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

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 (Ca(2+))-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 Ca(2+)-induced rubidium flux from human RBCs with an IC(50) value of 11 +/- 2 nM (CLT IC(50) = 100 +/- 12 nM) and inhibited RBC dehydration with an IC(50) 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.     

Modulation of erythrocyte potassium chloride cotransport, potassium content, and density by dietary magnesium intake in transgenic SAD mouse

Prevention of erythrocyte dehydration is a potential therapeutic strategy for sickle cell disease. Increasing erythrocyte magnesium (Mg) could inhibit sickle cell dehydration by increasing chloride (CI) and water content and by inhibiting potassium chloride (K-CI) cotransport. In transgenic SAD 1 and (control) C57BL/6 normal mice, we investigated the effect of 2 weeks of diet with either low Mg (6 +/- 2 mg/kg body weight/d) or high Mg (1,000 +/- 20 mg/kg body weight/ d), in comparison with a diet of standard Mg (400 +/- 20 mg/ kg body weight/d). The high- Mg diet increased SAD 1 erythrocyte Mg and K contents and reduced K-CI cotransport activity, mean corpuscular hemoglobin concentration (MCHC), cell density, and reticulocyte count. SAD 1 mice treated with low-Mg diet showed a significant reduction in erythrocyte Mg and K contents and increases in K-CI cotransport, MCHC, cell density, and reticulocyte counts. In SAD 1 mice, hematocrit (Hct) and hemoglobin (Hb) decreased significantly with low Mg diet and increased significantly with high-Mg diet. The C57BL/6 controls showed significant changes only in erythrocyte Mg and K content, and K-CI cotransport activities, similar to those observed in SAD 1 mice. Thus, in the SAD 1 mouse, changes in dietary Mg modulate K-CI cotransport, modify erythrocyte dehydration, and ultimately affect Hb levels.     

Formation of dense erythrocytes in SAD mice exposed to chronic hypoxia: evaluation of different therapeutic regimens and of a combination of oral clotrimazole and magnesium therapies

We have examined the effect of hydroxyurea (HU), clotrimazole (CLT), magnesium oxide (Mg), and combined CLT+Mg therapies on the erythrocyte characteristics and their response to chronic hypoxia in a transgenic sickle mouse (SAD) model. SAD mice were treated for 21 days with 1 of the following regimens (administered by gavage): control (n = 6), HU (200 mg/d; n = 6), CLT (80 mg/kg/d, n = 5), Mg (1,000 mg/kg/d, n = 5), and CLT+Mg (80 and 1,000 mg/kg/d, respectively, n = 6). Nine normal mice were also treated as controls (n = 3), HU (n = 3), and CLT+Mg (n = 3). Treatment with HU induced a significant increase in mean corpuscular volume and cell K content and a decrease in density in SAD mice. Treatment with the CLT and Mg, either alone or in combination, also increased cell K and reduced density in SAD mice. After 21 days of treatment, the animals were exposed to hypoxia (48 hours at 8% O(2)) maintaining the same treatment. In the SAD mice, hypoxia induced significant cell dehydration. These hypoxia-induced changes were blunted in either HU- or Mg-treated SAD mice and were completely abolished by either CLT or CLT+Mg treatment, suggesting a major role for the Gardos channel in hypoxia-induced dehydration in vivo.     

Erythrocyte adhesion is modified by alterations in cellular tonicity and volume

We tested the hypothesis that dehydration-induced alterations in red blood cell (RBC) membrane organisation or composition contribute to sickle cell adhesion in sickle cell disease (SCD). To examine the role of RBC hydration in adhesion to the subendothelial matrix protein thrombospondin-1 (TSP), normal and sickle RBCs were incubated in buffers of varying tonicity and tested for adhesion to immobilised TSP under flow conditions. Sickle RBCs exhibited a decrease in TSP binding with increasing cell hydration (P<0.005), suggesting that cellular dehydration may contribute to TSP adhesion. Consistent with this hypothesis, normal RBCs showed an increase in TSP adhesion with increasing dehydration (P<0.01). Furthermore, increased TSP adhesion of normal RBCs could also be induced by isotonic dehydration using nystatin-sucrose buffers. Finally, TSP adhesion of both sickle RBCs and dehydrated normal RBCs was inhibited by the anionic polysaccharides, chondroitin sulphate A and high molecular weight dextran sulphate, but not by competitors of CD47-, band 3-, or RBC phosphatidylserine-mediated adhesion. More importantly, we found increased adhesion of nystatin-sucrose dehydrated normal mouse RBCs to kidney capillaries following re-infusion in vivo. In summary, these findings demonstrate that changes in hydration can significantly impact adhesion, causing normal erythrocytes to display adhesive properties similar to those of sickle cells and vice versa.     

Characterization of hyaluronan synthase expression and hyaluronan synthesis in bone marrow mesenchymal progenitor cells: predominant expression of HAS1 mRNA and up-regulated hyaluronan synthesis in bone marrow cells derived from multiple myeloma patients

Interaction of hemoglobin S polymers with the red blood cell (RBC) membrane induces a reversible increase in permeability ("P(sickle)") to (at least) Na(+), K(+), Ca(2+), and Mg(2+). Resulting changes in [Ca(2+)] and [H(+)] in susceptible cells activate 2 transporters involved in sickle cell dehydration, the Ca(2+)-sensitive K(+) ("Gardos") channel (K(Ca)) and the acid- and volume-sensitive K:Cl cotransport. We investigated the distribution of P(sickle) expression among deoxygenated sickle cell anemia (SS) RBCs using new experimental designs in which the RBC Ca(2+) 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 Ca(2+))-induced dehydration rates within populations of deoxygenated SS cells, with complex distributions, reflecting a broad heterogeneity of their P(sickle) values. P(sickle)-mediated dehydration was inhibited by clotrimazole, verifying the role of K(Ca), and also by elevated [Ca(2+)](o), above 2 mM. Very high P(sickle) 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 P(sickle) 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 P(sickle)-triggered dehydration suggests that they form these pathways more efficiently than discocytes despite their lower cell hemoglobin concentrations.     

Efficacy and safety of the Gardos channel blocker, senicapoc (ICA-17043), in patients with sickle cell anemia

Senicapoc, a novel Gardos channel inhibitor, limits solute and water loss, thereby preserving sickle red blood cell (RBC) hydration. Because hemoglobin S polymerization is profoundly influenced by intracellular hemoglobin concentration, senicapoc could improve sickle RBC survival. In a 12-week, multicenter, phase 2, randomized, double-blind, dose-finding study, we evaluated senicapoc's safety and its effect on hemoglobin level and markers of RBC hemolysis in sickle cell anemia patients. The patients were randomized into 3 treatment arms: placebo; low-dose (6 mg/day) senicapoc; and high-dose (10 mg/day) senicapoc. For the primary efficacy end point (change in hemoglobin level from baseline), the mean response to high-dose senicapoc treatment exceeded placebo (6.8 g/L [0.68 g/dL] vs 0.1 g/L [0.01 g/dL], P < .001). Treatment with high-dose senicapoc also produced significant decreases in such secondary end points as percentage of dense RBCs (-2.41 vs -0.08, P < .001); reticulocytes (-4.12 vs -0.46, P < .001); lactate dehydrogenase (-121 U/L vs -15 U/L, P = .002); and indirect bilirubin (-1.18 mg/dL vs 0.12 mg/dL, P < .001). Finally, senicapoc was safe and well tolerated. The increased hemoglobin concentration and concomitant decrease in the total number of reticulocytes and various markers of RBC destruction following senicapoc administration suggests a possible increase in the survival of sickle RBCs. This study is registered at http://clinicaltrials.gov as NCT00040677.     

Exaggerated cation leak from oxygenated sickle red blood cells during deformation: evidence for a unique leak pathway.

An abnormal susceptibility of the sickle red blood cell (RBC) membrane to deformation could compromise its permeability barrier function and contribute to the exuberant cation leakiness occurring during the sickling phenomenon. We examined this hypothesis by subjecting RBCs at ambient oxygen tension to elliptical deformation, applying shear stress in a viscous medium under physiologic conditions. Compared with normal and high-reticulocyte control RBCs, sickle RBCs manifest an exaggerated K leak response to deformation. This leak is fully reversible, is both Cl and Ca independent, and at pHe 7.4 is fully balanced so that Kefflux equals Nainflux. This abnormal susceptibility is also evident in that the K leak in response to deformation occurs at an applied shear stress of only 141 dyne/cm2 for sickle RBCs, as compared to 204 dyne/cm2 for normal RBCs. Fresh sickle RBC membranes contain elevated amounts of lipid hydroperoxide, the presence of which is believed to provide the biochemical basis for enhanced deformation susceptibility. When examined at pHe 6.8, oxygenated sickle RBCs acquire an additional, unbalanced (Kefflux > Nainflux) component to the K leak increment specifically ascribable to deformation. Studies with inhibitors suggest that this additional component is not caused by a known leak pathway (eg, either K:Cl cotransport or the Gardos channel). This abnormal susceptibility of the sickle membrane to development of cation leakiness during deformation probably contributes to the exuberant cation leak taking place during RBC sickling.     

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.     

The contribution of endothelial cell P-selectin to the microvascular flow of mouse sickle erythrocytes in vivo

Microvascular occlusion in sickle cell disease can be initiated by adhesion of sickle red blood cells (RBCs) to the endothelium. Our objective in this study was to verify the relevance in vivo of our discovery that sickle RBCs adhere abnormally to endothelial P-selectin in vitro. We used computer-assisted intravital microscopy to characterize RBC flow velocity (V(RBC)) in mice. We found faster V(RBC) of sickle RBCs in P-selectin knock-out and control mice than in sickle cell mice, which have increased endothelial cell P-selectin expression. Agonist peptide for murine protease-activated receptor-1 (PAR-1), which selectively activates mouse endothelial cells but not platelets, was used to assess the effects of endothelial cell P-selectin on microvascular flow. Suffusion of venules with this agonist stopped flow promptly in normal and sickle mice but not in P-selectin knock-out mice or in control mice pretreated with anti-P-selectin monoclonal antibody or unfractionated heparin (UFH). Agonist-induced slowing of flow was reversed rapidly by suffusion with UFH, provided flow had not already stopped. We conclude that endothelial cell P-selectin contributes to the microcirculatory abnormalities in sickle cell disease and that blocking P-selectin may be useful for preventing painful vasoocclusion in sickle cell disease.     

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

The Ca(2+)-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 Ca(2+)-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.     

Chimerism and cure: hematologic and pathologic correction of murine sickle cell disease

Bone marrow transplantation (BMT) is the only curative therapy for sickle cell disease (SCD). However, the morbidity and mortality related to pretransplantation myeloablative chemotherapy often outweighs the morbidity of SCD itself, thus severely limiting the number of patients eligible for transplantation. Although nonmyeloablative transplantation is expected to reduce the risk of BMT, it will likely result in mixed-chimerism rather than complete replacement with donor stem cells. Clinical application of nonmyeloablative transplantation thus requires knowledge of the effect of mixed chimerism on SCD pathophysiology. We have, therefore, created a panel of transplanted SCD mice that received transplants displaying an array of red blood cell (RBC) and white blood cell (WBC) chimerism. A significant enrichment of RBC over WBC chimerism occurred in these mice, because of the dramatic survival advantage of donor over sickle RBCs in the peripheral blood. Increasing levels of RBC chimerism provided progressive correction of hematologic and pathologic abnormalities. However, sickle bone marrow and splenic hematopoiesis was not corrected until peripheral blood sickle RBCs were fully replaced with donor RBCs. These results have important and unexpected implications for nonmyeloablative BMT for SCD. As the critical hematopoietic organs were not corrected without full RBC replacement, 100% peripheral blood RBC chimerism becomes the most important benchmark for cure after nonmyeloablative BMT.     

GBT440 increases haemoglobin oxygen affinity,reduces sickling and prolongs RBC half‐life in a murine model of sickle cell disease

A major driver of the pathophysiology of sickle cell disease (SCD) is polymerization of deoxygenated haemoglobin S (HbS), which leads to sickling and destruction of red blood cells (RBCs) and end‐organ damage. Pharmacologically increasing the proportion of oxygenated HbS in RBCs may inhibit polymerization, prevent sickling and provide long term disease modification. We report that GBT440, a small molecule which binds to the N‐terminal α chain of Hb, increases HbS affinity for oxygen, delays in vitro HbS polymerization and prevents sickling of RBCs. Moreover, in a murine model of SCD, GBT440 extends the half‐life of RBCs, reduces reticulocyte counts and prevents ex vivo RBC sickling. Importantly, oral dosing of GBT440 in animals demonstrates suitability for once daily dosing in humans and a highly selective partitioning into RBCs, which is a key therapeutic safety attribute. Thus, GBT440 has the potential for clinical use as a disease‐modifying agent in sickle cell patients.     

GBT021601 improves red blood cell health and the pathophysiology of sickle cell disease in a murine model

The pathophysiologic mechanism of sickle cell disease (SCD) involves polymerization of deoxygenated haemoglobin S (HbS), leading to red blood cell (RBC) sickling, decreased RBC deformability, microvascular obstruction, haemolysis, anaemia and downstream clinical complications. Pharmacological increase in the concentration of oxygenated HbS in RBCs has been shown to be a novel approach to inhibit HbS polymerization and reduce RBC sickling and haemolysis. We report that GBT021601, a small molecule that increases HbS-oxygen affinity, inhibits HbS polymerization and prevents RBC sickling in blood from patients with SCD. Moreover, in a murine model of SCD (SS mice), GBT021601 reduces RBC sickling, improves RBC deformability, prolongs RBC half-life and restores haemoglobin levels to the normal range, while improving oxygen delivery and increasing tolerance to severe hypoxia. Notably, oral dosing of GBT021601 in animals results in higher levels of Hb occupancy than voxelotor and suggests the feasibility of once-daily dosing in humans. In summary, GBT021601 improves RBC health and normalizes haemoglobin in SS mice, suggesting that it may be useful for the treatment of SCD. These data are being used as a foundation for clinical research and development of GBT021601.     

Oxidation-induced changes in microrheologic properties of the red blood cell membrane

It has been hypothesized that some of the irreversible microrheologic abnormalities of sickle red blood cell (RBC) membranes could result from autoxidative perturbation. To model this possibility, we used micromechanical manipulation to examine the static extensional rigidity and inelastic or plastic behavior of normal RBCs exposed to phenazine methosulfate (PMS), an agent that generates superoxide from within the cell. In response to this stress, RBC membranes became stiff as evidenced by increasing extensional rigidity. At 50 mumol/L PMS they were as stiff as the membranes of most dense, dehydrated sickle RBCs; and at 25 mumols/L PMS the membranes were similar to somewhat less dense sickle RBCs. When examined for inelastic behavior, RBCs exposed to PMS even at 10 mumols/L showed hysteresis in loading and unloading phases of the curve relating aspiration length to suction pressure, and they developed membrane bumps that persisted after RBC release from the pipette. Examination of single cells in both isotonic and hypotonic buffers showed that the effect of PMS on RBC microheology is not mediated by cellular dehydration. Independent confirmation of the membrane stiffening effect of PMS was obtained by ektacytometric analysis of resealed RBC ghosts, with sickle-like increases in membrane rigidity observed between 50 and 100 mumol/L PMS. The rigidity of these ghosts was partially ameliorated by exposure to a thiol reductant. In terms of biochemical abnormalities, treated RBCs became significantly different from control RBCs at 25 mumol/L PMS, at which point they just began to enter the sickle range for amounts of membrane thiol oxidation and membrane-associated heme. The sickle average was achieved at 50 mumol/L PMS (for thiol oxidation) to 100 mumol/L PMS (for membrane heme). Thus, micromolar concentrations of PMS induce abnormalities of membrane microrheology that closely mimic those of unmanipulated sickle RBCs while reproducing similar degrees of oxidative biochemical change. We conclude that membrane protein oxidation could explain existence of an irreversible component to the abnormal rheology of the sickle membrane.     

Comparison of mechanisms of anemia in mice with sickle cell disease and beta-thalassemia: peripheral destruction,ineffective erythropoiesis,and phospholipid scramblase-mediated phosphatidylserine exposure

OBJECTIVES: 1). To study the mechanisms of anemia, erythroid hyperplasia, and red blood cell (RBC) clearance in murine models of sickle cell disease (Sickle) and beta-thalassemia (Th1/Th1); 2) To determine the contribution of the phospholipid scramblase enzyme to phosphatidylserine (PS) exposure and RBC death in Sickle and Th1/Th1 mice. METHODS: We used a combination of flow-cytometric analysis and assays for phospholipid remodeling to determine the extent and sites of erythroid hyperplasia, PS exposure, and cell death. RESULTS: 1) Sickle RBCs have a much shorter half-life than Th1/Th1 RBCs (0.8 days vs. 11 days). A significant proportion of Th1/Th1 peripheral reticulocytes mature into erythrocytes, however, approximately fivefold fewer Sickle reticulocytes mature. While erythroid hyperplasia exists in both Sickle and Th1/Th1 mice, Th1/Th1 produce fourfold more RBCs than necessary to maintain steady state, while Sickle produce no excess RBCs. 2) 61% of Sickle and 34% of Th1/Th1 RBCs are scramblase(+) as measured by internalization assays of the fluorescent phospholipid NBD-PC. The majority of NBD-PC(+) RBCs are also annexin-V(+), supporting a mechanistic link between scramblase activity and PS exposure. A proportion of both reticulocytes and older RBCs in Sickle and Th1/Th1 mice have active scramblase, and the degree of scramblase activation in these strains correlates with the propensity for RBC death. CONCLUSIONS: Sickle and Th1/Th1 mice are both anemic, with significant erythroid hyperplasia. Th1/Th1 mice display ineffective erythropoiesis while Sickle mice show rapid peripheral destruction of RBCs. PS exposure and phospholipid scramblase activity serve as markers of RBCs with altered phospholipid asymmetry and greater propensity for cell death.     

The unique red cell heterogeneity of SC disease: crystal formation, dense reticulocytes, and unusual morphology

Knowledge concerning SS (homozygous for the beta s gene) red blood cell (RBC) heterogeneity has been useful for understanding the pathophysiology of sickle cell anemia. No equivalent information exists for RBCs of the compound heterozygote for the beta s and beta c genes (SC) RBCs. These RBCs are known to be denser than most cells in normal blood and even most cells in SS blood (Fabry et al, J Clin Invest 70:1284, 1981). We have analyzed the characteristics of SC RBC heterogeneity and find that: (1) SC cells exhibit unusual morphologic features, particularly the tendency for membrane "folding" (multifolded, unifolded, and triangular shapes are all common); (2) SC RBCs containing crystals and some containing round hemoglobin (Hb) aggregates (billiard-ball cells) are detectable in circulating SC blood; (3) in contrast to normal reticulocytes, which are found mainly in a low-density RBC fraction, SC reticulocytes are found in the densest SC RBC fraction; and (4) both deoxygenation and replacement of extracellular Cl- by NO3- (both inhibitors of K:Cl cotransport) led to moderate depopulation of the dense fraction and a dramatic shift of the reticulocytes to lower density fractions. We conclude that the RBC heterogeneity of SC disease is very different from that of SS disease. The major contributions of properties introduced by HbC are "folded" RBCs, intracellular crystal formation in circulating SC cells, and apparently a very active K:Cl cotransporter that leads to unusually dense reticulocytes.     

Phosphatidylserine externalization in sickle red blood cells: associations with cell age,density, and hemoglobin F

Phosphatidylserine (PS) is normally confined to the cytoplasmic leaflet of the red blood cell (RBC) membrane, but some sickle RBCs expose PS in the outer leaflet (PS+ cells). This study examined the relationships among PS externalization, fetal hemoglobin content, hydration state, and cell age. Sickle RBCs exhibit a wide range of PS externalization. Those with low-level exposure (type 1 PS+) include many young transferrin-receptor-positive (TfR+) cells. This is not specific for sickle cell disease because many nonsickle TfR+ cells are also PS+. RBCs with higher PS exposure (type 2 PS+) appear to be more specific for sickle cell disease. Their formation is most likely sickling dependent because type 2 PS+ dense sickle cells have a lower percentage of fetal hemoglobin (HbF) than PS- cells in the same density fraction (1.7 vs 2.9; n = 8; P <.01). In vivo experiments using biotin-labeled sickle cells showed a sharp decrease in the percentage of circulating, labeled PS+ cells in the first 24 hours after reinfusion. This decrease was confined to type 1 PS+ cells and was thus consistent with the reversal of PS exposure in very young cells. As the labeled cells aged in the circulation, the percentages of type 1 and type 2 PS+ cells increased. These studies indicate that PS externalization in sickle cells may be low level, as observed in many immature cells, or high level, which is associated with dehydration and appears to be more specific for sickle RBCs.     

Increased adhesion of erythrocytes to components of the extracellular matrix: isolation and characterization of a red blood cell lipid that binds thrombospondin and laminin

Red blood cell (RBC) adhesion to the vascular endothelium is increased in several pathologic conditions, including sickle cell disease and malaria. However, RBC interactions with components of the subendothelial matrix are not well-characterized. Under in vitro flow conditions of 1 dyne/cm2, washed RBCs bound to the purified adhesive molecules thrombospondin (TSP) and laminin. Sickle RBCs had the greatest adhesion of all tested RBCs. The adhesion of sickle RBCs to immobilized TSP was inhibited by the anionic polysaccharides high molecular weight (MW) dextran sulfate and chondroitin sulfate A, but not other anionic polysaccharides of similar structure and/or charge density. These data were consistent with the RBC adhesive molecule being a sulfated glycolipid. Therefore, TSP-binding lipids from normal and sickle RBCs were isolated and characterized. The TSP-binding lipid was purified by alkaline methanolysis, anion exchange chromatography and preparative thin layer chromatography (TLC). A homogeneous band on TLC was identified using a specific overlay TSP-binding assay. TSP binding to the purified lipid was stable to bass and neuraminidase treatment, labile to acid treatment, and was inhibited by high MW dextran sulfate, similar to that seen with intact RBCs binding to immobilized TSP under conditions of flow. In addition, soluble laminin bound to the purified RBC lipid. This acidic TSP- and laminin-binding lipid(s) isolated from both sickle and normal RBC membranes may contribute to erythrocyte interactions with the subendothelial matrix, hereby participating in the pathogenesis of vaso-occlusive diseases.     

Senescence of canine biotinylated erythrocytes: increased autologous immunoglobulin binding occurs on erythrocytes aged in vivo for 104 to 110 days

We have evaluated senescence related changes in canine red blood cells (RBCs) using the biotinylation system, where RBCs are labeled in vivo with biotin at the beginning of their life span, and retrieved from circulation on immobilized avidin at the end of their life span. This approach avoids the controversial use of density gradient centrifugation to collect presumably old RBCs. Furthermore, the dog is an appropriate model for human RBC senescence because it has a low degree of random RBC loss and a similarly long RBC life span (approximately 110 days). Two dogs had 97% to 100% of their circulating RBCs biotinylated by infusion of N-hydroxysuccinimido biotin (Clontech, Palo Alto, CA; Calbiochem, La Jolla, CA) dissolved in dimethyl sulfoxide. At postbiotinylation days 104 and 107 for one dog and day 110 for the other dog, biotinylated RBCs were isolated by magnetic cell sorting and analyzed for the presence of autologous IgG using 125I- labeled sheep-antidog IgG (SAD IgG). On all 3 days, there were at least three times more SAD IgG molecules per RBC on senescent biotinylated RBCs than on control (unfractionated) RBCs (day 104: 11,677 v 3,399; day 107: 6,710 v 2,115; day 110: 6,042 v 1,838 molecules of SAD IgG per senescent v control RBC). Furthermore, it is unlikely that an immune response to the conjugated biotin had been elicited, because fresh in vitro biotinylated RBCs that were incubated in autologous plasma (taken after exposure to circulating biotinylated RBCs for 113 days) and then exposed to the SAD IgG showed no increase in antibody binding over control (non-biotinylated) RBCs (1,431 v 1,378 cpm/10(8) biotinylated v control RBCs; P > .20). These results suggest that senescence of canine biotinylated RBCs is characterized by binding of autologous IgG and that antibiotin antibodies do not contribute to this process.     

The effect of cell hydration on the deformability of normal and sickle erythrocytes

The deformability of the erythrocyte (RBC) is greatly influenced by its state of hydration. The purpose of this investigation is to quantitate this relationship by measuring the deformability of an RBC population over a broad range of cell water content. By manipulation of the ion content of the RBC, we performed all of the experiments in media which were isotonic with plasma. To raise ion and water content, RBC were incubated in a Li₂CO₃ medium. To lower cell ion and water content, RBC were exposed to the K ionophore, valinomycin. The range of cell water content achieved during the entire experiment was 900–3200 g/kg cell solid (normal in vivo cell water content being 1800–1950 g/kg cell solid). By using the Ektacytometer, an automated cylindrical viscometer, we were able to measure deformability of the RBC sampled at various points along this range of cell water content. We found that optimal rheologic behavior was exhibited by normal RBC when their water content was in the normal range. A rise or a fall in cell hydration resulted in a decrease in cell deformability. By contrast, the deformability of freshly drawn, well-oxygenated sickle RBC was well below that found for normal RBC. Upon volume expansion, however, the deformability of these sickle RBC improved markedly. This observation suggests that sickle RBC are suboptimally hydrated and that their abnormal rheology is at least in part a consequence of cell dehydration.