Biomechanics and biorheology of red blood cells in sickle cell anemia

Sickle cell anemia (SCA) is an inherited blood disorder that causes painful crises due to vaso-occlusion of small blood vessels. The primary cause of the clinical phenotype of SCA is the intracellular polymerization of sickle hemoglobin resulting in sickling of red blood cells (RBCs) in deoxygenated conditions. In this review, we discuss the biomechanical and biorheological characteristics of sickle RBCs and sickle blood as well as their implications toward a better understanding of the pathophysiology and pathogenesis of SCA. Additionally, we highlight the adhesive heterogeneity of RBCs in SCA and their specific contribution to vaso-occlusive crisis.     

A Ca2+-refractory state of the Ca-sensitive K+ permeability mechanism in sickle cell anaemia red cells

Simultaneous measurements of Ca content and ⁴²K⁺ influx in sickle cell anaemia red cells confirm predictions from earlier data in the literature that the increased Ca content of sickle cell anaemia cells which are not metabolically depleted does not cause a quinine-sensitive increase in K⁺ permeability.It is shown that the ionophore, A23187, can cause the Ca contained inside sickle cell anaemia cells to activate the quinine-sensitive K⁺-permeability mechanism. This demonstrates the existence of a Ca²⁺-refractory state of the K⁺ channel in sickle cell anaemia cells and a direct stimulatory effect of the ionophore A23187 on its Ca sensitivity.     

Linus Pauling and sickle cell disease

The 1949 paper by Linus Pauling et al. [Science 110 (1949) 543–548] describing the discovery of sickle cell anemia as the first molecular disease had a major impact on biology and medicine. Inspired by the scholarly works of John Edsall on the history of hemoglobin research, I present a brief retrospective analysis of Pauling's paper. This is followed by some personal recollections of Edsall and Pauling.     

Erythrocytic ecdysis in smears of EDTA venous blood in eight patients with sickle cell anemia

The light-microscope finding of red cell membrane fragments in the form of long filamentous processes and myelin bodies in the blood smears of a patient with sickle cell anemia has recently been described. This phenomenon has been termed erythrocytic ecdysis. We examined the blood smears of all sickle cell anemia patients admitted to the Cook County Hospital and those attending the hemoglobinopathy clinic between October 1979 and December 1981. Nine instances of erythrocytic ecdysis were uncovered. Associated clinical conditions included congestive heart failure, acute viral syndrome, pneumonia, and metastatic malignancy. Transient ecdysis associated with congestive heart failure was noted for one patient during two separate admissions one year apart. Ecdysis is a transient form of erythrocytic fragmentation occurring in sickle cell anemia. Its pathogenesis is unknown. The role of regional circulatory stasis and hypoxia in the induction of erythrocyte membrane damage in sickle cell anemia needs investigation.     

Effects of membrane acting-drugs on plasmodium species and sickle cell erythrocytes

The effects of several membrane-acting drugs on malaria and sickle cell anemia was studied. In the initial experiments, propranolol and W-7 were shown to increase red cell density.In vitro, these drugs inhibited the growth ofP. falciparum. However,in vivo experiments using the murine malarial parasite,P. vinckei, demonstrated little, if any, anti-parasite activity with the doses of drugs employed. Subsequently, prostaglandin oligomeric derivatives were found to inhibit the growth ofP. falciparum in vitro andP. vinckei in vivo. Since prostaglandin oligomers inhibited the formation of dense, dehydrated cells (irreversible sickle cells), they may also have therapeutic efficacy in sickle cell anemia.     

Membrane components in the red cells of patients with sickle cell anemia Relationship to cell aging and to irreversibility of sickling

Cholesterol, phospholipid and sialic acid were measured in red cells from patients with sickle cell anemia to determine whether the cells had abnormal concentrations of these components and whether the amounts of these compounds differed in irreversibly sickled cells as compared to non-irreversibly sickled cells. Sickle cells had significantly higher levels of both lipids than similar populations of normal cells, however, comparisons to populations of young control cells showed that the differences were generally not significant. Sialic acid levels in sickle cells were not significantly different from normal cells. Irreversibly sickled cells had lower lipid and sialic acid concentrations than those not irreversibly sickled, but the differences were either not significant or did not occur when compared to young control cells. The studies show that the increased lipid concentrations in the membrane of sickle cells are not abnormal but are related to cell age and that the decrease in membrane components in irreversibly sickled cells is no greater than would be predicted for similarly aged populations of cells.     

Pyridyl derivatives of benzaldehyde as potential antisickling agents

Compounds that bind to sickle hemoglobin (Hb S) producing an allosteric shift to the high-affinity Hb S that does not polymerize are being developed to treat sickle cell anemia (SCA). In this study, three series of pyridyl derivatives of substituted benzaldehydes (Classes I-III) that combine structural features of two previously determined potent antisickling agents, vanillin and pyridoxal, were synthesized. When analyzed with normal human whole blood, the compounds form Schiff-base adducts with Hb and left shift the oxygen equilibrium curve (OEC) to the more soluble high-affinity Hb, more than vanillin or pyridoxal. Generally, Class-I compounds with an aromatic aldehyde located ortho to the pyridyl substituent are the most potent, followed by the Class-II compounds with the aldehyde at the meta-position. Class-III compounds with the aldehyde at the para position show the weakest activity. The structure-activity studies of these pyridyl derivatives of substituted benzaldehydes demonstrate significant allosteric potency that may be useful for treating SCA.     

The effects of hydrazine on sickle cells

The response of the red cells from patients with sickle cell disease to hydrazine treatment in vitro is to inhibit the sickled morphology, while the metabolic characteristics and the osmotic fragility of the cells remain unaltered. However, the oxygen affinity of the sickle cell haemoglobin is decreased.     

Prevalence of diabetes mellitus in individuals heterozygous and homozygous for sickle cell anemia

Colorimetric determinations of glycosylated Hb were carried out in a sample (n = 97) of sickle cell anemia patients, and in an age- and sex-matched group of individuals (n = 45) heterozygous for sickle cell anemia, from the Eastern Province of Saudi Arabia. A statistically significant increase in the value of glycosylated Hb was found in sickle cell trait (HbAS) group, when compared with those of sickle cell anemia (HbSS) and normal (HbAA) groups. Since glycosylated Hb is considered a valid indicator of long-term blood glucose, and assuming normal red blood cell survival in HbAS carriers, the increased value of glycosylated Hb may suggest that there exists a higher incidence of undiagnosed diabetes mellitus in individuals with heterozygous inheritance for sickle cell hemoglobin than homozygous sickle cell patients and normal individuals. The mechanism underlying this observation remains to be defined.     

Comparative study of human red blood cell analysis with three different field-flow fractionation systems

An extensive multi-laboratory study was conducted to compare three different field-flow fractionation (FFF) systems for use in the analysis of human erythrocytes. The object of this study was to determine the relationship between the FFF elution properties for each system and the traditional hematological blood cell parameters. One centrifugal system (Utah) and two gravitational systems (Paris and Abbott) were compared. In order to analyze erythrocyte populations with a broad range of hematological indices, blood samples were collected from individuals heterozygous for sickle cell anemia (A/S) and also from normal controls (A/A), and these were analyzed at each site. Identical samples were analyzed by the Abbott and Utah sites. With all three systems, blood samples from each category produced narrow, overlapping distributions of FFF retention ratios, with the Abbott and Utah systems showing slight elevations in the mean retention ratios for the sickle cell samples. Blood cell elution peak characteristics were compared with standard hematological parameters for each of the FFF systems, and negative correlations were consistently found between mean corpuscular volume (MCV) and retention ratios. Positive correlations were found between red cell distribution width (RDW) and retention ratios. Elevated FFF retention ratios were frequently found with blood samples having abnormal hematological profiles. These results demonstrate that the three differently configured systems all produce similar analysis profiles for erythrocytes from the classes studied here. The relationships between FFF parameters and hematological indices were consistent for all systems.     

Oxidative status of valinomycin-resistant normal, β-thalassemia and sickle red blood cells

Exposure of red blood cells (RBC) to the K⁺-ionophore valinomycin (val), causes loss of KCl and water, resulting in cell dehydration, manifested by increased cell density. While almost all normal val-treated RBC dehydrate, in sickle cell anemia (SCA) a portion of the RBC fail to dehydrate and maintain a light density, indicating the existence of val-resistant (val-res) RBC. In thalassemia and sickle cell disease (SCD), although the primary lesion is in the globin genes, damage to the RBC is partly mediated by oxidative stress. We previously showed that such RBC are under oxidative stress, having more reactive oxygen species (ROS) and less reduced glutathione than normal RBC. We now report a relationship between the phenomenon of val-res and the RBC oxidative status: Treatment with oxidants that increase ROS, also increased the frequency of val-res cells. Val-res cells had higher oxidative status than other RBC in the sample. Similar to SCA, thalassemic blood has more val-res cells than does normal blood. Val-res cells in thalassemic and sickle blood showed a higher oxidative status than normal val-res cells. Thus, oxidative stress might be involved in generation of val-res cells. Further studies are required to elucidate the origin and significance of these cells.     

Bantu beta s cluster haplotype predominates among Brazilian blacks.

We describe the combination of polymorphic restriction-enzyme sites in the beta globin gene cluster (haplotypes) for 74 chromosomes from Brazilian Blacks bearing the sickle hemoglobin gene (beta s). The three most common African beta s haplotypes account for 67 chromosomes: 49/74 (66.2%) were identified as Central African Republic (CAR or Bantu) type, 17 (23.0%) as Benin, and one as Senegal; seven chromosomes (9.5%) had minor atypical haplotypes. This distribution is different from that observed in the United States or Jamaica, where the Benin haplotype predominates, and results from different patterns of slave trades to North and South Americas. Since the beta s gene cluster polymorphisms modulate the severity of sickle cell anemia, this heterogeneity may explain differences of the clinical behavior of the disease in the United States and South America, and should also be considered in relation to other features and diseases.     

Hematological and hemorheological Determinants of the Six-Minute Walk Test Performance in Children with Sickle Cell Anemia

The six-minute walk test is a well-established submaximal exercise reflecting the functional status and the clinical severity of sickle cell patients. The aim of the present cross-sectional study was to investigate the biological determinants of the six-minute walk test performance in children with sickle cell anemia. Hematological and hemorheological parameters, pulmonary function and the six-minute walk test performance were determined in 42 children with sickle cell anemia at steady state. The performance during the six-minute walk test was normalized for age, sex and height and expressed as percentage of the predicted six-minute walk distance. We showed that a high level of anemia, a low fetal hemoglobin expression and low red blood cell deformability were independent predictors of a low six-minute walk test performance. This study describes for the first time the impact of blood rheology in the six-minute walk test performance in children with sickle cell anemia.     

Activation state of alpha4beta1 integrin on sickle red blood cells is linked to the duffy antigen receptor for chemokines (DARC) expression

In sickle cell anemia, reticulocytes express enhanced levels of α4β1 integrin that interact mainly with vascular cell adhesion molecule-1 and fibronectin, promoting vaso-occlusion. These interactions are known to be highly sensitive to the inflammatory chemokine IL-8. The Duffy antigen receptor for chemokines (DARC) modulates the function of inflammatory processes. However, the link between α4β1 activation by chemokines and DARC erythroid expression is not or poorly explored. Therefore, the capacity of α4β1 to mediate Duffy-negative and Duffy-positive sickle reticulocyte (SRe) adhesion to immobilized vascular cell adhesion molecule-1 and fibronectin was evaluated. Using static adhesion assays, we found that, under basal conditions, Duffy-positive SRe adhesion was 2-fold higher than that of Duffy-negative SRes. Incubating the cells with IL-8 or RANTES (regulated on activation normal T cell expressed and secreted) increased Duffy-positive SRe adhesion only, whereas Mn(2+) increased cell adhesion independently of the Duffy phenotype. Flow cytometry analyses performed with anti-β1 and anti-α4 antibodies, including a conformation-sensitive one, in the presence or absence of IL-8, revealed that Duffy-positive and Duffy-negative SRes displayed similar erythroid α4β1 expression levels, but with distinct activation states. IL-8 did not affect α4β1 affinity in Duffy-positive SRes but induced its clustering as corroborated by immunofluorescence microscopy. Our results indicate that in Duffy-negative SRes α4β1 integrin is constitutively expressed in a low affinity state, whereas in Duffy-positive SRes α4β1 is expressed in a higher chemokine-sensitive affinity state. This activation state associated with DARC RBC expression may influence the intensity of the inflammatory responses encountered in sickle cell anemia and participate in its interindividual clinical expression variability.     

Hepatic sequestration in sickle cell anaemia.

Several episodes of acute hepatic enlargement associated with a dramatic fall in haemoglobin concentration were observed in two patients with sickle cell anaemia. No appreciable disturbances of liver function or signs of cardiac failure were evident. The most likely mechanism was sequestration of sickled erythrocytes in the liver. This complication, which may have a basis similar to that of splenic sequestration and the sickle lung syndrome, may be easily overlooked unless the size of the liver is regularly monitored in patients with sickle cell crisis.     

Hydroxycarbamide: Clinical aspects

Due to its oral route of administration and mild toxicity profile, as well as its potent laboratory and clinical effects, hydroxyurea (or hydroxycarbamide) has been the primary focus of fetal hemoglobin (HbF) induction strategies for the treatment of children with sickle cell anemia (SCA). When administered orally once a day, hydroxyurea treatment is very well tolerated with little short-term toxicity. Hydroxyurea has documented laboratory efficacy with increases in Hb and HbF; treatment also significantly reduces the number of painful episodes, acute chest syndrome, transfusions, and hospitalizations. Most young patients reach a maximum tolerated dose of hydroxyurea at 25–30 mg/kg/d, where they will achieve key laboratory thresholds (Hb ≥ 9 g/dL and HbF ≥ 20%) without excessive myelosuppression. Potential long-term toxicities continue to be of great concern and should be monitored in all patients with SCA who receive hydroxyurea therapy. To date, however, no increases in stroke, myelodysplasia, or carcinogenicity have been detected in SCA patient cohorts, with drug exposure now reaching 15 years for some treated children. Taken together, available evidence suggests that hydroxyurea represents an inexpensive and effective treatment option that should be offered to most, if not all, patients with SCA. As countries in Africa develop newborn screening programs to identify SCA, the widespread use of hydroxyurea may prove to be a useful treatment to help ameliorate the disease in resource-limited settings. Hydroxyurea is the only currently available disease-modifying therapy for SCA, and is emerging as a safe and effective treatment for all patients with SCA, in both developed and developing countries.     

Evaluation of Th17 related cytokines associated with clinical and laboratorial parameters in sickle cell anemia patients with leg ulcers

Leg ulcers (LUs) represent one of the main causes of morbidity in sickle cell anemia (SCA). This manifestation has been related to hemolysis, infections predisposition and inflammation that leads cytokines secretion. In this context, our study aimed to evaluate Th17 related cytokines (IL-6, IL-17A, IL-22 and IL-23) in serum and peripheral mononuclear cells culture supernatants with and without lymphoproliferative stimulation (anti-human CD3 and anti-human CD28). The cytokines levels were also correlated to clinical, hematological and biochemical parameters in SCA patients with and without LUs history (SCALU and SCAWH) as well as in healthy controls. In SCALU patients, high levels of IL-17A were associated with absence of acute chest syndrome (ACS, p = 0.0328). The other clinical parameters analyzed (osteonecrosis, stroke, priapism, splenectomy and blood transfusions history) were not significantly related with other cytokine levels. In SCALU patients was also observed that IL-17A increased levels were associated with high levels of LDH (p = 0.0130), the same association pattern was found for IL-6 (0.0160) and IL-22 (p = 0.0165) in the SCALU group. Interestingly, we did not find statistical correlations with these parameters in SCAWH group. The other hematological parameters (hemoglobin, leucocyte and reticulocyte count) and indirect bilirrubin did not show any correlation with analyzed cytokines in both groups. So, for the first time, we show that IL-17A present in SCALU patients may exert a preventive role in the ACS development. Furthermore, IL-6, IL-17A and IL-22 accompanied the LDH levels only in SCALU patients suggesting to serve as additional markers of hemolysis or to be related with immunity response against extracellular pathogens.     

Chronic inflammatory state in sickle cell anemia patients is associated with HBB*S haplotype

The chronic inflammatory state in sickle cell anemia (SCA) is associated with several factors such as the following: endothelial damage; increased production of reactive oxygen species; hemolysis; increased expression of adhesion molecules by leukocytes, erythrocytes, and platelets; and increased production of proinflammatory cytokines. Genetic characteristics affecting the clinical severity of SCA include variations in the hemoglobin F (HbF) level, coexistence of alpha-thalassemia, and the haplotype associated with the HbS gene. The different haplotypes of SCA are Bantu, Benin, Senegal, Cameroon, and Arab-Indian. These haplotypes are associated with ethnic groups and also based on the geographical origin. Studies have shown that the Bantu haplotype is associated with higher incidence of clinical complications than the other haplotypes and is therefore considered to have the worst prognosis. This study aimed to evaluate the profile of the proinflammatory cytokines interleukin-6, tumor necrosis factor-α, and interleukin-17 in patients with SCA and also to assess the haplotypes associated with beta globin cluster S (HBB^*S). We analyzed a total of 62 patients who had SCA and had been treated with hydroxyurea; they had received a dose ranging between 15 and 25 (20.0 ± 0.6) mg/kg/day for 6–60 (18 ± 3.4) months; their data were compared with those for 30 normal individuals. The presence of HbS was detected and the haplotypes of the beta S gene cluster were analyzed by polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP). Our study demonstrated that SCA patients have increased inflammatory profile when compared to the healthy individuals. Further, analysis of the association between the haplotypes and inflammatory profile showed that the levels of IL-6 and TNF-α were greater in subjects with the Bantu/Bantu haplotype than in subjects with the Benin/Benin haplotype. The Bantu/Benin haplotype individuals had lower levels of cytokines than those with the Bantu/Bantu haplotype and greater levels than those of subjects with the Benin/Benin haplotype. For IL-17, a slight trend toward decreased levels was observed in the subjects with the Benin/Benin haplotype, when compared to those with the Bantu/Bantu and Bantu/Benin haplotypes; however, this difference was not statistically significant. Our results show that genetic polymorphisms in sickle cell anemia are associated with the inflammatory profile.     

Measurement of fluxes through the pentose phosphate pathway in erythrocytes from individuals with sickle cell anemia by carbon-13 nuclear magnetic resonance spectroscopy

Erythrocytes from individuals with sickle cell anemia have previously been shown to have increased levels of intracellular oxidants and increased oxidative damage. Oxidative damage has been implicated in the events leading to the painful crises and hemolytic anemia found in sickle cell anemia. Since the pentose phosphate pathway (PPP) is an important source of reducing capacity in erythrocytes, we have investigated the fluxes through the PPP in normal and sickle cell erythrocytes using [2-¹³C]D-glucose and carbon-13 nuclear magnetic resonance (NMR) spectroscopy. Our results indicate that sickle cell erythrocytes have a flux through the PPP of 0.13±0.02 μmol/h per ml erythrocytes that is comparable to that in normal erythrocytes, 0.21±0.02 μmol/h per ml erythrocytes. However, when stimulated with methylene blue, sickle cell erythrocytes show a decreased response, 0.59±0.10 μmol/h per ml erythrocytes, compared to normal erythrocytes, 1.64±0.10 μmol/h per ml erythrocytes. When homogeneous populations of sickle cell erythrocytes are isolated by density gradient centrifugation, the rate of flux through the PPP in methylene blue-stimulated sickle cell erythrocytes, 1.16±0.16 μmol/h per ml erythrocytes, approaches that in methylene blue-stimulated normal erythrocytes. In addition, by analyzing the dose response to methylene blue, we have found that the decreased stimulation of the PPP by methylene blue in heterogeneous populations of sickle cell erythrocytes is a failure of methylene blue to simulate the PPP rather than a deficiency in the PPP in sickle cell erythrocytes.     

Sickle red cells as danger signals on proinflammatory gene expression,leukotriene B4 and interleukin-1 beta production in peripheral blood mononuclear cell

This study tested the hypothesis that sickle red blood cell (SS-RBC) induce Toll-like receptors (TLR) and Nod-like receptor family, pyrin domain containing 3 (NLRP3)- inflammasome expression in peripheral blood mononuclear cells (PBMC). TLR and NLRP3 inflammasome could contribute to the maintenance of the inflammatory status in sickle cell anemia (SCA) patients, since SS-RBC act as danger signals activating these pathways. In this study, first, we evaluated TLR (2, 4, 5 and 9), NLRP3, Caspase-1, interleukin (IL)-1β and IL-18 expression in PBMC freshly isolated from SCA patients (SS-PBMC) in comparison with PBMC from healthy individuals (AA-PBMC). In the second moment, we investigated whether SS-RBC could interfere with the expression of these molecules in PBMC from healthy donor, in the absence or presence of hydroxyurea (HU) in vitro. TLRs and NLRP3 inflammasome expression were investigated by qPCR. IL-1β, Leukotriene-B4 (LTB₄) and nitrite production were measured in PBMC (from healthy donor) culture supernatants. TLR2, TLR4, TLR5, NLRP3 and IL-1β were highly expressed in SS-PBMC when compared to AA-PBMC. Additionally, SS-RBC induced TLR9, NLRP3, Caspase-1, IL-1β and IL-18 expression and induced IL-1β, LTB₄ and nitrite production in PBMC cultures. HU did not prevent TLR and NLRP3 inflammasome expression, but increased TLR2 and IL-18 expression and reduced nitrite production. In conclusion, our data suggest that TLR and inflammasome complexes may be key inducers of inflammation in SCA patients, probably through SS-RBC; also, HU does not prevent NLRP3 inflammasome- and TLR-dependent inflammation, indicating the need to develop new therapeutic strategies to SCA patients that act with different mechanisms of those observed for HU.