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 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.
Highlights
Transfusion of red blood cells (RBCs) is often a life-saving therapy for which no substitute exists
The library of pharmacologically active compounds (LOPAC) and the Selleckchem Kinase Inhibitor Library, we confirmed the importance of well-known pathways such as calcium signalling [26], caspase activity [27] and protein kinase C (PKC) signalling [28], but we discovered several cascades not described previously to play a role in RBC vesiculation
We performed inhibition and induction screens in parallel since we aimed at identifying molecules that induce RBC vesiculation and molecules that inhibit vesiculation induced by ionomycin
Summary
Transfusion of red blood cells (RBCs) is often a life-saving therapy for which no substitute exists. RBC undergo considerable changes during storage, including lactate accumulation, decrease in ATP and 2,3DPG (2,3-diphosphoglycerate) levels [1] and vesicle release [2]. These modifications, known as the storage lesion, lead to alterations in the function and lifespan of RBC in vivo post transfusion [3,4], which can have detrimental side effects in the recipient [5]. Vesicles released during storage induce the production of pro-inflammatory cytokines by monocytes promoting T-cell proliferation [12]. The vesicles that are released are subsequently engulfed by monocytes promoting the production of pro-inflammatory cytokines and endothelial cell adhesion [13]
Sign-in/Register to view highlights & summary Sign-in/Register to access full text options 
Talk to us
Join us for a 30 min session where you can share your feedback and ask us any queries you have
Schedule a callDisclaimer: All third-party content on this website/platform is and will remain the property of their respective owners and is provided on "as is" basis without any warranties, express or implied. Use of third-party content does not indicate any affiliation, sponsorship with or endorsement by them. Any references to third-party content is to identify the corresponding services and shall be considered fair use under The CopyrightLaw.
