Missense mutations in PIEZO1, which encodes the Piezo1 mechanosensor protein, define Er red blood cell antigens.

Despite the identification of the high-incidence red cell antigen Era nearly 40 years ago, the molecular background of this antigen, together with the other 2 members of the Er blood group collection, has yet to be elucidated. Whole exome and Sanger sequencing of individuals with serologically defined Er alloantibodies identified several missense mutations within the PIEZO1 gene, encoding amino acid substitutions within the extracellular domain of the Piezo1 mechanosensor ion channel. Confirmation of Piezo1 as the carrier molecule for the Er blood group antigens was demonstrated using immunoprecipitation, CRISPR/Cas9-mediated gene knockout, and expression studies in an erythroblast cell line. We report the molecular bases of 5 Er blood group antigens: the recognized Era, Erb, and Er3 antigens and 2 novel high-incidence Er antigens, described here as Er4 and Er5, establishing a new blood group system. Anti-Er4 and anti-Er5 are implicated in severe hemolytic disease of the fetus and newborn. Demonstration of Piezo1, present at just a few hundred copies on the surface of the red blood cell, as the site of a new blood group system highlights the potential antigenicity of even low-abundance membrane proteins and contributes to our understanding of the in vivo characteristics of this important and widely studied protein in transfusion biology and beyond.

Disruption of the tumour-associated EMP3 enhances erythroid proliferation and causes the MAM-negative phenotype.

The clinically important MAM blood group antigen is present on haematopoietic cells of all humans except rare MAM-negative individuals. Its molecular basis is unknown. By whole-exome sequencing we identify EMP3, encoding epithelial membrane protein 3 (EMP3), as a candidate gene, then demonstrate inactivating mutations in ten known MAM-negative individuals. We show that EMP3, a purported tumour suppressor in various solid tumours, is expressed in erythroid cells. Disruption of EMP3 by CRISPR/Cas9 gene editing in an immortalised human erythroid cell line (BEL-A2) abolishes MAM expression. We find EMP3 to associate with, and stabilise, CD44 in the plasma membrane. Furthermore, cultured erythroid progenitor cells from MAM-negative individuals show markedly increased proliferation and higher reticulocyte yields, suggesting an important regulatory role for EMP3 in erythropoiesis and control of cell production. Our data establish MAM as a new blood group system and demonstrate an interaction of EMP3 with the cell surface signalling molecule CD44.

Tetraspanins CD81 and CD82 facilitate α4ß1-mediated adhesion of human erythroblasts to vascular cell adhesion molecule-1.

The proliferation and terminal differentiation of erythroid progenitors occurs in human bone marrow within erythroblastic islands, specialised structures consisting of a central macrophage surrounded by developing erythroid cells. Many cell-cell and cell-matrix adhesive interactions maintain and regulate the co-ordinated daily production of reticulocytes. Erythroid cells express only one integrin, α4ß1, throughout differentiation, and its interactions with both macrophage Vascular Cell Adhesion Molecule-1 and with extracellular matrix fibronectin are critical for erythropoiesis. We observed that proerythroblasts expressed a broad tetraspanin phenotype, and investigated whether any tetraspanin could modulate integrin function. A specific association between α4ß1 and CD81, CD82 and CD151 was demonstrated by confocal microscopy and co-immune precipitation. We observed that antibodies to CD81 and CD82 augmented adhesion of proerythroblasts to Vascular Cell Adhesion Molecule-1 but not to the fibronectin spliceoforms FnIII12-IIICS-15 and FnIII12-15. In contrast, different anti-CD151 antibodies augmented or inhibited adhesion of proerythroblasts to Vascular Cell Adhesion Molecule-1 and the fibronectin spliceoform FnIII12-IIICS-15 but not to FnIII12-15. These results strongly suggest that tetraspanins have a functional role in terminal erythropoiesis by modulating interactions of erythroblast α4ß1 with both macrophages and extracellular matrix.

The Laminin 511/521-binding site on the Lutheran blood group glycoprotein is located at the flexible junction of Ig domains 2 and 3.

The Lutheran blood group glycoprotein, first discovered on erythrocytes, is widely expressed in human tissues. It is a ligand for the alpha5 subunit of Laminin 511/521, an extracellular matrix protein. This interaction may contribute to vaso-occlusive events that are an important cause of morbidity in sickle cell disease. Using x-ray crystallography, small-angle x-ray scattering, and site-directed mutagenesis, we show that the extracellular region of Lutheran forms an extended structure with a distinctive bend between the second and third immunoglobulin-like domains. The linker between domains 2 and 3 appears to be flexible and is a critical determinant in maintaining an overall conformation for Lutheran that is capable of binding to Laminin. Mutagenesis studies indicate that Asp312 of Lutheran and the surrounding cluster of negatively charged residues in this linker region form the Laminin-binding site. Unusually, receptor binding is therefore not a function of the domains expected to be furthermost from the plasma membrane. These studies imply that structural flexibility of Lutheran may be essential for its interaction with Laminin and present a novel opportunity for the development of therapeutics for sickle cell disease.

Two missense mutations in the CD44 gene encode two new antigens of the Indian blood group system.

BACKGROUND: Blood samples were referred over a 10-year period from five patients whose serum samples contained antibodies to unidentified high-incidence antigens. Three patients (A, B, C) were of Moroccan origin and their antibodies and red blood cells (RBCs) were mutually compatible, but incompatible with those of the other two patients (D, E), who were of Pakistani origin. The antibodies and RBCs of D and E were mutually compatible, but incompatible with those of Patients A, B, and C. All the antibodies were detected during pregnancy. STUDY DESIGN AND METHODS: Serologic tests, including the use of enzyme-treated and chemically modified RBCs, suggested a relationship to CD44 (Indian blood group system). The monoclonal antibody immobilization of erythrocyte antigens (MAIEA) assay with monoclonal CD44 antibodies, immunoblotting of RBC membranes, and CD44 gene sequencing were carried out. RESULTS: Positive reactions in the MAIEA assay confirmed that the patients' antibodies are directed at CD44. Immunoblotting with two of the antibodies gave positive reactions of identical size to monoclonal anti-CD44 and failed to react with the RBCs of a CD44-deficient patient. One of the antibodies reacted with purified CD44. Sequencing of Exons 1 to 5 of CD44 revealed 255C>G in Exon 3 for A, B, and C encoding H85Q and 488C>A in Exon 5 for D and E encoding T163K [corrected] CONCLUSION: Two novel CD44 antigens of high incidence have been identified: IN3 (INFI) and IN4 (INJA) in the IN (Indian) blood group system. Lack of IN3 and IN4 results from homozygosity for mutations encoding H85Q and T163R in CD44, respectively.

Peptides based on alphaV-binding domains of erythrocyte ICAM-4 inhibit sickle red cell-endothelial interactions and vaso-occlusion in the microcirculation.

Growing evidence shows that adhesion molecules on sickle erythrocytes interact with vascular endothelium leading to vaso-occlusion. Erythrocyte intercellular adhesion molecule-4 (ICAM-4) binds alphaV-integrins, including alphaVbeta3 on endothelial cells. To explore the contribution of ICAM-4 to vascular pathology of sickle cell disease, we tested the effects of synthetic peptides, V(16)PFWVRMS (FWV) and T(91)RWATSRI (ATSR), based on alphaV-binding domains of ICAM-4 and capable of inhibiting ICAM-4 and alphaV-binding in vitro. For these studies, we utilized an established ex vivo microvascular model system that enables intravital microscopy and quantitation of adhesion under shear flow. In this model, the use of platelet-activating factor, which causes endothelial oxidant generation and endothelial activation, mimicked physiological states known to occur in sickle cell disease. Infusion of sickle erythrocytes into platelet-activating factor-treated ex vivo rat mesocecum vasculature produced pronounced adhesion of erythrocytes; small-diameter venules were sites of maximal adhesion and frequent blockage. Both FWV and ATSR peptides markedly decreased adhesion, and no vessel blockage was observed with either of the peptides, resulting in improved hemodynamics. ATSR also inhibited adhesion in unactivated microvasculature. Although infused fluoresceinated ATSR colocalized with vascular endothelium, pretreatment with function-blocking antibody to alphaVbeta3-integrin markedly inhibited this interaction. Our data strengthen the thesis that ICAM-4 on sickle erythrocytes binds endothelium via alphaVbeta3 and that this interaction contributes to vaso-occlusion. Thus peptides or small molecule mimetics of ICAM-4 may have therapeutic potential.

Targeted gene deletion demonstrates that the cell adhesion molecule ICAM-4 is critical for erythroblastic island formation.

Erythroid progenitors differentiate in erythroblastic islands, bone marrow niches composed of erythroblasts surrounding a central macrophage. Evidence suggests that within islands adhesive interactions regulate erythropoiesis and apoptosis. We are exploring whether erythroid intercellular adhesion molecule 4 (ICAM-4), an immunoglobulin superfamily member, participates in island formation. Earlier, we identified alpha(V) integrins as ICAM-4 counterreceptors. Because macrophages express alpha(V), ICAM-4 potentially mediates island attachments. To test this, we generated ICAM-4 knock-out mice and developed quantitative, live cell techniques for harvesting intact islands and for re-forming islands in vitro. We observed a 47% decrease in islands reconstituted from ICAM-4 null marrow compared to wild-type marrow. We also found a striking decrease in islands formed in vivo in knock-out mice. Further, peptides that block ICAM-4/alpha(V) adhesion produced a 53% to 57% decrease in reconstituted islands, strongly suggesting that ICAM-4 binding to macrophage alpha(V) functions in island integrity. Importantly, we documented that alpha(V) integrin is expressed in macrophages isolated from erythroblastic islands. Collectively, these data provide convincing evidence that ICAM-4 is critical in erythroblastic island formation via ICAM-4/alpha(V) adhesion and also demonstrate that the novel experimental strategies we developed will be valuable in exploring molecular mechanisms of erythroblastic island formation and their functional role in regulating erythropoiesis.

The effects of leukodepletion on the generation and removal of microvesicles and prion protein in blood components.

BACKGROUND: Universal leukodepletion (LD) has been implemented in the United Kingdom to reduce the risk of transfusion-transmitted variant Creutzfeldt-Jakob disease. If LD causes microvesiculation of blood cells, however, potentially infectious membrane-associated prion could reach the final products. STUDY DESIGN AND METHODS: We have measured microvesicles (MV) derived from red cells (RBC-MV), platelets (PLT-MV), and white blood cells (WBC-MV) and cellular prion protein (PrP(c)) in blood components produced by four whole-blood, five RBC, three PLT, and two plasma LD filters and three plateletpheresis techniques. RESULTS: RBC-MV and PLT-MV were either unaltered or reduced by all processes, with PLT-MV reduced 10-fold by RBC LD and greater than 300-fold by plasma LD. WBC-MV were reduced or unchanged by RBC and PLT LD and reduced by plasma LD. Whole-blood filtration appeared to increase MVs derived from granulocytes, but the load in the final components was comparable to that in processed RBCs in additive solution. PrP(c) was reduced by whole-blood, RBC, and plasma LD and unchanged by PLT techniques. There were differences between various filters and techniques, which were generally minor compared to the overall effects. CONCLUSION: These findings suggest no detrimental effects of LD processes in terms of generation of MVs or PrP(c) release.

Identification of critical amino-acid residues on the erythroid intercellular adhesion molecule-4 (ICAM-4) mediating adhesion to alpha V integrins.

Intercellular adhesion molecule-4 (ICAM-4, syn. LW glycoprotein) interacts with the integrins alpha(L)beta(2), alpha(M)beta(2), A(4)beta(1), the alpha(V) family, and alpha(IIb)beta(3). Systematic mutagenesis of surface-exposed residues conserved between human and murine ICAM-4 defined 12 single amino-acid changes that affect the interaction of ICAM-4 with alpha(V) integrins. Mutation of 10 of these residues, 8 of which are spatially close on the surface of the molecule, led to a reduction in adhesion. Moreover, peptides corresponding to regions of ICAM-4 involved in its interaction with alpha(V) integrins inhibited these interactions. The other 2 mutations increased the extent of interaction of ICAM-4 with alpha(V) integrins. These mutations appear to prevent glycosylation of N160, suggesting that changes in glycosylation may modulate ICAM-4-alpha(V) integrin interactions. The region of ICAM-4 identified as the binding site for alpha(V) integrins is adjacent to the binding sites for alpha(L)beta(2) and alpha(M)beta(2). Selective binding of ICAM-4 to different integrins may be important for a variety of normal red cell functions and also relevant to the pathology of thrombotic disorders and vasoocclusive events in sickle cell disease. Our findings suggest the feasibility of developing selective inhibitors of ICAM-4-integrin adhesion of therapeutic value in these diseases.

Novel secreted isoform of adhesion molecule ICAM-4: potential regulator of membrane-associated ICAM-4 interactions.

Intercellular adhesion molecule-4 (ICAM-4), a newly characterized adhesion molecule, is expressed early in human erythropoiesis and functions as a ligand for binding alpha4beta1 and alphaV integrin-expressing cells. Within the bone marrow, erythroblasts surround central macrophages forming erythroblastic islands. Evidence suggests that these islands are highly specialized subcompartments where cell adhesion events, in concert with cytokines, play critical roles in regulating erythropoiesis and apoptosis. Since erythroblasts express alpha4beta1 and ICAM-4 and macrophages exhibit alphaV, ICAM-4 is an attractive candidate for mediating cellular interactions within erythroblastic islands. To determine whether ICAM-4 binding properties are conserved across species, we first cloned and sequenced the murine homologue. The translated amino acid sequence showed 68% overall identity with human ICAM-4. Using recombinant murine ICAM-4 extracellular domains, we discovered that hematopoietic alpha4beta1- expressing HEL cells and nonhematopoietic alphaV-expressing FLY cells adhered to mouse ICAM-4. Cell adhesion studies showed that FLY and HEL cells bound to mouse and human proteins with similar avidity. These data strongly suggest conservation of integrin-binding properties across species. Importantly, we characterized a novel second splice cDNA that would be predicted to encode an ICAM-4 isoform, lacking the membrane-spanning domain. Erythroblasts express both isoforms of ICAM-4. COS-7 cells transfected with green flourescent protein constructs of prototypic or novel ICAM-4 cDNA showed different cellular localization patterns. Moreover, analysis of tissue culture medium revealed that the novel ICAM-4 cDNA encodes a secreted protein. We postulate that secretion of this newly described isoform, ICAM-4S, may modulate binding of membrane-associated ICAM-4 and could thus play a critical regulatory role in erythroblast molecular attachments.