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BackgroundFontan survivors have depressed cardiac index that worsens over time. Serum biomarker measurement is minimally invasive, rapid, widely available, and may be useful for serial monitoring. The purpose of this study was to identify biomarkers that correlate with lower cardiac index in Fontan patients.Methods and resultsThis study was a multi-centre case series assessing the correlations between biomarkers and cardiac magnetic resonance-derived cardiac index in Fontan patients ⩾6 years of age with biochemical and haematopoietic biomarkers obtained ±12 months from cardiac magnetic resonance. Medical history and biomarker values were obtained by chart review. Spearman’s Rank correlation assessed associations between biomarker z-scores and cardiac index. Biomarkers with significant correlations had receiver operating characteristic curves and area under the curve estimated. In total, 97 cardiac magnetic resonances in 87 patients met inclusion criteria: median age at cardiac magnetic resonance was 15 (6–33) years. Significant correlations were found between cardiac index and total alkaline phosphatase (−0.26, p=0.04), estimated creatinine clearance (0.26, p=0.02), and mean corpuscular volume (−0.32, p2versus lower-risk group=2.96 L/minute/m2, (pConclusionsHigher total alkaline phosphatase and mean corpuscular volume as well as lower estimated creatinine clearance identify Fontan patients with lower cardiac index. Using biomarkers to monitor haemodynamics and organ-specific effects warrants prospective investigation.

Background Although sickle cell trait (SCT) is largely a benign carrier state, it may increase risk for certain clinical outcomes. Purpose To evaluate associations between SCT and clinical outcomes in children and adults. Data Sources English-language searches of PubMed, CINAHL, the Cochrane Library, Current Contents Connect, Scopus, and Embase (1 January 1970 to 30 June 2018) and bibliographies of review articles. Study Selection Observational controlled studies (published in English) in children or adults that examined an association between SCT and any of 24 clinical outcomes specified a priori in the following 6 categories: exertion-related injury; renal, vascular, pediatric, and surgery- or trauma-related outcomes; and overall mortality. Data Extraction A single reviewer extracted study data, which was checked by another; 2 reviewers independently assessed study quality; and strength of evidence was assessed by consensus. Data Synthesis Of 7083 screened studies, 41 met inclusion criteria. High-strength evidence supported a positive association between SCT and risk for pulmonary embolism, proteinuria, and chronic kidney disease. Moderate-strength evidence supported a positive association between SCT and exertional rhabdomyolysis and a null association between SCT and deep venous thrombosis, heart failure or cardiomyopathy, stroke, and pediatric height or weight. Absolute risks for thromboembolism and rhabdomyolysis were small. For the remaining 15 clinical outcomes, data were insufficient or strength of evidence was low. Limitation Publication bias was possible, and high-quality evidence was scant. Conclusion Sickle cell trait is a risk factor for a few adverse health outcomes, such as pulmonary embolism, kidney disease, and exertional rhabdomyolysis, but does not seem to be associated with such complications as heart failure and stroke. Insufficient data or low-strength evidence exists for most speculated complications of SCT. Primary Funding Source National Human Genome Research Institute.

Externalization of phosphatidylserine (PS) is thought to contribute to sickle cell disease (SCD) pathophysiology. The red blood cell (RBC) aminophospholipid translocase (APLT) mediates the transport of PS from the outer to the inner RBC membrane leaflet to maintain an asymmetric distribution of PL, while phospholipid scramblase (PLSCR) equilibrates PL across the RBC membrane, promoting PS externalization. We previously identified an association between PS externalization level and PLSCR activity in sickle RBC under basal conditions. Other studies showed that activation of protein kinase C (PKC) by PMA (phorbol-12-myristate-13-acetate) causes increased external PS on RBC. Therefore, we hypothesized that PMA-activated PKC stimulates PLSCR activity in RBC and thereby contributes to increased PS externalization. In the current studies, we show that PMA treatment causes immediate and variable PLSCR activation and subsequent PS externalization in control and sickle RBC. While TfR+ sickle reticulocytes display some endogenous PLSCR activity, we observed a robust activation of PLSCR in sickle reticulocytes treated with PMA. The PKC inhibitor, chelerythrine (Chel), significantly inhibited PMA-dependent PLSCR activation and PS externalization. Chel also inhibited endogenous PLSCR activity in sickle reticulocytes. These data provide evidence that PKC mediates PS externalization in RBC through activation of PLSCR.

Sickle cell nephropathy begins with hyperfiltration and microalbuminuria and may progress to renal failure. The aim of this study was to determine the effects of losartan on glomerular function and albumin excretion in sickle cell anemia (SCA). Individuals with SCA on hydroxyurea with persistent albuminuria were enrolled in a 1-year study of losartan. Glomerular filtration rate (GFR) measured by iohexol clearance, albumin excretion rate (AER), and fractional clearance of dextran were assessed at baseline, short-term (1-2month), and long-term (≥12month) intervals. Twelve subjects (6 microalbuminuria, 6 macroalbuminuria) completed short-term studies; 8 completed long-term studies. Baseline GFR was 112ml/min/1.73m2 (71-147ml/min/1.73m2). AER decreased significantly at the short-term (median decrease -134 mcg/min, p=0.0063). GFR was not significantly-different at short-term or long-term intervals. Dextran clearance improved for diameters smaller than albumin (

Abnormal sickle red blood cell (sRBC) biomechanics, including pathological deformability and adhesion, correlate with clinical severity in sickle cell disease (SCD). Clinical intravenous fluids (IVFs) of various tonicities are often used during treatment of vaso-occlusive pain episodes (VOE), the major cause of morbidity in SCD. However, evidence-based guidelines are lacking, and there is no consensus regarding which IVFs to use during VOE. Further, it is unknown how altering extracellular fluid tonicity with IVFs affects sRBC biomechanics in the microcirculation, where vaso-occlusion takes place. Here, we report how altering extracellular fluid tonicity with admixtures of clinical IVFs affects sRBC biomechanical properties by leveraging novel in vitro microfluidic models of the microcirculation, including 1 capable of deoxygenating the sRBC environment to monitor changes in microchannel occlusion risk and an “endothelialized” microvascular model that measures alterations in sRBC/endothelium adhesion under postcapillary venular conditions. Admixtures with higher tonicities (sodium = 141 mEq/L) affected sRBC biomechanics by decreasing sRBC deformability, increasing sRBC occlusion under normoxic and hypoxic conditions, and increasing sRBC adhesion in our microfluidic human microvasculature models. Admixtures with excessive hypotonicity (sodium = 103 mEq/L), in contrast, decreased sRBC adhesion, but overswelling prolonged sRBC transit times in capillary-sized microchannels. Admixtures with intermediate tonicities (sodium = 111-122 mEq/L) resulted in optimal changes in sRBC biomechanics, thereby reducing the risk for vaso-occlusion in our models. These results have significant translational implications for patients with SCD and warrant a large-scale prospective clinical study addressing optimal IVF management during VOE in SCD.

Background Renal damage is a progressive complication of sickle cell disease (SCD) that begins in childhood and may progress to renal failure and early mortality in 12% of adults with hemoglobin SS (HbSS) SCD. Early sickle nephropathy is characterized by hyperfiltration and microalbuminuria; therefore, urine albumin to creatinine ratio (ACR) is an effective screening tool for its detection. Procedure This study investigated the effect of hydroxyurea (HU) therapy on urine ACR levels among children with SCD. A retrospective review was conducted to identify all patients with HbSS or HbSβ0 thalassemia of age 7–18 years who began HU therapy in 2011–2013; a control group of patients not on HU were matched by age and baseline hemoglobin. All urine ACR measurements ≤24 months prior to and ≥24 months after HU initiation were recorded. Results There were 63 eligible patients on HU and 13 (25%) with albuminuria prior to HU initiation. Among those with baseline albuminuria, the median ACR was 96 mg/g prior to HU, 39 mg/g at 1 year (P = 0.02), and 25 mg/g at 2 years (P = 0.03). Albuminuria normalized in 37.5% (6/16) after 1 year and 61% (8/13) after 2 years of HU therapy. Among those without albuminuria prior to HU, 13% (6/47) developed albuminuria during HU therapy. Sixteen percent (13/80) of control patients had albuminuria in the beginning of study period, which normalized in 15% (two of 13) of patients at 1-year follow up. Conclusion Introduction of HU is associated with significant decreases in urine ACR in children with SCD and albuminuria.

Vaso-occlusive crisis (VOC) is a complex process that occurs in patients with sickle cell disease (SCD) and is often associated with pain and urgent hospitalization. A major instigator of VOC is microvascular obstruction by pathologically stiffened sickle red blood cells (RBCs), and thus therapy relies heavily on optimizing intravenous fluid (IVF) hydration to increase RBC deformability. However, no evidence-based guidelines regarding the choice of IVF currently exist. We therefore analyzed alterations in biomechanical properties of sickle RBCs isolated from patients with homozygous SCD (hemoglobin SS) after exposure to different osmolarities of clinical IVF formulations. Atomic force microscopy (AFM) was used to assess stiffness of RBCs after exposure to different IVFs. A microfluidic model of the human capillary system was used to assess transit time and propensity to occlusion after exposure to the different IVF formulations. Sickle RBCs exposed to normal saline (NS) had increased stiffness, transit times, and propensity to microchannel occlusion compared to other osmolarities. Normal saline, an IVF formulation often used to treat patients with SCD during VOC, may induce localized microvascular obstruction due to alterations of sickle RBC biomechanical properties. This article is protected by copyright. All rights reserved.

Hemolysis is a key feature of sickle cell anemia (HbSS). Direct quantitation of hemolysis could be used as an objective outcome in clinical trials of new therapeutics for HbSS and would also enable better human studies of the pathogenesis of complications of HbSS that are ostensibly hemolysis-related, such as pulmonary hypertension. However, contemporary human studies in HbSS have used only surrogate markers of hemolysis rather than direct measurements of RBC survival. We directly quantified hemolysis in HbSS by measuring survival of an age cohort of RBCs labeled with a stable isotope, administered orally as 15N-glycine, a metabolic precursor of heme. The atomic excess of 15N in heme extracted from blood was monitored by mass spectrometry over time. We performed 13 labeling experiments in 11 individuals with HbSS. Mean RBC survival was 31.9 days (range 14.1 - 53.6). Both HbF level, a known determinant of hemolysis, and absolute reticulocyte count (ARC), an index of the marrow's response to hemolysis, correlated with directly measured RBC survival (r=0.61, P

Chronic inflammation has emerged as an important pathogenic mechanism in sickle cell disease (SCD). One component of this inflammatory response is oxidant stress mediated by reactive oxygen species (ROS) generated by leukocytes, endothelial cells, plasma enzymes, and sickle red blood cells (RBC). Sickle RBC ROS generation has been attributed to sickle hemoglobin auto-oxidation and Fenton chemistry reactions catalyzed by denatured heme moieties bound to the RBC membrane. In this study, we demonstrate that a significant part of ROS production in sickle cells is mediated enzymatically by NADPH oxidase, which is regulated by protein kinase C, Rac GTPase, and intracellular Ca(2+) signaling within the sickle RBC. Moreover, plasma from patients with SCD and isolated cytokines, such as transforming growth factor β1 and endothelin-1, enhance RBC NADPH oxidase activity and increase ROS generation. ROS-mediated damage to RBC membrane components is known to contribute to erythrocyte rigidity and fragility in SCD. Erythrocyte ROS generation, hemolysis, vaso-occlusion, and the inflammatory response to tissue damage may therefore act in a positive-feedback loop to drive the pathophysiology of sickle cell disease. These findings suggest a novel pathogenic mechanism in SCD and may offer new therapeutic targets to counteract inflammation and RBC rigidity and fragility in SCD.

The changes in red blood cells (RBC) as they age and the mechanisms for their eventual removal have been of interest for many years. Proposed age-related changes include dehydration with increased density and decreased size, increased membrane IgG, loss of membrane phospholipid asymmetry, and decreased activity of KCl cotransport. The biotin RBC label allows unambiguous identification of older cells and exploration of their properties as they age. Autologous normal human RBC were labeled ex vivo and, after reinfusion, compared with unlabeled RBC throughout their lifespan. RBC density increased with age, with most of the change in the first weeks. Near the end of their lifespan, RBC had increased surface IgG. However, there was no evidence for elevated external phosphatidylserine (PS) even though older RBC had significantly lower activity of aminophospholipid translocase (APLT). KCl cotransport activity persisted well past the reticulocyte stage, but eventually decreased as the RBC became older. These studies place limitations on the use of density fractionation for the study of older human RBC, and do not support loss of phospholipid asymmetry as a mechanism for human RBC senescence. However, increased levels of IgG were associated with older RBC, and may contribute to their removal from the circulation.

Sickle Cell Trait (HbAS), the heterozygous state for the sickle hemoglobin beta globin gene is carried by as many as 100 million individuals including up to 25% of the population in some regions of the world (World Health Organization, Provisional agenda item 4.8, EB117/34 (22 December 2005) or World Health Organization, Provisional agenda item 11.4 (24 April 2006)). Persons with HbAS have some resistance to falciparum malaria infection in early childhood (Piel FB, Patil AP, Howes RE, et al., Nat Commun 2010;1104:1–7 and Aidoo M, Terlouw DJ, Kolczak M, et al., Lancet 2002;359:1311–1312) and as a result individuals with HbAS living in malarial endemic regions of Africa have a survival advantage over individuals with HbAA. Reports from the US emphasize possible health risks for individuals with HbAS including increased incidence of renal failure and malignancy, thromboembolic disorders, splenic infarction as a high altitude complication, and exercise-related sudden death. The National Heart, Lung, and Blood Institute, National Institutes of Health convened a workshop in Bethesda, Maryland on June 3–4, 2010, Framing the Research Agenda for Sickle Cell Trait, to review the clinical manifestations of HbAS, discuss the exercise-related sudden death reports in HbAS, and examine the public health, societal, and ethical implications of policies regarding HbAS. The goal of the workshop was to identify potential research questions to address knowledge gaps.

The potassium chloride co-transporter (KCC) is a member of the electroneutral cation chloride family of cotransporters found in multiple tissues that are involved in transepithelial ion transport and regulation of intracellular ion content and cell volume. We have shown previously that three of the four KCC genes -- KCC1, KCC3, and KCC4 -- are expressed in red blood cells (RBC) (Exp.Hem. 33:624, 2005). Functionally, the KCC mediates volume reduction of reticulocytes that establishes the higher cellular hemoglobin concentration (CHC) of mature RBC. KCC activity is higher in reticulocytes and diminishes with age. KCC activity in RBC containing sickle hemoglobin (SS RBC) is elevated compared to normal (AA RBC) in part due to reticulocytosis in SS blood. However, we have demonstrated that SS reticulocytes have abnormal regulation of KCC activity leading to increased CHC upon activation of KCC compared to AA reticulocytes (Blood 104:2954, 2004; Blood 109:1734, 2007). These findings implicate KCC as a factor in the dehydration of SS RBC, which leads to elevated Hb S concentration and enhances Hb S polymerization and hemolysis. Because KCC activity correlates with cell age, standard flux measurements on blood samples with different numbers of reticulocytes or young non-reticulocytes are not comparable. The Advia automated cell counter measures cell volume (MCV) and cellular hemoglobin concentration (CHC) in reticulocytes, an age-defined population of cells, and thus circumvents the problem of variable reticulocyte counts among SS and AA blood samples. In this study, reticulocyte CHC measurements on fresh blood demonstrated a clear difference between AA and SS cells, reflecting in vivo dehydration of SS reticulocytes, although there was significant inter-individual variation, and the CHC distributions of the two groups overlapped. After KCC activation in vitro by cell swelling using the nystatin method, the initial changes in reticulocyte MCV and CHC with time were used to estimate flux rates mediated by KCC, assuming that changes were associated with isotonic KCl movements. After 20-30 minutes a final steady state MCV/CHC (set point) was achieved and maintained, reflecting inactivation of the transporter. CHC set points were 26.5-29 g/dl in SS reticulocytes compared to 25-26.5 g/dl in AA reticulocytes, reflecting abnormal regulation in SS cells. These results were reproducible in the same individual over time. KCC flux derived from CHC ranged from 5-10.3 mmolK/kgHb/min in SS reticulocytes, compared to 2.9-7.2 mmolK/kgHb/min in AA reticulocytes. Such measures of KCC activity in red cell populations controlled for cell age will facilitate further studies correlating KCC activity with phenotypic or genetic variability in sickle cell disease.

This article describes the planning, implementation, and evaluation of Research and Education Day, an event designed to promote education and awareness of sickle cell disease (SCD), while also fostering and increasing participation by families in research studies. Quantitative and qualitative evaluation data (tracking of participant attendance and participation in research studies; program evaluation surveys) indicate that program goals were achieved. Results show that event attendance, research participation and retention, and community volunteerism has increased exponentially over the 8-year history of the event. Parents report that their top-rated reasons for attending are learning about research studies and participating in SCD-related educational activities. Data also show that families learn about the event in multiple ways (i.e., flyers, clinic staff, word of mouth, etc.). Research and Education Day is a model for educating participants with sickle cell and their families, engaging them in research studies, and enhancing community awareness and support.

Eukaryotic core promoters are often characterized by the presence of consensus motifs such as the TATA box or initiator elements, which attract and direct the transcriptional machinery to the transcription start site. However, many human promoters have none of the known core promoter motifs, suggesting that undiscovered promoter motifs exist in the genome. We previously identified a mutation in the human Ankyrin-1 (ANK-1) promoter that causes the disease ankyrin-deficient Hereditary Spherocytosis (HS). Although the ANK-1 promoter is CpG rich, no discernable basal promoter elements had been identified. We showed that the HS mutation disrupted the binding of the transcription factor TFIID, the major component of the pre-initiation complex. We hypothesized that the mutation identified a candidate promoter element with a more widespread role in gene regulation. We examined 17,181 human promoters for the experimentally validated binding site, called the TFIID localization sequence (DLS) and found three times as many promoters containing DLS than TATA motifs. Mutational analyses of DLS sequences confirmed their functional significance, as did the addition of a DLS site to a minimal Sp1 promoter. Our results demonstrate that novel promoter elements can be identified on a genome-wide scale through observations of regulatory disruptions that cause human disease.

Sickle 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.

Modulation of intracellular chloride concentration ([Cl(-)](i)) plays a fundamental role in cell volume regulation and neuronal response to GABA. Cl(-) exit via K-Cl cotransporters (KCCs) is a major determinant of [Cl(-)](I); however, mechanisms governing KCC activities are poorly understood. We identified two sites in KCC3 that are rapidly dephosphorylated in hypotonic conditions in cultured cells and human red blood cells in parallel with increased transport activity. Alanine substitutions at these sites result in constitutively active cotransport. These sites are highly phosphorylated in plasma membrane KCC3 in isotonic conditions, suggesting that dephosphorylation increases KCC3's intrinsic transport activity. Reduction of WNK1 expression via RNA interference reduces phosphorylation at these sites. Homologous sites are phosphorylated in all human KCCs. KCC2 is partially phosphorylated in neonatal mouse brain and dephosphorylated in parallel with KCC2 activation. These findings provide insight into regulation of [Cl(-)](i) and have implications for control of cell volume and neuronal function.

Although red blood cell (RBC) life span is a known determinant of percentage hemoglobin A1c (HbA1c), its variation has been considered insufficient to affect clinical decisions in hematologically normal persons. However, an unexplained discordance between HbA1c and other measures of glycemic control can be observed that could be, in part, the result of differences in RBC life span. To explore the hypothesis that variation in RBC life span could alter measured HbA1c sufficiently to explain some of this discordance, we determined RBC life span using a biotin label in 6 people with diabetes and 6 nondiabetic controls. Mean RBC age was calculated from the RBC survival curve for all circulating RBCs and for labeled RBCs at multiple time points as they aged. In addition, HbA1c in magnetically isolated labeled RBCs and in isolated transferrin receptor-positivereticulocytes was used to determine the in vivo synthetic rate of HbA1c. The mean age of circulating RBCs ranged from 39 to 56 days in diabetic subjects and 38 to 60 days in nondiabetic controls. HbA1c synthesis was linear and correlated with mean whole blood HbA1c (R2 = 0.91). The observed variation in RBC survival was large enough to cause clinically important differences in HbA1c for a given mean blood glucose.

OBJECTIVE—To determine whether interindividual heterogeneity in the erythrocyte (red blood cell [RBC]) transmembrane glucose gradient might explain discordances between A1C and glycemic control based on measured fructosamine. RESEARCH DESIGN AND METHODS—We modeled the relationship between plasma glucose and RBC glucose as the concentration distribution (Ci-to-Co ratio) of a nonmetabolizable glucose analog 14C-3-O-methyl glucose (14C-3OMG) inside (Ci) and outside (Co) RBCs in vitro. We examined the relationship between that distribution and the degree of glycation of hemoglobin in comparison with glycation of serum proteins (fructosamine), the glycation gap. A1C, fructosamine, and in vitro determination of the 14C-3OMG distribution in glucose-depleted RBCs were measured in 26 fasted subjects. RESULTS—The Ci-to-Co ratio 0.89 ± 0.07 for 3-O-methyl-d-glucopyranose (3OMG) ranged widely (0.72–1.04, n = 26). In contrast, urea Ci-to-Co (1.015 ± 0.022 [range 0.98–1.07], P < 0.0001) did not. Concerning mechanism, in a representative subset of subjects, the Ci-to-Co ratio was retained in RBC ghosts, was not dependent on ATP or external cations, and was reestablished after reversal of the glucose gradient. The 3OMG Ci-to-Co ratio was not correlated with serum fructosamine, suggesting that it was independent of mean plasma glucose. However, Ci-to-Co did correlate with A1C (R2 = 0.19) and with the glycation gap (R2 = 0.20), consistent with a model in which differences in internal glucose concentration at a given mean plasma glucose contribute to differences in A1C for given level of glycemic control. CONCLUSIONS—The data demonstrate interindividual heterogeneity in glucose gradients across RBC membranes that may affect hemoglobin glycation and have implications for diabetes complications risk and risk assessment.

KCl cotransport (KCC) activity contributes to pathologic dehydration in sickle (SS) red blood cells (RBCs). KCC activation by urea was measured in SS and normal (AA) RBCs as Cl-dependent Rb influx. KCC-mediated volume reduction was assessed by measuring reticulocyte cellular hemoglobin concentration (CHC) cytometrically. Urea activated KCC fluxes in fresh RBCs to levels seen in swollen cells, although SS RBCs required lower urea concentrations than did normal (AA) RBCs. Little additional KCC stimulation by urea occurred in swollen AA or SS RBCs. The pH dependence of KCC in “euvolemic” SS RBCs treated with urea was similar to that in swollen cells. Urea triggered volume reduction in SS and AA reticulocytes, establishing a higher CHC. Volume reduction was Cl dependent and was limited by the KCC inhibitor, dihydro-indenyl-oxyalkanoic acid. Final CHC depended on urea concentration, but not on initial CHC. Under all activation conditions, volume reduction was exaggerated in SS reticulocytes and produced higher CHCs than in AA reticulocytes. The sulfhydryl-reducing agent, dithiothreitol, normalized the sensitivity of KCC activation to urea in SS RBCs and mitigated the urea-stimulated volume decrease in SS reticulocytes, suggesting that the dysfunctional activity of KCC in SS RBCs was due in part to reversible sulfhydryl oxidation.

The determinants of sickle red blood cell (RBC) life span have not been well-defined but may include both intrinsic factors (eg, the tendency to sickle) and extrinsic factors (eg, the capacity of the reticuloendothelial system to remove defective RBCs). Fetal hemoglobin (HbF) is heterogeneously distributed among sickle RBCs; F cells contain 20% to 25% HbF, whereas the remainder have no detectable HbF (non-F cells). Autologous sickle RBCs were labeled with biotin and reinfused to determine overall survival, non–F- and F-cell survival, and time-dependent changes in HbF content (%HbF) for the surviving F cells. A total of 10 patients were enrolled, including 2 who were studied before and after the percentage of F cells was increased by treatment with hydroxyurea. As expected, F cells survived longer in all subjects. Non–F-cell survival correlated inversely with the percentage of F cells, with the time for 30% cell survival ranging from 6 days in patients with more than 88% F cells to 16 days in patients with less than 16% F cells. As the biotin-labeled RBCs aged in the circulation, the HbF content of the surviving F-cell population increased by 0.28%/d ± 0.21%/d, indicating that within the F-cell population those with higher HbF content survived longer.

Recent studies have identified older, low-density sickle red blood cells (SSRBCs) that were resistant to dehydration by valinomycin, a K+ ionophore. These cells, thought to derive from dense SSRBCs that have rehydrated, may represent a terminal cellular phase. To study rehydration, we subjected dense SSRBCs (ρ > 1.107 g/cc) to either oxygenated incubation or rapid oxygenated/deoxygenated (oxy/deoxy) cycling (70 seconds per cycle). Light cells (ρ

Sickling-induced cation fluxes contribute to cellular dehydration of sickle red blood cells (SS RBCs), which in turn potentiates sickling. This study examined the inhibition by dipyridamole of the sickling-induced fluxes of Na(+), K(+), and Ca(++) in vitro. At 2% hematocrit, 10 microM dipyridamole inhibited 65% of the increase in net fluxes of Na(+) and K(+) produced by deoxygenation of SS RBCs. Sickle-induced Ca(++) influx, assayed as (45)Ca(++) uptake in quin-2-loaded SS RBCs, was also partially blocked by dipyridamole, with a dose response similar to that of Na(+) and K(+) fluxes. In addition, dipyridamole inhibited the Ca(++)-activated K(+) flux (via the Gardos pathway) in SS RBCs, measured as net K(+) efflux in oxygenated cells exposed to ionophore A23187 in the presence of external Ca(++), but this effect resulted from reduced anion conductance, rather than from a direct effect on the K(+) channel. The degree of inhibition of sickling-induced fluxes was dependent on hematocrit, and up to 30% of dipyridamole was bound to RBC membranes at 2% hematocrit. RBC membrane content of dipyridamole was measured fluorometrically and correlated with sickling-induced flux inhibition at various concentrations of drug. Membrane drug content in patients taking dipyridamole for other clinical indications was similar to that producing inhibition of sickling-induced fluxes in vitro. These data suggest that dipyridamole might inhibit sickling-induced fluxes of Na(+), K(+), and Ca(++) in vivo and therefore have potential as a pharmacological agent to reduce SS RBC dehydration. (Blood. 2001;97:3976-3983)

Sickle red blood cells (RBC) are subject to a number of important cellular changes and selection pressures. In this study, we validated a biotin RBC label by comparison to the standard 51Cr label, and used it to study changes that occur in sickle cells as they age. Sickle RBC had a much shorter lifespan than normal RBC, but the two labels gave equivalent results for each cell type. A variable number of sickle, but not normal, RBC disappeared from the circulation during the first few hours after reinfusion. The number of biotinylated sickle reticulocytes was decreased by 50% after 24 h and 75% after 48 h, with a gradual decrease in the amount of reticulum per cell. The labeled sickle cells exhibited major density increases during the first 4-6 d after reinfusion, with smaller changes thereafter. A small population of very light, labeled sickle RBC was essentially constant in number after the first few days. Fetal hemoglobin (HbF) content was determined in isolated biotinylated sickle RBC after reinfusion, allowing an estimate of lifespan for RBC containing HbF (F cells) and non-F cells. The lifespan of sickle biotinylated RBC lacking HbF was estimated to be approximately 2 wk, whereas F cells survived 6-8 wk.

Sickle cell disease (SCD) is a globally distributed hereditary red blood cell (RBC) disorder. One of the hallmarks of SCD is the presence of circulating dense RBCs, which are important in SCD-related clinical manifestations. In human dense sickle cells, we found reduced calpastatin activity and protein expression compared to either healthy RBCs or unfractionated sickle cells, suggesting an imbalance between activator and inhibitor of calpain-1 in favor of activator in dense sickle cells. Calpain-1 is a nonlysosomal cysteine proteinase that modulates multiple cell functions through the selective cleavage of proteins. To investigate the relevance of this observation in vivo, we evaluated the effects of the orally active inhibitor of calpain-1, BDA-410 (30 mg/kg/d), on RBCs from SAD mice, a mouse model for SCD. In SAD mice, BDA-410 improved RBC morphology, reduced RBC density (D20; from 1106±0.001 to 1100±0.001 g/ml; P

A subset of sickle cells becomes K(+)-depleted and dehydrated before or soon after leaving the bone marrow. These young cells may be identified in blood as transferrin receptor-positive (TfR+) dense reticulocytes. KCl cotransport, which is normally active in young erythroid cells with a maximum at pH 6.8, is a candidate pathway for K+ depletion of sickle reticulocytes. In this investigation, KCl cotransport activity was evaluated in young, TfR+ cells which had become dense in vivo and in age-matched cells which had retained normal hydration. Sickle erythrocytes were first separated into three primary density fractions, with care taken to preserve the in vivo hydration state. After normalization of intracellular hemoglobin concentration with nystatin, the cells were incubated at 37 degrees C for 20 min at pH 6.8 and 7.4. Before and after incubation, each primary fraction was separated into four secondary density fractions. The percentage of TfR+ cells in each secondary fraction was measured and a density distribution for TfR+ cells was determined for each primary fraction before and after incubation. The density shift during incubation was a measure of KCl cotransport. TfR+ cells from the denser primary fractions II and III had significantly more density shift than TfR+ cells from the light fraction I. Although the shifts were larger at low pH, differences between primary fractions were also observed at pH 7.4. These data indicate that the cells which become dense quickly in vivo have more KCl cotransport activity than those which remain light in vivo, and support this pathway as a primary mechanism for dehydration of young sickle cells.

Membrane cholesterol is distributed asymmetrically both within the cell or within cellular membranes. Elaboration of intracellular cholesterol trafficking, targeting and intramembrane distribution has been spurred by both molecular and structural approaches. The expression of recombinant sterol carrier proteins in L-cell fibroblasts has been especially useful in demonstrating for the first time that such proteins actually elicit intracellular and intraplasma membrane redistribution of sterol. Additional advances in the use of native fluorescent sterols allowed resolution of transbilayer and lateral cholesterol domains in plasma membranes from cultured fibroblasts, brain synaptosomes and erythrocytes. In all three cell surface membranes, cholesterol is enriched in the inner, cytofacial leaflet. Up to three different cholesterol domains have been identified in the lateral plane of the plasma membrane: a fast exchanging domain comprising less than 10% of cholesterol, a slowly exchanging domain comprising about 30% of cholesterol, and a very slowly or non-exchangeable sterol domain comprising 50-60% of plasma membrane cholesterol. Factors modulating plasma membrane cholesterol domains include polyunsaturated fatty acids, expression of intracellular sterol carrier proteins, drugs such as ethanol, and several membrane pathologies (systemic lupus erythematosus, sickle cell anaemia and aging). Disturbances in plasma membrane cholesterol domains alter transbilayer fluidity gradients in plasma membranes. Such changes are associated with decreased Ca(2+)-ATPase and Na+, K(+)-ATPase activity. Thus, the size, dynamics and distribution of cholesterol domains within membranes not only regulate cholesterol efflux/influx but also modulate plasma membrane protein functions and receptor-effector coupled systems.

Structural domains of cholesterol and their regulation in the erythrocyte membrane are poorly understood. Dehydroergosterol fluorescence polarization change was used to continuously monitor the kinetics of sterol exchange and sterol domain size in erythrocyte ghost membranes. Direct correlation between molecular sterol exchange and steady-state dehydroergosterol fluorescence polarization measurements was obtained without separation of donor and acceptor membranes. Three important observations were made. First, sterol exchange between small unilamellar vesicles (SUV) with the same cholesterol/phospholipid ratio as the erythrocyte membrane (1-palmitoyl-2-oleoylphosphatidylcholine/cholesterol = 1:1) was resolved into three kinetic cholesterol domains: 23 +/- 9% of total sterol was rapidly exchangeable, with t1/2 = 23 +/- 6 min; 59 +/- 9% of total sterol was slowly exchangeable, with t1/2 = 135 +/- 3 min; and 19 +/- 9% of total sterol was essentially nonexchangeable, with a t1/2 of days. Second, the substitution of erythrocyte ghosts for SUV as an acceptor significantly altered the kinetic parameters of sterol exchange from donor SUV, graphically showing that both the properties of the acceptor and spontaneous desorption of cholesterol from the donor SUV influenced spontaneous cholesterol transfer. Third, studies of exchange between erythrocyte ghosts revealed multiple kinetic pools of sterol differing from those in the SUV: 4 +/- 2% of total sterol was rapidly exchangeable, with t1/2 = 32 +/- 9 min; 29 +/- 3% of total sterol was very slowly exchangeable, with t1/2 = 23 +/- 7 h; and a surprisingly large 67 +/- 2% of total sterol was nonexchangeable, with a t1/2 of days.

The K-Cl cotransporter (KCC) regulates red blood cell (RBC) volume, especially in reticulocytes. Western blot analysis of RBC membranes revealed KCC1, KCC3, and KCC4 proteins in mouse and human cells, with higher levels in reticulocytes. KCC content was higher in sickle versus normal RBC, but the correlation with reticulocyte count was poor, with inter-individual variability in KCC isoform ratios. Messenger RNA for each isoform was measured by real time RT-quantitative PCR. In human reticulocytes, KCC3a mRNA levels were consistently the highest, 1–7-fold higher than KCC4, the second most abundant species. Message levels for KCC1 and KCC3b were low. The ratios of KCC RNA levels varied among individuals but were similar in sickle and normal RBC. During in vivo maturation of human erythroblasts, KCC3a RNA was expressed consistently, whereas KCC1 and KCC3b levels declined, and KCC4 message first increased and then decreased. In mouse erythroblasts, a similar pattern for KCC3 and KCC1 expression during in vivo differentiation was observed, with low KCC4 RNA throughout despite the presence of KCC4 protein in mature RBC. During differentiation of mouse erythroleukemia cells, protein levels of KCCs paralleled increasing mRNA levels. Functional properties of KCCs expressed in HEK293 cells were similar to each other and to those in human RBC. However, the anion dependence of KCC in RBC resembled most closely that of KCC3. The results suggest that KCC3 is the dominant isoform in erythrocytes, with variable expression of KCC1 and KCC4 among individuals that could result in modulation of KCC activity.

Cellular dehydration is one of several pathological features of the sickle cell. Cation depletion is quite severe in certain populations of sickle cells and contributes to the rheological dysfunction that is the root cause of vascular occlusion in this disease. The mechanism of dehydration of sickle cells in vivo has not been ascertained, but three transport pathways may play important roles in this process. These include the deoxygenation-induced pathway that permits passive K+ loss and entry of Na+ and Ca2+; the K(+)-Cl- cotransport pathway, activated by acidification or cell swelling; and the Ca(2+)-activated K+ channel, or Gardos pathway, presumably activated by deoxygenation-induced Ca2+ influx. Recent evidence suggests that these pathways may interact in vivo. Heterogeneity exists among sickle cells as to the rate at which they become dense, suggesting that other factors may affect the activity or interactions of these pathways. Understanding the mechanism of dehydration of sickle cells may provide opportunities for pharmacological manipulation of cell volume to mitigate some of the symptoms of sickle cell disease.

Respiratory infections are associated with clinically significant illness in patients with sickle cell disease (SCD). The 2009 H1N1 pandemic was perceived as a significant threat to this population.We undertook a chart review of all patients with SCD followed at our institution to identify those with confirmed H1N1 infection. Further chart and laboratory data was collected on affected patients to analyze clinical courses and the factors that correlated with disease severity.Approximately half of the patients with confirmed H1N1 infection were managed successfully on an outpatient basis with oseltamivir therapy. Among the patients admitted, the most common diagnosis was acute chest syndrome (ACS). Most admitted patients had uncomplicated clinical courses, with a median length of admission of 3 days and no mortality or requirement for mechanical ventilation. A past history of ACS or reactive airway disease correlated with a higher rate of admission and of ACS incidence during the acute illness. Chronic transfusion therapy or hydroxyurea therapy with high hemoglobin F levels had a strong inverse correlation with incidence of ACS.Our results indicate that that in general the impact of the H1N1 influenza pandemic on patients with SCD was mild but that past clinical history correlated with the severity of illness. Additionally, effective hydroxyurea therapy and chronic transfusion therapy appeared to be protective against the incidence of ACS. Our results suggest guidelines for the management of patients with SCD during future influenza pandemics as well as during seasonal influenza epidemics.

The small Rho GTPases Rac1 and Rac2 regulate actin structures and mediate reactive oxygen species (ROS) production via NADPH oxidase in a variety of cells. We have demonstrated that deficiency of Rac1 and Rac2 GTPases in mice disrupts the normal hexagonal organization of the RBC cytoskeleton and reduces erythrocyte deformability. This is associated with increased phosphorylation of adducin at Ser-724, (corresponding to Ser-726 in human erythrocytes), a domain target of protein kinase C (PKC). PKC phosphorylates adducin and leads to decreased F-actin capping and dissociation of spectrin from actin, implicating a significant role of such phosphorylation in cytoskeletal remodeling. We evaluated adducin phosphorylation in erythrocytes from patients with sickle cell disease and found it consistently increased at Ser-726. In addition, ROS concentration is elevated in sickle erythrocytes by 150-250% compared to erythrocytes from normal control individuals. Here, we review previous studies demonstrating that altered phosphorylation of erythrocyte cytoskeletal proteins and increased ROS production result in disruption of cytoskeleton stability in healthy and sickle cell erythrocytes. We discuss in particular the known and potential roles of protein kinase C and the Rac GTPases in these two processes.

A 7-year-old girl with HbSE experienced fever and pain in her legs and back while on a car trip with her family, but did not seek medical attention. She complained of sudden respiratory distress and then collapsed. An emergency squad responded within minutes, and the child was evacuated by helicopter to a local tertiary care pediatric emergency room, where she was admitted in full cardiopulmonary arrest. After prolonged attempts at resuscitation, she was pronounced dead. An autopsy was performed. Hemoglobin E (HbE) is a beta chain variant characterized by lysine substitution for glutamic acid at amino acid 26 [b] [1]. The GAG to AAG mutation in the b-globin exon 1 also results in abnormal RNA splicing that produces an unstable mRNA [2], resulting in a mild thalassemic phenotype in individuals with homozygous HbE and a more severe phenotype when combined with beta thalassemia (HbE/beta thal) [3–7]. Due to a weakened interface between the a-globin and b-globin chains, HbE is also unstable [8], causing hemolysis that may be exacerbated by fever or oxidant stress [9]. HbE is common in Southeast Asia, where the gene frequency may be as high as 60% in some localities [8]. HbS is widely distributed in Africa and to a lesser degree in the eastern Mediterranean and India [10]. Compound heterozygosity for HbE and HbS (HbSE) is relatively rare, but there are a significant number of individuals with HbSE in India [11] and in Arab countries such as Oman [12]. With population admixture and racial intermarriages, the incidence of HbSE is increasing in the West [3,7,13]. The current literature generally associates HbSE with a benign clinical course, although vaso-occlusive complications have been reported [3–7]. The child in this case was born to an African-American father and a mother of mixed Cambodian and European descent, with no family history of sickle cell anemia, bleeding or clotting disorders, or pulmonary embolism. A newborn screen performed showed HbSE. Records of counseling the family received at the time of diagnosis are not available; however, they believed that the condition was benign and did not require follow-up care. The patient was not seen by a hematologist and was not placed on penicillin prophylaxis in infancy. At the age of five the child was referred to a pediatric orthopedic surgery clinic for complaints of intermittent leg pain that had begun six months earlier. A bone scan showed mildly increased uptake in the physis of the left tibia and femur (not shown). MRI evaluation revealed two areas of abnormal signal within the mid and distal left femoral diaphysis (see Fig 1), interpreted as most consistent with bone infarction. Review of her medical records at that time revealed the diagnosis of HbSE, and she was referred to a comprehensive sickle cell center. When first seen in the hematology clinic at age 5 [1/2], she appeared well, with growth parameters in the 50th percentile. Physical examination was normal, except for a palpable spleen tip. There was no jaundice and no cardiac murmur. Over the next 2 years, the patient’s hemoglobin averaged 11.4 gm/dl (114 gm/L) ranging from 10.5 to 11.5 gm/dl. Reticulocyte count was slightly elevated, typically 1.5–1.7% (ranging from 1.1 to 3.0%). Bilirubin was 0.9 to 1.4 mg/dL (normal: 0.0–1.1) and urine urobilinogen typically was 1.0 to 2.0 mg/dL (normal: 0.2–1.0). These findings suggested slight, but variable hemolysis; LDH was not measured. The red blood cells were microcytic, with a mean cell volume ranging from 68.8 to 74.2 fL (nl 77.0– 95.0 fL) and with mean cell hemoglobin averaging 26.4 pg. (nl 25.0–33.0 pg) Blood smear showed microcytosis, occasional target cells, rare sickle cells, mild hypochromia, and mild polychromasia, no Howell–Jolly bodies. Platelet counts ranged from 180,000 to 220,000 per lL. Electrolytes and urinalysis were normal. Quantification of hemoglobin by high performance liquid chromatography showed 55% HbS, 31% HbE, and 1.3% HbF, with normal amounts of hemoglobin A2 and minor hemoglobins. Between the ages of 5 [1/2] and 7 [1/2] years, the child was hospitalized twice for pain management of vaso-occlusive events. On one of these occasions, a repeat bone scan revealed decreased uptake in the proximal and distal left femur and distal right femur consistent with a new bone infarction (not shown). Cerebral MRI/MRA was normal (not shown). She was treated with narcotics and nonsteroidal anti-inflammatory agents for pain relief. During several comprehensive care visits over this time, she exhibited no jaundice or splenomegaly on physical exam. In the days before her death the patient complained intermittently of pain in her back and legs. By parental report, the patient was febrile the day she died, but it is not clear when the fever began. The onset of respiratory distress was acute and severe, and loss of consciousness occurred very shortly thereafter. On arrival

The transbilayer cholesterol distribution of human erythrocytes was examined by two independent techniques, quenching of dehydroergosterol fluorescence and fluorescence photobleaching of NBD-cholesterol. Dehydroergosterol in conjunction with leaflet selective quenching showed that, at equilibrium, 75% of the sterol was localized to the inner leaflet of resealed erythrocyte ghosts. NBD-cholesterol and fluorescence photobleaching displayed two diffusion values in both resealed ghosts and intact erythrocytes. The fractional contribution of the fast and slow diffusion constants of NBD-labelled cholesterol represent its inner and outer leaflet distribution. At room temperature the plasma membrane inner leaflet of erythrocyte ghosts as well as intact erythrocytes cells contained 78% of the plasma membrane sterol. The erythrocyte membrane transbilayer distribution of sterol was independent of temperature. In conclusion, dehydroergosterol and NBD-cholesterol data are consistent with an enrichment of cholesterol in the inner leaflet of the human erythrocyte.

New methods for labeling red blood cells (RBC) and monitoring their survival have made it possible to explore changes in the properties of RBC as they age in the circulation. We have adapted a method, originally developed for studying wild animals, to calculate the age of a random sample of labeled RBC from their survival curve. We also show how this method can be expanded to allow continuous calculation of the mean age of the labeled RBC population at any time after labeling. It is expected that this analytical approach will be useful in the study of age-dependent RBC changes.

A major assumption in the interpretation of HbA1c as a measure of glycemic control is that the duration of Hb exposure to glucose does not vary among patients who are hematologically normal. In this issue of Diabetes Care , Virtue et al. (1) use an appealingly simple method to quantitate erythrocyte lifespan (2,3) and report an inverse correlation with glycohemoglobin concentration in patients with diabetes. They interpret this as a reduced erythrocyte lifespan at higher blood glucose concentrations. The calculation of erythrocyte lifespan depends on Hb concentration and the amount of carbon monoxide (CO) in exhaled air and in atmospheric air collected at the subject’s home just after awakening. The principles behind the method are that heme catabolism is the only endogenous source of CO and that erythrocytes removed from the circulation are the major source of heme. However, there are multiple assumptions behind the methodology, which may not be met, in the complicated scenario of diabetes. These all come together in Eq. 1 of Virtue et al. (see [1] for the definition of terms) \batchmode \documentclass[fleqn,10pt,legalpaper]{article} \usepackage{amssymb} \usepackage{amsfonts} \usepackage{amsmath} \pagestyle{empty} \begin{document} \[\mathrm{Erythrocyte\ lifespan}{=}\frac{{[}\mathrm{Hb}{]}(22,400)(\mathrm{blood\ volume})}{0.7(\mathrm{endogenous}\ P\mathrm{\mbox{\textsc{co}}}(64,400)(1,440)(\mathrm{alveolar\ ventilation})}\] \end{document} which determines erythrocyte lifespan as the inverse of the CO excretion rate, estimating from CO the amount of heme metabolized, and then the amounts of Hb and, finally, red blood cells removed. Heme is a component not only of Hb but also of cytochromes and muscle myoglobin, with cytochromes being ubiquitous but particularly enriched in liver. The Hb concentration depends not only on erythrocyte mass but also on plasma volume; hence, it is sensitive to variations in …

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. (Blood. 2003;102: 365-370)

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.

KCl cotransport activated by swelling of sickle red blood cells (SS RBC) is inhibited by deoxygenation. Yet recent studies found a Cl−-dependent increase in sickle reticulocyte density with cyclic deoxygenation. This study sought to demonstrate cotransporter stimulation by deoxygenation of SS RBC in isotonic media with normal pH. Low-density SS RBC exhibited a Cl−-dependent component of the deoxygenation-induced net K+efflux, which was blocked by two inhibitors of KCl cotransport, [(dihydroindenyl)oxy]alkanoic acid and okadaic acid. Cl−-dependent K+efflux stimulated by deoxygenation was enhanced 2.5-fold by clamping of cellular Mg2+at the level in oxygenated cells using ionophore A-23187. Incubating cells in high external K+or Rb+minimized inhibition of KCl cotransport by internal Mg2+, and under these conditions deoxygenation markedly stimulated KCl cotransport in the absence of ionophore. Activation of KCl cotransport by deoxygenation of SS RBC in isotonic media at normal pH is consistent with the generalized dephosphorylation of membrane proteins induced by deoxygenation and activation of the cotransporter by a dephosphorylation mechanism. Na+/H+exchange activity, known to be modulated by cytosolic Ca2+elevation and cell shrinkage, remained silent under deoxygenation conditions.

Changes in a membrane sterol exchange of sickle red blood cells (SS RBC) induced by deoxygenation were studied using the fluorescent cholesterol analogue dehydroergosterol (DHE). DHE uptake by SS RBC membrane was measured by the incubation of SS RBC with small unilamellar vesicles (SUV) containing DHE. Deoxygenation of SS RBC, but not normal RBC, increased the rate of DHE uptake. DHE membrane content after 5 h of incubation with SUV in the cell-to-SUV ratio of 1:1 (mol lipid) was 16.25 +/- 0.94 and 12.22 +/- 0.85% of total sterol for deoxygenated and oxygenated cells, respectively. Membrane spicules isolated from these deoxygenated SS RBC had three-fold higher DHE content, suggesting that the increased sterol exchange was localized to spicules. When isolated spicules were incubated with DHE-SUV directly, 91 +/- 3% of membrane sterol was rapidly exchanged, in contrast to intact RBC, in which a maximum of 33% of sterol could be exchanged. The results suggest that spicule formation in SS RBC alters membrane cholesterol structure, such that a domain of cholesterol that is normally nonexchangeable becomes readily exchangeable with exogenous sterol.

Net cation movements were measured in low-density sickle red blood cells (SS RBC) in the presence and absence of oxygen. External Ca2+ (Ca2+o) partially inhibited deoxygenation-induced fluxes of both Na+ and K+. Deoxygenation-induced Na+ influx was reduced by 2 mM Ca2+o to 0.71 +/- 0.04 (SE) of its value in Ca(2+)-free solutions, whereas this ratio was 0.90 +/- 0.05 for K+ efflux (P < 0.01 by paired t-test). Because Ca2+o inhibited Na+ influx more than K+ efflux, net cation loss in deoxygenated SS RBC was higher in the presence of Ca2+o. In separate experiments, Ca2+o reduced deoxygenation-induced Na+ influx to 0.66 +/- 0.03 of its Ca(2+)-free value compared with 0.77 +/- 0.03 for Rb+ influx (P < 0.001), indicating relative selectivity of this effect for Na+ over Rb+. However, this effect is not specific for Ca2+ because other divalent cations also inhibited deoxygenation-induced Na+ and K+ fluxes. Under the conditions of these experiments, no evidence for K+ channel activation was found, indicating that K+ loss measured in deoxygenated SS RBC was mediated by the deoxygenation-induced pathway. These studies show that in the presence of Ca2+o deoxygenation-induced Na+ influx and K+ efflux are unbalanced. This pathway can, therefore, mediate cation loss and contribute directly to cellular dehydration in SS RBC.

Deoxygenation of sickle cells increases membrane permeability to Na, K, and Ca and contributes to cellular cation depletion. This study examines the physiological nature of the pathway that mediates deoxygenation-induced movements of monovalent cations. Deoxygenation-induced Rb influx was a linear function of external Rb concentration, with no evidence of saturation. Activation of the deoxygenation-induced pathway was fostered by alkaline pH (7.5), whereas ion movements via the activated pathway were optimal between pH 6.9 and 7.0 in cells incubated in media in which NO3 replaced Cl to eliminate KCl cotransport. The deoxygenation-induced pathway exhibited no selectivity among the alkali metal cations Li, Na, K, Rb, or Cs, but the monovalent organic cations tetramethylammonium, tetraethylammonium, and N-methylglucamine were excluded. Sickle cells incubated in low-Cl media (external Cl, 40 mM) to depolarize the membrane exhibited increased deoxygenation-induced K efflux and reduced Na influx. Cells treated with valinomycin to hyperpolarize the membrane showed increased deoxygenation-induced Na influx. These characteristics of the deoxygenation-induced transport pathway, linear concentration dependence, lack of cation selectivity, and response to membrane potential, argue against a carrier-mediated mechanism in favor of a diffusional process. The exclusion of small organic cations, however, suggests that factors other than ion size influence deoxygenation-induced permeability.