Erythrocytes from patients with myeloproliferative neoplasms and splanchnic venous thrombosis show greater expression of Lu/BCAM.

INTRODUCTION: Lutheran/BCAM protein (Lu) on the surface of erythrocytes is key for their adhesion to the endothelium, and erythrocytes from individuals with JAK2V617F-mutated myeloproliferative neoplasms (MPN) have increased endothelial adhesion. Splanchnic vein thrombosis (SVT) is a devastating thrombotic complication of MPN, and frequently, the only diagnostic feature is the JAK2V617F mutation. We sought to examine whether erythrocytes from patients with JAK2V617F mutated SVT (MPN-SVT) exhibited increased Lu expression, thereby supporting a mechanistic contribution to the development of thrombosis. METHODS: We report the validation of a novel flow cytometry assay for Lu expression on erythrocytes. We examined the expression of Lu on erythrocytes from a cohort of MPN patients with and without SVT, and healthy controls. Samples were obtained from 20 normal individuals, 22 with MPN (both JAK2V617F-mutated and wild-type) and 8 with JAK2V617F-mutated MPN-SVT. RESULTS: Lu expression by erythrocytes from patients with MPN and MPN-SVT is significantly increased compared to erythrocytes from healthy individuals (P < .05), but there was no significant difference between patients with MPN-SVT and MPN. CONCLUSIONS: Patients with MPN have increased expression of the red cell Lu/BCAM adhesion molecule. Further work is required to determine the role of the increased Lu/BCAM adhesion to the endothelium in the development of thrombosis in MPN of all genotypes.

The functional importance of blood group-active molecules in human red blood cells.

Antigens of 23 of the 30 human blood group systems are defined by the amino acid sequence of red cell membrane proteins. The antigens of DI, RH, RHAG, MNS, GE and CO systems are carried on blood group-active proteins (Band 3, D and CE polypeptides, RhAG, Glycophorins A and B, Glycophorins C and D and Aquaporin 1, respectively) which are expressed at high levels (>200,000 copies/red cell). These major proteins contribute to essential red cell functions either directly as membrane transporters and by providing linkage to the underlying red cell skeleton or by facilitating the membrane assembly of the protein complexes involved in these processes. The proteins expressing antigens of the remaining 17 blood group systems are much less abundant (<20,000 copies/red cell) and their functional importance for the circulating red cell is largely unknown. Human gene knock-outs (null phenotypes) have been described for many of these minor blood group-active proteins, but only absence of Kx glycoprotein has been clearly linked with pathology directly related to the function of circulating red cells. Recent evidence suggesting the normal quality control system for glycoprotein synthesis is altered during the latter stages of red cell production raises the possibility that many of these low abundance blood group-active proteins are vestigial. In sickle cell disease and polycythaemia vera, elevated Lutheran glycoprotein expression may contribute to pathology. Dyserythropoiesis with reduced antigen expression can result from mutations in the erythroid transcription factors GATA-1 and EKLF.

Production of erythroid cells from human embryonic stem cells (hESC) and human induced pluripotent stem cells (hiPS).

Erythroid progenitors can be generated ex vivo from human embryonic stem cells (hESC) or human induced pluripotent stem cells (hiPS). Development of laboratory scale culture conditions capable of generating mature functional erythrocytes from human embryonic stem cells or human induced pluripotent stem cells would open the possibility for manufacture of therapeutic quantities of red cells and thereby new clinical transfusion products. Current attempts to produce erythrocytes from human embryonic stem cells reveal the need for greater understanding of the process whereby primitive erythropoiesis switches to definitive fetal and adult erythropoiesis and the factors driving erythrocyte maturation. Studies with human embryonic stem cells have already yielded encouraging results but functional mature biconcave erythrocytes have yet to be generated from these cells.

Intercellular adhesion molecule-4 binds alpha(4)beta(1) and alpha(V)-family integrins through novel integrin-binding mechanisms.

The LW blood group glycoprotein, ICAM-4, is a member of the intercellular adhesion molecule (ICAM) family expressed in erythroid cells. To begin to address the function of this molecule, ligands for ICAM-4 on hemopoietic and nonhemopoietic cell lines were identified. Peptide inhibition studies suggest that adhesion of cell lines to an ICAM-4-Fc construct is mediated by an LDV-inhibitable integrin on hemopoietic cells and an RGD-inhibitable integrin on nonhemopoietic cells. Antibody inhibition studies identified the hemopoietic integrin as alpha(4)beta(1.) Antibody inhibition studies on alpha(4)beta(1)-negative, nonhemopoietic cell lines suggested that adhesion of these cells is mediated by alpha(V) integrins (notably alpha(V)beta(1) and alpha(V)beta(5)). The structure of ICAM-4 modeled on the crystal structure of ICAM-2 was used to identify surface-exposed amino acid residues for site-directed mutagenesis. Neither an unusual LETS nor an LDV motif in the first domain of ICAM-4 was critical for integrin binding. ICAM-4 is the first ICAM family member shown to be a ligand for integrins other than those of the beta(2) family, and the data suggest that ICAM-4 has a novel integrin-binding site(s). These findings suggest a role for ICAM-4 in normal erythropoiesis and may also be relevant to the adhesive interactions of sickle cells.

Coexpression of band 3 mutants and Rh polypeptides: differential effects of band 3 on the expression of the Rh complex containing D polypeptide and the Rh complex containing CcEe polypeptide.

K562 cells were stably transfected with cDNAs encoding the band 3 found in Southeast Asian ovalocytosis (B3SAO, deletion of residues 400-408), band 3 with a transport-inactivating E681Q point mutation (B3EQ), or normal band 3 (B3). Flow cytometric analysis and quantitative immunoblotting revealed that B3SAO expressed alone was translocated to the plasma membrane, at levels similar to B3 or B3EQ. Nine monoclonal antibodies that reacted with extracellular loops of B3 also reacted with B3SAO, although the affinity of most antibodies for the mutant protein was reduced. Both known Wr(b) epitopes were expressed on K562/B3SAO cells, demonstrating that B3SAO interacts with glycophorin A. The growth rates of K562 clones expressing equivalent amounts of B3 and B3EQ were the same, suggesting that the potentially toxic transport function of band 3 may be regulated in K562 cells. The band 3-mediated enhancement of Rh antigen reactivity and the depression of Rh epitopes on SAO erythrocytes were investigated by comparing the coexpression of B3, B3SAO, or B3EQ in K562 clones expressing exogenous RhcE or RhD polypeptides. The results are consistent with an interaction between band 3 and the Rh polypeptide-Rh glycoprotein (RhAG) complex, which may enhance translocation of the complex or affect its conformation in the plasma membrane. The data suggest that the interaction between band 3 and the RhD-RhAG complex is weaker than it is between band 3 and the RhCcEe-RhAG complex.

Expression of C antigen in transduced K562 cells.

BACKGROUND: The Rh blood group system is involved in HDN and transfusion reactions. A retrovirus-expression system was previously used to show that polypeptides carrying the Rh blood group antigens are encoded by the RHD and RHCE genes. This study investigated the structure of the C antigen. STUDY DESIGN AND METHODS: K562 cells were transduced with full-length cDNA encoding Ce and CE antigens, and the expression of C, e, and E antigens was examined by flow cytometry using MoAbs. The importance of Cys16 in C antigen expression was examined by utilizing site-directed mutagenesis to convert Cys16 to Trp in cDNA encoding Ce and CE before expression in K562 cells. RESULTS: When K562 cells were transduced with cDNA that was predicted to encode Ce antigens, clear reactivity with anti-e and anti-C was obtained. In contrast, K562 cells transduced with cDNA that was predicted to encode CE antigens gave strong reactivity with anti-E but failed to react with two examples of anti-C. A third example of anti-C gave weak reactivity. When cDNA encoding Ce antigens was mutated to encode Trp16, one example of anti-C had the same reactivity with the mutated polypeptide as with the wild-type molecule, but reactivity with two other anti-C examples was reduced by 50 percent. CONCLUSIONS: The nature of polymorphic residue 226 (proline when E is expressed, alanine when e is expressed) has a marked effect on the epitopes recognized by the three C MoAbs studied. The presence of Cys16 in Ce polypeptides influences the presentation of the C epitope recognized by two of the three MoAbs. These experiments provide the first direct demonstration that C and E/e antigens can be expressed on the same polypeptide.

Lutheran blood group glycoprotein and its newly characterized mouse homologue specifically bind alpha5 chain-containing human laminin with high affinity.

Lutheran blood group glycoproteins (Lu gps) are receptors for the extracellular matrix protein, laminin. Studies suggest that Lu gps may contribute to vaso-occlusion in sickle cell disease and it has recently been shown that sickle cells adhere to laminin isoforms containing the alpha5 chain (laminin 10/11). Laminin alpha5 is present in the subendothelium and is also a constituent of bone marrow sinusoids, suggesting a role for the Lu/laminin interaction in erythropoiesis. The objectives of the current study were to define more precisely the molecular interactions of the extracellular and intracellular regions of human Lu and to clone and characterize a mouse homologue. To this end, complementary DNA and genomic clones for the mouse homologue were sequenced and the mouse Lu gene mapped to a region on chromosome 7 with conserved synteny with human 19q13.2. Mouse and human Lu gps are highly conserved (72% identity) at the amino acid sequence level and both mouse and human Lu gps specifically bind laminin 10/11 with high affinity. Furthermore, the first 3, N-terminal, immunoglobulin superfamily domains of human Lu are critical for this interaction. The results indicated that the cytoplasmic domain of BRIC 221-labeled human Lu gp is linked with the spectrin-based skeleton, affording the speculation that this interaction may be critical for signal transduction. These results further support a role for Lu gps in sickle cell disease and indicate the utility of mouse models to explore the function of Lu gp-laminin 10/11 interaction in normal erythropoiesis and in sickle cell disease.

Epitope mapping of four novel CD44 monoclonal antibodies using surface plasmon resonance and soluble CD44.

CD44 is a ubiquitous multistructural and multifunctional cell surface adhesion molecule. The molecular diversity of this glycoprotein is generated by both post-translational modification and the differential use of alternatively spliced exons which play a critical role in determining the exact conformation of the molecule. CD44 isoforms are found in many tissues and in soluble form in plasma. Soluble CD44 was purified by a two-step purification combining ion exchange and immuno-affinity chromatography. Our aim was to check that all the known antigenic epitopes are present on sCD44, which could thus be used for the mapping of new anti-CD44 antibodies. Competitive binding assays using reference antibodies and novel anti-CD44 monoclonal antibodies were performed by real-time biospecific interaction analysis. Reference mAbs identified on soluble CD44 the three distinct epitopes previously defined using red blood cell membrane CD44. From the four novel CD44 mAbs, two mAbs (NaM198-6B5, NaM198-10B4) mapped to epitope group 1, whereas the others (NaM10-8F4, NaM77-9D6) mapped to epitope group 2. Immunopurified sCD44 obtained from the plasma of healthy donors appears to be a usable tool for the mapping of anti-CD44 mAbs.

The expression of human blood group antigens during erythropoiesis in a cell culture system.

Phenotypic analysis of hematopoietic stem and progenitor cells has been an invaluable tool in defining the biology of stem cell populations. We use here flow cytometry to examine the expression of human erythroid-specific surface markers during the maturation of early committed erythroid cells derived from cord blood in vitro. The temporal order of the expression of erythroid specific markers was as follows: Kell glycoprotein (gp), Rh gp, Landsteiner Wiener (LW) gp, glycophorin A (GPA), Band 3, Lutheran (Lu) gp, and Duffy (Fy) gp. The time at which some of these markers appeared suggests possible roles for some of these erythroid-specific polypeptides during the differentiation of these committed progenitors. The early appearance of Kell gp raises the possibility that it may have an important role in the early stages of hematopoiesis or cell lineage determination. Kell gp may also be a useful marker for the diagnosis of erythroleukemia. The late expression of Lu gp suggests it may be involved in the migration of erythroid precursors from the marrow. Fy gp is also expressed late consistent with a role as a scavenger receptor for cytokines in the bone marrow and circulation. Rh c antigen appeared before Rh D antigen, and it is suggested that this may reflect a reorganization of the developing erythroid cell membrane involving the Rh polypeptides and other components, including GPA and Band 3.

Reductions in finger doses for radiopharmaceutical dispensing afforded by a syringe shield and an automatic dose dispenser.

A gamma extremity monitoring system (GEMS) has been used to measure finger doses during radio-nuclide dispensing procedures. GEMS uses a small semi-conductor probe that can be attached to a finger from which a continuous read-out can be obtained that is related to dose rate. The pattern of dose accumulation can be analysed to allow doses received from individual operations within a procedure to be evaluated. GEMS has been used to compare the radiation dose reduction afforded by a syringe shield and an automatic dose dispenser. Dispensing procedures were simulated using 10% of the activity normally administered in order to avoid problems with detector saturation. Results show that the syringe shield and the automatic dose dispenser reduced finger doses by more than a factor of 10 for the procedures tested. The disadvantages of the automatic dispenser tested were a failure to transfer activity in 10% of cases and a longer time taken for the dispensing process. GEMS has the potential to facilitate greater optimization of finger doses through analysis of finger dose patterns.

Erythroid cell adhesion molecules Lutheran and LW in health and disease.

The Lutheran and LW glycoproteins are blood group-active proteins found at the surface of human red cells. The Lutheran glycoprotein (Lu gp) is a member of the immunoglobulin superfamily (IgSF) that binds the extracellular matrix protein laminin, in particular, laminin isoforms containing the alpha 5 subunit. The LW glycoprotein (LW gp), also an IgSF member, has substantial sequence homology with the family of intercellular adhesion molecules (ICAMs). LW gp binds the integrin very late antigen-4 (VLA-4, alpha 4 beta 1) and alpha V-containing integrins. Studies on the expression of LW and Lu gps during erythropoiesis utilizing in vitro cultures of haemopoietic progenitor cells have shown that LW gp expression precedes that of Lu gp. These observations have led to the suggestion that LW gp on erythroblasts may interact with VLA-4 on macrophages to stabilize erythroblastic islands in normal bone marrow and that Lu gp may facilitate trafficking of more mature erythroid cells to the sinusoidal endothelium where alpha 5-containing laminins are known to be expressed. Levels of Lu gp and LW gp expression on sickle red cells are greater than on normal red cells and sickle red cells adhere to alpha 5-containing laminins. These data suggest that the Lu and LW molecules may contribute to the vaso-occlusive events associated with episodes of acute pain in sickle cell disease.

Functional cell surface expression of band 3, the human red blood cell anion exchange protein (AE1), in K562 erythroleukemia cells: band 3 enhances the cell surface reactivity of Rh antigens.

Human K562 erythroleukemia cells were transfected with human band 3 (anion exchanger 1 [AE1]) cDNA, using the pBabe retroviral vector. Stable K562 clones expressing band 3 were isolated by flow cytometry, and surface expression was quantified by immunoblotting. The function of band 3 expressed at the cell surface was demonstrated in chloride transport assays. K562 cells expressing band 3 also displayed high levels of the Wrb blood group antigen, confirming the role of band 3 in Wrb expression, and an increase in the low levels of endogenous Rh antigen activity. We also performed coexpression experiments with K562 clones that had previously been transduced with cDNAs encoding RhD or RhcE polypeptides. The transfection and expression of band 3 in these clones substantially increased the levels of RhD and cE antigen activity expressed on the cells and also increased the reactivity of the cells with antibody to the endogenous Rh glycoprotein (RhGP, Rh50). The increased reactivity of Rh antigens may result from cell surface or intracellular interactions of band 3 with the protein complex which contains the Rh polypeptides and RhGP, or from indirect effects of band 3 on the membrane environment. This work establishes a system for cell surface expression of band 3 in a mammalian cell line, which will enable further studies of the protein and its interactions with other membrane components.

Application of a gamma extremity monitoring system in a radiopharmaceutical dispensary.

A new gamma extremity monitoring system (GEMS) was used to assess finger doses of staff working in a hospital radionuclide dispensary. The system is designed to give a continuous readout of dose rate from a small probe which may be attached to a finger. It allows the contributions to radiation dose to the fingers from different parts of a procedure to be measured for the first time and the detailed pattern of radiation exposure to be determined. The dose reduction benefits of small changes in procedure and the use of syringe shields were easily demonstrated after monitoring staff for a few sessions using GEMS, which would not have been possible using thermoluminescent or other integrating dosemeters. GEMS was calibrated against 99Tc(m), 241Am, 137Cs and 131I. The main disadvantage of the system is that the response of the detector increases significantly at lower radiation energies, with that at 60 keV being approximately 70 times the response at 662 keV. In addition, the response of the current detector is nonlinear and saturates around 7000 counts s(-1), which corresponds to a dose rate of 2200 microGy h(-1) for 99Tc(m). Despite these drawbacks, GEMS can play a significant part in the analysis of finger dose patterns and assist in dose reduction.