Determinants of Sickle Cell Density Distribution: A Study of Benin Twins.

Blood ◽  
2006 ◽  
Vol 108 (11) ◽  
pp. 1244-1244
Author(s):  
Mary E. Fabry ◽  
Anne C. Rybicki ◽  
Sandra M. Suzuka ◽  
M. Cherif Rahimy ◽  
Rajagopal Krishnamoorthy ◽  
...  
Keyword(s):  
Density Distribution ◽  
Cell Density ◽  
Sickle Cell ◽  
Dna Analysis ◽  
Complex Mixture ◽  
Density Gradients ◽  
Red Cell ◽  
Alpha Thalassemia ◽  
Gardos Channel ◽  
One Year

Abstract Red cell density distribution affects both hemolysis and vaso-occlusion; however, currently recognized factors cannot account for all of the variation seen. We hypothesized that a range of genetically controlled factors contributes to red cell density distribution and hemolysis, which has recently received a great deal of attention from Gladwin et al for its role in sickle cell anemia (SCA) and its impact on NO metabolism. Our previous studies have demonstrated that although RBC density distribution varies significantly from patient to patient, the pattern for individual patients is stable in the absence of disease. Some genetically determined factors that affect red cell density distribution have been defined, such as alpha-thalassemia and % HbF (Fabry et al, Blood, 1982); however, neither of these factors completely predicts density distribution. The study of identical twins offers the unique opportunity to minimize some of the genetic variability between individuals that may not be relevant to RBC density while allowing the remaining differences to be detected. Because SCA patients from the US have a complex mixture of Caucasian, other ethnicities, and genes from all parts of Africa including all of the sickle haplotypes, we have chosen to recruit our population from Benin that has a single beta-globin haplotype, thus further minimizing differences arising from admixture from inside and outside of Africa. We have collected samples from six sets of monozygous twins from Benin and validated their monozygosity by DNA analysis. Of the 6 twin sets analyzed to date, 4 have alpha-thalassemia. We compared density gradients on two separate occasions, approximately one year apart, for these twins and found that density gradients for both members of all twin sets without medical complications (malaria, painful crisis) were indistinguishable. This is not true for pairs of randomly chosen individuals even after their % HbF and alpha-thalassemia status has been determined and matched. After elimination of WBCs, we were able to isolate sufficient RNA to obtain microarray data without amplification. We compared subjects with a low % dense cells vs a high % dense cells. In the combinations that were analyzed, we found a consistent pattern of up- and down-regulated genes. Down-regulated genes included the Gardos channel (KCNN4), K-Cl cotransporter (KCC1), NOS3, CAIV, and PKC. Up-regulated genes included ferritin heavy chain (probably in mitochondria that are present in reticulocytes) and 2,3-bisphosphomutase that was elevated in twins with higher MCHC (density). The latter is consistent with our previous observations and those of Poillon et al that DPG can affect polymer formation and RBC density. We conclude that the study of twins demonstrates that there is a strong genetic component in the control of sickle cell density distribution and that a better understanding of factors controlling density distribution may lead to new forms of treatment.

Effects of Hydroxyurea (HU) and Magnesium Pidolate (Mg) in Hemoglobin SC Disease (HbSC): the “CHAMPS” Trial.

Blood ◽  
2009 ◽  
Vol 114 (22) ◽  
pp. 819-819 ◽  
Author(s):  
Winfred C. Wang ◽  
Cathie Snyder ◽  
Carlo Brugnara ◽  
Marilyn J. Telen ◽  
Martin H Steinberg ◽  
...  
Keyword(s):  
Red Blood Cells ◽  
Cell Density ◽  
Primary Endpoint ◽  
Blood Cells ◽  
Maximum Tolerated Dose ◽  
Phosphatidyl Serine ◽  
Primary Objective ◽  
Red Cell ◽  
Channel Activity ◽  
Gardos Channel

Abstract Abstract 819 Background. HbSC is characterized by increased red cell density and clinical complications but has rarely been the target of therapeutic trials. The CHAMPS Trial was a prospective, randomized, double-blinded, multi-center Phase II study of HU and Mg in children and adults with HbSC conducted by the Comprehensive Sickle Cell Centers Clinical Trials Consortium. HU is clinically efficacious in HbSS, but its value has not been demonstrated for HbSC; Mg reduces cation transport activity and improves hydration of HbSS erythrocytes. The primary objective of the trial was to measure the ability of HU and Mg individually and in combination to reduce the density of HbSC erythrocytes. Secondary objectives were to determine the effects of the 2 agents on hematologic parameters and red cell pathobiology, to identify toxicities of HU and Mg, and to record adverse events. Methods. Eligible subjects had HbSC and at least 1 vaso-occlusive event in the previous 12 months (but none in the 4 weeks before study entry) and were ≥ age 5 years. After providing informed consent and baseline evaluations, subjects were randomized to 1 of 4 arms: (A) HU (20 mg/kg/d PO) and Mg (0.6 mEq/kg/d PO in 2 doses), (B) HU and placebo for Mg, (C) HU placebo and Mg, or (D) placebos for both. Subjects were evaluated at 2 or 4 week intervals for 11 months (15 visits). In addition to physical exams, blood counts and chemistry profiles, subjects had central lab evaluations [including measurements of red cell density (by Advia instrument), HbF, red cell cation content, KCl co-transport and Gardos channel activity, cell adhesion to endothelial cells and laminin, and erythrocyte membrane phosphatidyl serine (PS exposure)] at baseline (twice) and weeks 8, 16, 24, and 44. The primary endpoint was red cell density measured at week 8. Results. Forty-four subjects (median age 13.6 years, range 5-53, 73% < age 16) had baseline evaluations and were randomized, 36 reached the primary endpoint and 22 completed 11 months on study drugs. The trial was halted prematurely because of slow enrollment. Subjects in the HU groups (A and B) had increased red cell MCV, cell Hb and HbF compared to baseline and to groups C and D at week 8 (p<0.001); these differences were further increased at week 24 (Table). Mg did not have measurable effects. No differences were seen among the groups in Hb level, hyperdense red cells, erythrocyte Na, K, and Mg, KCl co-transport and Gardos channel activity, plasma magnesium, serum LDH, red cell PS exposure and adhesion to endothelium. Adults did not differ significantly from children. Ten acute events required ER visits/hospitalizations in group A, 15 in group B, 15 in group C, and 19 in group D (differences not significant). Average compliance with HU/HU placebo was approximately 83% and Mg/Mg placebo 77%. No significant toxicity was associated with HU or Mg alone or in combination. Conclusions. HU had significant effects on HbSC erythrocytes, including increased HbF and MCV, with increasing response over 6 months. Mg at a dose of 0.6 mEq/kg/d had no measurable effect on red cell properties or endothelial interactions, either alone or in combination with HU. This may have been related to suboptimal dosing, since the maximum tolerated dose was recently found to be 0.9 mEq/kg/d. Differences in acute events, although not significant, suggest a need for studies with larger enrollment. These data provide a basis for performing clinical efficacy trials using HU, perhaps at higher doses, in subjects with HbSC disease. Disclosures: Off Label Use: Hydroxyurea is being tested for its effects on red blood cells in persons with HbSC disease. Magnesium pidolate is being tested for its effects on red blood cells in persons with HbSC disease.

Pharmacological Inhibition of Calpain-1 Prevents Red Cell Dehydration and Reduces Gardos Channel Activity in a Mouse Model of Sickle Cell Disease. Identification of Druggable Protease Target

Blood ◽  
2011 ◽  
Vol 118 (21) ◽  
pp. 852-852
Author(s):  
Lucia De Franceschi ◽  
Alessandro Mattè ◽  
Carlo Brugnara ◽  
Angela Siciliano ◽  
Mariarita Bertoldi ◽  
...  
Keyword(s):  
Sickle Cell Disease ◽  
Sickle Cell ◽  
Cysteine Proteases ◽  
Calcium Pump ◽  
Membrane Association ◽  
Red Cell ◽  
Channel Activity ◽  
Cell Disease ◽  
Left Shift ◽  
Gardos Channel

Abstract Abstract 852 Sickle cell disease (SCD) is a hereditary red cell disorder characterized by the presence of pathological HbS, which polymerizes upon deoxygenation promoting red blood cell (RBC) dehydration and sickling. In SCD, the dense RBCs play a crucial role in the pathogenesis of sickle cell related organ damage and clinical manifestations. Although progress has been made in pathogenesis of SCD, the treatment options for SCD have limited pharmacological tools for clinical practice. Calpains are ubiquitous calcium-activated cysteine proteases, causing controlled proteolysis of protein substrates with regulatory functions. RBCs express only calpain-1, whose physiological function remains poorly understood. Gene inactivation of mouse calpain-1 revealed differential regulation of RBC calcium pump and enhanced RBC hydration. To investigate the relevance of these findings in SCD, we used BDA-410, a novel orally active inhibitor of calpain-1. Using the sickle (SAD) mouse, a model for human sickle cell disease, BDA-410 was administrated at the dosage of 30 mg/K/d by gavage to wild-type (WT) and sickle cell (SAD) mice. Animals were divided into 4 groups of 6 mice each: two groups from each strain were treated with BDA-410 for 14 days along with vehicle controls. Mice at baseline, day 7, and day 14 of BDA-410 treatment were evaluated for hematological parameters including the RBC density profile with phthalate density curves, RBC cation content, and Ca2+ activated K+ channel (Gardos channel) activity. BDA-410 induced a significant increase in Hct in both WT and SAD mice with no significant changes in Hb levels and an associated increased in MCV. The red cell K+ content increased significantly in SAD RBCs at day 7 and 14 of inhibitor treatment as compared to untreated SAD mice; whereas no major changes were observed in the WT RBCs. The mean corpuscular Hb concentration (CHCM) decreased in both WT and SAD mice treated with BDA-410. A left-shift in the RBC density curves was observed in SAD mice; whereas this left-shift was limited to a sub-fraction of denser red cells in the WT mice. The activity of the Gardos channel was significantly reduced in BDA-410 treated SAD mice compared to the untreated SAD group, while no significant differences were observed in the WT mice. Since the membrane-association of Peroxiredoxin-2 (Prx2) is increased in SCD RBCs, and has been correlated with Gardos channel activity, we evaluated Prx2-membrane association. BDA-410 treatment induced a significant reduction in the amount of Prx2 translocated to the membrane in both WT and SAD mice. Moreover, when we exposed WT and SAD mice to hypoxia (8% oxygen) for 48 hours, followed by 2 hours of re-oxygenation to mimic sickle cell related vaso-occlusive events, BDA-410 treatment prevented the hypoxia induced K+ loss and RBC dehydration in SAD mice. Identification of RBC membrane substrates in the calpain-1 null mice suggests that proteolytic modification of clapain targets as calcium pump, Prx2, and the Gardos channel protein may underpin some of the protective effects of BDA-410 in SAD mice. These results suggest that the inhibition of calpain-1 may offer a new therapeutic strategy to ameliorate hematological phenotype of SCD. Disclosures: No relevant conflicts of interest to declare.

Training-dependent changes of red cell density and erythrocytic oxygen transport

1983 ◽  
Vol 55 (5) ◽  
pp. 1403-1407 ◽  
Author(s):  
H. Mairbaurl ◽  
E. Humpeler ◽  
G. Schwaberger ◽  
H. Pessenhofer
Keyword(s):  
Density Distribution ◽  
Cell Density ◽  
Training Status ◽  
Red Cell ◽  
Transport Parameters ◽  
Trained Subjects ◽  
O2 Transport ◽  
The Mean ◽  
Fitness Training ◽  
2,3 Dpg

Prolonged endurance training causes a decreased O2 affinity of Hb, which is due to an increase in erythrocyte 2,3-diphosphoglycerate (2,3-DPG) concentration. Possible mechanisms were studied in 20 males with varying degrees of fitness. Training status was tested by ergometry. Red cell density and O2 transport parameters were determined before this test. The O2 tension at 50% O2 saturation of Hb (P50) was higher in the more fit subjects (+1.3 mmHg) and the 2,3-DPG concentration was higher (+2.3 mumol/g Hb) in this group. The mean density was significantly lower in fit subjects (1.1002 g/ml) as compared with less fit subjects (1.1056 g/ml), indicating a lower mean age. Density distribution curves show that in the fit subjects more young erythrocytes were in blood and that the very old erythrocytes were missing. After correction for the differences in the density distribution, no differences in the P50 value and 2,3-DPG concentration between less fit and fit subjects were found. Therefore, the decreased Hb-O2 affinity after training can be explained by the presence of more young erythrocytes in the blood of trained subjects. The magnitude of this effect correlates with the training status.

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

Arginine supplementation of sickle transgenic mice reduces red cell density and Gardos channel activity

Blood ◽  
2002 ◽  
Vol 99 (4) ◽  
pp. 1103-1108 ◽  
Author(s):  
José R. Romero ◽  
Sandra M. Suzuka ◽  
Ronald L. Nagel ◽  
Mary E. Fabry
Keyword(s):  
Nitric Oxide ◽  
Cell Density ◽  
Sickle Cell ◽  
T Test ◽  
Red Cells ◽  
K Channel ◽  
Mean Corpuscular Hemoglobin ◽  
Red Cell ◽  
Plasma Arginine ◽  
Arginine Supplementation

Nitric oxide (NO), essential for maintaining vascular tone, is produced from arginine by nitric oxide synthase. Plasma arginine levels are low in sickle cell anemia, and it is reported here that low plasma arginine is also found in our sickle transgenic mouse model that expresses human α, human βS, and human βS-Antilles and is homozygous for the mouse βmajor deletion (S+S-Antilles). S+S-Antilles mice were supplemented with a 4-fold increase in arginine that was maintained for several months. Mean corpuscular hemoglobin concentration (MCHC) decreased and the percent high-density red cells was reduced. Deoxy K+ efflux is characteristic of red cells in sickle cell disease and contributes to the disease process by increasing the MCHC and rendering the cells more susceptible to polymer formation. This flux versus the room air flux was reduced in S+S-Antilles red cells from an average value of 1.6 ± 0.3 mmol per liter of red cells × minute (FU) in nonsupplemented mice to 0.9 ± 0.3 FU (n = 4, P &lt; .02, paired t test) in supplemented mice. In room air, Vmax of the Ca++-activated K+ channel (Gardos) was reduced from 4.1 ± 0.6 FU (off diet) to 2.6 ± 0.4 FU (n = 7 and 8,P &lt; .04, t test) in arginine-supplemented mice versus clotrimazole. In conclusion, the major mechanism by which arginine supplementation reduces red cell density (MCHC) in S+S-Antilles mice is by inhibiting the Ca++-activated K+ channel.

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.

The orientation of sickle hemoglobin fibers within fascicles

1988 ◽  
Vol 46 ◽  
pp. 400-401
Author(s):  
Christopher A. Miller ◽  
Bridget Carragher ◽  
William A. McDade ◽  
Robert Josephs
Keyword(s):  
Sickle Cell ◽  
Sickle Cell Anemia ◽  
Relative Orientation ◽  
Unit Cell ◽  
Red Cell ◽  
High Ph ◽  
Sickle Hemoglobin ◽  
Polar Crystal ◽  
Helical Configuration

Highly ordered bundles of deoxyhemoglobin S (HbS) fibers, termed fascicles, are intermediates in the high pH crystallization pathway of HbS. These fibers consist of 7 Wishner-Love double strands in a helical configuration. Since each double strand has a polarity, the odd number of double strands in the fiber imparts a net polarity to the structure. HbS crystals have a unit cell containing two double strands, one of each polarity, resulting in a net polarity of zero. Therefore a rearrangement of the double strands must occur to form a non-polar crystal from the polar fibers. To determine the role of fascicles as an intermediate in the crystallization pathway it is important to understand the relative orientation of fibers within fascicles. Furthermore, an understanding of fascicle structure may have implications for the design of potential sickling inhibitors, since it is bundles of fibers which cause the red cell distortion responsible for the vaso-occlusive complications characteristic of sickle cell anemia.

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