SS Erythrocytes Research Articles

Evaluation the in vitro Anti-Sickle Cell Potential of Two Medicinal Plant Species, Rhynchospora corymbosa and Olax subscorpioïdea on the Inhibition of Falciformation of SS Genotype Erythrocytes

Aims: Sickle cell disease is one of the world's leading genetic disorders. It is characterized by the presence of an abnormal hemoglobin HbS in the blood. The anomaly affects the primary structure of hemoglobin, through the substitution of valine for glutamic acid. In traditional medicine, two plant species, Rhynchospora corymbosa and Olax subscorpioïdea, are used to treat this condition.
 Place and Duration of Study: Agro-valuation Laboratory, Hematology Clinic laboratory of Cocody Hospital University (CHU), between June-Aout 2021 and June-July 2022.
 Methodology: Following a triphytochemical test, the effect of each species on the in vitro inhibition of SS erythrocyte falciformation was evaluated.
 Results: Thus an abundance of alkaloids, tannins, saponins, polyterpenes and sterols were found in the families of molecules identified in Rhynchospora corymbosa. As for the decocted Olax subscorpioïdea, in addition to the families of molecules identified in Rhynchospora corymbosa the polyphenols, leuco-anthocyanins and flavonoids were found. In addition, at an equal concentration of 0.312 mg/mL, decoctions of Rhynchospora corymbosa and Olax subscorpioïdea gave an inhibition rates of 80 and 84 % respectively. Both extracts showed activity on in vitro falciformation inhibition. This activity is thought to be due to the presence of chemical groups with anti-oxidant and anti-inflammatory properties.
 Conclusion: The use of Rhynchospora corymbosa and Olax subscorpioïdea in the treatment of sickle cell disease in Côte d'Ivoire would be justified.

Effects of Flavonoid Quercetin on Thrombo-Inflammatory Processes in Patients with Sickle Cell Disease

Introduction:Altered erythrocyte physiology in Sickle Cell Disease (SCD) results in severe vascular complications, including vaso-occlusion (VOC) along with various other manifestations, such as thrombosis and end organ damage. Moreover, platelet activation endothelial dysfunction and fibrin formation, are critical processes implicated in thrombo-inflammatory vasculopathy in SCD. Since flavonoids intake is associated with lowered adverse cardiovascular disease outcomes and cancer associated thrombosis, we are evaluating the effects of the flavonoid Isoquercetin (IQ) on thrombo-inflammatory biomarkers in patients with SCD (NCT:04514510). Here we report the mechanistic effects of Quercetin (Que), an aglycone that differs from IQ by the absence of 3-linked glucoside moiety, on markers of erythrocyte and platelet activation in patients with SCD.Methods:We studied patients with SCD (Genotype SS, mean age: 39 yrs, N=8) and ethnic matched controls (Genotype AA, mean age: 42 yrs, N=8). Erythrocyte reactive oxygen species (ROS) was measured in washed red cells by flow cytometry using the fluorescent probe 2‘, 7‘-dichlorofluorescein diacetate (DCFDA) in the presence or absence of Quercetin (Que) (100 µM). Effects of in vitro erythrocyte ROS induction by hydrogen peroxide (H 2O 2) and ROS inhibition by N-acetyl-L-cysteine (NAC) were evaluated. Baseline and agonist-induced (ADP 20 µM) platelet P-selectin expression in vitro in the presence or absence of Que (100 µM) was assessed by flow cytometry.Results:At baseline, SS erythrocytes demonstrated elevated levels of ROS reflected by greater florescent intensity with DCFDA when compared with erythrocytes from ethnic matched controls (SS mean= 1884, SD: 508.8 vs. AA mean: 1604, SD: 554.5; p value= 0.3798). Stimulation of AA erythrocytes in vitro with H 2O 2 led to accumulation of ROS that approached levels similar to those exhibited by SS erythrocytes at baseline (AA mean: 1890, SD: 748.3). Pre-treatment of SS and AA erythrocytes in vitro with the antioxidant NAC exhibited minimal effects on ROS in SS erythrocytes (SS baseline+NAC mean: 1942, SD: 526.8, p value 0.4609 and SS H 2O 2+NAC mean: 1889, SD: 437.4, p value 0.5469) but lowered ROS levels in both baseline and H 2O 2 treated AA erythrocytes (AA baseline+NAC mean: 1442, SD: 453.9, p value 0.1563 and AA H 2O 2+NAC mean: 1581, SD: 518.7, p value 0.078). Pre-treatment of SS and AA erythrocytes in vitro with Que substantially lowered erythrocyte ROS in patients and controls at the baseline (SS baseline ROS with Que mean: 1582, SD: 542.0, p value 0.0078; AA baseline ROS with Que mean: 1341, SD: 490.9, p value 0.0313; Fig. 1A), and after H 2O 2 stimulation (SS H 2O 2 induced ROS+Que mean: 1677, SD: 572.5, p value 0.0156; AA H 2O 2 induced ROS+Que mean: 1417, SD: 512, p value 0.0078). Pre-treatment of erythrocytes with a combination of Que and NAC did not reveal synergistic anti-oxidant activity (SS NAC+Que mean: 1616, SD: 464.8; AA NAC+Que mean: 1333, SD: 524.1).We simultaneously investigated the effects of Que on platelet P-selectin expression in the same subjects. As expected, baseline platelet surface P-selectin expression was higher in SS patients compared with controls (SS N=5, mean: 1.48%, Max: 4.9%, Min: 0.03%; AA N=4, mean: 0.89%, Max: 2.3%, Min: 0%; p value 0.55). Stimulation of platelet rich plasma (PRP) with ADP increased surface P-selectin expression in comparison with baseline, in both patients and controls (SS N=5, mean: 18.04%, Max: 40.4%, Min: 5.8%, p value 0.0047; AA N=4, mean: 20.60%, Max: 43.8%, Min: 5.4%; p value 0.0140). Pre-treatment of PRP with Que prior to ADP stimulation revealed a trend toward decreased platelet surface P-selectin expression in both patients and controls (SS N=5, mean: 10.49%, Max: 24.8%, Min: 3.1%; AA N=4, mean: 12.09%, Max: 21.9%, Min: 3.4%; Fig 1B) although this did not return to baseline.Conclusion:The flavonoid Quercetin appears to have a beneficial effect in lowering erythrocyte ROS and platelet surface P-selectin and warrant further evaluation as a strategy to ameliorate thrombo-inflammatory pathophysiology in sickle hemoglobinopathies. [Display omitted] DisclosuresNo relevant conflicts of interest to declare.

Mutations in the Hemoglobin Binding Domain of Band 3 Perturb Cellular Metabolism and Alter the Sickling of SS Erythrocytes

▪Under oxygenated conditions, 4 glycolytic enzymes that perform the terminal steps of glycolysis (phospho-fructoKinase [PFK], lactate dehydrogenase [LDH], aldolase [ALD] and glygeraldehyde 3 phosphate dehydrogenase [GAPDH]) bind to the cytoplasmic domain of band 3. Under deoxy conditions deoxy hemoglobin (Hb) is bound to band 3 and PFK, LDH, ALD and GAPDH are displaced (Campanella et al. PNAS 102, 2005; Blood 112, 2008). We generated transgenic mice in which the sequence encoding the first 35 amino acids of the wild type human band 3 cytoplasmic domain replaced the endogenous mouse band 3 sequences in the Slc4a1 gene, a mutant line in which human amino acids 12-21 were deleted removing the deoxy Hb binding site (-Hb) and a third line in which amino acids 1-11 were deleted creating a high affinity binding site for deoxyHb (++Hb). Erythrocytes from the mutant lines were insensitive to Oxygen concentration resulting in changes in oxygen dependent deformability and other physical properties compared to the wild type line (Chu et al. Blood 128, 2016, Zheng et al. JBC 294, 2019, Zhou et al. Sci. Adv. 5, 2019).We crossed our humanized band 3 mouse strains to the Townes Sickle Cell Disease (SCD) mouse model, maintaining both the human βA and βS alleles to generate human AA, AS and SS mice homozygous for each of the human band 3 cytoplasmic domain sequences. Using an assay in which SS red cells in phosphate buffer are deoxygenated to 6% oxygen over time (Dunkelberger et al., J. Phys. Chem. B 122, 2018), we observed that -Hb band 3/SS mice showed an accelerated rate of sickle cell formation and a higher percent of sickled cells than wild type band 3/SS mice (p<0.01). Conversely, ++Hb band 3/SS mice showed an inhibition of both the rate of sickling and the precent of sickled cells compared to wild type band 3/SS mice (p<0.05).We hypothesized that the inability of the glycolytic enzymes to reversibly bind to band 3 in the mutant mice were responsible for the differences in sickling. To test this hypothesis, we analyzed a panel of 28 cellular metabolites in 12 mice (6 female, 6 male) of each genotype: wild type band 3/AA, -AS and -SS, -Hb band 3/AA, -AS, -SS and ++Hb/AA, -AS, -SS. The metabolites were quantified by LC-MS/MS using an API 4500 triple quadrupole mass spectrometer (AB Sciex), with chromatographic resolution enabled on a polymeric amino column (apHera by Supelco) under alkaline mobile phase conditions (pH ~9.3). Stable isotope dilution and 8pt calibration curves allowed the absolute quantification of each metabolite.Consistent with the constitutive binding of the terminal glycolytic enzymes to band 3 in -Hb erythrocytes, glycolysis was inhibited after the phosphoenol pyruvate step, as evidenced by significant accumulation of the intermediates at top of the glycolysis pathway, including fructose 1,6 biphosphate (FBP; p<0.01), dihydroxyacetone phosphate/ glyceraldehyde-3-phosphate (G3P; p<0.01), and 3-phosphoglycerate/2-phosphoglycerate (PG; p<0.01). In the ++Hb mutant where the terminal glycolytic enzymes are constitutively displaced from band 3, significantly lower levels of FBP, G3P and PG were observed (p<0.01). The levels of these metabolites in wild type band 3/SS erythrocytes were intermediate between the two mutant strains.We hypothesized that the accumulation of FBP, G3P and PG contributed to the increased rate of sickling in the -Hb band 3/SS mice. To test this, we incubated wild type band 3/SS cells with either FBP or PG. Both intermediates increased the rate of sickle cell formation and percentage of sickled cells in a dose dependent fashion with no alteration in any RBC indices including MCV and osmotic fragility. We next hypothesized that reduction of the levels of glycolytic intermediates would have an antisickling effect. To test this, we incubated wild type band 3/SS cells with 2,3 diphosphoglycerol (DPG), which is a potent inhibitor of glycolysis. We found that DPG treatment led to a dose dependent decrease in the rate of sickle cell formation and percentage of sickled cells, again with no alteration in any RBC indices including MCV and osmotic fragility.We conclude that the accumulation of glycolytic intermediates leads to increased sickle cell formation. We propose that reduction in the levels of glycolytic intermediates either by accelerating the terminal stages of glycolysis or by redirection to the pentose phosphate pathway may offer a means to treat SCD. DisclosuresNo relevant conflicts of interest to declare.

Antisickling effect of chrysin is associated with modulation of oxygenated and deoxygenated haemoglobin via alteration of functional chemistry and metabolic pathways of human sickle erythrocytes.

Sickle cell disease (SCD) treatment and management remain a challenging puzzle especially among developing Nations. Chrysin's sickling-suppressive properties in human sickle (SS) erythrocytes in addition to its effect on AA-genotype erythrocytes were evaluated. Sickling was induced (76%) with 2% sodium metabisulphite at 3 h. Chrysin prevented (81.19%) the sickling and reversed same (84.63%) with strong IC50s (0.0257 µM and 0.00275 µM, respectively). The levels of oxygenated haemoglobin in the two groups (before and after induction approaches) were similar but significantly (P < 0.05) higher than that of SS erythrocytes (the 'induced' control), with chrysin-treated AA-genotype showing no effects relative to the untreated. The level of deoxygenated haemoglobin in the 'induced' control group was significantly (P < 0.05) higher than those of the chrysin-treated SS erythrocytes. Normal and chrysin-untreated erythrocytes (AA-untreated) were significantly more resistant to osmotic fragility than the SS-untreated. However, treatment with chrysin significantly reduced the osmotic fragility of the cells relative to the untreated cells. Furthermore, chrysin treatment significantly lowers the high level of 2,3-diphosphoglycerate (2,3-DPG) observed in the sickle erythrocytes, with no effects on AA-genotype erythrocytes. Based on functional chemistry, chrysin treatment alters the functional groups in favour of its antisickling effects judging from the observed bends and shifts. From metabolomics analysis, it was observed that chrysin treatment favors fatty acid alkyl monoesters (FAMEs) production with concomitant shutting down-effects on selenocompound metabolism. Thus, sickling-suppressive effects of chrysin could potentially be associated with modulation of oxygenated and deoxygenated haemoglobin via alteration of human sickle erythrocyte's functional chemistry and metabolic pathways implicated in SCD crisis.

Decreased erythrocyte binding of Siglec-9 increases neutrophil activation in sickle cell disease

Oxidative stress and inflammation promote vaso-occlusion in sickle cell disease (SCD). CD33-related Sialic acid-binding immunoglobulin-type lectins (CD33rSiglecs) are cell surface proteins that recognize sialic acids inhibit innate immune cell functions. We have shown that Siglec-9 on human neutrophils interact with erythrocyte sialic acids (prominently glycophorin-A (GYPA) to suppress neutrophil reactive oxygen species (ROS). We hypothesized that altered sickle erythrocyte membrane sialic acid leads to decreased Siglec-9 binding capability, and thus a decreased neutrophil oxidative burst. SS erythrocytes express significantly more sialic acid than AA erythrocytes (p = 0.02). SS erythrocytes displayed significantly less Siglec-9-Fc binding 39% ± 11 (mean ± SEM) compared to AA erythrocytes 78% ± 5 (p = 0.009). Treatment of AA erythrocytes with sialidase to remove sialic acid decreased binding to 3% ± 7.9 (p ≤ 0.001). When freshly isolated neutrophils were incubated with AA erythrocytes, neutrophils achieved 16% ± 6 of the oxidative burst exhibited by a stimulated neutrophil without erythrocytes. In contrast, neutrophils incubated with SS erythrocytes achieved 47% ± 6 of the oxidative burst (AA versus SS, p = 0.03). Stimulated neutrophils incubated with AA erythrocytes showed minimal NET formation while with SS erythrocytes NETs increased. SS erythrocytes are deficient in binding to neutrophil Siglec-9 which may contribute to the increased oxidative stress in SCD.

Determination of the potentials of aqueous and ethanolic extracts of Buchholzia coriacea in preventing sodium metabisulphite-induced oxidation of some hemoglobin SS erythrocyte membrane proteins

BACKGROUND: The sustenance of the erythrocyte membrane integrity is crucial to ensuring a continuous lamina flow of erythrocytes through narrow blood vessels. So it is imperative to find a substance to attenuate the oxidation of erythrocytes that cause the membrane structure remodelling. OBJECTIVES: To investigate the hydrating strength and modulatory effect of Buchholzia coriacea seed extracts on erythrocyte membrane proteins degradation. MATERIALS AND METHODS: Aqueous and ethanolic extracts of B. coriacea were obtained following standard procedures. Blood samples (5.00 mL) from five known hemoglobin SS individuals in steady state were subjected to electrophoresis to establish the genotype of the individuals. The osmotic fragility assay was performed with 2.50, 5.00, 10.00 and 20.00 mg/mL of the extracts in the presence of different concentrations of phosphate-buffered saline. The graph of percentage hemolysis was plotted against saline concentration, and the mean corpuscular fragility (MCF) determined. Sodium dodecyl sulfate polyacrylamide gel electrophoresis was also done using the same concentrations of extracts. RESULTS: There was no statistically significant difference (P > 0.01) between the average MCF in all the extract concentration and the control. Protein 4.1 and Ankyrin did not appear on the electrophoretic lanes of all the test samples when compared with the control. However, spectrin and ankyrin bands were visible with regular size and intensity. CONCLUSION: B. coriacea seed extracts possess antihemolytic character. However, it does not have the ability to maintain the erythrocyte membrane model despite its acclaimed antioxidant character.

Decreased Erythrocyte Binding Capability for Neutrophil Siglec-9 Is a Source of Oxidative Stress in Sickle Cell Disease

IntroductionOxidative stress and inflammation promote hemolysis and vaso-occlusion in sickle cell disease (SCD). Erythrocytes can play a pro-oxidative and anti-oxidative role in disease-associated inflammation through iron-driven free radical production and endogenous anti-oxidants. In SCD, HbS accelerated auto-oxidation, iron de-compartmentalization, and inflammatory cell-derived oxidants drive this oxidative stress. CD33-related Sialic acid-binding immunoglobulin-type lectins (CD33rSiglecs) are cell surface proteins that recognize sialic acids in “Self-Associated Molecular Patterns” (SAMPs) and typically inhibit innate immune cell functions via cytosolic signaling. Recent studies have shown that Siglec-9 on human neutrophils in circulating blood interact with erythrocyte sialic acids (prominently glycophorin-A (GYPA) to suppress neutrophil reactivity, including reactive oxygen species (ROS) production. Modification of erythrocyte membrane sialic acids interferes with their ability to inhibit neutrophil activation and oxidative burst. As erythrocytes age and undergo cellular damage there is a loss of membrane bound sialoglycoproteins. Several studies have indicated that this reduction in the sialome is accelerated on the sickle erythrocyte. We hypothesize that altered sickle erythrocyte membrane sialic acid leads to decreased Siglec-9 binding capability, and that decreased binding of neutrophil Siglec-9 to sickle erythrocyte sialic acid enhances neutrophil activation and oxidative burst.Methods & ResultsBinding of recombinant Siglec-9-Fc protein to AA (n=9) and SS erythrocytes (n=7) was measured using flow cytometry. SS erythrocytes displayed significantly less Siglec-9-Fc binding 45% ± 11.9 (mean ± SEM) compared to AA erythrocytes 82% ± 5.4 (p=.03). Treatment of AA erythrocytes with neuraminidase to remove sialic acid decreased binding to 4% ± 7.9.Neutrophil ROS production was measured by flow cytometry using dihydrorhodamine 123 (DHR123). Neutrophils were purified from AA donors (n=5) and SS (n=11) or AA erythrocytes (n=5) were added to the neutrophils at a ratio of 50 erythrocytes to 1 neutrophil. Oxidative burst was stimulated using phorbol 12-myristate 13-acetate (PMA). AA erythrocytes decreased PMA-stimulated neutrophil ROS production by 84% ± 6.7. In contrast, SS erythrocytes decreased PMA-stimulated neutrophil ROS production by 53% ± 6.8 (p=0.03,).Recent studies have shown that neutrophil extracellular trap (NET) formation is pathogenic in SCD. We added AA and SS erythrocytes (50:1) to neutrophils stimulated with PMA and NET formation was assessed using Syto Orange, Syto Green and confocal microscopy. PMA-stimulated neutrophils incubated with AA erythrocytes showed minimal NET formation. In contrast, AA erythrocytes treated with neuraminidase to remove sialic acid had increased NET formation. PMA-stimulated neutrophils incubated with SS erythrocytes showed increased NET formation.ConclusionsA constant disease state of oxidative stress and inflammation underlies SCD pathophysiology. These data demonstrate that SS erythrocytes with decreased membrane sialic acid are deficient in binding to neutrophil Siglec-9. The decreased binding of SS erythrocytes to neutrophil Siglec-9 diminishes the ability of SS erythrocytes to properly modulate neutrophil activation, which may contribute to the oxidative stress and increased state of basal inflammation inherent to SCD. The overall reduction in number of RBCs in SS may also be a factor? [Display omitted] DisclosuresBelcher:CSL Behring: Research Funding. Vercellotti:CSL Behring: Research Funding.

Vzhe-039, a Novel Structurally-Enhanced Allosteric Hemoglobin Effector Inhibits Sickling of SS Erythrocytes In Vitro, and Exhibits Improved Pharmacologic Properties In Vivo

Allosteric effectors of hemoglobin (AEH) remain promising as a viable therapeutic approach for the management of sickle cell disease (SCD). AEH bind to hemoglobin (Hb) in a transiently covalently manner, increasing Hb affinity for oxygen (O2), with concomitant inhibition of polymerization of deoxygenated sickle Hb (HbS) and erythrocyte sickling. As part of our ongoing efforts to surmount well-known AEH druggability challenges, we have designed and synthesized VZHE-039, a novel substituted benzaldehyde with potent in-vitro anti-polymerization activity, as well as more favorable in-vivopharmacokinetic and pharmacodynamic (PK/PD) properties. Here, we report the results of our early findings.The anti-sickling properties of VZHE-039 were tested in-vitro by incubating 0.5, 1, and 2 mM concentrations with blood suspensions from a subject with homozygous SCD (hematocrit: 20%) under hypoxic conditions (4% O2/96% N2) at 37°C for 2 h. At conclusion, aliquots were drawn into a fixative (2% glutaraldehyde solution), and sickling was assessed by microscopy. Aliquot samples were also subjected to cation-exchange HPLC analyses to measure the degree of Hb modification (Hb adduct formation), as well as standard O2 equilibrium curves (OEC) to assess p50 shifts. Subsequently, we conducted in-vivo PK/PD studies in wild-type mice that received single doses of VZHE-039 via the intravenous (I.V.: 25 and 50 mg/kg), intraperitoneal (I.P.: 100-150 mg/kg) and oral (P.O.: 100-200 mg/kg) routes. Serial blood samples were collected up to 24 h after I.V. and P.O., and up to 6 h after I.P. administration, respectively; aliquots were hemolyzed, de-proteinized and subjected to reverse-phase HPLC-UV assay to quantify VZH-039 blood concentrations. Residual blood samples were also hemolyzed, and clarified lysates were assayed for in-vivoHb adduct formation, and the corresponding change in Hb oxygen affinity (Δp50, %).The results of our in-vitrostudies demonstrated a concentration-dependent inhibition of SS cell sickling: 39±1.4%, 68.6±4.9%, and 89.9±5.2%, at 0.5, 1 and 2 mM of VZHE-039, respectively. HbS was modified correspondingly (46±7.4%, 83.6±6.4%, and 96.4±6.2%), and this adduct formation was correlated linearly with the left shift in OEC (Δp50 values of 34.3±8.7%, 63.6±2.4%, and 76.6±2.5%).Noncompartmental PK analysis showed that, after I.V. administration, systemic PK was dose-independent with a CLtot of 0.5 mL/min/kg, resulting in a terminal half-life of 9 h. After I.P. administration, AUCtrap increased supra-proportionally with dose; the limited sampling schedule suggested an I.P. bioavailability of >20%. After P.O. administration, AUCtrap increased supra-proportionally with dose as well due to an increase in oral bioavailability to ~ 10%, with peak blood concentrations up to ~ 0.25 mM; the terminal half-lives were prolonged (relative to I.V.) to 14 h. The PD effect-time course of change in %Hb adduct paralleled (and was reasonably linearly related to) blood concentrations for all routes. Baseline-corrected peak PD effects after I.V. administration were 15.7% and 31.9%, after 25 and 50 mg/kg respectively; after P.O. administration of 200 mg/kg, the peak change was 17.6%. Overall, VZHE-039 exhibited a low (metabolic) clearance, presumably reflecting high-affinity and sustained Hb (and possibly albumin) binding, leading to the observed extended terminal half-life and duration of action. While its oral bioavailability is low (likely due to limited GI solubility) and there is evidence of saturable first-pass effects and possibly enterohepatic recycling, oral in-vivo exposures were associated with PD effects that were consistent with the results from the in-vitrostudies.In conclusion, the results of our current studies establish VZHE-039 as a novel, potent anti-sickling agent, confirm its proposed mechanism of action in-vitro and in-vivo, and suggest that therapeutically-relevant blood concentrations may be achieved after daily oral doses. Therefore, VZHE-039 is our new lead drug candidate that may require further modifications and studies to (a) improve its GI solubility and oral bioavailability; (b) elucidate atomic level structural interactions and kinetics of its Hb binding; and (c) formally investigate its biological activity after repeat-doses in a SCD mouse model. DisclosuresNo relevant conflicts of interest to declare.

Identification of a small molecule that increases hemoglobin oxygen affinity and reduces SS erythrocyte sickling.

Small molecules that increase the oxygen affinity of human hemoglobin may reduce sickling of red blood cells in patients with sickle cell disease. We screened 38 700 compounds using small molecule microarrays and identified 427 molecules that bind to hemoglobin. We developed a high-throughput assay for evaluating the ability of the 427 small molecules to modulate the oxygen affinity of hemoglobin. We identified a novel allosteric effector of hemoglobin, di(5-(2,3-dihydro-1,4-benzodioxin-2-yl)-4H-1,2,4-triazol-3-yl)disulfide (TD-1). TD-1 induced a greater increase in oxygen affinity of human hemoglobin in solution and in red blood cells than did 5-hydroxymethyl-2-furfural (5-HMF), N-ethylmaleimide (NEM), or diformamidine disulfide. The three-dimensional structure of hemoglobin complexed with TD-1 revealed that monomeric units of TD-1 bound covalently to β-Cys93 and β-Cys112, as well as noncovalently to the central water cavity of the hemoglobin tetramer. The binding of TD-1 to hemoglobin stabilized the relaxed state (R3-state) of hemoglobin. TD-1 increased the oxygen affinity of sickle hemoglobin and inhibited in vitro hypoxia-induced sickling of red blood cells in patients with sickle cell disease without causing hemolysis. Our study indicates that TD-1 represents a novel lead molecule for the treatment of patients with sickle cell disease.

Ginkgo biloba extract (EGb 761) attenuates oxidative stress induction in erythrocytes of sickle cell disease patients

Sickle cell disease promotes hemolytic anemia and occlusion of small blood vessels due to the presence of high concentrations of hemoglobin S, resulting in increased production of reactive oxygen species and decreased antioxidant defense capacity. The aim of this study was to evaluate the protective action of a standardized extract of Ginkgo biloba (EGb 761), selected due to its high content of flavonoids and terpenoids, in erythrocytes of patients with sickle cell anemia (HbSS, SS erythrocytes) subjected to oxidative stress using tert-butylhydroperoxide or 2,2-azobis-(amidinepropane)-dihydrochloride, in vitro. Hemolysis indexes, reduced glutathione, methemoglobin concentrations, lipid peroxidation, and intracellular reactive oxygen species were determined. SS erythrocytes displayed increased rates of oxidation of hemoglobin and membrane lipid peroxidation compared to normal erythrocytes (HbAA, AA erythrocytes), and the concentration of EGb 761 necessary to achieve the same antioxidant effect in SS erythrocytes was at least two times higher than in normal ones, inhibiting the formation of intracellular reactive oxygen species (IC50 of 13.6 µg/mL), partially preventing lipid peroxidation (IC50 of 242.5 µg/mL) and preventing hemolysis (IC50 of 10.5 µg/mL). Thus, EGb 761 has a beneficial effect on the oxidative status of SS erythrocytes. Moreover, EGb 761 failed to prevent oxidation of hemoglobin and reduced glutathione at the concentrations examined.

Sickle cell disease resulting from uniparental disomy in a child who inherited sickle cell trait

AbstractSickle cell disease (SCD) is a classic example of a disorder with recessive Mendelian inheritance, in which each parent contributes one mutant allele to an affected offspring. However, there are exceptions to that rule. We describe here the first reported case of conversion of inherited sickle cell trait to SCD by uniparental disomy (UPD) resulting in mosaicism for SS and AS erythrocytes. A 14-year-old boy presented with splenomegaly and hemolysis. Although his father has sickle cell trait, his mother has no abnormal hemoglobin (Hb). DNA sequencing, performed to rule out Hb S/β-thalassemia, detected homozygous Hb SS. Further studies revealed mosaic UPD of the β-globin locus, more SS erythroid progenitors than AS, but a reverse ratio of erythrocytes resulting from the survival advantage of AS erythrocytes. This report exemplifies non-Mendelian genetics wherein a patient who inherited sickle cell trait has mild SCD resulting from postzygotic mitotic recombination leading to UPD.

Anti-Sickling Effect Of Abrus Seed Spasmolytic Substance

A spasmolytic substance BN, extractable from the seeds of Abrus precatorius Linn Fabaceae has been found to inhibit both metabisulphite- and calcium- induced sickling of human haemoglobin (Hb) SS erythrocytes in vitro. It also inhibited human platelet aggregation elicited by adrenaline (epinephrine) and ADP. In addition, it stabilised Hb SS erythrocyte membrane. The results substantiate the use of the seeds in sickle cell disease. Keywords: Anti-Sickling, Abrus seed, Spasmolytic substance, Induced sickling, HaemoglobinPlant Product Research Journal Vol. 12 2008: pp. 6-9

Oxidative process in erythrocytes of individuals with hemoglobin S

The understanding of the oxidative stress mechanisms helps to explain many of the processes of cellular lesion and death, especially those related to the hemolytic diseases. Sickle cell anemia, thalassemias and G6-PD deficiency are among the more frequent genetic anomalies accompanied by oxidative stress. In the sickle cells, one of the factors that predisposes to the hemolytic process is the oxidative degradation of the hemoglobin S due to its deoxigenation leading to hemichrome formation and precipitation as Heinz bodies. The oxidative stress contributes to the sickle process and shortening of the erythrocyte survival. Here we analyzed the oxidative process in erythrocytes of patients with two different genotypes for HbS (AS and SS). Units of blood from donors of the Center of Hematology and Hemotherapy of Paraná (HEMEPAR), from normal individuals (AA) and from heterozygote individuals (AS), and venous blood collected from patients with sickle cell anemia (SS) were analyzed. In order to evaluate the protective action of the vitamins C and E in oxidative stress, erythrocytes were treated with antioxidant substances, vitamin C and vitamin E, and then treated with the oxidant tert-butilhydroperoxide (TBHP). The oxidative action induced by TBHP was observed in erythrocytes AA<AS<SS, by the increase in the content of Heinz bodies, methemoglobin, hemolysis, GSH depletion and lowering activities of the enzymes G6-PD and GR. The protective actions of the vitamins C and E for the oxidative stress induced by TBHP were observed for the erythrocytes in the lowering Heinz bodies, methemoglobin, hemolysis, and partial recovery of GSH more efficiently in AS and SS erythrocytes. Recovery was not observed in the levels of the activities of the enzymes G6-PD and GR, under the vitamins actions. The results obtained confirmed the higher susceptibility of the sickle erythrocyte to oxidation which necessitates precaution in the transfusion adequacy of AS erythrocytes. On the other hand, the protective effect of the vitamins C and E over the oxidative stress observed in erythrocytes AS and SS open perspectives for their use for treatment of patients with sickle cell anemias, as well as in the preservation of transfusional erythrocyte bag units.

Mixed haematopoietic chimerism for sickle cell disease prevents intravascular haemolysis

Mixed haematopoietic chimerism for sickle cell disease prevents intravascular haemolysis

Hydroxyurea Therapy Reduces the Gene Expression of Adhesion Molecules VLA-4 and CD36 in Sickle Erythrocytes, with a Concomitant Decrease in Adhesion to Fibronectin.

Adhesive interactions between sickle red cells, leukocytes and the endothelium are thought to initiate vaso-oclusive events in the microcirculation. An increased expression of adhesion molecules such as CD 36 (thrombospondin receptor) and VLA-4 integrin (α4β1, CD49d/CD29) has been observed on the surface of sickle reticulocytes and the increased adhesive properties of sickle erythrocytes are well documented. Gene expression of adhesion molecules and their modulation by drugs such as hydroxyurea (HU) have yet to be fully explored. We compared the expression of the CD36 gene and the VLA-4 integrin subunit genes, CD29 and CD49d, in the reticulocytes of steady-state SCD patients and patients on HU therapy (20–30 mg/Kg/day) using Real Time PCR and also compared the basal adhesion of erythrocytes from these subjects using static adhesion assays. Basal adhesion of sickle erythrocytes (2x108 cells/ml) to fibronectin (20mg/ml) - coated plates was significantly higher than normal erythrocyte adhesion (15.4 ± 2.8% compared to 5.9 ± 1.1%, n=9, p=0.008). In contrast, the adhesion of erythrocytes from SS patients on HU therapy to fibronectin was significantly lower than that of SS erythrocytes of patients not on HU (5.8 ± 0.7%, n=9, p=0.006). Pre-treatment of cells, in vitro, with varying concentrations of HU (0.1 → 250μM, 30 minutes, 37oC), had no significant effect on either normal or sickle cell erythrocyte adhesion under our experimental conditions, suggesting that HU may not reduce sickle cell adhesion by altering adhesion molecule function due to affinity changes. We next used real time PCR to investigate the expression of the mRNA of the CD36 gene and the genes for the VLA-4 subunits, CD49d and CD29, in the reticulocytes of steady-state SCD patients and patients on HU therapy. CD 36 gene expression was reduced by 62.5% ± 10.4% in the reticulocytes of patients on HU compared to the reticulocytes of SCD patients not on HU (n=8, p < 0.001). The β1 (CD29) integrin subunit expression was reduced by 87.9 ± 8.7% (n=8, p < 0.001) in patients on HU compared to SS reticulocytes; likewise α4 integrin (CD49d), expression was reduced by 50.0% ± 2.6% (n=8, p <0.001). In addition, reticulocyte mRNA was collected from one steady-state SCD patient before and 3 months after initiation of HU therapy and decreases of 99.5%, 98.0% and 98.0% were observed in the expressions of CD36, CD29 and CD49d mRNA, respectively, in the reticulocytes of this patient following HU therapy. We speculate that the decrease in adhesive properties observed in the erythrocytes of sickle cell patients during HU therapy may be the consequence of a down-regulation of the gene expressions of adhesion molecules and, consequently, protein expression by sickle cells rather than as a result of alterations in adhesion molecule function.

Spectrin's E2/E3 ubiquitin conjugating/ligating activity is diminished in sickle cells

Erythrocyte spectrin contains E2/E3 ubiquitin conjugating/ligating activity in its alpha subunit. Ankyrin is a target of spectrin's E2/E3 ubiquitin conjugating/ligating activity in vitro and in vivo. We compare the ubiquitination levels of ankyrin mediated by control and sickle cell spectrin using a biotinylated ubiquitin cell-free assay. Sickle cell spectrin has diminished ability to transfer ubiquitin from an intermediate spectrin-ubiquitin thioester adduct (alpha' spectrin) to ankyrin, which may be due to glutathiolation of spectrin's E2 and/or E3 active site cysteines. There is also a diminished ability of the sickle cell ankyrin to serve as target of spectrin's E2/E3 activity, probably due to oxidative damage to ankyrin. A direct correlation exists between the alpha'/alpha spectrin ratio and spectrin's ability to ubiquitinate ankyrin. There is also an inverse correlation between severity of the disease and the alpha'/alpha spectrin ratio in SS erythrocytes. These results suggest that reduced spectrin E2/E3 activity is an important determinant of sickle cell severity.

Vasoactive factors in sickle cell disease: in vitro evidence for endothelin-1-mediated vasoconstriction.

While systemic plasma endothelin-1 (ET-1) levels are increased during acute crisis in sickle cell disease, the relative levels of potent vasoactive factors that contribute to the regulation of vascular function, such as ET-1, NO, and cell-free hemoglobin, during the course of periodic vaso-occlusive episodes remain unclear. Moreover, whether and to what extent sickling-induced release of ET-1 alters vascular tone is not completely understood. To investigate the sequential changes in circulating vasoactive factors, we measured plasma ET-1, NO metabolites (NOx), and cell-free hemoglobin (Hb) before (steady-state), during (crisis), and after a vaso-occlusive (post-crisis) episode. Steady-state ET-1 levels (fmol/mL) increased from 2.3 +/- 0.4 to 11.0 +/- 1.4 and 4.2 +/- 1.0 during crisis and post-crisis periods, respectively. There was no significant difference in plasma NOx levels. Cell-free Hb levels were significantly higher in sickle cell patients in all phases as compared to the control group, and especially during crisis cell-free Hb levels were elevated by 4-fold (209,000 +/- 31,000 vs. 46,000 +/- 5,300 ng/mL in steady-state). Conditioned medium from human pulmonary artery endothelial cells exposed to sickled erythrocytes prepared by deoxygenation induced contraction of aortic rings, and this effect was blocked by an ET(A) receptor antagonist. These findings indicate that ET-1 is the predominant contractile factor released by cultured endothelial cells upon exposure to deoxygenated sickled SS erythrocytes and ET-1-NO-NO scavenger balance is altered in favor of vasoconstriction during an acute episode in SCD.

Fluorescence measurements of the labile iron pool of sickle erythrocytes

AbstractSickle erythrocytes have increased ferritin and increased molecular iron on the inner membrane leaflet, and we postulated that cytosolic labile iron is also elevated. We used the fluorescent metal-losensor, calcein, and a permeant Fe2+ chelator to estimate labile cytoslic Fe2+, and calcein plus an Fe3+ chelator to estimate total cytosolic labile iron (Fe2+ + Fe3+). We measured membrane nonheme iron by its reactivity with ferrozine. As estimated by calcein and Fe2+ chelator, the mean ± SD labile Fe2+ concentration was significantly lower in hemoglobin (Hb) SS (n = 29) than hemoglobin AA (n = 17) erythrocytes (0.56 ± 0.35 μM versus 1.25 ± 0.65 μM; P < .001). In contrast, as estimated by calcein and Fe3+ chelator, total erythrocyte labile iron was similar in hemoglobin SS (n = 12) and hemoglobin AA (n = 10) participants (1.75 ± 0.41 μM versus 2.14 ± 0.93 μM; P = .2). Mean membrane nonheme iron levels were higher in hemoglobin SS cells than hemoglobin AA cells (0.0016 × 10-4 versus 0.0004 × 10-4 fmol/cell; P = .01), but much lower than the mean amounts of total labile iron (1.6-1.8 × 10-4 fmol/cell) or hemoglobin iron (18 000-19 000 × 10-4 fmol/cell). Both membrane iron and total labile iron were much less than the mean amount of iron potentially present in erythrocyte ferritin as calculated from results of other investigators (15 × 10-4 versus 34 × 10-4 fmol/cell in HbAA versus HbSS erythrocytes). We conclude that cytosolic labile iron is not elevated in hemoglobin SS erythrocytes and that elemental membrane iron is present in only trace amounts. (Blood. 2003;102:357-364)

N‐acetylcysteine and clotrimazole inhibit sickle erythrocyte dehydration induced by 1‐chloro‐2,4‐dinitrobenzene

Clotrimazole, a specific inhibitor of the Ca2+ activated potassium (Gardos) channel, and the antioxidant N-acetylcysteine were found to inhibit the in vitro formation of high-density sickle cells induced by treatment with 1-chloro-2,4-dinitrobenzene (CDNB). The CDNB induced leakage of K+ can be inhibited by treatment of SS erythrocytes with 20 mM N-acetylcysteine. We conclude that the effect of N-acetylcysteine in preventing K+ efflux and formation of high-density sickle cells is related to its ability to protect the Gardos channel from oxidative damage caused by diminished levels of reduced glutathione. This effect is due to the ability of N-acetylcysteine to maintain an appropriate level of reduced glutathione and its direct antioxidant activity. Am. J. Hematol. 62:19–24, 1999. © 1999 Wiley-Liss, Inc.

N-acetylcysteine and clotrimazole inhibit sickle erythrocyte dehydration induced by 1-chloro-2,4-dinitrobenzene

Clotrimazole, a specific inhibitor of the Ca(2+) activated potassium (Gardos) channel, and the antioxidant N-acetylcysteine were found to inhibit the in vitro formation of high-density sickle cells induced by treatment with 1-chloro-2,4-dinitrobenzene (CDNB). The CDNB induced leakage of K(+) can be inhibited by treatment of SS erythrocytes with 20 mM N-acetylcysteine. We conclude that the effect of N-acetylcysteine in preventing K(+) efflux and formation of high-density sickle cells is related to its ability to protect the Gardos channel from oxidative damage caused by diminished levels of reduced glutathione. This effect is due to the ability of N-acetylcysteine to maintain an appropriate level of reduced glutathione and its direct antioxidant activity.