title: Identification of signalling cascades involved in red blood cell shrinkage and vesiculation.
abstract: 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 Ca(2+) 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.
authors.affiliation: *Department of Blood Cell Research, Sanquin Research, Plesmanlaan 125, 1066CX, Amsterdam, The Netherlands.
authors.forename: Elena B
authors.initials: EB
authors.lastname: Kostova
authors.affiliation: *Department of Blood Cell Research, Sanquin Research, Plesmanlaan 125, 1066CX, Amsterdam, The Netherlands.
authors.forename: Boukje M
authors.initials: BM
authors.lastname: Beuger
authors.affiliation: *Department of Blood Cell Research, Sanquin Research, Plesmanlaan 125, 1066CX, Amsterdam, The Netherlands.
authors.forename: Thomas R L
authors.initials: TR
authors.lastname: Klei
authors.affiliation: †Division of Molecular Carcinogenesis, NKI Robotics and Screening Center, Plesmanlaan 121, 1066CX, Amsterdam, The Netherlands.
authors.forename: Pasi
authors.initials: P
authors.lastname: Halonen
authors.affiliation: †Division of Molecular Carcinogenesis, NKI Robotics and Screening Center, Plesmanlaan 121, 1066CX, Amsterdam, The Netherlands.
authors.forename: Cor
authors.initials: C
authors.lastname: Lieftink
authors.affiliation: †Division of Molecular Carcinogenesis, NKI Robotics and Screening Center, Plesmanlaan 121, 1066CX, Amsterdam, The Netherlands.
authors.forename: Roderick
authors.initials: R
authors.lastname: Beijersbergen
authors.affiliation: *Department of Blood Cell Research, Sanquin Research, Plesmanlaan 125, 1066CX, Amsterdam, The Netherlands.
authors.forename: Timo K
authors.initials: TK
authors.lastname: van den Berg
authors.affiliation: *Department of Blood Cell Research, Sanquin Research, Plesmanlaan 125, 1066CX, Amsterdam, The Netherlands.
authors.forename: Robin
authors.initials: R
authors.lastname: van Bruggen
mesh_terms: D061207:Calcium Ionophores / Q000494:pharmacology*; D047390:Casein Kinase II / Q000378:metabolism; D055252:Cell-Derived Microparticles / Q000378:metabolism*; D016922:Cellular Senescence / Q000187:drug effects; D004912:Erythrocytes / Q000166:cytology / Q000378:metabolism*; D006801:Humans; D015759:Ionomycin / Q000494:pharmacology*; D048490:raf Kinases / Q000378:metabolism
chemical_list: D061207:Calcium Ionophores; D015759:Ionomycin; D047390:Casein Kinase II; D048490:raf Kinases