Glycophorin B-PfEMP1 interaction mediates robust rosetting in Plasmodium falciparum.

P. falciparumerythrocyte membrane protein 1 (PfEMP1) is the major parasite protein responsible for rosetting by binding to host receptors such as heparan sulfate, CR1 on RBC surface. Usually monomeric protein-carbohydrate interactions are weak [1], therefore PfEMP1 binds to plasma proteins like IgM or α2-macroglobulin that facilitate its clustering on parasitized RBC surface and augment rosetting [2,3]. We show that 3D7A expresses PfEMP1, PF3D7_0412900, and employs its CIDRγ2 domain to interact with glycophorin B on uninfected RBC to form large rosettes but more importantly even in the absence of plasma proteins. Overall, we established the role of PF3D7_0412900 in rosetting as antibodies against CIDRγ2 domain reduced rosetting and also identified its receptor, glycophorin B which could provide clue why glycophorin B null phenotype, S-s-U- RBCs prevalent in malaria endemic areas is protective against severe malaria.

Aberrant GPA expression and regulatory function of red blood cells in sickle cell disease.

ABSTRACT: Glycophorin A (GPA), a red blood cell (RBC) surface glycoprotein, can maintain peripheral blood leukocyte quiescence through interaction with a sialic acid-binding Ig-like lectin (Siglec-9). Under inflammatory conditions such as sickle cell disease (SCD), the GPA of RBCs undergo structural changes that affect this interaction. Peripheral blood samples from patients with SCD before and after RBC transfusions were probed for neutrophil and monocyte activation markers and analyzed by fluorescence-activated cell sorting (FACS). RBCs were purified and tested by FACS for Siglec-9 binding and GPA expression, and incubated with cultured endothelial cells to evaluate their effect on barrier function. Activated leukocytes from healthy subjects (HS) were coincubated with healthy RBCs (RBCH), GPA-altered RBCs, or GPA-overexpressing (OE) cells and analyzed using FACS. Monocyte CD63 and neutrophil CD66b from patients with SCD at baseline were increased 47% and 27%, respectively, as compared with HS (P = .0017, P = .0162). After transfusion, these markers were suppressed by 22% and 17% (P = .0084, P = .0633). GPA expression in RBCSCD was 38% higher (P = .0291) with decreased Siglec-9 binding compared with RBCH (0.0266). Monocyte CD63 and neutrophil CD66b were suppressed after incubation with RBCH and GPA-OE cells, but not with GPA-altered RBCs. Endothelial barrier dysfunction after lipopolysaccharide challenge was restored fully with exposure to RBCH, but not with RBCSCD, from patients in pain crisis, or with RBCH with altered GPA. Pretransfusion RBCSCD do not effectively maintain the quiescence of leukocytes and endothelium, but quiescence is restored through RBC transfusion, likely by reestablished GPA-Siglec-9 interactions.

Radiofrequency dielectric spectroscopy study: Effects of pH, hydrogen bond donors and acceptors on the attachment of spectrin skeleton to the lipid membrane of erythrocytes.

Band 3 protein and glycophorin C are the two major integral proteins of the lipid membrane of human red blood cells (RBCs). They are attached from below to a network of elastic filamentous spectrin, the third major RBC membrane protein. The binding properties of the attachments to spectrin affect the shape and deformability of RBCs. We addressed band 3 and glycophorin C attachments to spectrin by measuring the strength of two recently discovered radiofrequency dielectric relaxations, ßsp (1.4 MHz) and γ1sp (9 MHz), that are observable as changes in the complex admittance of RBCs in medium. In medium at pH 5.2, and also in media with protic substances (formamide, methylformamide, or urea), the ßsp relaxation became inhibited that is attributable to detachment of glycophorin C from spectrin. In medium at pH 9.2, we observed inhibition of γ1sp relaxation attributable to detachment of band 3 from spectrin, as also was seen in media with aprotic substances difluoropyridine, dimethylsolfoxide, dimethylformamide, acetone, sodium tetrakis(4-fluorophenyl)borate), chlorpromazine, thioridazine and trifluopiperazine. The viscogenic cosolvents (glycerol, ethylene glycol, or i-erythritol) inhibited both the ßsp and γ1sp relaxations and significantly lowered their characteristic frequencies. Our observations indicate that the glycophorin C attachment to spectrin has nucleophilic centers whose saturation disconnects this attachment and inhibits the ßsp relaxation, whereas at band 3-spectrin attachment site, it is the saturation of electrophilic centers that weakens this attachment and inhibits the γ1sp relaxation.

Alpha hemolysin of E. coli induces hemolysis of human erythrocytes independently of toxin interaction with membrane proteins.

Alpha hemolysin (HlyA) is a hemolytic and cytotoxic protein secreted by uropathogenic strains of E. coli. The role of glycophorins (GPs) as putative receptors for HlyA binding to red blood cells (RBCs) has been debated. Experiments using anti-GPA/GPB antibodies and a GPA-specific epitope nanobody to block HlyA-GP binding on hRBCs, showed no effect on hemolytic activity. Similarly, the hemolysis induced by HlyA remained unaffected when hRBCs from a GPAnull/GPBnull variant were used. Surface Plasmon Resonance experiments revealed similar values of the dissociation constant between GPA and either HlyA, ProHlyA (inactive protoxin), HlyAΔ914-936 (mutant of HlyA lacking the binding domain to GPA) or human serum albumin, indicating that the binding between the proteins and GPA is not specific. Although far Western blot followed by mass spectroscopy analyses suggested that HlyA interacts with Band 3 and spectrins, hemolytic experiments on spectrin-depleted hRBCs and spherocytes, indicated these proteins do not mediate the hemolytic process. Our results unequivocally demonstrate that neither glycophorins, nor Band 3 and spectrins mediate the cytotoxic activity of HlyA on hRBCs, thereby challenging the HlyA-receptor hypothesis. This finding holds significant relevance for the design of anti-toxin therapeutic strategies, particularly in light of the growing antibiotic resistance exhibited by bacteria.

Folding and modulation of the helical conformation of Glycophorin A by point mutations.

Transmembrane helix folding and self-association play important roles in biological signaling and transportation pathways across biomembranes. With molecular simulations, studies to explore the structural biochemistry of this process have been limited to focusing on individual fragments of this process - either helix formation or dimerization. While at an atomistic resolution, it can be prohibitive to access long spatio-temporal scales, at the coarse grained (CG) level, current methods either employ additional constraints to prevent spontaneous unfolding or have a low resolution on sidechain beads that restricts the study of dimer disruption caused by mutations. To address these research gaps, in this work, we apply our recent, in-house developed CG model (ProMPT) to study the folding and dimerization of Glycophorin A (GpA) and its mutants in the presence of Dodecyl-phosphocholine (DPC) micelles. Our results first validate the two-stage model that folding and dimerization are independent events for transmembrane helices and found a positive correlation between helix folding and DPC-peptide contacts. The wild type (WT) GpA is observed to be a right-handed dimer with specific GxxxG contacts, which agrees with experimental findings. Specific point mutations reveal several features responsible for the structural stability of GpA. While the T87L mutant forms anti-parallel dimers due to an absence of T87 interhelical hydrogen bonds, a slight loss in helicity and a hinge-like feature at the GxxxG region develops for the G79L mutant. We note that the local changes in the hydrophobic environment, affected by the point mutation, contribute to the development of this helical bend. This work presents a holistic overview of the structural stability of GpA in a micellar environment, while taking secondary structural fluctuations into account. Moreover, it presents opportunities for applications of computationally efficient CG models to study conformational alterations of transmembrane proteins that have physiological relevance.

Differential enzymatic deglycosylation reveals attachment of red cell B antigen onto the carbohydrate moiety of glycophorin A and glycophorin B.

BACKGROUND AND OBJECTIVES: Early studies indicate that red cell A and B antigens are attached primarily onto band 3 and GLUT1 on the erythrocyte membrane and little onto glycophorin A (GPA) and glycophorin B (GPB). But as GPA and band 3 form stable protein complexes and GPA is much more heavily glycosylated than band 3, this study re-examined the association between ABO antigens and GPA/GPB. MATERIALS AND METHODS: Band 3/GPA-associated protein complexes were first immunoprecipitated, followed by differential enzymatic deglycosylation that removed sialic acids, N-glycans and O-glycans. Serological anti-A (BIRMA 1) and anti-B IgM (GAMA 110) could be used for western blot (WB); however, only the anti-B IgM showed significant reactivity for the immunoprecipitates isolated by anti-band 3. The expression of the B antigen in un-deglycosylated and differentially deglycosylated band 3 immunoprecipitates was thus compared. RESULTS: Besides attachment to band 3, red cell B antigen expressed substantially on GPA monomer and homodimer, GPA*GPB heterodimer, and GPB monomer and dimer via attachments through the N- and O-glycans. CONCLUSION: Immunoprecipitation (IP), as a means of protein separation and concentration, was used in combination with a WB to differentiate glycosylation on different proteins and oligomers. This study implemented differential enzymatic deglycosylation during IP of the band 3 complexes. This combined approach allowed separate identification of the B antigen on GPA/GPB monomer and dimer and GPA*GPB heterodimer, and band 3 on the WB and verified non-trivial expression of the B antigen on GPA and GPB on the erythrocyte surface.

Genetic variation of glycophorins and infectious disease.

Glycophorins are transmembrane proteins of red blood cells (RBCs), heavily glycosylated on their external-facing surface. In humans, there are four glycophorin proteins, glycophorins A, B, C and D. Glycophorins A and B are encoded by two similar genes GYPA and GYPB, and glycophorin C and glycophorin D are encoded by a single gene, GYPC. The exact function of glycophorins remains unclear. However, given their abundance on the surface of RBCs, it is likely that they serve as a substrate for glycosylation, giving the RBC a negatively charged, complex glycan "coat". GYPB and GYPE (a closely related pseudogene) were generated from GYPA by two duplication events involving a 120-kb genomic segment between 10 and 15 million years ago. Non-allelic homologous recombination between these 120-kb repeats generates a variety of duplication alleles and deletion alleles, which have been systematically catalogued from genomic sequence data. One allele, called DUP4, encodes the Dantu NE blood type and is strongly protective against malaria as it alters the surface tension of the RBC membrane. Glycophorins interact with other infectious pathogens, including viruses, as well as the malarial parasite Plasmodium falciparum, but the role of glycophorin variation in mediating the effects of these pathogens remains underexplored.

Red Blood Cells Oligosaccharides as Targets for Plasmodium Invasion.

The key element in developing a successful malaria treatment is a good understanding of molecular mechanisms engaged in human host infection. It is assumed that oligosaccharides play a significant role in Plasmodium parasites binding to RBCs at different steps of host infection. The formation of a tight junction between EBL merozoite ligands and glycophorin receptors is the crucial interaction in ensuring merozoite entry into RBCs. It was proposed that sialic acid residues of O/N-linked glycans form clusters on a human glycophorins polypeptide chain, which facilitates the binding. Therefore, specific carbohydrate drugs have been suggested as possible malaria treatments. It was shown that the sugar moieties of N-acetylneuraminyl-N-acetate-lactosamine and 2,3-didehydro-2-deoxy-N-acetylneuraminic acid (DANA), which is its structural analog, can inhibit P. falciparum EBA-175-GPA interaction. Moreover, heparin-like molecules might be used as antimalarial drugs with some modifications to overcome their anticoagulant properties. Assuming that the principal interactions of Plasmodium merozoites and host cells are mediated by carbohydrates or glycan moieties, glycobiology-based approaches may lead to new malaria therapeutic targets.

A Balance between Transmembrane-Mediated ER/Golgi Retention and Forward Trafficking Signals in Glycophorin-Anion Exchanger-1 Interaction.

Anion exchanger-1 (AE1) is the main erythroid Cl-/HCO3- transporter that supports CO2 transport. Glycophorin A (GPA), a component of the AE1 complexes, facilitates AE1 expression and anion transport, but Glycophorin B (GPB) does not. Here, we dissected the structural components of GPA/GPB involved in glycophorin-AE1 trafficking by comparing them with three GPB variants-GPBhead (lacking the transmembrane domain [TMD]), GPBtail (mainly the TMD), and GP.Mur (glycophorin B-A-B hybrid). GPB-derived GP.Mur bears an O-glycopeptide that encompasses the R18 epitope, which is present in GPA but not GPB. By flow cytometry, AE1 expression in the control erythrocytes increased with the GPA-R18 expression; GYP.Mur+/+ erythrocytes bearing both GP.Mur and GPA expressed more R18 epitopes and more AE1 proteins. In contrast, heterologously expressed GPBtail and GPB were predominantly localized in the Golgi apparatus of HEK-293 cells, whereas GBhead was diffuse throughout the cytosol, suggesting that glycophorin transmembrane encoded an ER/Golgi retention signal. AE1 coexpression could reduce the ER/Golgi retention of GPB, but not of GPBtail or GPBhead. Thus, there are forward-trafficking and transmembrane-driven ER/Golgi retention signals encoded in the glycophorin sequences. How the balance between these opposite trafficking signals could affect glycophorin sorting into AE1 complexes and influence erythroid anion transport remains to be explored.

Human Cell-Camouflaged Nanomagnetic Scavengers Restore Immune Homeostasis in a Rodent Model with Bacteremia.

Bloodstream infection caused by antimicrobial resistance pathogens is a global concern because it is difficult to treat with conventional therapy. Here, scavenger magnetic nanoparticles enveloped by nanovesicles derived from blood cells (MNVs) are reported, which magnetically eradicate an extreme range of pathogens in an extracorporeal circuit. It is quantitatively revealed that glycophorin A and complement receptor (CR) 1 on red blood cell (RBC)-MNVs predominantly capture human fecal bacteria, carbapenem-resistant (CR) Escherichia  coli, and extended-spectrum beta-lactamases-positive (ESBL-positive) E. coli, vancomycin-intermediate Staphylococcus aureus (VISA), endotoxins, and proinflammatory cytokines in human blood. Additionally, CR3 and CR1 on white blood cell-MNVs mainly contribute to depleting the virus envelope proteins of Zika, SARS-CoV-2, and their variants in human blood. Supplementing opsonins into the blood significantly augments the pathogen removal efficiency due to its combinatorial interactions between pathogens and CR1 and CR3 on MNVs. The extracorporeal blood cleansing enables full recovery of lethally infected rodent animals within 7 days by treating them twice in series. It is also validated that parameters reflecting immune homeostasis, such as blood cell counts, cytokine levels, and transcriptomics changes, are restored in blood of the fatally infected rats after treatment.

Effects of cholesterol and PIP2 on interactions between glycophorin A and Band 3 in lipid bilayers.

In the erythrocyte membrane, the interactions between glycophorin A (GPA) and Band 3 are associated strongly with the biological function of the membrane and several blood disorders. In this work, using coarse-grained molecular-dynamics simulations, we systematically investigate the effects of cholesterol and phosphatidylinositol-4,5-bisphosphate (PIP2) on the interactions of GPA with Band 3 in the model erythrocyte membranes. We examine the dynamics of the interactions of GPA with Band 3 in different lipid bilayers on the microsecond time scale and calculate the binding free energy between GPA and Band 3. The results indicate that cholesterols thermodynamically favor the binding of GPA to Band 3 by increasing the thickness of the lipid bilayer and by producing an effective attraction between the proteins due to the depletion effect. Cholesterols also slow the kinetics of the binding of GPA to Band 3 by reducing the lateral mobility of the lipids and proteins and may influence the binding sites between the proteins. The anionic PIP2 lipids prefer binding to the surface of the proteins through electrostatic attraction between the PIP2 headgroup and the positively charged residues on the protein surface. Ions in the solvent facilitate PIP2 aggregation, which promotes the binding of GPA to Band 3.

CD1d expression demarcates CDX4+ hemogenic mesoderm with definitive hematopoietic potential.

To achieve efficient, reproducible differentiation of human pluripotent stem cells (hPSCs) towards specific hematopoietic cell-types, a comprehensive understanding of the necessary cell signaling and developmental trajectories involved is required. Previous studies have identified the mesodermal progenitors of extra-embryonic-like and intra-embryonic-like hemogenic endothelium (HE), via stage-specific WNT and ACTIVIN/NODAL, with GYPA/GYPB (CD235a/b) expression serving as a positive selection marker for mesoderm harboring exclusively extra-embryonic-like hemogenic potential. However, a positive mesodermal cell-surface marker with exclusively intra-embryonic-like hemogenic potential has not been identified. Recently, we reported that early mesodermal expression of CDX4 critically regulates definitive HE specification, suggesting that CDX4 may act in a cell-autonomous manner during hematopoietic development. To identify CDX4+ mesoderm, we performed single cell (sc)RNAseq on hPSC-derived mesodermal cultures, revealing CDX4hi expressing mesodermal populations were uniquely enriched in the non-classical MHC-Class-1 receptor CD1D. Flow cytometry demonstrated approximately 60% of KDR+CD34-CD235a- mesoderm was CD1d+, and CDX4 was robustly enriched within CD1d+ mesoderm. Critically, only CD1d+ mesoderm harbored CD34+ HOXA+ HE with multilineage erythroid-myeloid-lymphoid potential. Thus, CDX4+CD1d+ expression within early mesoderm demarcates an early progenitor of HE. These insights may be used for further study of human hematopoietic development and improve hematopoietic differentiation conditions for regenerative medicine applications.

Characterization of alternatively spliced transcript variants of glycophorin A and glycophorin B genes in Chinese blood donors.

BACKGROUND AND OBJECTIVES: The molecular basis of MNS blood group variants is not fully clear yet. In this study, we have characterized mRNA variants of GYPA and GYPB genes to reveal whether alternative RNA splicing may cause antigenic diversity of the MNS system. MATERIALS AND METHODS: Total RNA was extracted from peripheral blood of Chinese blood donors and full-length cDNA products were generated. A nested polymerase chain reaction (PCR)-based method was established for fragment amplification and Sanger sequencing. Resulted full-length mRNA sequences were aligned with GYPA or GYPB genomic sequences respectively for exon identification. Amino acid (AA) sequences of GPA and GPB proteins were extrapolated and GYPA-EGFP, GYPB-EGFP fusion genes were generated to monitor subcellular distribution of the encoded glycophorin (GP) proteins. RESULTS: Totally 10 blood samples were analysed. GYPB mRNAs of all the subjects demonstrated frequent exon insertion or deletion whereas this kind of variation was only observed in 3 of 10 GYPA mRNA samples. None of the reported Miltenberger hybrids was detected in any of the mRNA samples. The alternative splicing resulted in changes of AA sequences in N-terminal domains where the MNS antigenic motifs resided; however, subcellular localizations of GP-EGFP fusion proteins showed that the above-mentioned AA changes did not affect cell surface distribution of the encoded GP proteins. CONCLUSIONS: Alternative RNA splicing may influence the antigenic features of GP proteins but not their cell surface distribution. Therefore, GYPA and GYPB mRNA characterization might be an invaluable supplement to serological phenotyping and DNA-based genotyping in MNS blood grouping.

Arsenite exposure inhibits the erythroid differentiation of human hematopoietic progenitor CD34+ cells and causes decreased levels of hemoglobin.

Arsenic exposure poses numerous threats to human health. Our previous work in mice has shown that arsenic causes anemia by inhibiting erythropoiesis. However, the impacts of arsenic exposure on human erythropoiesis remain largely unclear. We report here that low-dose arsenic exposure inhibits the erythroid differentiation of human hematopoietic progenitor cells (HPCs). The impacts of arsenic (in the form of arsenite; As3+) on red blood cell (RBC) development was evaluated using a long-term culture of normal human bone marrow CD34+-HPCs stimulated in vitro to undergo erythropoiesis. Over the time course studied, we analyzed the expression of the cell surface antigens CD34, CD71 and CD235a, which are markers commonly used to monitor the progression of HPCs through the stages of erythropoiesis. Simultaneously, we measured hemoglobin content, which is an important criterion used clinically for diagnosing anemia. As compared to control, low-dose As3+ exposure (100 nM and 500 nM) inhibited the expansion of CD34+-HPCs over the time course investigated; decreased the number of committed erythroid progenitors (BFU-E and CFU-E) and erythroblast differentiation in the subsequent stages; and caused a reduction of hemoglobin content. These findings demonstrate that low-dose arsenic exposure impairs human erythropoiesis, likely by combined effects on various stages of RBC formation.

Comprehensive transcriptome analysis of erythroid differentiation potential of olive leaf in haematopoietic stem cells.

Anaemia is one of the leading causes of disability in young adults and is associated with increased morbidity and mortality in elderly. With a global target to reduce the disease burden of anaemia, recent researches focus on novel compounds with the ability to induce erythropoiesis and regulate iron homeostasis. We aimed to explore the biological events and potential polypharmacological effects of water-extracted olive leaf (WOL) on human bone marrow-derived haematopoietic stem cells (hHSCs) using a comprehensive gene expression analysis. HPLC analysis identifies six bioactive polyphenols in the WOL. Treatment with WOL for 12 days regulated gene expressions related to erythroid differentiation, oxygen homeostasis, iron homeostasis, haem metabolism and Hb biosynthesis in hHSCs. Functional clustering analysis reveals several major functions of WOL such as ribosomal biogenesis and mitochondrial translation machinery, glycolytic process, ATP biosynthesis and immune response. Additionally, the colonies of both primitive and mature erythroid progenitors, CFU-E and BFU-E, were significantly increased in WOL-treated hHSCs. The expressions of erythroid markers, CD47, glycophorin A (GYPA), and transferrin receptor (TFRC) and adult Hb subunits-HBA and HBB were also confirmed in immunofluorescent staining and flow cytometer analysis in WOL-treated hHSCs. It is well known that induction of lineage-specific differentiation, as well as the maturation of early haematopoietic precursors into fully mature erythrocytes, involves multiple simultaneous biological events and complex signalling networks. In this regard, our genome-wide transcriptome profiling with microarray study on WOL-treated hHSCs provides general insights into the multitarget prophylactic and/or therapeutic potential of WOL in anaemia and other haematological disorders.

Diagnostic value of glycophorin-A in comparison with P57 immunohistochemical staining method in differentiating complete and partial molar pregnancies.

INTRODUCTION: Current histomorphological criteria in distinguishing two subtypes of hydatidiform moles has considerable inter-observer variability and limitations. In this regard, ancillary studies can aid pathologist to obtain an accurate diagnosis. Herein, we evaluated the utility of Glycophorin-A (GLA) in differentiating complete and partial moles. MATERIALS AND METHODS: In this case-control study, formalin-fixed paraffin-embedded blocks of 47 patients with pathologic diagnosis of complete and 42 partial hydatidiform moles were included and the diagnoses were confirmed by immunohistochemistry (IHC) for P57. Sections from all samples were stained for GLA using IHC method. Using 2 × 2 tables, the sensitivity, specifity, Positive and Negative Predictive Values (PPV and NPV) as well as accuracy of GLA were determined. RESULTS: Primary pathologic diagnosis was changed in 7.1% and types of hydatidiform mole were specified in 11.9% of the cases after review of the slides and IHC study for P57. NRBCs were found in 52.7% of the PM cases and none of CMs by pathologist in H&E sections. IHC study for GLA revealed positive result in one case of complete moles (2%) and 31 case of partial mole samples (73.8%). It was negative in 98% of the complete mole and 11 (26.2%) of partial mole cases. DISCUSSION: The results of this study showed a significant association between GLA immunoreactivity and type of molar pregnancy. Diagnostic sensitivity, specificity and accuracy of this marker for discrimination of molar pregnancy were 73.8%, 98% and 86.5%, respectively. Therefore, this marker can be utilized in differentiating partial and complete hydatidiform mole.

Hotspots in Plasmodium and RBC Receptor-Ligand Interactions: Key Pieces for Inhibiting Malarial Parasite Invasion.

Protein-protein interactions (IPP) play an essential role in practically all biological processes, including those related to microorganism invasion of their host cells. It has been found that a broad repertoire of receptor-ligand interactions takes place in the binding interphase with host cells in malaria, these being vital interactions for successful parasite invasion. Several trials have been conducted for elucidating the molecular interface of interactions between some Plasmodium falciparum and Plasmodium vivax antigens with receptors on erythrocytes and/or reticulocytes. Structural information concerning these complexes is available; however, deeper analysis is required for correlating structural, functional (binding, invasion, and inhibition), and polymorphism data for elucidating new interaction hotspots to which malaria control methods can be directed. This review describes and discusses recent structural and functional details regarding three relevant interactions during erythrocyte invasion: Duffy-binding protein 1 (DBP1)-Duffy antigen receptor for chemokines (DARC); reticulocyte-binding protein homolog 5 (PfRh5)-basigin, and erythrocyte binding antigen 175 (EBA175)-glycophorin A (GPA).

Glycophorin A-based exclusion of red blood cells for flow cytometric analysis of platelet glycoprotein expression in citrated whole blood.

Objectives Analysis of platelet glycoprotein (GP) expression by flow cytometry is applied for diagnostic confirmation of GP-associated thrombocytopathies. While platelet-rich plasma may be used for distinct identification of target events, this strategy is not feasible for small sample volumes or for patients showing low platelet counts and/or giant platelets. However, also the use of whole blood (WB) is hampered by the difficulty to discriminate platelets from red blood cells (RBC) in such patients. To circumvent these limitations, we evaluated the feasibility of a RBC gating-out strategy. Methods In addition to platelet GPIb, GPIIa/IIIa, as well as P-selectin (CD62P), citrated whole blood (CWB) samples were stained for RBC-specific glycophorin A (CD235a). CD235a-negative platelet events were further discriminated by forward-/side-scatter characteristics and platelet GP expressions analyzed relative to that of a healthy control sample processed in parallel. Results Established reference intervals allowed for clear identification of decreased GPIIb/IIIa- or GPIb expression pattern in samples of patients with confirmed Glanzmann thrombasthenia or Bernard-Soulier syndrome, respectively. It could be shown that the analysis of 2,500 platelet events is sufficient for reliable GP expression analysis, rendering the proposed method applicable to samples with low platelet counts. Conclusions This study demonstrates the feasibility of CD235a-based exclusion of RBC for platelet GP expression analysis in CWB. In contrast to direct staining of platelet-specific antigens for target identification, this indirect gating out approach is generally applicable independent of any underlying platelet GP expression deficiency.

An unusual variant glycophorin expressing protease-resistant M antigen encoded by the GYPB-E(2-4)-B hybrid gene.

BACKGROUND AND OBJECTIVES: MNS is a highly polymorphic blood group comprising 49 antigens recognized by International Society of Blood Transfusion, some of which may have been generated by genomic recombination among the closely linked genes GYPA, GYPB and GYPE. The GYPE gene has an almost identical sequence to GYPA*01 allele in exon 2 (99% homology), which accounts for M antigen. We investigated an unusual glycophorin molecule with protease-resistant M antigen. METHODS: Blood samples were screened by an automated blood typing system (PK7300) using bromelain-treated red blood cells (RBCs) and murine monoclonal anti-M. The M-positive RBC samples were analysed by immunoblotting using anti-M as the primary antibody. GYPA, GYPB and GYPE genes were analysed by polymerase chain reaction (PCR), cloning and sequencing using reticulocyte mRNA and genomic DNA. RESULTS: Serological tests and immunoblotting revealed that 103 of the 193 009 individuals (0·0534%) expressed protease-resistant M-active glycophorin having a molecular weight of 20 kDa. All the 103 individuals were S+ s- or S- s+. When reticulocyte mRNA from the individuals with M-active glycophorin (20 kDa) was examined by PCR and cloning followed by sequencing, a novel GYPE-B hybrid transcript was identified. Long-range PCR and sequencing using genomic DNA revealed that the individuals had a GYPB-E(2-4)-B hybrid gene. This hybrid gene was predicted to encode a 59-amino-acid mature glycoprotein that expresses no S or s antigens CONCLUSIONS: The prevalence of the M-active glycophorin (20 kDa) in the Japanese population is 0·0534%. This glycophorin is predicted to be a 59 amino acids polypeptide encoded by the novel GYPB-E(2-4)-B hybrid gene.