Transposing sequences between fetal and adult hemoglobins indicates which subunits and regulatory molecule interfaces are functionally related.

To correlate amino acid sequence changes with hemoglobin function we are carrying out a detailed recombinant analysis of the adult hemoglobin/fetal hemoglobin (HbA/HbF) systems. The important physiological differences between these two tetramers lie at unspecified sites in the 39 sequence substitutions of the 146 amino acids in their beta and gamma chains. In this paper, significant differences in the tetramer-dimer dissociation constants (referred to as tetramer "strength" or "stability") of adult (HbA) and fetal (HbF) hemoglobin tetramers have been used to probe the relationship between the allosteric, sliding interface and the effects of the allosteric regulator, 2,3-DPG, in promoting oxygen release. The single amino acid difference at the allosteric interfaces of these two hemoglobins, Glu-43(beta) --> Asp-43(gamma), which is not near the DPG binding site, leads to a significantly lower DPG response, approaching that of HbF. The results are inconsistent with the long-held idea that the replacement of His-143(beta) in HbA to Ser-143(gamma) in HbF is solely responsible for the lowered DPG response in HbF. On the other hand, the Val-1(beta) --> Gly-1(gamma) replacement near the DPG binding site has no effect on the DPG response. The replacement of His-116(beta) by the hydrophobic Ile-116(gamma) at the rigid alpha(1)beta(1) interface has a marginal yet detectable effect on the allosteric alpha(1)beta(2) interface. The results, overall, are interpreted using a model involving electrostatic coupling between certain side chains and extend the concept of a long-range relationship between some distant regions of the tetramer that are likely mediated through the central cavity.

Safety of hydroxyurea in children with sickle cell anemia: results of the HUG-KIDS study, a phase I/II trial. Pediatric Hydroxyurea Group.

Previous studies have determined the short-term toxicity profile, laboratory changes, and clinical efficacy associated with hydroxyurea (HU) therapy in adults with sickle cell anemia. The safety and efficacy of this agent in pediatric patients with sickle cell anemia has not been determined. Children with sickle cell anemia, age 5 to 15 years, were eligible for this multicenter Phase I/II trial. HU was started at 15 mg/kg/d and escalated to 30 mg/kg/d unless the patient experienced laboratory toxicity. Patients were monitored by 2-week visits to assess compliance, toxicity, clinical adverse events, growth parameters, and laboratory efficacy associated with HU treatment. Eighty-four children were enrolled between December 1994 and March 1996. Sixty-eight children reached maximum tolerated dose (MTD) and 52 were treated at MTD for 1 year. Significant hematologic changes included increases in hemoglobin concentration, mean corpuscular volume, mean corpuscular hemoglobin, and fetal hemoglobin parameters, and decreases in white blood cell, neutrophil, platelet, and reticulocyte counts. Laboratory toxicities typically were mild, transient, and were reversible upon temporary discontinuation of HU. No life-threatening clinical adverse events occurred and no child experienced growth failure. This Phase I/II trial shows that HU therapy is safe for children with sickle cell anemia when treatment was directed by a pediatric hematologist. HU in children induces similar laboratory changes as in adults. Phase III trials to determine if HU can prevent chronic organ damage in children with sickle cell anemia are warranted.

Silent cerebral infarcts in sickle cell anemia: a risk factor analysis. The Cooperative Study of Sickle Cell Disease.

BACKGROUND: Silent infarcts have been reported in 17% of young patients with sickle cell disease and are associated with impaired performance on standardized psychometric tests. Risk factors for the development of these lesions have not been identified. METHODS: Investigators in the Cooperative Study of Sickle Cell Disease performed a brain magnetic resonance imaging scan on sickle cell anemia patients age 5.9 years and older who had been followed according to the protocols of the Cooperative Study since birth. Individuals with a known history of cerebrovascular accident were excluded from this analysis. Patients with and without silent infarctions were compared with regard to clinical and laboratory parameters. RESULTS: The study sample included 42 patients (18.3%) with silent infarcts. Patients who had silent infarcts were significantly more likely to have a clinical history of seizure and a lower painful event rate. Lower hemoglobin level, increased leukocyte count, elevated pocked red blood cell count, and SEN betaS globin gene haplotype were associated also with the presence of silent infarcts. There was no relationship between silent infarcts and platelet count, fetal hemoglobin level, reticulocyte percentage, serum aspartate aminotransferase level, total bilirubin concentration, blood pressure, growth parameters, or presence of alpha-thalassemia. A multivariate model for silent infarction identified the following as risk factors: low pain event rate, history of seizure, leukocyte count >/=11.8 x 10(9)/L, and the SEN betaS globin gene haplotype. CONCLUSIONS: Patients with risk factors for silent infarcts should be evaluated for cerebrovascular disease. If evidence of infarction is found, consideration must be given to therapeutic intervention. At present, the appropriate treatment has not been determined.

Therapy with oral clotrimazole induces inhibition of the Gardos channel and reduction of erythrocyte dehydration in patients with sickle cell disease.

Pathologic water loss from sickle erythrocytes concentrates the abnormal hemoglobin and promotes sickling. The Ca2+-activated K+ channel (Gardos channel) contributes to this deleterious dehydration in vitro, and blockade of K+ and water loss via this channel could be a potential therapy in vivo. We treated five subjects who have sickle cell anemia with oral clotrimazole, a specific Gardos channel inhibitor. Patients were started on a dose of 10 mg clotrimazole/kg/d for one week. Protocol design allowed the daily dose to be escalated by 10 mg/kg each week until significant changes in erythrocyte density and K+ transport were achieved. Blood was sampled three times a week for hematological and chemical assays, erythrocyte density, cation content, and K+ transport. At dosages of 20 mg clotrimazole/kg/d, all subjects showed Gardos channel inhibition, reduced erythrocyte dehydration, increased cell K+ content, and somewhat increased hemoglobin levels. Adverse effects were limited to mild/moderate dysuria in all subjects, and a reversible increase in plasma alanine transaminase and aspartic transaminase levels in two subjects treated with 30 mg clotrimazole/kg/d. This is the first in vivo evidence that the Gardos channel causes dehydration of sickle erythrocytes, and that its pharmacologic inhibition provides a realistic antisickling strategy.

Membrane protein interactions in sickle red blood cells: evidence of abnormal protein 3 function.

The pattern of membrane abnormalities in sickle red blood cells suggests that sickle hemoglobin damages membrane proteins. We have previously shown a functional defect in sickle ankyrin, poor spectrin-binding ability. Here we examine the other major binding interactions of sickle membrane proteins including spectrin self-association, binding of ankyrin and protein 4.1 to protein 3, and the formation of the spectrin-actin-protein 4.1 complex. We found that sickle spectrin was normal in self-association and ability to participate in the spectrin-actin-protein 4.1 complex. Sickle protein 4.1 bound normally to protein 3 and formed normal complexes with actin and spectrin, even when sickle spectrin was used. The only major abnormality we found was a reduced ability of sickle protein 3 to bind ankyrin. This functional defect could not be explained experimentally on the basis of cysteine modification or enhanced tyrosine phosphorylation. We conclude that damage of sickle membrane proteins is not a diffuse scattershot process, but is largely confined to regions near membrane-associated hemoglobin, the spectrin-binding domain of ankyrin and the ankyrin-binding domain of protein 3. The mechanism and consequences of this damage continues to be investigated.

Sickle reticulocytes adhere to VCAM-1.

Adherence of sickle (SS) erythrocytes to endothelial cells represents interactions between red blood cell (RBC) and endothelial cell surface molecules. To enhance our understanding of the ligands involved, we transfected COS cells with the cDNAs of two endothelial cell adhesion molecules, VCAM-1 and E-selectin, and measured the binding of normal and sickle RBCs after static incubation. The percentage of COS cells with rosettes (five or more adherent RBCs) was determined. Normal RBCs did not adhere to VCAM-1-transfected COS cells. Unfractionated SS RBCs formed rosettes on the VCAM-1-transfected COS cells (mean +/- SD, 5.85% +/- 4.98%). Low-density SS RBCs were more adherent than high-density SS RBCs (P < .005). The adherent SS RBCs were a young reticulocyte population, staining positively for transferrin receptor. Another measure of reticulocyte age, RNA content, also correlated with adherence. SS RBC binding was specific--inhibitable by antibodies to either VCAM-1 or the alpha 4 integrin chain of VLA-4. In contrast, there was no significant adherence of normal or SS RBCs to E-selectin-transfected COS cells. These results suggest that young reticulocytes bind to endothelial cell VCAM-1 via VLA-4 integrin.

Mortality in sickle cell disease. Life expectancy and risk factors for early death.

BACKGROUND: Information on life expectancy and risk factors for early death among patients with sickle cell disease (sickle cell anemia, sickle cell-hemoglobin C disease, and the sickle cell-beta-thalassemias) is needed to counsel patients, target therapy, and design clinical trials. METHODS: We followed 3764 patients who ranged from birth to 66 years of age at enrollment to determine the life expectancy and calculate the median age at death. In addition, we investigated the circumstances of death for all 209 adult patients who died during the study, and used proportional-hazards regression analysis to identify risk factors for early death among 964 adults with sickle cell anemia who were followed for at least two years. RESULTS: Among children and adults with sickle cell anemia (homozygous for sickle hemoglobin), the median age at death was 42 years for males and 48 years for females. Among those with sickle cell-hemoglobin C disease, the median age at death was 60 years for males and 68 years for females. Among adults with sickle cell disease, 18 percent of the deaths occurred in patients with overt organ failure, predominantly renal. Thirty-three percent were clinically free of organ failure but died during an acute sickle crisis (78 percent had pain, the chest syndrome, or both; 22 percent had stroke). Modeling revealed that in patients with sickle cell anemia, the acute chest syndrome, renal failure, seizures, a base-line white-cell count above 15,000 cells per cubic millimeter, and a low level of fetal hemoglobin were associated with an increased risk of early death. CONCLUSIONS: Fifty percent of patients with sickle cell anemia survived beyond the fifth decade. A large proportion of those who died had no overt chronic organ failure but died during an acute episode of pain, chest syndrome, or stroke. Early mortality was highest among patients whose disease was symptomatic. A high level of fetal hemoglobin predicted improved survival and is probably a reliable childhood forecaster of adult life expectancy.

A highly conserved region of human erythrocyte ankyrin contains the capacity to bind spectrin.

Ankyrin has a spectrin-binding region within a central 62-kDa chymotryptic peptide. We examined the spectrin binding ability of a series of smaller ankyrin fragments and recombinant peptides within the 62-kDa domain using a ligand blot assay. The smallest proteolytic fragment that bound was a 12-kDa tryptic peptide starting at amino acid 1068. Peptides containing this region expressed as glutathione S-transferase fusion products also bound spectrin and suggested that residues 1101-1192 were important. In contrast, a fusion protein containing residues 826-898 did not bind spectrin, a surprising finding since this region is known to influence binding affinity. Proteins that bound spectrin on ligand blots also competed for binding in solution, but did so with one-tenth the affinity of the native peptide. Comparing the 62-kDa domains of erythrocyte and brain ankyrins (species that bind spectrin but with 10-fold differences in affinity), the NH2-terminal regions are 0-40% identical, while the regions (1136-1160) common to all binding peptides are 80-90% identical. We hypothesize that the highly conserved region contains an important spectrin-binding site, while the poorly conserved region controls the binding affinity. We speculate that this unique NH2-terminal region is what gives different members of the ankyrin family their signature set of affinities, and accordingly their distinctive cellular localization.

Hydroxyurea: effects on hemoglobin F production in patients with sickle cell anemia.

Patients with sickle cell anemia were treated with daily doses of hydroxyurea, to assess pharmacokinetics, toxicity, and increase in fetal hemoglobin (Hb) production in response to the drug. Plasma hydroxyurea clearances were not a useful guide to maximum tolerated doses of the drug. The mean daily single oral dose that could be maintained for at least 16 weeks was 21 mg/kg (range, 10 to 35 mg/kg). Among 32 patients, last HbF levels were 1.9% to 26.3% (mean, 14.9%) with increases in HbF over initial values of 1.4% to 20.2% (mean, 11.2%). The most significant predictors of last HbF were last plasma hydroxyurea level, initial white blood count and initial HbF concentration. Last HbF was not related to beta globin haplotype or alpha globin gene number. No serious toxicity was encountered. Clinically significant bone marrow depression was avoided, and chromosome abnormalities after 2 years of treatment were no greater than those observed before treatment. The period of observation has been too short to evaluate the risk of carcinogenesis. Patient's red cells developed striking macrocytosis. Median red cell Hb concentrations did not change. Hb concentrations increased, on average 1.2 g/dL, but serum erythropoietin levels increased. Patients' body weights increased, and some returned to work or school, but no conclusions regarding therapeutic efficacy could be drawn from this uncontrolled open-label study.

Pain in sickle cell disease. Rates and risk factors.

BACKGROUND AND METHODS: Acute episodes of pain are the principal symptom of sickle cell disease, but little is known about the epidemiologic features of these episodes or risk factors for them, nor is it known whether patients with high rates of such episodes die prematurely. We prospectively studied the natural history of sickle cell disease in 3578 patients ranging from newborns to persons up to 66 years old who were followed at clinical centers across the United States. RESULTS: There were 12,290 episodes of pain in 18,356 patient-years. The average rate was 0.8 episode per patient-year in sickle cell anemia, 1.0 episode per patient-year in sickle beta 0-thalassemia, and 0.4 episode per patient-year in hemoglobin SC disease and sickle beta(+)-thalassemia. The rate varied widely within each of these four groups--e.g., 39 percent of patients with sickle cell anemia had no episodes of pain, and 1 percent had more than six episodes per year. The 5.2 percent of patients with 3 to 10 episodes per year had 32.9 percent of all episodes. Among patients with sickle cell anemia who were more than 20 years old, those with high rates of pain episodes tended to die earlier than those with low rates. High rates were associated with a high hematocrit and low fetal hemoglobin levels. alpha-Thalassemia had no effect on pain apart from its association with an increased hematocrit. CONCLUSIONS: The "pain rate" (episodes per year) is a measure of clinical severity and correlates with early death in patients with sickle cell anemia over the age of 20. Even when the fetal hemoglobin level is low, one can predict that small increments in the level may have an ameliorating effect on the pain rate and may ultimately improve survival. This outcome is particularly encouraging to investigators studying hydroxyurea and other treatments designed to increase the fetal hemoglobin level.

Membrane protein lesions in erythrocytes with Heinz bodies.

We studied Heinz body-containing erythrocytes with three different unstable hemoglobins: Nottingham, Brockton, and unclassified. We demonstrated two classes of membrane protein defects in unstable hemoglobin-containing cells (UH-RBCs), a defect of the spectrin-depleted inside-out vesicle (UH-IOV), and a defect of spectrin (UH-spectrin) itself. The composition of UH-IOVs is the same as control with respect to quantity of ankyrin and proportion inside-out. However, UH-IOVs bind even less spectrin than IOVs derived from sickle erythrocytes (SS-IOVs), suggesting a severe functional defect in the ankyrin of UH-RBCs (UH-ankyrin). Further evidence that UH-ankyrin is abnormal is demonstrated by the virtual absence of ankyrin in isotonic membrane shells of UH-RBCs (UH-shells), and abnormal mobility and decreased binding of the 72-kD (spectrin-binding) alpha-chymotryptic fragment of UH-ankyrin (UH-72-kD) to control spectrin. All UH-RBC membranes were spectrin-deficient (60% of control). In addition, spectrin isolated from UH-RBCs (UH-spectrin) was abnormal in two respects: (a) presence of a fast-moving band on nondenaturing polyacrylamide gels of both 0 degree C and 37 degrees C extracts, and (b) decreased binding to actin in the presence of protein 4.1. UH-spectrin did exhibit normal self-association, binding to IOVs and binding to actin in the absence of protein 4.1. This pattern of normal and abnormal spectrin functions has been described for spectrin subjected to mild diamide oxidation, suggesting the role of oxidation is the pathogenesis of membrane defect(s) of erythrocytes with abnormal hemoglobins.

Cation depletion by the sodium pump in red cells with pathologic cation leaks. Sickle cells and xerocytes.

The mechanism by which sickle cells and xerocytic red cells become depleted of cations in vivo has not been identified previously. Both types of cells exhibit elevated permeabilities to sodium and potassium, in the case of sickle cells, when deoxygenated. The ouabain-insensitive fluxes of sodium and potassium were equivalent, however, in both cell types under these conditions. When incubated 18 hours in vitro, sickle cells lost cations but only when deoxygenated. This cation depletion was blocked by ouabain, removal of external potassium, or pretreatment with 4,4'-diisothiocyanostilbene-2,2'-disulfonate, which blocks the increase in cation permeability induced by deoxygenation. The loss of cation exhibited by oxygenated xerocytes similarly incubated was also blocked by ouabain. These data support the hypothesis that the elevated "passive" cation fluxes of xerocytes and deoxygenated sickle cells are not directly responsible for cation depletion of these cells; rather, these pathologic leaks interact with the sodium pump to produce a net loss of cellular cation.