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.

Molecular defect in the sickle erythrocyte skeleton. Abnormal spectrin binding to sickle inside-our vesicles.

Although functional abnormalities of the sickle erythrocyte membrane skeleton have been described, there is little quantitative data on the function of the proteins that compose the skeleton. We have examined the association of spectrin, the major skeletal protein, with ankyrin, its high-affinity membrane binding site, and found sickle erythrocytes to have markedly reduced binding. Binding is assayed by incubation of purified 125I-spectrin with spectrin-depleted inside-out vesicles (IOVs) and measurement of the label bound to IOVs. Sickle IOVs bind approximately 50% less ankyrin than do controls IOVs (P less than 0.001). Control experiments show that this reduced binding is not a function of faulty composition or orientation of sickle IOVs, or of reticulocytosis per se. Our least symptomatic patient has the highest binding capacity, suggesting that this abnormality may be related to clinical severity. This trend is supported by experiments showing that asymptomatic subjects with sickle trait, sickle cell anemia and high fetal hemoglobin, and sickle beta +-thalassemia have normal binding, whereas a symptomatic patient with sickle beta zero-thalassemia has abnormal binding. In contrast to what we see with ankyrin in situ on the IOV, when isolated and studied in solution, sickle ankyrin binds normally to spectrin. This discrepancy may be related to preferential purification of the normal ankyrin species or to an abnormal topography of the membrane near the spectrin attachment site. We hypothesize that sickle hemoglobin or perhaps the metabolic consequences of sickling damage the protein skeleton. This damage may alter the surface of the erythrocyte and result in abnormal cell-cell interactions which may be related to clinical severity.

Exercise-induced hemolysis in xerocytosis. Erythrocyte dehydration and shear sensitivity.

A patient with xerocytosis was found to have swimming-induced intravascular hemolysis and shortening of erythrocyte life-span. In a microviscometer, xerocytes were more susceptible than normal erythrocytes to hemolysis by shear stress. Fractionation of normal and abnormal cells on discontinuous Stractan density gradients revealed that increasingly dehydrated cells were increasingly more shear sensitive. This sensitivity was partially corrected by rehydrating xerocytic erythrocytes by means of the cation-ionophore nystatin in a high potassium buffer. Conversely, normal erythrocytes were rendered shear sensitive by dehydrating them with nystatin in a low potassium buffer. This effect of dehydration was entirely reversible if normal cells were dehydrated for less than 4 h but was only partially reversed after more prolonged dehydration. It is likely that dehydration of erythrocytes results in shear sensitivity primarily because of concentration of cell contents and reduced cellular deformability. With prolonged dehydration, secondary membrane changes may potentiate the primary effect. This increased shear sensitivity of dehydrated cells may explain atraumatic exercise-induced hemolysis in xerocytosis as cardiac output is shifted to vessels of exercising muscles with small diameters and high shear rates.

Oral magnesium supplements reduce erythrocyte dehydration in patients with sickle cell disease.

Intracellular polymerization and sickling depend markedly on the cellular concentration of sickle hemoglobin (Hb S). A possible therapeutic strategy for sickle cell disease is based on reducing the cellular concentration of Hb S through prevention of erythrocyte dehydration. The K-Cl cotransporter is a major determinant of sickle cell dehydration and is inhibited by increasing erythrocyte Mg content. We studied 10 patients with sickle cell disease before treatment and after 2 and 4 wk of treatment with oral Mg supplements (0.6 meq/kg/d Mg pidolate). Hematological parameters, erythrocyte Na, K, and Mg content, erythrocyte density, membrane transport of Na and K, and osmotic gradient ektacytometry were measured. We found significant increases in sickle erythrocyte Mg and K content and reduction in the number of dense sickle erythrocytes. Erythrocyte K-Cl cotransport was reduced significantly. We also observed a significant reduction in the absolute reticulocyte count and in the number of immature reticulocytes. Ektacytometric analysis showed changes indicative of improved hydration of the erythrocytes. There were no laboratory or clinical signs of hypermagnesemia. Mild, transient diarrhea was the only reported side effect. We conclude that oral Mg supplementation reduces the number of dense erythrocytes and improves the erythrocyte membrane transport abnormalities of patients with sickle cell disease.

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.

Hydroxyurea enhances fetal hemoglobin production in sickle cell anemia.

Hydroxyurea, a widely used cytotoxic/cytostatic agent that does not influence methylation of DNA bases, increases fetal hemoglobin production in anemic monkeys. To determine its effect in sickle cell anemia, we treated two patients with a total of four, 5-d courses (50 mg/kg per d, divided into three oral doses). With each course, fetal reticulocytes increased within 48-72 h, peaked in 7-11 d, and fell by 18-21 d. In patient I, fetal reticulocytes increased from 16.0 +/- 2.0% to peaks of 37.7 +/- 1.2, 40.0 +/- 2.0, and 32.0 +/- 1.4% in three successive courses. In patient II the increase was from 8.7 +/- 1.2 to 50.0 +/- 2.0%. Fetal hemoglobin increased from 7.9 to 12.3% in patient I and from 5.3 to 7.4% in patient II. Hemoglobin of patient I increased from 9.0 to 10.5 g/dl and in patient II from 6.7 to 9.9 g/dl. Additional single-day courses of hydroxyurea every 7-20 d maintained the fetal hemoglobin of patient I t 10.8-14.4%, and the total hemoglobin at 8.7-10.8 g/dl for an additional 60 d. The lowest absolute granulocyte count was 1,600/mm3; the lowest platelet count was 390,000/mm3. The amount of fetal hemoglobin per erythroid burst colony-forming unit (BFU-E)-derived colony cell was unchanged, but the number of cells per BFU-E-derived colony increased. Although examination of DNA synthesis in erythroid marrow cells in vitro revealed no decreased methylcytidine incorporation, Eco RI + Hpa II digestion of DNA revealed that hypomethylation of gamma-genes had taken place in vivo after treatment. This observation suggests that hydroxyurea is a potentially useful agent for the treatment of sickle cell anemia and that demethylation of the gamma-globin genes accompanies increased gamma-globin gene activity.

Glycosylation of variant hemoglobins in normal and diabetic subjects.

The extent of in vivo glycosylation of variant hemoglobins was examined in individuals with S-, C-, and D-trait. Chromatographic estimates of glycosylation for nondiabetic individuals with S-trait were significantly lower than those for nondiabetic black subjects with normal hemoglobin (P < 0.001). However, chemical determinations of glycosylation (thiobarbituric acid or TBA technique) were similar for these groups (P > 0.10). The chromatographic elution pattern of hemoglobin S (HbS) was determined, and on this basis an adjustment procedure was performed for chromatographic data. A regression line was calculated for the relationship between chromatographic and colorimetric estimates of glycosylated hemoglobin in S-trait individuals with and without diabetes. The slope of this line was significantly different (P < 0.001) from that for the relationship in individuals with normal hemoglobin. However, after adjustment of chromatographic values from S-trait individuals, the slopes were similar (P < 0.10). Findings from individuals heterozygous for HbC and D were similar to those for individuals with S-trait. These data indicate that the extent of glycosylation of HbS, C, and D is similar to that of HbA in both the normoglycemic and hyperglycemic range. The TBA technique is the most direct method for determining the extent of glycosylation in individuals with HbS, C, or D. However, adjustment of column chromatographic values is feasible.

Reticulocyte hemoglobin: an integrated parameter for evaluation of erythropoietic activity.

Traditional reticulocyte counts provide only a partial estimate of the erythropoietic bone marrow activity and do not account for qualitative variations of reticulocyte cellular indexes and hemoglobin content in particular. We have studied a new integrated parameter, reticulocyte hemoglobin (retHb), that quantifies in grams per liter the hemoglobin contained in the circulating reticulocyte compartment and is obtained by multiplying the absolute reticulocyte count and the reticulocyte cell hemoglobin content. In 50 normal control subjects, retHb values were 1.76 +/- 0.59 g/L. The retHb values were lower in patients double heterozygous for HbS and HbC (SC disease) (3.33 +/- 1.52 g/L, n = 13) compared with homozygous HbS disease (SS) with concomitant alpha-thalassemia (5.27 +/- 1.51 g/L and 5.48 +/- 1.06 g/L for 12 patients with 3 alpha-genes and 3 patients with 2 alpha-genes, respectively) and to SS disease with no alpha-thalassemia (6.47 +/- 3.05, n = 20). The hemoglobin contained in the red blood cell pool (rbcHb) also can be calculated by subtracting retHb from the total hemoglobin. The ratio between the two pools (rbcHb/retHb, normal value 76.6 +/- 21.9, n = 50) provides a rough estimate of red blood cell survival. It was 9.8 +/- 4.1 in SS disease, 16.2 +/- 10.1 and 14.7 +/- 5.0 in SS disease with 3 and 2 normal alpha-genes, respectively, and 36.6 +/- 17.8 in SC disease with no alpha-thalassemia. We also studied retHb in patients receiving hydroxyurea therapy for SS disease, intravenous or oral iron for iron deficiency, or recombinant human erythropoietin (r-HuEPO) therapy. All these conditions are characterized by changes in reticulocyte counts and marked variations in reticulocyte cellular hemoglobin contents, which can be integrated into the retHb parameter. Measurement of retHb and the rbcHb/retHb ratio may provide an estimate of the reduction in red blood cell survival and the severity of hemolysis in various anemias and allow more precise monitoring of the response to hydroxyurea, iron, r-HuEPO, or other therapies.

Predicting medical student success in a clinical clerkship by rating students' nonverbal behavior.

OBJECTIVE: To evaluate the relative contribution of affective and cognitive skills to ratings of students' clinical performance by their supervisors. METHODS: Each students' nonverbal behavior was analyzed by examining 33 nonverbal behavioral characteristics in videotape of each of 36 students interviewing a parent or patient three times during a 4-week pediatric clerkship. These ratings were then compared with the student's formal academic evaluation. RESULTS: The 33 nonverbal behavioral characteristics rated for each student were reduced to five composite variables. Three of these correlated significantly with the final grade, providing an affective profile of the highly rated student. Regression analysis of the five composite variables revealed that affective skills accounted for at least 46% of the variance in the students' final grades (multiple R = .68, P = .0015). CONCLUSIONS: Ratings of students' affective characteristics were highly related to clinical evaluations in this pediatric setting. Students who were evaluated highly had a specific affective profile that could be described by analysis of their nonverbal behaviors.

Chemotherapy to increase fetal hemoglobin in patients with sickle cell anemia.

The obvious beneficial effects of hemoglobin F on sickling have motivated numerous investigators to increase this type of hemoglobin artificially in patients with sickle cell anemia. Various chemotherapeutic agents including 5-azacytidine, hydroxyurea, and cytosine arabinoside, have been used successfully in patients. All of these drugs can increase the level of hemoglobin F in sickle cell anemia (SS) patients, but the kinetics and magnitude of the responses are highly individual and variable. The mechanism or mechanisms responsible for the increased synthesis of hemoglobin F remain unknown. Further controlled studies in a limited number of patients with severe sickle cell disease will be necessary in order to work out a rational, safe treatment program suitable for wider use.

Exercise-induced hemolysis in sickle cell anemia: shear sensitivity and erythrocyte dehydration.

We describe a steady-state patient with sickle cell anemia (SS disease) who developed sporadic hemoglobinuria, historically related to vigorous exercise. We studied him and four other patients with SS disease and demonstrated exercise-induced hemoglobinemia. To see if SS erythrocytes were abnormally fragile when exposed to shear forces that could be generated in small vessels of exercising muscles, we exposed them to physiologic shear rates in a cone-plate viscometer. We show that SS erythrocytes are more shear sensitive than normal erythrocytes. This phenomenon is directly related to the presence of dehydrated cells as demonstrated by the increasing shear sensitivity of increasingly dehydrated cells separated on Stractan density gradients. Normal shear sensitivity could be restored to dehydrated layers by restoring normal hydration. Restoration of shear stability was complete in all layers except for the most dense ISC layer. A control group of patients with SC disease exhibited no exercise-induced hemoglobinemia, no abnormal shear sensitivity of whole blood, and only rare dehydrated ISCs. These studies suggest that the exercise-induced hemolysis in SS patients is related to the lysis of dehydrated, shear-sensitive cells. This same process may also contribute to the chronic hemolysis of SS disease--a phenomenon known to correlate with the numbers of dehydrated ISCs.

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.

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.