Boukje M. Beuger

Identification of signalling cascades involved in red blood cell shrinkage and vesiculation

Even though red blood cell (RBC) vesiculation is a well-documented phenomenon, notably in the context of RBC aging and blood transfusion, the exact signalling pathways and kinases involved in this process remain largely unknown. We have established a screening method for RBC vesicle shedding using the Ca2+ ionophore ionomycin which is a rapid and efficient method to promote vesiculation. In order to identify novel pathways stimulating vesiculation in RBC, we screened two libraries: the Library of Pharmacologically Active Compounds (LOPAC) and the Selleckchem Kinase Inhibitor Library for their effects on RBC from healthy donors. We investigated compounds triggering vesiculation and compounds inhibiting vesiculation induced by ionomycin. We identified 12 LOPAC compounds, nine kinase inhibitors and one kinase activator which induced RBC shrinkage and vesiculation. Thus, we discovered several novel pathways involved in vesiculation including G protein-coupled receptor (GPCR) signalling, the phosphoinositide 3-kinase (PI3K)–Akt (protein kinase B) pathway, the Jak–STAT (Janus kinase–signal transducer and activator of transcription) pathway and the Raf–MEK (mitogen-activated protein kinase kinase)–ERK (extracellular signal-regulated kinase) pathway. Moreover, we demonstrated a link between casein kinase 2 (CK2) and RBC shrinkage via regulation of the Gardos channel activity. In addition, our data showed that inhibition of several kinases with unknown functions in mature RBC, including Alk (anaplastic lymphoma kinase) kinase and vascular endothelial growth factor receptor 2 (VEGFR-2), induced RBC shrinkage and vesiculation.

Clearance of stored red blood cells is not increased compared with fresh red blood cells in a human endotoxemia model

It is thought that the clearance of transfused red blood cells (RBCs) is related both to the storage time of the transfusion product and to the inflammatory status of the recipient. We investigated these effects in a randomized, “two‐hit,” healthy volunteer transfusion model, comparing autologous RBCs that were stored for 35 days with those that were stored for 2 days.

Intrinsic defects in erythroid cells from familial hemophagocytic lymphohistiocytosis type 5 patients identify a role for STXBP2/Munc18-2 in erythropoiesis and phospholipid scrambling

Familial hemophagocytic lymphohistiocytosis type 5 (FHL-5) is a rare genetic disorder caused by mutations in STXBP2/Munc18-2. Munc18-2 plays a role in the degranulation machinery of natural killer cells and cytotoxic T lymphocytes. Mutations in STXBP2/Munc18-2 lead to impaired killing of target cells by natural killer cells and cytotoxic T lymphocytes, which in turn results in elevated levels of the inflammatory cytokine interferon γ, macrophage activation, and hemophagocytosis. Even though patients with FHL-5 present with anemia and hemolysis, no link between the disease and the erythroid lineage has been established. Here we report that red blood cells express Munc18-2 and that erythroid cells from patients with FHL-5 exhibit intrinsic defects caused by STXBP2/Munc18-2 mutations. Red blood cells from patients with FHL-5 expose less phosphatidylserine on their surface upon Ca(2+) ionophore ionomycin treatment. Furthermore, cultured erythroblasts from patients with FHL-5 display defective erythropoiesis characterized by decreased CD235a expression and aberrant cell morphology.

A method for red blood cell biotinylation in a closed system: A CLOSED SYSTEM TO PRODUCE BIOTINYLATED RBCs

Several circumstances require the accurate measurement of red blood cell (RBC) survival and clearance, such as determination of posttransfusion recovery of stored RBCs to investigate the effect of new additive solutions. To this end, biotin as a marker of RBCs to track donor RBCs in the blood of the recipient has been used in many studies. However, so far only experimental, nonvalidated, biotin‐labeled red cell concentrates (RCCs) are transfused. The goal of this study was to produce a standardized biotin‐labeled RCC product in a fast, simple, and sterile manner that can be used for clinical research and for the evaluation of new blood products according to Good Practice Guidelines (GPG) for blood establishments.

Glycophorin-C sialylation regulates Lu/BCAM adhesive capacity during erythrocyte aging

Lutheran/basal cell adhesion molecule (Lu/BCAM) is a transmembrane adhesion molecule expressed by erythrocytes and endothelial cells that can interact with the extracellular matrix protein laminin-α5. In sickle cell disease, Lu/BCAM is thought to contribute to adhesion of sickle erythrocytes to the vascular wall, especially during vaso-occlusive crises. On healthy erythrocytes however, its function is unclear. Here we report that Lu/BCAM is activated during erythrocyte aging. We show that Lu/BCAM-mediated binding to laminin-α5 is restricted by interacting, in cis, with glycophorin-C-derived sialic acid residues. Following loss of sialic acid during erythrocyte aging, Lu/BCAM is released from glycophorin-C and allowed to interact with sialic acid residues on laminin-α5. Decreased glycophorin-C sialylation, as observed in individuals lacking exon 3 of glycophorin-C, the so-called Gerbich phenotype, was found to correlate with increased Lu/BCAM-dependent binding to laminin-α5. In addition, we identified the sialic acid-binding site within the third immunoglobulin-like domain within Lu/BCAM that accounts for the interaction with glycophorin-C and laminin-α5. Last, we present evidence that neuraminidase-expressing pathogens, such as Streptococcus pneumoniae, can similarly induce Lu/BCAM-mediated binding to laminin-α5, by cleaving terminal sialic acid residues from the erythrocyte membrane. These results shed new light on the mechanisms contributing to increased adhesiveness of erythrocytes at the end of their lifespan, possibly facilitating their clearance. Furthermore, this work may contribute to understanding the pathology induced by neuraminidase-positive bacteria, because they are especially harmful to patients suffering from sickle cell disease and are associated with the occurrence of vaso-occlusive crises.Lutheran/basal cell adhesion molecule (Lu/BCAM) is a transmembrane adhesion molecule expressed by erythrocytes and endothelial cells that can interact with the extracellular matrix protein laminin-α5. In sickle cell disease, Lu/BCAM is thought to contribute to adhesion of sickle erythrocytes to the vascular wall, especially during vaso-occlusive crises. On healthy erythrocytes however, its function is unclear. Here we report that Lu/BCAM is activated during erythrocyte aging. We show that Lu/BCAM-mediated binding to laminin-α5 is restricted by interacting, in cis, with glycophorin-C-derived sialic acid residues. Following loss of sialic acid during erythrocyte aging, Lu/BCAM is released from glycophorin-C and allowed to interact with sialic acid residues on laminin-α5. Decreased glycophorin-C sialylation, as observed in individuals lacking exon 3 of glycophorin-C, the so-called Gerbich phenotype, was found to correlate with increased Lu/BCAM-dependent binding to laminin-α5. In addition, we identified the sialic acid-binding site within the third immunoglobulin-like domain within Lu/BCAM that accounts for the interaction with glycophorin-C and laminin-α5. Last, we present evidence that neuraminidase-expressing pathogens, such as Streptococcus pneumoniae, can similarly induce Lu/BCAM-mediated binding to laminin-α5, by cleaving terminal sialic acid residues from the erythrocyte membrane. These results shed new light on the mechanisms contributing to increased adhesiveness of erythrocytes at the end of their lifespan, possibly facilitating their clearance. Furthermore, this work may contribute to understanding the pathology induced by neuraminidase-positive bacteria, because they are especially harmful to patients suffering from sickle cell disease and are associated with the occurrence of vaso-occlusive crises.

Residual pyruvate kinase activity in PKLR-deficient erythroid precursors of a patient suffering from severe haemolytic anaemia

Here, we present a 7‐year‐old patient suffering from severe haemolytic anaemia. The most common cause of chronic hereditary non‐spherocytic haemolytic anaemia is red blood cell pyruvate kinase (PK‐R) deficiency. Because red blood cells rely solely on glycolysis to generate ATP, PK‐R deficiency can severely impact energy supply and cause reduction in red blood cell lifespan. We determined the underlying cause of the anaemia and investigated how erythroid precursors in the patient survive.

Apheresis causes complement deposition on red blood cells (RBCs) and RBC antigen alterations, possibly inducing enhanced clearance: THE EFFECTS OF APHERESIS ON DONOR RBCs

Apheresis is increasingly being applied to collect cells or plasma, even allowing the collection of multiple blood components during one procedure. Although the quality of the cellular and plasma products that are obtained by apheresis have been extensively studied and shown to be of high quality, the impact of apheresis on the red blood cells (RBCs) that are returned to the donor has not been investigated.