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Sitosterolemia is a rare autosomal recessive disease that lead to an increase in the intestinal absorption and decreased biliary excretion plant sterols. It is caused by mutations in ABCG5 and ABCG8 genes, encoring sterolin‐1 and sterolin‐2 protein. The main clinical manifestations are xanthomas, premature atherosclerosis, arthralgia and, of note, hematological alterations. As in many other systemic diseases, hematological manifestations may be the only notable finding, for this reason we want to highlight the importance of multidisciplinary work and raise awareness of this rare disease that can lead to serious consequences if not treated prematurely. Here we present a case of this disease as well as its entire diagnostic process developed from a simple analytical alteration. [ABSTRACT FROM AUTHOR]

The K+ channel activated by the Ca2+, KCNN4, has been shown to contribute to red blood cell dehydration in the rare hereditary hemolytic anemia, the dehydrated hereditary stomatocytosis. We report two de novo mutations on KCNN4, We reported two de novo mutations on KCNN4, V222L and H340N, characterized at the molecular, cellular and clinical levels. Whereas both mutations were shown to increase the calcium sensitivity of the K+ channel, leading to channel opening for lower calcium concentrations compared to WT KCNN4 channel, there was no obvious red blood cell dehydration in patients carrying one or the other mutation. The clinical phenotype was greatly different between carriers of the mutated gene ranging from severe anemia for one patient to a single episode of anemia for the other patient or no documented sign of anemia for the parents who also carried the mutation. These data compared to already published KCNN4 mutations question the role of KCNN4 gain-of-function mutations in hydration status and viability of red blood cells in bloodstream. [ABSTRACT FROM AUTHOR]

The K+ channel activated by the Ca2+, KCNN4, has been shown to contribute to red blood cell dehydration in the rare hereditary hemolytic anemia, the dehydrated hereditary stomatocytosis. We report two de novo mutations on KCNN4, We reported two de novo mutations on KCNN4, V222L and H340N, characterized at the molecular, cellular and clinical levels. Whereas both mutations were shown to increase the calcium sensitivity of the K+ channel, leading to channel opening for lower calcium concentrations compared to WT KCNN4 channel, there was no obvious red blood cell dehydration in patients carrying one or the other mutation. The clinical phenotype was greatly different between carriers of the mutated gene ranging from severe anemia for one patient to a single episode of anemia for the other patient or no documented sign of anemia for the parents who also carried the mutation. These data compared to already published KCNN4 mutations question the role of KCNN4 gain-of-function mutations in hydration status and viability of red blood cells in bloodstream.

Sitosterolemia, also known as phytosterolemia, results from increased intestinal absorption of plant sterols and decreased intestinal and biliary excretion of sterols, resulting in increased levels of plant sterols in the plasma. The most common symptoms include xanthomas, premature atherosclerosis, hemolytic anemia and macrothrombocytopenia, however delayed diagnosis or misdiagnosis also occur. Clinical exome sequencing was performed on a 10-year-old boy whom we followed up with signs of pancytopenia accompanied by macrothrombocytopenia and stomatocytosis. In addition, the blood sterol levels of the patient and his family were studied. A novel homozygous c.904 + 5G > C intronic variant was detected in ABCG5 gene in index case. The mother and father were identified as carriers. The blood plant sterol levels of the patient and his family were studied, and the levels in the patient confirmed Sitosterolemia. Sitosterol levels decreased dramatically with restricted diet and ezetimibe treatment. In children, signs of Sitosterolemia may be subtle and the only symptom may be hematological. Therefore, Sitosterolemia should be kept in mind in children with stomatocytosis and macrothrombocytopenia. [ABSTRACT FROM AUTHOR]

The bone marrow produces billions of reticulocytes daily. These reticulocytes mature into red blood cells by reducing their plasma membrane by 20% and ejecting or degrading residual internal organelles, membranes and proteins not required by the mature cell. This process occurs by autophagy, protein degradation and vesiculation but is not well understood. We previously reported that Southeast Asian Ovalocytic RBCs demonstrate incomplete reticulocyte maturation and we have now extended this study to a number of other variant RBCs. By comparing the profile of a pure reticulocyte preparation of cultured red cells with these variant cells, we show that the largest of these cells, the overhydrated hereditary stomatocytosis cells, are the least mature, they barely reduced their plasma membrane and contain large amounts of proteins that should have been reduced or removed. Intermediate sized variant RBCs appear to be more mature but retain some endoplasmic reticulum and residual membrane proteins. We propose that the size and composition of these variant cell types correlate with the different stages of reticulocyte maturation and provide insight into the reticulocyte maturation process. [ABSTRACT FROM AUTHOR]

The bone marrow produces billions of reticulocytes daily. These reticulocytes mature into red blood cells by reducing their plasma membrane by 20% and ejecting or degrading residual internal organelles, membranes and proteins not required by the mature cell. This process occurs by autophagy, protein degradation and vesiculation but is not well understood. We previously reported that Southeast Asian Ovalocytic RBCs demonstrate incomplete reticulocyte maturation and we have now extended this study to a number of other variant RBCs. By comparing the profile of a pure reticulocyte preparation of cultured red cells with these variant cells, we show that the largest of these cells, the overhydrated hereditary stomatocytosis cells, are the least mature, they barely reduced their plasma membrane and contain large amounts of proteins that should have been reduced or removed. Intermediate sized variant RBCs appear to be more mature but retain some endoplasmic reticulum and residual membrane proteins. We propose that the size and composition of these variant cell types correlate with the different stages of reticulocyte maturation and provide insight into the reticulocyte maturation process.

The aim of this study was to investigate the beneficial effects of vitamin K relate to protection against detrimental effects of bromadiolone. Wistar rats (n = 30) were divided in three groups (n = 10): control group and two groups treated with bromadiolone (0.12 mg/kg) and bromadiolone + vitamin K (0.12 mg/kg + 100 mg/kg) over the period of four days. The main findings in the bromadiolone-exposed rats, such as damaged hepatocytes, high levels of globulin, total proteins and lymphocytes, and altered albumin/globulin ratio, collectively indicate an acute inflammatory process. Morphological changes in erythrocytes include microcytosis, hypochromia, hyperchromia, hemolysis, stomatocytosis, and spherocytosis. Significantly low values of RBC, Hct, and hemoglobin concentrations indicate impairments of the hematopoietic pathway causing combined anemia. The selected dose of bromadiolone caused a non-significant increase of catalase activity and a significant increase of the total protein content in brain tissue homogenates. Vitamin K supplementation reduced many of the harmful effects of bromadiolone. The cytoprotective role of vitamin K was proved to be of great importance for the preservation of structural changes on the membranes of hepatocytes and erythrocytes, in addition to the known role in the treatment of coagulopathies. The results of the study suggest valuable properties of vitamin K in the prevention and treatment of various types of anemia caused by bromadiolone toxicity. Future research is necessary to determine the adequate dose and treatment duration with vitamin K in disorders caused by the cumulative action of bromadiolone and possibly other pesticides. [ABSTRACT FROM AUTHOR]

Hereditary Xerocytosis, a rare hemolytic anemia, is due to gain of function mutations in PIEZO1, a non-selective cation channel activated by mechanical stress. How these PIEZO1 mutations impair channel function and alter red blood cell (RBC) physiology, is not completely understood. Here, we report the characterization of mutations in the N-terminal part of the protein (V598M, F681S and the double mutation G782S/R808Q), a part of the channel that was subject of many investigations to decipher its role in channel gating. Our data show that the electrophysiological features of these PIEZO1 mutants expressed in HEK293T cells are different from previously characterized PIEZO1 mutations that are located in the pore or at the C-terminal extracellular domain of the protein. Although RBC with PIEZO1 mutations showed a dehydrated phenotype, the activity of V598M, F681S or R808Q in response to stretch was not significantly different from the WT channels. In contrast, the G782S mutant showed larger currents compared to the WT PIEZO1. Interestingly, basal activity of all the mutated channels was not significantly altered at the opposite of what was expected according to the decreased water and cation contents of resting RBC. In addition, the features of mutant PIEZO1 expressed in HEK293 cells do not always correlate with the observation in RBC where PIEZO1 mutations induced a cation leak associated with an increased conductance. Our work emphasizes the role of the membrane environment in PIEZO1 activity and the need to characterize RBC permeability to assess pathogenicity to PIEZO1 mutants associated with erythrocyte diseases.

Hereditary Xerocytosis, a rare hemolytic anemia, is due to gain of function mutations in PIEZO1, a non-selective cation channel activated by mechanical stress. How these PIEZO1 mutations impair channel function and alter red blood cell (RBC) physiology, is not completely understood. Here, we report the characterization of mutations in the N-terminal part of the protein (V598M, F681S and the double mutation G782S/R808Q), a part of the channel that was subject of many investigations to decipher its role in channel gating. Our data show that the electrophysiological features of these PIEZO1 mutants expressed in HEK293T cells are different from previously characterized PIEZO1 mutations that are located in the pore or at the C-terminal extracellular domain of the protein. Although RBC with PIEZO1 mutations showed a dehydrated phenotype, the activity of V598M, F681S or R808Q in response to stretch was not significantly different from the WT channels. In contrast, the G782S mutant showed larger currents compared to the WT PIEZO1. Interestingly, basal activity of all the mutated channels was not significantly altered at the opposite of what was expected according to the decreased water and cation contents of resting RBC. In addition, the features of mutant PIEZO1 expressed in HEK293 cells do not always correlate with the observation in RBC where PIEZO1 mutations induced a cation leak associated with an increased conductance. Our work emphasizes the role of the membrane environment in PIEZO1 activity and the need to characterize RBC permeability to assess pathogenicity to PIEZO1 mutants associated with erythrocyte diseases. [ABSTRACT FROM AUTHOR]

- Sitosterolemia should be considered in patients with unexplained macrothrombocytopenia. - Sitosterolemia should be diagnosed in patients with sitosterol level (>15 μg/mL). - Once an index case is identified, family members should also undergo sterol testing. - Four novel variants may expand the genetic spectrum of sitosterolemia. - Ezetimibe can be a good choice for hematologic abnormalities in sitosterolemia. Sitosterolemia is a rare lipid disorder caused by mutations in adenosine triphosphate-binding cassette genes (ABCG) 5 and 8. To evaluate the phenotypic/genotypic features of sitosterolemia in a group of Turkish patients. Seven probands with unexplained hematologic abnormalities and their 13 relatives were enrolled. Sterol levels were measured by gas chromatography and genetic studies were performed using Sanger sequencing. Individuals were diagnosed with sitosterolemia if they were found to have frankly elevated sitosterol level >15 μg/mL and/or pathogenic variants of the ABCG 5/ ABCG 8. The seven probands and their six relatives were diagnosed with frank sitosterolemia, and all these patients had hematologic abnormalities. The remaining seven relatives were asymptomatic heterozygous carriers. Three novel variants in the ABCG 5 gene (c.161G> A , c.1375C> T , IVS10–1G> T), one novel variant in the ABCG 8 gene (c.1762G> C) and one known variant in the ABCG 5 gene (c.1336 C > T) were identified. No variant was identified in one case. The mean sitosterol level was significantly higher and mean platelet count was significantly lower in patients with homozygous variants compared to heterozygous variants (p <0.05, for all). Diets low in plant sterols were recommended for 13 symptomatic cases. Four homozygotes received ezetimibe, and their splenomegaly, anemia, and thrombocytopenia completely resolved except one. The five pathogenic variants identified in this study indicate the genetic heterogeneity of sitosterolemia in Turkish population. Patients with unexplained hematologic abnormalities (specifically macrothrombocytopenia) should have their sterol level measured as initial testing. Ezetimibe can be a good choice for sitosterolemia. [ABSTRACT FROM AUTHOR]

Glucose transport is intimately linked to red blood cell physiology. Glucose is the unique energy source for these cells, and defects in glucose metabolism or transport activity are associated with impaired red blood cell morphology and deformability leading to reduced lifespan. In vertebrate erythrocytes, glucose transport is mediated by GLUT1 (in humans) or GLUT4 transporters. These proteins also account for dehydroascorbic acid (DHA) transport through erythrocyte membrane. The peculiarities of glucose transporters and the red blood cell pathologies involving GLUT1 are summarized in the present review. [ABSTRACT FROM AUTHOR]

PIEZO1 is a cation channel activated by mechanical force. It plays an important physiological role in several biological processes such as cardiovascular, renal, endothelial and hematopoietic systems. Two different diseases are associated with alteration in the DNA sequence of PIEZO1: (i) dehydrated hereditary stomatocytosis (DHS1, #194380), an autosomal dominant hemolytic anemia caused by gain-of-function mutations; (ii) lymphatic dysplasia with non-immune fetal hydrops (LMPH3, #616843), an autosomal recessive condition caused by biallelic loss-of-function mutations. We analyzed a 14-year-old boy affected by severe lymphatic dysplasia already present prenatally, with peripheral edema, hydrocele, and chylothoraces. By whole exome sequencing, we identified compound heterozygosity for PIEZO1, with one splicing and one deletion mutation, the latter causing the formation of a premature stop codon that leads to mRNA decay. The functional analysis of the erythrocytes of the patient highlighted altered hydration with the intracellular loss of the potassium content and structural abnormalities with anisopoikolocytosis and presence of both spherocytes and stomatocytes. This novel erythrocyte trait, sharing features with both hereditary spherocytosis and overhydrated hereditary stomatocytosis, complements the clinical features associated with loss-of-function mutations of PIEZO1 in the context of the generalized lymphatic dysplasia of LMPH3 type.

Piezo1, encoded by the gene PIEZO1, is an erythrocytic cellular membrane mechanoactivated cation channel. Mutations have been implicated in erythrocyte volume disorders (EVDs)—especially hereditary xerocytosis (HX)/dehydrated stomatocytosis (DHS). We identified three patients, all with novel PIEZO1 mutations, but only one displaying the HX/DHS phenotype. Retrospective review of three cases. Osmotic gradient red cell deformability (Osmoscan) was assessed via the Technicon Ektacytometer. Red cell band 3 content was estimated using Eosin-5′-Maleimide staining. Patient 1 was evaluated for polycythemia. Osmoscans suggested mild spherocytosis; a novel PIEZO1 mutation (p.Thr1589Ile, exon 35) was identified, causing mild erythrocytosis without hemolytic anemia. Patient 2 was evaluated for macrocytosis/reticulocytosis, normal-to-high hemoglobin, and indirect hyperbilirubinemia. Osmoscans suggested increased cellular hydration; a second novel PIEZO1 mutation (p.Arg1728Cys, exon 37) was identified, resulting in overhydrated stomatocytosis with well-compensated hemolysis. Patient 3 was evaluated for indirect hyperbilirubinemia only. Osmoscans suggested dehydrated stomatocytosis (DHS, xerocytosis); a third novel PIEZO1 mutation (p.Arg2279Cys, exon 47) was identified. All three patients' blood smears demonstrated stomatocytes and spherocytes. EVDs may be underdiagnosed due to the lack of "expected" anemia in a hemolytic disorder; two of three patients had high hemoglobin and red cell counts and one had high normal values for both parameters and the presence of stomatocytes/dehydrated cells lead to identification of causative PIEZO1 mutations. PIEZO1-associated EVDs may be more common than previously suspected and should be included in the diagnostic algorithms for mild erythrocytosis/unexplained jaundice. [ABSTRACT FROM AUTHOR]

PIEZO1 is a cation channel activated by mechanical force. It plays an important physiological role in several biological processes such as cardiovascular, renal, endothelial and hematopoietic systems. Two different diseases are associated with alteration in the DNA sequence of PIEZO1 : (i) dehydrated hereditary stomatocytosis (DHS1, #194380), an autosomal dominant hemolytic anemia caused by gain-of-function mutations; (ii) lymphatic dysplasia with non-immune fetal hydrops (LMPH3, #616843), an autosomal recessive condition caused by biallelic loss-of-function mutations. We analyzed a 14-year-old boy affected by severe lymphatic dysplasia already present prenatally, with peripheral edema, hydrocele, and chylothoraces. By whole exome sequencing, we identified compound heterozygosity for PIEZO1 , with one splicing and one deletion mutation, the latter causing the formation of a premature stop codon that leads to mRNA decay. The functional analysis of the erythrocytes of the patient highlighted altered hydration with the intracellular loss of the potassium content and structural abnormalities with anisopoikolocytosis and presence of both spherocytes and stomatocytes. This novel erythrocyte trait, sharing features with both hereditary spherocytosis and overhydrated hereditary stomatocytosis, complements the clinical features associated with loss-of-function mutations of PIEZO1 in the context of the generalized lymphatic dysplasia of LMPH3 type. [ABSTRACT FROM AUTHOR]

Abstract: Hydration status is critical for erythrocyte survival and is mainly determined by intracellular cation content. Active pumps, passive transporters, and ion channels are the key components of volume homeostasis, whereas water passively fits ionic movements. Whenever cation content increases, erythrocyte swells, whereas it shrinks when cation content decreases. Thus, inappropriate cation leak causes erythrocyte hydration disorders, hemolytic anemia, and characteristic red cell shape abnormalities named stomatocytosis. All types of stomatocytosis either overhydrated or dehydrated are linked to inherited or de novo mutations in genes encoding ion transporters or channels. Although intracellular ion content can be assessed by experimental methods, laboratory diagnosis is guided by a combination of red blood cell parameters and deformability measurement when possible, and confirmed by sequencing of the putative genes. A better knowledge of the mechanisms underlying erythrocyte hydration imbalance will further lead to therapeutic improvements. [ABSTRACT FROM AUTHOR]

It is proposed that the specific reversal by serum albumin of the erythrocyte echinocytosis in an inorganic phosphate buffer saline or in a saline, either after 24 h in blood or after a storage of 6-7 weeks in SGAM or PAGGSM media, is due to a cell dehydration by a decrease of the total NaCl and KCl concentrations favoring the stomatocytogenic slow outward transport of inorganic phosphate with a hydrogen ion by band 3 anion exchanger, which was previously proposed to control the erythrocyte shape. This proposal would indicate that the opposition of the erythrocyte echinocytosis by serum albumin is not limited to binding to echinocytogenic amphiphiles, supported by the ability of the band 3-based mechanism of control of the erythrocyte shape to explain a variety of observations on the erythrocyte shape. It would also imply that this mechanism is a determinant of the erythrocyte rheological properties since influenced by cell shape and volume. It is shown that the above process of stomatocytosis can explain stomatocytoses by different agents as well as a knizocytosis induced in vitro and occurring in acquired and inherited disorders and other situations. Lastly, it can also explain the opposition of hemolysis by mannitol in SGAM and PAGGSM media. [ABSTRACT FROM AUTHOR]

MFSD2B has been identified as the exclusive sphingosine-1-phosphate (S1P) transporter in red blood cells (RBC) and platelets. MFSD2B-mediated S1P export from platelets is required for aggregation and thrombus formation, whereas RBC MFSD2B maintains plasma S1P levels in concert with SPNS2, the vascular and lymphatic endothelial cell S1P exporter, to control endothelial permeability and ensure normal vascular development. However, the physiological function of MFSD2B in RBC remains rather elusive despite mounting evidence that the intracellular S1P pool plays important roles in RBC glycolysis, adaptation to hypoxia and the regulation of cell shape, hydration, and cytoskeletal organisation. The large accumulation of S1P and sphingosine in MFSD2B-deficient RBC coincides with stomatocytosis and membrane abnormalities, the reasons for which have remained obscure. MFS family members transport substrates in a cation-dependent manner along electrochemical gradients, and disturbances in cation permeability are known to alter cell hydration and shape in RBC. Furthermore, the mfsd2 gene is a transcriptional target of GATA together with mylk3 , the gene encoding myosin light chain kinase (MYLK). S1P is known to activate MYLK and thereby impact on myosin phosphorylation and cytoskeletal architecture. This suggests that metabolic, transcriptional and functional interactions may exist between MFSD2B-mediated S1P transport and RBC deformability. Here, we review the evidence for such interactions and the implications for RBC homeostasis. [ABSTRACT FROM AUTHOR]

Sitosterolemia is a metabolic disorder characterized by increased intestinal absorption and tissue accumulation of phytosterols. Although sitosterolemia is considered a rare disease, its prevalence may be significantly higher than initially thought. Indeed, accumulating evidence suggests that patients with unexplained hematologic abnormalities or premature cardiovascular disease in the absence of classic risk factors may exhibit disordered phytosterol metabolism. In this review, we present a patient with sitosterolemia, describe the pathophysiology and the clinical picture of this disorder, and discuss the clinical value of phytosterol supplementation in patients with primary dyslipidemias. [ABSTRACT FROM AUTHOR]

Genetic defects of erythrocyte transport proteins cause disorders of red blood cell volume that are characterized by abnormal permeability to the cations Na+ and K+ and, consequently, by changes in red cell hydration. Clinically, these disorders are associated with chronic haemolytic anaemia of variable severity and significant co-morbidities, such as iron overload. This review provides an overview of recent insights into the molecular basis of this group of rare anaemias involving cation channels and transporters dysfunction. To date, a total of 5 different membrane proteins have been reported to be responsible for volume homeostasis alteration when mutated, 3 of them leading to overhydrated cells (AE1 [also termed SLC4A1], RHAG and GLUT1 [also termed SCL2A1) and 2 others to dehydrated cells (PIEZO1 and the Gardos Channel). These findings are not only of basic scientific interest, but also of direct clinical significance for improving diagnostic procedures and identify potential approaches for novel therapeutic strategies. [ABSTRACT FROM AUTHOR]

Hereditary xerocytosis (HX) or dehydrated hereditary stomatocytosis (DHS) [OMIM 194380], in which PIEZO1 gene mutation has recently been identified, is difficult to diagnose. We report here the discovery of a PIEZO1 gene mutation in a Japanese family (father, daughter, and son) who were previously diagnosed with hereditary high phosphatidylcholine hemolytic anemia (HPCHA). All of the affected family members had non-spherocytic hemolytic anemia associated with severe hemochromatosis-related diabetes mellitus. Although the causative correlation between HPCHA and PIEZO1-gene mutated HX/DHS remains to be clarified, our findings raise an important question as to whether any of the HPCHA cases previously diagnosed in Japan may have in fact been the form of hemolytic anemia known as HX/DHS with PIEZO1 gene mutation. [ABSTRACT FROM AUTHOR]

Aim. To evaluate erythrocyte surface architectonics and cytoskeleton in bronchial asthma (BA) under modulation of adrenergic agentsMaterial and methods. 24 healthy persons, 61 patients with bronchial asthma (BA) were examined. Of them, 28 patients had allergic BA (ABA) and 33 ones had nonallergic BA (NABA). Erythrocyte surface architectonics was studied by phase-contrast microscopy. Fixation in 0.5% solution of glutar aldehyde was used, preparation "squashed drop" was prepared. In all preparations adrenalin hydrochloride and obzidan were used in equimolar final concentrations of 4 10~5M. The mean morphologic index of transformation that reflects the shift to stomatocytosis or to echinocytosis was estimated to characterize integrally erythrocyte surface architectonics. The cytoskeleton was studied by the modified method of Chentsov's et al. The integral optic density was evaluated on imaging analyzer of Ista-Videotest company (St-Petersburg). Results. In healthy persons there was a correlation between erythrocyte surface architectonics and cytoskeleton under modulation of adrenergic system. Adrenalin-induced decrease of cytoskeletal proteins correlated with the shift to stomatocytosis. In NABA erythrocyte surface architectonics was characterized by a pronounced shift to stomatocytosis. This shift was accompanied with the most pronounced decrease of cytoskeletal proteins and it reduced in remission. In ABA manifestation of the shift was minimal and did not depend on the phase of the disease. In ABA no changes in erythrocyte surface architectonics in the presence of adrenalin were revealed. In the same conditions a decrease in cytoskeletal proteins was found. Conclusion. A correlation was found between erythrocyte surface architectonics and cytoskeleton in healthy persons. In ABA, under modulation of adrenergic system by adrenalin dissociation was revealed between receptor and cytoskeletal mechanisms of cell form induction. This dissociation does not depend on the phase of the disease and is thought to be one of the important postreceptor disorders typical for this variant of the disease.

Inherited disorders of erythrocyte volume homeostasis are a heterogeneous group of rare disorders with phenotypes ranging from dehydrated to overhydrated erythrocytes. Clinical, laboratory, physiologic, and genetic heterogeneities characterize this group of disorders. A series of recent reports have provided novel insights into our understanding of the genetic bases underlying some of these disorders of red cell volume regulation. This report reviews this progress in understanding determinants that influence erythrocyte hydration and how they have yielded a better understanding of the pathways that influence cellular water and solute homeostasis. [ABSTRACT FROM AUTHOR]

Severe side effects of cocaine consumption are vasoocclusive events such as myocardial infarction and stroke. We have hypothesized that cocaine could affect red blood cells (RBCs) and alter the rheological behaviour of blood. Heparinized blood from healthy volunteers was incubated with a final hematocrit of 45% with increasing cocaine concentrations: 0, 10, 100, 1000, and 10'000 μmol/L plasma. Time dependence of the shape change was tested in phosphate buffered saline containing cocaine. RBCs were fixed in 1% glutaraldehyde for morphological analysis. Blood viscosity was measured with a Couette Viscometer (Contraves LS 30) at 37°C and a shear rate of 69.5 s-1. RBC aggregation was assessed with a Myrenne aggregometer. Cocaine induced a dose-dependent stomatocytic shape transformation of RBCs, which was more pronounced in buffer than in plasma (plasma protein binding of the drug). Stomatocytosis occurs when a drug intercalates preferentially in the inner half of the membrane lipid bilayer. It was a time-dependent process with two components, an almost instant shape change occurring within 1 s, followed by a gradual further shape change during 10 min. Stomatocytosis was reversible by resuspension of the RBCs in cocaine-free buffer. This stomatocytic shape change increased whole blood viscosity at high shear rate from 5.69 ± 0.31 mPa.s to 6.39 ± 0.34 mPa.s for control and 10'000 μmol/L cocaine, respectively (p < 0.01). RBC aggregation was not affected by the shape change. These effects occurred at a cocaine concentration, which is several-fold above those measured in vivo. Therefore, it is unlikely that hemorheological factors are involved in vascular events after cocaine consumption. [ABSTRACT FROM AUTHOR]

Abstract: Hereditary spherocytosis and elliptocytosis are the two most common inherited red cell membrane disorders resulting from mutations in genes encoding various red cell membrane and skeletal proteins. Red cell membrane, a composite structure composed of lipid bilayer linked to spectrin-based membrane skeleton is responsible for the unique features of flexibility and mechanical stability of the cell. Defects in various proteins involved in linking the lipid bilayer to membrane skeleton result in loss in membrane cohesion leading to surface area loss and hereditary spherocytosis while defects in proteins involved in lateral interactions of the spectrin-based skeleton lead to decreased mechanical stability, membrane fragmentation and hereditary elliptocytosis. The disease severity is primarily dependent on the extent of membrane surface area loss. Both these diseases can be readily diagnosed by various laboratory approaches that include red blood cell cytology, flow cytometry, ektacytometry, electrophoresis of the red cell membrane proteins, and mutational analysis of gene encoding red cell membrane proteins. [Copyright &y& Elsevier]

Abstract: Autosomal dominant overhydrated cation-leak stomatocytosis in humans has been associated with missense mutations in the erythroid membrane transport genes AE1, RhAG, and GLUT1. Syndromic stomatocytosis has been reported in three dog breeds, but stomatocytosis in Standard Schnauzers is usually asymptomatic, and is accompanied by minimal if any anemia. We have extended the evaluation of a cohort of schnauzers. We found that low-level stomatocytosis was accompanied by increased MCV and increased red cell Na content, and minimal or no reticulocytosis. Red cells from two affected dogs exhibited increased currents in on-cell patches measured in symmetrical NaCl solutions, but Na,K-ATPase and NKCC-mediated cation flux was minimal. Three novel coding polymorphisms found in canine RhAG cDNA and three novel polymorphisms found in canine SLC4A1 cDNA did not cosegregate with MCV or Na content. The GLUT1 cDNA sequence was normal. We conclude that unlike human overhydrated cation-leak stomatocytosis, stomatocytosis in this cohort of Standard Schnauzers is not caused by mutations in the genes encoding RhAG, SLC4A1, or GLUT1. [Copyright &y& Elsevier]

In red cells, Rh-associated glycoprotein (RhAG) acts as an ammonia channel, as demonstrated by stopped-flow analysis of ghost intracellular pH (pHi) changes. Recently, overhydrated hereditary stomatocytosis (OHSt), a rare dominantly inherited hemolytic anemia, was found to be associated with a mutation (Phe65Ser or Ile61Arg) in RHAG. Ghosts from the erythrocytes of four of the OHSt patients with a Phe65Ser mutation were resealed with a pH-sensitive probe and submitted to ammonium gradients. Alkalinization rate constants, reflecting NH3 transport through the channel and NH3 diffusion unmediated by RhAG, were deduced from time courses of fluorescence changes. After subtraction of the constant value found for Rhnull lacking RhAG, we observed that alkalinization rate constant values decreased ~50% in OHSt compared with those of controls. Similar RhAG expression levels were found in control and OHSt. Since half of the expressed RhAG in OHSt most probably corresponds to the mutated form of RhAG, as expected from the OHSt heterozygous status, this dramatic decrease can be therefore related to the loss of function of the Phe65Ser-mutated RhAG monomer. [ABSTRACT FROM AUTHOR]

Plasmalemmal Cl-/HCO3- exchangers are encoded by the SLC4 and SLC26 gene superfamilies, and function to regulate intracellular pH, [Cl-] and cell volume. The Cl-/HCO3- exchangers of polarized epithelial cells also contribute to transepithelial secretion and reabsorption of acid-base equivalents and Cl-. This review focuses on Na+-independent electroneutral Cl-/HCO3- exchangers of the SLC4 family. Human SLC4A1/AE1 mutations cause the familial erythroid disorders of spherocytic anemia, stomatocytic anemia and ovalocytosis. A largely discrete set of AE1 mutations causes familial distal renal tubular acidosis. The SIc4a2/Ae2-/- mouse dies before weaning with achiorhydria and osteopetrosis. A hypomorphic Ae2-/- mouse survives to exhibit male infertility with defective spermatogenesis and a syndrome resembling primary biliary cirrhosis. A human SLC4A3/AE3 polymorphism is associated with seizure disorder, and the Ae3-/- mouse has increased seizure susceptibility. The transport mechanism of mammalian SLC4/AE polypeptides is that of electroneutral Cl-/anion exchange, but trout erythroid Ael also mediates Cl- conductance. Erythroid Ael may mediate the DIDS-sensitive Cl- conductance of mammalian erythrocytes, and, with a single missense mutation, can mediate electrogenic SO42-/Cl- exchange. AE1 trafficking in polarized cells is regulated by phosphorylation and by interaction with other proteins. AE2 exhibits isoform-specific patterns of acute Inhibition by acidic intracellular pH and independently by acidic extracellular pH. In contrast, AE2 is activated by hypertonicity and, in a pH-independent manner, by ammonium and by hypertonicity. A growing body of structure-function and interaction data, together with emerging information about physiological function and structure, is advancing our understanding of SLC4 anion exchangers. [ABSTRACT FROM AUTHOR]

Abstract: A number of situations that result in abnormal permeability pathways in human red blood cells (RBCs) have been investigated. In sickle cell disease (SCD), RBCs contain HbS, rather than the normal HbA. When deoxygenated, an abnormal conductance pathway, termed Psickle, is activated, which contributes to cell dehydration, largely through allowing Ca2+ entry and subsequent activation of the Gardos channel. Whole-cell patch-clamp recordings from sickle RBCs show a deoxygenated-induced conductance, absent from normal RBCs, which shares some of the properties of Psickle: equivalent Na+ and K+ permeability, significant Ca2+ conductance, partial inhibition by DIDS and also Zn2+. Gd3+ markedly attenuates conductance in both normal and sickle RBCs. In addition, deoxygenated sickle cells, but not oxygenated ones or normal RBCs regardless of the oxygen tension, undergo haemolysis in isosmotic non-electrolyte solutions. Non-electrolyte entry was confirmed radioisotopically whilst haemolysis was inhibited by DIDS. These findings suggest that under certain circumstances Psickle may also be permeable to non-electrolytes. Finally, RBCs from certain patients with hereditary stomatocytosis have a mutated band 3, which appears able to act as a conductance pathway for univalent cations. These results extend our understanding of the abnormal permeability pathways of RBCs. [Copyright &y& Elsevier]

Stomatocytosis resembles human overhydrated hereditary stomatocytosis (OHSt), a disease characterised by a reduced or absent stomatin expression. The objective of this report was to investigate the expression level of stomatin in erythrocytes from Standard Schnauzers with stomatocytosis. Routine haematology, intraerythrocytic Na+/K+ concentration and stomatin expression were evaluated in blood from twelve Standard Schnauzers and from three controls. SDS-PAGE and Western blotting on isolated integral membrane proteins were used to investigate stomatin expression. Circulating stomatocytes, macrocytosis, anisocytosis, increased erythrocyte fragility and high intracellular sodium and potassium concentrations were found in 10/12 dogs from the same breeding line although stomatin levels were similar to those of controls. In spite of the clinico-pathological similarities between human and canine stomatocytosis, erythrocytes from affected dogs do not lack stomatin and the expression level of this protein cannot therefore be used to diagnose hereditary stomatocytosis in Standard Schnauzers. [ABSTRACT FROM AUTHOR]

Abstract: We have recently shown that amino acid substitutions in the membrane domain of band 3 (anion exchanger 1, SLC4A1) are associated with hereditary stomatocytosis (HSt), a red cell condition in which the cells leak sodium and potassium ions. These substitutions appear to convert band 3 from an anion exchanger into a cation channel. In this review, I will first give some background on the structure and function of normal band 3 and describe our findings in red cells from HSt patients. Then I will compare the properties of the HSt band 3 to those of Southeast Asian Ovalocytosis (SAO) band 3 and discuss the implications for the structure of band 3, the quality control of protein expression in red cells and the cation permeability of normal human red cells. [Copyright &y& Elsevier]

Phytosterolaemia (sitosterolaemia) is a recessively inherited metabolic condition in which the absorption of both cholesterol and plant-derived cholesterol-like molecules at the gut is unselective and unrestricted. In haematology, Mediterranean stomatocytosis or Mediterranean macrothrombocytopenia is a poorly understood haematological condition that combines stomatocytic haemolysis with the presence of very large platelets. Five pedigrees showing this haematology were identified. Gas chromatography mass spectrometry (GC-MS) showed that all of the patients with this highly specific haematology had grossly elevated levels of phytosterols in the blood, diagnostic of phytosterolaemia. All showed mutations in the ABCG5 and ABCG8 previously linked to phytosterolaemia. Three pedigrees showed five new mutations, while two pedigrees showed the common W361X mutation in ABCG8. We draw the following four conclusions: (i) that Mediterranean stomatocytosis/macrothrombocytopenia is caused by an excess of phytosterols in the blood; (ii) that phytosterolaemia, which does not respond to standard statin treatment, can be diagnosed via the distinctive haematology described here, even when the cholesterol is normal; (iii) that phytosterolaemia should be considered in the differential diagnosis of all patients with large platelets; and (iv) that the platelet size should be noted in patients with hypercholesterolaemia. [ABSTRACT FROM AUTHOR]

Summary. The tendency for thrombosis to occur if haemolysis persists after splenectomy is especially marked in ‘hereditary stomatocytosis’, in which the red cell membrane ‘leaks’ Na and K. A 21-year-old woman, who was splenectomized in childhood for a congenital haemolytic state, presented with major pulmonary embolism that recurred despite anticoagulation. Tests showed a significant cation leak with a ‘shallow-slope’ abnormality in temperature dependence. Allogeneic bone marrow transplantation caused the thrombophilic state to cease and subsequently anticoagulation was stopped without recurrence of thromboembolism. However, she died 9 months after transplantation: iron overload, intensified by the transfusion demands of the transplant, was a major factor. [ABSTRACT FROM AUTHOR]

Summary. The hereditary stomatocytoses are a group of dominant haemolytic anaemias that show two main features: invaginated, ‘stomatocytic’ morphology; and a membrane leak to the univalent cations Na and K. A patient with the most severe variant of these conditions was reported to show a defect in an in vitro process of ATP-dependent endocytic vesiculation (ADEV), which is found in normal red cells. We have examined this endocytosis process in 11 leaky red cell pedigrees available to us in the UK. ADEV in broken membranes was absent only in the two most severely affected, ‘overhydrated’ pedigrees studied, both of which showed a deficiency in the membrane raft protein, stomatin. The process was present, although typically diminished by about 10–20% compared with normal red cells, in all others. The cross-linker dimethyl adipimate (DMA), which could correct the cation leak in some of these patients, also corrected the ADEV defect in the same patients. In those patients in whom DMA had no effect on the ion leak, ADEV was not absent. In normal cells, this process of vesiculation was inhibited by inhibitors of membrane ‘raft’ function, by an antistomatin antibody and by vanadate and N-ethyl maleimide, but not by inhibitors of a number of kinases. These data highlight the heterogeneity of these conditions. A mechanism is discussed by which a defect in raft-based endocytosis could lead to the exaggerated surface exposure of an ion channel, which could then function constitutively, i.e. ‘leak’. [ABSTRACT FROM AUTHOR]

We describe two families with the ‘cryohydrocytosis’ form of stomatocytosis. Both show a mild stomatocytic anaemia with Hb levels of 12–16 g/dl and reticulocyte counts of 4·3–24%, with very marked autohaemolysis at refrigerator temperatures and pseudohyperkalaemia as a result of loss of K from red cells on storage at room temperature. The ouabain + bumetanide-insensitive ‘passive leak’ K influx showed a ‘U’-shaped temperature dependence, with a minimum at 23°C. In one family, there was consistent variation in haematological severity within the pedigree. In the other, the parents of the proposita were normal, but all three of her children were affected, consistent with a new mutation of a dominant condition. Cold storage of the red cells led to a very marked increase in osmotic fragility and macrospherocytosis, explaining why a diagnosis of ‘hereditary spherocytosis’ can easily be reached in these pedigrees. [ABSTRACT FROM AUTHOR]