheme iron as detected by its formation of a colored complex with ferrozine. Sickle ghosts have 33.8 +/- 18.2 nmol nonheme iron/mg membrane protein, and sickle IOM have 4.3 +/- 3.0 nmol/mg. In contrast, normal RBC ghosts and IOM have no detectable nonheme iron. The combination of heme and nonheme iron in sickle IOM averages nine times the amount of membrane- associated iron in normal IOM. Kinetics of the ferrozine reaction show that some of this nonheme iron on IOM reacts slowly and is probably in the form of ferritin, but most (72% +/- 18%) reacts rapidly and is in the form of some other biologic chelate. The latter iron compartment is removed by deferoxamine and by treatment of IOM with phospholipase D, which suggests that it represents an abnormal association of iron with polar head groups of aminophospholipids. The biologic feasibility of such a chelate was demonstrated by using an admixture of iron with model liposomes. Even in the presence of tenfold excess adenosine diphosphate, iron partitions readily into phosphatidylserine liposomes; there is no detectable association with phosphatidylcholine liposomes. To examine the bioavailability of membrane iron, we admixed membranes and t-butylhydroperoxide and found that sickle membranes show a tenfold greater peroxidation response than do normal membranes. This is not due simply to a deficiency of vitamin E, and this is profoundly inhibited by deferoxamine. Thus, while thiol oxidation in sickle membranes previously was shown to correlate with heme iron, the present data suggest that lipid peroxidation is related to nonheme iron. In control studies, we did not find this pathologic association of nonferritin, nonheme iron with IOM prepared from sickle trait, high-reticulocyte, postsplenectomy, or iron-overloaded individuals. These data provide additional support for the concept that iron decompartmentalization is a characteristic of sickle RBCs.     

Interactions of hydroxycarbamide (hydroxyurea) with iron and copper: implications on toxicity and therapeutic strategies

Presented at the 19th International Conference on Chelation, London, UK, 13-16 November 2009 Preliminary spectrophotometric and potentiometric studies have shown that hydroxycarbamide or hydroxyurea (HU) can interact with copper(II) [Cu(II)], iron(II) [Fe(II)] and Fe(III) ions and form complexes, for example, a ratio of 1 HU:1 metal at pH 5. The affinity for Cu (log β1 = 3.1) and Fe (log β1 = 5) by HU is much lower than that of the Fe and Cu chelating drug deferiprone (L1), which is used for the treatment of iron overload. It is anticipated that under certain conditions of high concentrations of these metal ions such as in transfusional iron overload, the therapeutic, pharmacological and toxicological properties of HU could be affected. It is also suggested that excess chelatable and labile forms of Fe or Cu ions, such as non transferrin-bound iron (NTBI) or intracellular low molecular weight labile iron, are among the main factors that may cause variations in the therapeutic response to HU in cancer, sickle cell anemia, thalassemia intermedia and other groups of patients. Further studies are needed to clarify the interaction mechanisms of HU with metal ions in vitro, in vivo and in clinical conditions.     

Antimalarial properties of orally active iron chelators

The appearance of widespread multiple drug resistance in human malaria has intensified the search for new antimalarial compounds. Metal chelators, especially those with high affinity for iron, represent one presently unexploited class of antimalarials. Unfortunately the use of previously identified chelators as antimalarials has been precluded by their toxicity and, in the case of desferrioxamine, the necessity for parenteral administration. The investigators now report that a new class of orally active iron chelators, namely the derivatives of alpha-ketohydroxypyridines (KHPs), are potent antimalarials against cultured Plasmodium falciparum. The KHPs evidently exert this effect by sequestering iron because a preformed chelator:iron complex has no antimalarial action. The pool(s) of iron being sequestered by the chelators have not been identified but may not include serum transferrin. Preincubation of human serum with KHPs followed by removal of the drug results in the removal of greater than 97% of total serum iron. Nonetheless, this serum effectively supports the growth of P falciparum cultures. Therefore the KHPs may exert antimalarial effect through chelation of erythrocytic rather than serum iron pool(s). The investigators conclude that these powerful, orally active iron chelators may form the basis of a new class of antimalarial drugs.     

On the evolutionary significance and metal-binding characteristics of a monolobal transferrin from Ciona intestinalis

Transferrins are a family of proteins that bind and transport Fe(III). Modern transferrins are typically bilobal and are believed to have evolved from an ancient gene duplication of a monolobal form. A novel monolobal transferrin, nicatransferrin (nicaTf), was identified in the primitive ascidian species Ciona intestinalis that possesses the characteristic features of the proposed ancestral Tf protein. In this work, nicaTf was expressed in Pichia pastoris. Extensive solution studies were performed on nicaTf, including UV-vis, fluorescence, CD, EPR and NMR spectroscopies, and electrospray time-of-flight mass spectrometry. The expressed protein is nonglycosylated, unlike the protein isolated from the organism. This property does not affect its ability to bind Fe(III). However, Fe(III)-bound nicaTf displays important spectral differences from other Fe(III)-bound transferrins, which are likely the consequence of differences in metal coordination. Coordination differences could also account for the weaker affinity of nicaTf for Fe(III) (log K = 18.5) compared with bilobal human serum transferrin (HsTf) (log K = 22.5 and 21.4). The Fe-nicaTf complex is not labile, as indicated by slow metal removal kinetics by the high-affinity chelator tiron at pH 7.4. The protein alternatively binds up to one equivalent of Ti(IV) or V(V), which suggests that it may transport nonferric metals. These solution studies provide insight into the structure and function of the primitive monolobal transferrin of C. intestinalis for comparison with higher order bilobal transferrins. They suggest that a major advantage for the evolution of modern transferrins, dominantly of bilobal form, is stronger Fe(III) affinity because of cooperativity.     

Novel di-2-pyridyl-derived iron chelators with marked and selective antitumor activity: in vitro and in vivo assessment

Aroylhydrazone and thiosemicarbazone iron (Fe) chelators have potent antitumor activity. The aim of the current study was to examine the antitumor effects and mechanisms of action of a novel series of Fe chelators, the di-2-pyridyl thiosemicarbazones. Of 7 new chelators synthesized, 4 showed pronounced antiproliferative effects. The most active chelator was Dp44mT, which had marked and selective antitumor activity-for example, an IC(50) of 0.03 microM in neuroepithelioma cells compared with more than 25 microM in mortal fibroblasts. Indeed, this antiproliferative activity was the greatest yet observed for an Fe chelator. Efficacy was greater than it was for the cytotoxic ligand 311 and comparable to that of the antitumor agent doxorubicin. Strikingly, Dp44mT significantly (P <.01) decreased tumor weight in mice to 47% of the weight in the control after only 5 days, whereas there was no marked change in animal weight or hematologic indices. Terminal deoxyribonucleotidyl transferase (TdT)-mediated dUTP nick end-labeling (TUNEL) staining demonstrated apoptosis in tumors taken from mice treated with Dp44mT. This chelator caused a marked increase of caspase-3 activity in murine Madison-109 (M109) cells. Caspase activation was at least partially mediated by the release of mitochondrial holo-cytochrome c (h-cytc) after incubation with Dp44mT. In conclusion, Dp44mT is a novel, highly effective antitumor agent in vitro and in vivo that induces apoptosis.     

Impaired vasodilation by red blood cells in sickle cell disease

Red blood cells (RBCs) have been ascribed a unique role in dilating blood vessels, which requires O2-regulated binding and bioactivation of NO by Hb and transfer of NO equivalents to the RBC membrane. Vasoocclusion in hypoxic tissues is the hallmark of sickle cell anemia. Here we show that sickle cell Hb variant S (HbS) is deficient both in the intramolecular transfer of NO from heme iron (iron nitrosyl, FeNO) to cysteine thiol (S-nitrosothiol, SNO) that subserves bioactivation, and in transfer of the NO moiety from S-nitrosohemoglobin (SNO-HbS) to the RBC membrane. As a result, sickle RBCs are deficient in membrane SNO and impaired in their ability to mediate hypoxic vasodilation. Further, the magnitudes of these impairments correlate with the clinical severity of disease. Thus, our results suggest that abnormal RBC vasoactivity contributes to the vasoocclusive pathophysiology of sickle cell anemia, and that the phenotypic variation in expression of the sickle genotype may be explained, in part, by variable deficiency in RBC processing of NO. More generally, our findings raise the idea that defective NO processing may characterize a new class of hemoglobinopathy.     

Piezo1 activation augments sickling propensity and the adhesive properties of sickle red blood cells in a calcium-dependent manner

Haemoglobin S polymerization in the red blood cells (RBCs) of individuals with sickle cell anaemia (SCA) can cause RBC sickling and cellular alterations. Piezo1 is a mechanosensitive protein that modulates intracellular calcium (Ca²⁺) influx, and its activation has been associated with increased RBC surface membrane phosphatidylserine (PS) exposure. Hypothesizing that Piezo1 activation, and ensuing Gárdos channel activity, alter sickle RBC properties, RBCs from patients with SCA were incubated with the Piezo1 agonist, Yoda1 (0.1–10 μM). Oxygen-gradient ektacytometry and membrane potential measurement showed that Piezo1 activation significantly decreased sickle RBC deformability, augmented sickling propensity, and triggered pronounced membrane hyperpolarization, in association with Gárdos channel activation and Ca²⁺ influx. Yoda1 induced Ca²⁺-dependent adhesion of sickle RBCs to laminin, in microfluidic assays, mediated by increased BCAM binding affinity. Furthermore, RBCs from SCA patients that were homo−/heterozygous for the rs59446030 gain-of-function Piezo1 variant demonstrated enhanced sickling under deoxygenation and increased PS exposure. Thus, Piezo1 stimulation decreases sickle RBC deformability, and increases the propensities of these cells to sickle upon deoxygenation and adhere to laminin. Results support a role of Piezo1 in some of the RBC properties that contribute to SCA vaso-occlusion, indicating that Piezo1 may represent a potential therapeutic target molecule for this disease.     

Erythropoietic activity in patients with sickle cell anaemia before and after treatment with hydroxyurea

In this project we have prospectively studied the erythropoietic activity in patients with sickle cell anaemia (SS) before and after treatment with hydroxyurea (HU). Some of the patients were enrolled in a double-blind placebo controlled trial of HU in patients with SS and others were enrolled in an open label study. Determinants of erythropoietic activity included the reticulocyte count, red blood cell (RBC) survival by the 51Cr method, plasma 59Fe clearance, plasma iron turnover (PIT), erythron transferrin uptake (ETU), RBC production/destruction rate, and RBC Fe utilization. Therapy with HU increased the mean corpuscular volume (MCV), haemoglobin (Hb)F, RBC survival and t1/2 59Fe clearance; it decreased the reticulocyte count, the white blood cell (WBC) count, ETU, and the PIT. Most of the changes in parameters of erythropoiesis could be explained by the increase in 51Cr RBC survival after therapy with HU. Together the data showed that in selected patients the net effect of HU on Hb level was a function of the difference between the suppressive effect of HU (decreased RBC production) and the increase in RBC survival. In the majority of patients who responded to HU, there was a preferential effect on RBC survival.