Dehydration response of sickle cells to sickling-induced Ca++ permeabilization

Blood ◽  
2002 ◽  
Vol 99 (7) ◽  
pp. 2578-2585 ◽  
Author(s):  
Virgilio L. Lew ◽  
Zipora Etzion ◽  
Robert M. Bookchin
Keyword(s):  
Sickle Cell ◽  
Fragility Curves ◽  
Initial Density ◽  
Osmotic Fragility ◽  
Sickle Cells ◽  
Rbc Membrane ◽  
Flow Cytometric ◽  
Gardos Channel ◽  
Cell Dehydration

Interaction of hemoglobin S polymers with the red blood cell (RBC) membrane induces a reversible increase in permeability (“Psickle”) to (at least) Na+, K+, Ca2+, and Mg2+. Resulting changes in [Ca2+] and [H+] in susceptible cells activate 2 transporters involved in sickle cell dehydration, the Ca2+-sensitive K+ (“Gardos”) channel (KCa) and the acid- and volume-sensitive K:Cl cotransport. We investigated the distribution of Psickle expression among deoxygenated sickle cell anemia (SS) RBCs using new experimental designs in which the RBC Ca2+ pumps were partially inhibited by vanadate, and the cells' dehydration rates were detected as progressive changes in the profiles of osmotic fragility curves and correlated with flow cytometric measurements. The results exposed marked variations in (sickling plus Ca2+)–induced dehydration rates within populations of deoxygenated SS cells, with complex distributions, reflecting a broad heterogeneity of their Psickle values. Psickle-mediated dehydration was inhibited by clotrimazole, verifying the role of KCa, and also by elevated [Ca2+]o, above 2 mM. Very high Psickle values occurred with some SS discocytes, which had a wide initial density (osmotic resistance) distribution. Together with its previously shown stochastic nature, the irregular distribution of Psickle documented here in discocytes is consistent with a mechanism involving low-probability, reversible interactions between sickle polymers and membrane or cytoskeletal components, affecting only a fraction of the RBCs during each deoxygenation event and a small number of activated pathways per RBC. A higher participation of SS reticulocytes in Psickle-triggered dehydration suggests that they form these pathways more efficiently than discocytes despite their lower cell hemoglobin concentrations.

scholarly journals Role of Calcium in Phosphatidylserine Externalisation in Red Blood Cells from Sickle Cell Patients

Anemia ◽  
2011 ◽  
Vol 2011 ◽  
pp. 1-8 ◽  
Author(s):  
Erwin Weiss ◽  
David Charles Rees ◽  
John Stanley Gibson
Keyword(s):  
Red Blood Cells ◽  
Sickle Cell ◽  
Blood Cells ◽  
Phosphatidylserine Exposure ◽  
Channel Inhibition ◽  
Cation Conductance ◽  
Gardos Channel ◽  
Cation Permeability

Phosphatidylserine exposure occurs in red blood cells (RBCs) from sickle cell disease (SCD) patients and is increased by deoxygenation. The mechanisms responsible remain unclear. RBCs from SCD patients also have elevated cation permeability, and, in particular, a deoxygenation-induced cation conductance which mediates entry, providing an obvious link with phosphatidylserine exposure. The role of was investigated using FITC-labelled annexin. Results confirmed high phosphatidylserine exposure in RBCs from SCD patients increasing upon deoxygenation. When deoxygenated, phosphatidylserine exposure was further elevated as extracellular [] was increased. This effect was inhibited by dipyridamole, intracellular chelation, and Gardos channel inhibition. Phosphatidylserine exposure was reduced in high saline. levels required to elicit phosphatidylserine exposure were in the low micromolar range. Findings are consistent with entry through the deoxygenation-induced pathway (), activating the Gardos channel. [] required for phosphatidylserine scrambling are in the range achievablein vivo.

The Flow Cytometric Osmotic Fragility Test Is an Effective Screening Method for Dehydrated Hereditary Stomatocytosis

Blood ◽  
2017 ◽  
Vol 130 (Suppl_1) ◽  
pp. 929-929
Author(s):  
Taiju Utsugisawa ◽  
Takuya Iwasaki ◽  
Takako Aoki ◽  
Yoshio Okamoto ◽  
Takahiro Kawakami ◽  
...  
Keyword(s):  
Hemolytic Anemia ◽  
Conflicts Of Interest ◽  
Screening Method ◽  
Osmotic Fragility ◽  
Target Cells ◽  
Next Generation ◽  
Causative Gene ◽  
Rbc Membrane ◽  
Flow Cytometric ◽  
Congenital Hemolytic Anemia

Abstract Introduction: Dehydrated hereditary stomatocytosis (DHSt) or hereditary xerocytosis (HX) is a form of congenital hemolytic anemia characterized by red blood cell (RBC) dehydration. Heterozygous mutations in PIEZO1, a mechanically-activated ion channel, cause DHSt. Recently, KCNN4, which encodes the Gardos channel, has been found to be the second pathogenic gene for DHSt. DHSt is characterized by an alteration in the RBC morphology in target cells, stomatocytes, and/or echinocytes, and RBC deformability assessments by ektacytometry as well as RBC ion flux measurements are currently the standard laboratory tests for DHSt, but their use in laboratories is limited. The flow cytometric osmotic fragility (FCM-OF) test is a useful diagnostic test for hereditary spherocytosis (HS) and also for hereditary elliptocytosis (HE). In this study, we showed that the FCM-OF test could also successfully diagnose DHSt. Subjects: A total of 46 cases of RBC membrane disorders were examined, and tentative diagnoses were made based on the RBC morphology, acid glycerol lysis time, and eosin 5'-maleimide binding tests, resulting in HS (n=31), HE (n=6), and DHSt (n=9). Methods: The number of RBCs in isotonic and hypotonic buffers were measured by flow cytometry. The degree of osmotic fragility was expressed as the "percentage residual RBCs (%RRC)". We confirmed the DHSt diagnosis by the massively paralleled sequencing using our custom panels targeting 68 hemolytic anemia-related genes with the next-generation sequencer. Results: Both HS and HE patients showed a decrease in %RRC; HS (18.0±8.9%, p<0.001) and HE (41.8±15.7%, p<0.001) compared to normal control (66.7±1.5%). DHSt patients showed a significant increase (112.6±34.5%, p<0.001) in FCM-OF. Additionally, next-generation sequencing revealed consistent causative gene mutations for DHSt; PIEZO1 (p.R2488Q and p.E2496ELE) or KCNN4 (p.P204R, p.A279T and p.R352H). Discussion: We examined 77 patients with congenital hemolytic anemia recently, and 59 cases were confirmed by diagnostic tests (76.6%). The results were as follows: 48 cases of RBC membrane abnormality (62.3%), 6 cases of RBC enzymopathy (7.8%), and 5 cases of hemoglobinopathy (6.5%). Of the cases of RBC membrane disorders, 31 cases of HS, 9 cases of DHSt, and 8 cases of HE were identified. These observations suggest that DHSt is the second-most common RBC membranopathy in Japan, and that the FCM-OF test and targeted sequencing efficiently discriminate DHSt from other RBC membrane disorders. Disclosures No relevant conflicts of interest to declare.

scholarly journals Pharmacological inhibition of calpain‐1 prevents red cell dehydration and reduces Gardos channel activity in a mouse model of sickle cell disease

2012 ◽  
Vol 27 (2) ◽  
pp. 750-759 ◽  
Author(s):  
Lucia De Franceschi ◽  
Robert S. Franco ◽  
Mariarita Bertoldi ◽  
Carlo Brugnara ◽  
Alessandro Matté ◽  
...  
Keyword(s):  
Sickle Cell Disease ◽  
Mouse Model ◽  
Sickle Cell ◽  
Red Cell ◽  
Channel Activity ◽  
Cell Disease ◽  
Pharmacological Inhibition ◽  
Gardos Channel ◽  
Cell Dehydration

scholarly journals Band 3 Peptides Block the Adherence of Sickle Cells to Endothelial Cells In Vitro

Blood ◽  
1997 ◽  
Vol 90 (10) ◽  
pp. 4172-4179 ◽  
Author(s):  
Bernard J.-M. Thevenin ◽  
Ian Crandall ◽  
Samir K. Ballas ◽  
Irwin W. Sherman ◽  
Stephen B. Shohet
Keyword(s):  
Sickle Cell ◽  
Low Dose ◽  
Band 3 ◽  
Active Peptides ◽  
Sickle Cells ◽  
Calcium Loading ◽  
Sickle Erythrocytes ◽  
Plasma Factors

Abstract Malaria-parasitized erythrocytes have increased endothelial adherence due to exposure of previously buried intramembranous sites of band 3. Because sickle erythrocytes also show increased adhesiveness and because the membrane portion of band 3 is aggregated in both types of cells, we examined the role of band 3 in sickle cell adhesiveness. Synthetic peptides derived from the second and third exofacial, interhelical regions of band 3 completely inhibited the abnormal adherence of sickle cells to an endothelial monolayer in a static assay. This effect was observed independently of plasma factors, required micromolar levels of peptide, was sequence-specific, and was found with both L- and D-isomers. The active peptides also inhibited the increased adherence induced by low-dose calcium loading of normal red blood cells. Finally, a monoclonal antibody against an active peptide specifically immunostained a fraction of sickle cells. These findings implicate a role for band 3 in at least one type of sickle cell adhesiveness via the exposure of normally cryptic membrane sites.

scholarly journals The role of WNK in modulation of KCl cotransport activity in red cells from normal individuals and patients with sickle cell anaemia

2019 ◽  
Vol 471 (11-12) ◽  
pp. 1539-1549
Author(s):  
David C.-Y. Lu ◽  
Anke Hannemann ◽  
Rasiqh Wadud ◽  
David C. Rees ◽  
John N. Brewin ◽  
...  
Keyword(s):  
Oxygen Tension ◽  
Sickle Cell ◽  
Sickle Cell Anaemia ◽  
Kinase Inhibitors ◽  
Red Cells ◽  
Red Cell ◽  
Sickle Cells ◽  
Kcl Cotransport ◽  
Pre Treatment

AbstractAbnormal activity of red cell KCl cotransport (KCC) is involved in pathogenesis of sickle cell anaemia (SCA). KCC-mediated solute loss causes shrinkage, concentrates HbS, and promotes HbS polymerisation. Red cell KCC also responds to various stimuli including pH, volume, urea, and oxygen tension, and regulation involves protein phosphorylation. The main aim of this study was to investigate the role of the WNK/SPAK/OSR1 pathway in sickle cells. The pan WNK inhibitor WNK463 stimulated KCC with an EC50 of 10.9 ± 1.1 nM and 7.9 ± 1.2 nM in sickle and normal red cells, respectively. SPAK/OSR1 inhibitors had little effect. The action of WNK463 was not additive with other kinase inhibitors (staurosporine and N-ethylmaleimide). Its effects were largely abrogated by pre-treatment with the phosphatase inhibitor calyculin A. WNK463 also reduced the effects of physiological KCC stimuli (pH, volume, urea) and abolished any response of KCC to changes in oxygen tension. Finally, although protein kinases have been implicated in regulation of phosphatidylserine exposure, WNK463 had no effect. Findings indicate a predominant role for WNKs in control of KCC in sickle cells but an apparent absence of downstream involvement of SPAK/OSR1. A more complete understanding of the mechanisms will inform pathogenesis whilst manipulation of WNK activity represents a potential therapeutic approach.

Distribution of dehydration rates generated by maximal Gardos-channel activation in normal and sickle red blood cells

Blood ◽  
2005 ◽  
Vol 105 (1) ◽  
pp. 361-367 ◽  
Author(s):  
Virgilio L. Lew ◽  
Teresa Tiffert ◽  
Zipora Etzion ◽  
Deisy Perdomo ◽  
Nuala Daw ◽  
...  
Keyword(s):  
Red Blood Cells ◽  
Blood Cells ◽  
Osmotic Fragility ◽  
Large Cell ◽  
Volume Distribution ◽  
Human Red Blood Cells ◽  
Gardos Channel ◽  
Cell Dehydration ◽  
Osmotic Lysis ◽  
Channel Variation

Abstract The Ca2+-activated K+ channels of human red blood cells (RBCs) (Gardos channels, hIK1, hSK4) can mediate rapid cell dehydration, of particular relevance to the pathophysiology of sickle cell disease. Previous investigations gave widely discrepant estimates of the number of Gardos channels per RBC, from as few as 1 to 3 to as many as 300, with large cell-to-cell differences, suggesting that RBCs could differ extensively in their susceptibility to dehydration by elevated Ca2+. Here we investigated the distribution of dehydration rates induced by maximal and uniform Ca2+ loads in normal (AA) and sickle (SS) RBCs by measuring the time-dependent changes in osmotic fragility and RBC volume distributions. We found a remarkable conservation of osmotic lysis and volume distribution profiles during Ca2+-induced dehydration, indicating overall uniformity of dehydration rates among AA and SS RBCs. In light of these results, alternative interpretations were suggested for the previously proposed low estimates and heterogeneity of channel numbers per cell. The results support the view that stochastic Ca2+ permeabilization rather than Gardos-channel variation is the main determinant selecting which SS cells dehydrate through Gardos channels in each sickling episode. (Blood. 2005;105:361-367)

Ion Transport Pathology in the Mechanism of Sickle Cell Dehydration

2005 ◽  
Vol 85 (1) ◽  
pp. 179-200 ◽  
Author(s):  
Virgilio L. Lew ◽  
Robert M. Bookchin
Keyword(s):  
Red Blood Cells ◽  
Sickle Cell ◽  
Blood Cells ◽  
Experimental Tests ◽  
Red Cell ◽  
Red Cell Membrane ◽  
Open Questions ◽  
Gardos Channel ◽  
Cell Dehydration ◽  
Experimental Findings

Polymers of deoxyhemoglobin S deform sickle cell anemia red blood cells into sickle shapes, leading to the formation of dense, dehydrated red blood cells with a markedly shortened life-span. Nearly four decades of intense research in many laboratories has led to a mechanistic understanding of the complex events leading from sickling-induced permeabilization of the red cell membrane to small cations, to the generation of the heterogeneity of age and hydration condition of circulating sickle cells. This review follows chronologically the major experimental findings and the evolution of guiding ideas for research in this field. Predictions derived from mathematical models of red cell and reticulocyte homeostasis led to the formulation of an alternative to prevailing gradualist views: a multitrack dehydration model based on interactive influences between the red cell anion exchanger and two K+transporters, the Gardos channel (hSK4, hIK1) and the K-Cl cotransporter (KCC), with differential effects dependent on red cell age and variability of KCC expression among reticulocytes. The experimental tests of the model predictions and the amply supportive results are discussed. The review concludes with a brief survey of the therapeutic strategies aimed at preventing sickle cell dehydration and with an analysis of the main open questions in the field.

scholarly journals Physical Properties of Red Cells as Related to Effects in Vivo. II. Effect of Thermal Treatment on Rigidity of Red Cells, Stroma and the Sickle Cell

Blood ◽  
1968 ◽  
Vol 32 (6) ◽  
pp. 862-871 ◽  
Author(s):  
THOMAS HALE HAM ◽  
RICHARD W. SAYRE ◽  
REBECCA F. DUNN ◽  
JOHN R. MURPHY
Keyword(s):  
Thermal Treatment ◽  
Sickle Cell ◽  
Osmotic Fragility ◽  
Red Cells ◽  
Red Cell ◽  
Sickle Cells ◽  
Sonic Treatment ◽  
Physiologic Saline ◽  
Shape Changes

Abstract Heating of human or dog red cells at 49 C. for 15 minutes or 60 minutes increased resistance to packing by a centrifugal force, increased viscosity and deformed them. After 15 minutes of heating, osmotic fragility was usually normal but was elevated after 60 minutes with the formation of spheroidal cells. The increase in viscosity caused by heating red cells at 49 C. for 60 minutes did not appear to result from the effect of heat on the hemoglobin or the hemolysis products of red cells disrupted by sonic treatment. The heating of intact human or dog red cell stroma in serum, or stroma washed multiple times in physiologic saline however, regularly increased their viscosity and produced buds and microstroma. The rigidity of stroma was increased by heating, probably accounting for the increased viscosity of heated intact red cells. The rigidity of heated stroma of red cells was further demonstrated by showing that the shape changes of the sickling process could be prevented in large part by the heating of sickle cells. Heating did not, however, prevent tactoid formation in stroma-free sickle-cell hemoglobin solutions.

scholarly journals Targeted deletion of α-adducin results in absent β- and γ-adducin, compensated hemolytic anemia, and lethal hydrocephalus in mice

Blood ◽  
2008 ◽  
Vol 112 (10) ◽  
pp. 4298-4307 ◽  
Author(s):  
Raymond F. Robledo ◽  
Steven L. Ciciotte ◽  
Babette Gwynn ◽  
Kenneth E. Sahr ◽  
Diana M. Gilligan ◽  
...  
Keyword(s):  
Hemolytic Anemia ◽  
Osmotic Fragility ◽  
Significant Loss ◽  
Communicating Hydrocephalus ◽  
Capping Protein ◽  
Rbc Membrane ◽  
Actin Capping Protein ◽  
Cell Dehydration ◽  
Actin Capping

Abstract In the red blood cell (RBC), adducin is present primarily as tetramers of α- and β-subunits at spectrin-actin junctions, or junctional complexes. Mouse RBCs also contain small amounts of γ-adducin. Platelets contain α- and γ-adducin only. Adducin functions as a barbed-end actin capping protein to regulate actin filament length and recruits spectrin to the ends of actin filaments. To further define adducin's role in vivo, we generated α-adducin knockout mice. α-Adducin is absent in all tissues examined in homozygous null mice. In RBCs, β- and γ-adducin are also absent, indicating that α-adducin is the limiting subunit in tetramer formation at the spectrin-actin junction. Similarly, γ-adducin is absent in α-null platelets. α-Adducin–null mice display compensated hemolytic anemia with features characteristic of RBCs in hereditary spherocytosis (HS), including spherocytes with significant loss of surface area, decreased mean corpuscular volume (MCV), cell dehydration, and increased osmotic fragility. Platelets maintain their normal discoid shape, and bleeding times are normal. α-Adducin–null mice show growth retardation at birth and throughout adulthood. Approximately 50% develop lethal communicating hydrocephalus with striking dilation of the lateral, third, and fourth ventricles. These data indicate that adducin plays a role in RBC membrane stability and in cerebrospinal fluid homeostasis.

ICA-17043, a novel Gardos channel blocker, prevents sickled red blood cell dehydration in vitro and in vivo in SAD mice

Blood ◽  
2003 ◽  
Vol 101 (6) ◽  
pp. 2412-2418 ◽  
Author(s):  
Jonathan W. Stocker ◽  
Lucia De Franceschi ◽  
Grant A. McNaughton-Smith ◽  
Roberto Corrocher ◽  
Yves Beuzard ◽  
...  
Keyword(s):  
Sickle Cell ◽  
Hemoglobin Concentration ◽  
K Channel ◽  
Mean Corpuscular Hemoglobin ◽  
Gardos Channel ◽  
Ic50 Value ◽  
Cell Dehydration ◽  
20 Nm

A prominent feature of sickle cell anemia is the presence of dehydrated red blood cells (RBCs) in circulation. Loss of potassium (K+), chloride (Cl−), and water from RBCs is thought to contribute to the production of these dehydrated cells. One main route of K+ loss in the RBC is the Gardos channel, a calcium (Ca2+)–activated K+ channel. Clotrimazole (CLT), an inhibitor of the Gardos channel, has been shown to reduce RBC dehydration in vitro and in vivo. We have developed a chemically novel compound, ICA-17043, that has greater potency and selectivity than CLT in inhibiting the Gardos channel. ICA-17043 blocked Ca2+-induced rubidium flux from human RBCs with an IC50 value of 11 ± 2 nM (CLT IC50 = 100 ± 12 nM) and inhibited RBC dehydration with an IC50 of 30 ± 20 nM. In a transgenic mouse model of sickle cell disease (SAD), treatment with ICA-17043 (10 mg/kg orally, twice a day) for 21 days showed a marked and constant inhibition of the Gardos channel activity (with an average inhibition of 90% ± 27%, P < .005), an increase in RBC K+ content (from 392 ± 19.9 to 479.2 ± 40 mmol/kg hemoglobin [Hb], P < .005), a significant increase in hematocrit (Hct) (from 0.435 ± 0.007 to 0.509 ± 0.022 [43.5% ± 0.7% to 50.9% ± 2.2%], P< .005), a decrease in mean corpuscular hemoglobin concentration (MCHC) (from 340 ± 9.0 to 300 ± 15 g/L [34.0 ± 0.9 to 30 ± 1.5 g/dL], P < .05), and a left-shift in RBC density curves. These data indicate that ICA-17043 is a potent inhibitor of the Gardos channel and ameliorates RBC dehydration in the SAD mouse.

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