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Sickle cell anaemia (SCA) is a monogenic disease with a highly variable clinical course. We aimed to investigate associations between microvascular function, haemolysis markers, blood viscosity and various types of SCA-related organ damage in a multicentric sub-Saharan African cohort of patients with SCA. In a cross-sectional study, we selected seven groups of adult patients with SS phenotype in Dakar and Bamako based on the following complications: leg ulcer, priapism, osteonecrosis, retinopathy, high tricuspid regurgitant jet velocity (TRV), macro-albuminuria or none. Clinical assessment, echocardiography, peripheral arterial tonometry, laboratory tests and blood viscosity measurement were performed. We explored statistical associations between the biological parameters and the six studied complications. Among 235 patients, 58 had high TRV, 46 osteonecrosis, 43 priapism, 33 leg ulcers, 31 retinopathy and 22 macroalbuminuria, whereas 36 had none of these complications. Multiple correspondence analysis revealed no cluster of complications. Lactate dehydrogenase levels were associated with high TRV, and blood viscosity was associated with retinopathy and the absence of macroalbuminuria. Despite extensive phenotyping of patients, no specific pattern of SCA-related complications was identified. New biomarkers are needed to predict SCA clinical expression to adapt patient management, especially in Africa, where healthcare resources are scarce., (© 2023 The Authors. British Journal of Haematology published by British Society for Haematology and John Wiley & Sons Ltd.)

Hypoxia-mediated red blood cell (RBC) sickling is central to the pathophysiology of sickle cell disease (SCD). The signalling nucleoside adenosine is thought to play a significant role in this process. This study investigated expression of the erythrocyte type 1 equilibrative nucleoside transporter (ENT1), a key regulator of plasma adenosine, in adult patients with SCD and carriers of sickle cell trait (SCT). Relative quantitative expression analysis of erythrocyte ENT1 was carried out by Western blot and flow cytometry. Patients with SCD with steady state conditions, either with SS or SC genotype, untreated or under hydroxycarbamide (HC) treatment, exhibited a relatively high variability of erythrocyte ENT1, but with levels not significantly different from normal controls. Most strikingly, expression of erythrocyte ENT1 was found to be significantly decreased in patients with SCD undergoing painful vaso-occlusive episode and, unexpectedly, also in healthy SCT carriers. Promoting hypoxia-induced adenosine signalling, the reduced expression of erythrocyte ENT1 might contribute to the pathophysiology of SCD and to the susceptibility of SCT individuals to altitude hypoxia or exercise to exhaustion., (© 2022 The Authors. British Journal of Haematology published by British Society for Haematology and John Wiley & Sons Ltd.)

COVID-19 has compelled scientists to better describe its pathophysiology to find new therapeutic approaches. While risk factors, such as older age, obesity, and diabetes mellitus, suggest a central role of endothelial cells (ECs), autopsies have revealed clots in the pulmonary microvasculature that are rich in neutrophils and DNA traps produced by these cells, called neutrophil extracellular traps (NETs.) Submicron extracellular vesicles, called microparticles (MPs), are described in several diseases as being involved in pro-inflammatory pathways. Therefore, in this study, we analyzed three patient groups: one for which intubation was not necessary, an intubated group, and one group after extubation. In the most severe group, the intubated group, platelet-derived MPs and endothelial cell (EC)-derived MPs exhibited increased concentration and size, when compared to uninfected controls. MPs of intubated COVID-19 patients triggered EC death and overexpression of two adhesion molecules: P-selectin and vascular cell adhesion molecule-1 (VCAM-1). Strikingly, neutrophil adhesion and NET production were increased following incubation with these ECs. Importantly, we also found that preincubation of these COVID-19 MPs with the phosphatidylserine capping endogenous protein, annexin A5, abolished cytotoxicity, P-selectin and VCAM-1 induction, all like increases in neutrophil adhesion and NET release. Taken together, our results reveal that MPs play a key role in COVID-19 pathophysiology and point to a potential therapeutic: annexin A5., (© 2021 British Society for Haematology and John Wiley & Sons Ltd.)

Sickle cell disease (SCD) is characterised by chronic haemolysis and oxidative stress. Herein, we investigated 30 SCD patients and found 40% with elevated mitochondria levels (SS-mito + ) in their mature red blood cells, while 60% exhibit similar mitochondria levels compared to the AA group (SS-mito - ). The SS-mito + patients are characterised by higher reticulocytosis and total bilirubin levels, lower foetal haemoglobin, and non-functional mitochondria. Interestingly, we demonstrated decreased levels of mitophagy inducers, PINK1 and NIX, and higher levels of HSP90 chaperone in their red cells. Our results highlighted for the first time an abnormal retention of mitochondria in SCD linked with mitophagy-related proteins., (© 2021 British Society for Haematology and John Wiley & Sons Ltd.)

Although most individuals with sickle cell disease (SCD) live in sub-Saharan Africa, the natural history of the disease on this continent remains largely unknown. Intravascular haemolysis results in activation of circulating blood cells and release of microparticles (MPs) that exert pro-inflammatory effects and contribute to vascular damage. We designed a case-control study nested in the CADRE cohort (Coeur-Artère-DRÉpanocytose, clinical trials.gov identifier NCTO3114137) and based on extreme phenotypes, to analyse blood cell-derived MPs in 232 adult SS patients at steady state in Bamako and Dakar. Thirty-six healthy adult controls matched by age and sex were recruited in Bamako. The MPs concentrations were higher in SS patients compared to AA controls with a predominance of erythrocyte- and reticulocyte-derived MPs. These erythroid-derived MPs were significantly lower in patients with retinopathy (P = 0·022). Reticulocyte-derived MPs were significantly negatively and positively associated with a history of priapism (P = 0·020) and leg ulcers (P = 0·041) respectively. We describe for the first time the comparative patterns of plasma MPs in healthy subjects and patients with SCD living in sub-Saharan Africa and exhibiting various complications. Because our present results show no clear pattern of correlation between erythroid MPs and the classical hyper-haemolytic complications, we hypothesise a weak relevance of the hyper-haemolysis versus hyper-viscous paradigm in Africa., (© 2020 British Society for Haematology and John Wiley & Sons Ltd.)

Vaso-occlusive crisis (VOC) is the main acute complication in sickle cell anaemia (SS) and several clinical trials are investigating different drugs to improve the clinical severity of SS patients. A phase III study is currently exploring the profit of Velopoloxamer in SS during VOCs. We analysed, in-vitro, the effect of poloxamer (P188) on red blood cell (RBC) properties by investigating haemorheology, mechanical and adhesion functions using ektacytometry, microfluidics and dynamic adhesion approaches, respectively. We show that poloxamer significantly reduces blood viscosity, RBC aggregation and adhesion to endothelial cells, supporting the beneficial use of this molecule in SS therapy., (© 2016 John Wiley & Sons Ltd.)

Protein 4.1R plays an important role in maintaining the mechanical properties of the erythrocyte membrane. We analysed the expression of Kell blood group protein in erythrocytes from a patient with hereditary elliptocytosis associated with complete 4.1R deficiency (4.1(-) HE). Flow cytometry and Western blot analyses revealed a severe reduction of Kell. In vitro pull down and co-immunoprecipitation experiments from erythrocyte membranes showed a direct interaction between Kell and 4.1R. Using different recombinant domains of 4.1R and the cytoplasmic domain of Kell, we demonstrated that the R(46) R motif in the juxta-membrane region of Kell binds to lobe B of the 4.1R FERM domain. We also observed that 4.1R deficiency is associated with a reduction of XK and DARC (also termed ACKR1) proteins, the absence of the glycosylated form of the urea transporter B and a slight decrease of band 3. The functional alteration of the 4.1(-) HE erythrocyte membranes was also determined by measuring various transport activities. We documented a slower rate of HCO3 (-) /Cl(-) exchange, but normal water and ammonia transport across erythrocyte membrane in the absence of 4.1. These findings provide novel insights into the structural organization of blood group antigen proteins into the 4.1R complex of the human red cell membrane., (© 2015 John Wiley & Sons Ltd.)

Lu/BCAM, the unique erythroid receptor for laminin 511/521, interacts with the erythrocyte membrane skeleton through spectrin binding. It has been reported that Hereditary Spherocytosis red blood cells (HS RBC) exhibit increased adhesion to laminin. We investigated the role of Lu/BCAM-spectrin interaction in the RBC adhesion properties of 2 splenectomised HS patients characterized by 40% spectrin deficiency. Under physiological flow conditions, HS RBC exhibited an exaggerated adhesion to laminin that was completely abolished by soluble Lu/BCAM. Triton extraction experiments revealed that a greater fraction of Lu/BCAM was unlinked to the membrane skeleton of HS RBC, as compared to normal RBC. Disruption of the spectrin interaction site in Lu/BCAM expressed in the transfected K562 cell line resulted in a weakened interaction to the skeleton and an enhanced interaction to laminin. These results demonstrated that the adhesion of HS RBC to laminin was mediated by Lu/BCAM and that its interaction with the spectrin-based skeleton negatively regulated cell adhesion to laminin. Finally, the results of this study strongly suggest that the reinforced adhesiveness of spectrin-deficient HS RBC to laminin is partly brought about by an impaired interaction between Lu/BCAM and the membrane skeleton.

Lutheran (Lu) and Lu(v13), two glycoprotein (gp) isoforms belonging to the immunoglobulin superfamily, represent adhesion molecules that act as erythrocyte receptors for laminin 10/11. These two gps, which differ only by the length of their cytoplasmic tail, carry both Lu blood group and Basal Cell Adhesion Molecule (B-CAM) antigens. Here, analysis of the Triton extractability of recombinant Lu and Lu(v13) gps in K562 transfected cells showed that both gps were mainly associated with the detergent-insoluble material. Patching experiments using Cholera Toxin subunit B indicated that Lu gps were not localized in lipid rafts. Glutathione-S-transferase capture assays showed that the cytoplasmic domain of Lu and Lu(v13) bound to erythroid spectrin, present in a low ionic strength extract from red cell ghosts. Direct interaction with spectrin was confirmed by plasmon resonance assays. Site-directed mutagenesis mapped a major interaction site with spectrin to the RK573-574 motif, located on the cytoplasmic tail of Lu gp, in close vicinity to the inner leaflet of the membrane lipid bilayer. The two Lu adhesion gps represent the first example of a direct link between transmembrane proteins and spectrin in red blood cells. Since Lu gps are low abundant proteins, we speculate that their interaction with spectrin might be critical for signalling and receptor function rather than for participating in the linkage of the lipid bilayer to the red cell skeleton.

The Duffy antigen/receptor for chemokines (DARC), a seven-transmembrane glycoprotein carrying the Duffy (Fy) blood group, acts as a widely expressed promiscuous chemokine receptor. In a structure-function study, we analysed the binding of chemokines and anti-Fy monoclonal antibodies (mAbs) to K562 cells expressing 39 mutant forms of DARC with alanine substitutions spread out on the four extracellular domains (ECDs). Using synthetic peptides, we defined previously the Fy6 epitope (22-FEDVW-26), and we characterized the Fya epitope as the linear sequence 41-YGANLE-46. In agreement with these results, mutations of F22-E23, V25 and Y41, G42, N44, L45 on ECD1 abolished the binding of anti-Fy6 and anti-Fya mAbs to K562 cells respectively, Anti-Fy3 binding was abolished by D58-D59 (ECD1), R124 (ECD2), D263 and D283 (ECD4) substitutions. Mutations of C51 (ECD1), C129 (ECD2), C195 (ECD3) and C276 (ECD4 severely reduced anti-Fy3 and CXC-chemokine ligand 8 (CXCL-8) binding. CXCL-8 binding was also abrogated by mutations of F22-E23, P50 (ECD1) and D263, R267, D283 (ECD4). These results defined the Fya epitope and suggested that (1) two disulphide bridges are involved in the creation of an active chemokine binding pocket; (2) a limited number of amino acids in ECDs 1-4 participate in CXCL-8 binding; and (3) Fy3 is a conformation-dependent epitope involving all ECDs. We also showed that N-glycosylation of DARC occurred on N16SS and did not influence antibody and chemokine binding.

The linkage between blood group-related cell surface proteins and the detergent-insoluble material (DIM) was estimated by flow cytometry using a panel of specific monoclonal antibodies (mAbs) as a comparison of the antibody-binding capacity of intact and Triton-X100-treated cells. Studies were performed with K562 cells expressing endogenous or recombinant proteins and with human erythroid progenitors during their proliferation and differentiation in vitro. Glycophorin C (GPC) was found to be Triton-insoluble in both cellular models. When expressed (erythroid progenitors), Band 3 remained Triton-insoluble. Glycophorin A (GPA), however, behaved as Triton-soluble or insoluble according to the absence (K562) or the presence (erythroid progenitors) of Band 3 respectively. Comparison of the cellular models regarding the proteins that compose the Rh complex also indicated that Rh(D), RhAG and CD47 were resistant to Triton extraction in cells lacking Band 3. Similarly, RhAG and CD47 remained predominantly Triton-insoluble in K562 cells and early progenitors before Rh and Band 3 expression. Further analysis showed that the Kell protein was DIM-associated. In contrast, CD99 and DARC (Fy) proteins were not, or were very poorly, DIM-associated. Additionally, the adhesion molecules CD44 and Lu were completely or partially resistant to detergent extraction respectively. Deletion of the Lu cytoplasmic tail or its replacement by the cytoplasmic domain of GPC resulted in significant increase or decrease of the Triton solubility of the transfected proteins respectively. These data suggest that Triton insolubility of Lu results in part from direct attachment of its cytoplasmic tail with the cytoskeleton. We assume that this method should provide a useful tool to map interaction sites localized in the cytoplasmic domain of recombinant transmembrane proteins.

We have compared the sensitivity and specificity of four PCR methods of RHD gene detection using different sets of primers located in the regions of highest divergence between the RHD and RHCE genes, notably exon 10 (method I), exon 7 (method II), exon 4 (method III) and intron 4 (method IV). Methods I-III were the most sensitive and gave a detectable signal with D-pos/D-neg mixtures containing only 0.001% D-positive cells. Moreover, method II could detect the equivalent DNA amount present in only three nucleated cells in the assay without hybridization of PCR products, whereas the sensitivity of the other methods was 10-50 times less. Investigation of D variants indicated that false-negative results were obtained with method II (D(IVb) variant), method III (D(VI) and DFR variants) and method IV (D(VI) variants), but not method I. Weak D (D(u)) was correctly detected as D-positive by all methods, but most cases of Rh(null) appeared as false-positives, as they carry normal RH genes that are not phenotypically expressed. Some false-positive results were obtained with method I in a few Caucasian DNA samples serotyped as RhD-neg but carrying a C- or E-allele, whereas a high incidence of false-positives was found among non-Caucasian Rh-negative samples by all methods. In the Caucasian population, however, we found a full correlation between the predicted genotype and observed phenotype at birth of 92 infants. Although we routinely use the four methods for RHD genotyping, a PCR strategy based on at least two methods is recommended.

The Kx protein is an erythrocyte membrane polypeptide which is deficient in rare individuals suffering from the McLeod syndrome. The gene encoding this protein has been recently cloned and the Kx protein independently purified as a covalent complex with the Kell blood group protein. To further study the Kx membrane protein, antisera raised in rabbits against six synthetic peptides derived from the primary sequence of this protein were characterized. All antisera but two precipitated the recombinant Kx protein synthesized in coupled transcription-translation in vitro. Three antisera reacted on immunoblots with the 37 kD Kx protein present in the purified Kell-Kx complex and in SDS red cell membrane lysates from variants with different Kell blood group phenotypes, including Ko, which lack the Kell protein of 93 kD. However, no reactivity was found with McLeod preparations lacking Kx protein, thus clearly indicating that these antibodies have a Kx specificity. Unexpectedly, the relative amount of Kx protein in Ko cells was found to be lower than in red cells with common Kell phenotypes, suggesting that the absence of the Kell protein may alter the amount of Kx in the membrane.

The Rh blood group antigens D, Cc and Ee are encoded by two related genes, RHD and RHCE. The RhG antigen (Rh12) is associated with the expression of RhC and/or RhD, except in rare variant red cells. Here we have determined the molecular basis of G expression in the absence of D and C in the rGr phenotype. Nucleotide sequence analysis revealed that the rG allele resulted either from a segmental DNA exchange between part of exon 2 of the RHce gene and the equivalent region of the RHCE or RHD genes or from a crossing over between positions nt150 and nt178 of the RHce and RHCe genes. The predicted protein encoded by the hybrid rG gene (c-C-e or c-D-e) carries Ile60, Ser68 and Ser103 (as C and D polypeptides); any of these positions appear to be critical in the formation of the G antigen. In addition, Cys16 was found to be important in the phenotypic expression of C.

The Rh blood group antigens D, Cc and Ee are encoded by two highly related genes, RHD and RHCE. Almost all red cells which carry D and all cells which carry C also express the G (Rh12) antigen. In this report we have determined the molecular basis of the DIIIb category phenotype which represents a very rare condition characterized by the presence of most of the D epitopes and the total absence of the antigen G. mRNA sequencing and Southern blot analysis of two unrelated samples indicated that the DIIIb category phenotype is associated with a segmental DNA exchange between exon 2 of the RHD and RHCE genes resulting in three D-->c amino acid substitutions (Ile60Leu, Ser68Asn and Ser103Pro).

After anti-RhD, anti-Rhc is the most important red cell alloantibody which can cause haemolytic disease of the newborn (HDN) when the mother is Rhc-negative and the fetus Rhc-positive. We report here the development of polymerase chain reaction (PCR) assays which detect the presence of the Rhc alleles in amniotic cells by the use of allele-specific primers (ASP). It is expected that such determination will help in the management of pregnancies at risk of Rhc haemolytic disease. In the course of this study we have similarly performed PCR-ASP experiments to detect fetal RHE alleles since, in rare cases, anti-RhE can also cause HDN.