Exosomes released during reticulocyte maturation bind to fibronectin via integrin alpha 4 beta 1

Exosomes are vesicles formed in the endosomal compartment and released in t he extracellular medium during reticulocyte maturation into erythrocytes. T hey have a clearing function because of their enrichment with some proteins known to decrease or disappear from the cell surface during maturation, e. g. acetylcholinesterase and transferrin receptor. We show here that integri n alpha 4 beta 1, present on the surface of erythroid precursors but absent from the mature red cell membrane, is at least partly cleared from the ret iculocyte plasma membrane by the exosomal pathway. Using flow cytometry, we found that the alpha 4 subunit disappears from the reticulocyte surface du ring in vitro maturation. Two different monoclonal antibodies (B-5G10 and H P 2/1) were used to demonstrate the presence of the alpha 4 chain on the ex osome surface. Moreover, membrane acetylcholinesterase and lumenal peroxida se-like (i.e. hemoglobin) enzymatic activities were assayed to demonstrate exosome binding to plates coated with increasing fibronectin (FN) concentra tions. This interaction was dependent on divalent cations (MnCl2 > MgCl2 > CaCl2). Similarly, vesicles bound to plates coated with the chymotryptic 40 K fragment (FN-40) containing the heparin-binding region of FN. This bindi ng was inhibited by exosome preincubation with fibronectin CS1 peptide and with a monoclonal antibody (HP 2/1) against the integrin alpha 4-chain, con firming an alpha 4 beta 1-induced interaction. The importance of the exosom e clearance function is highlighted here, since the presence of VLA-4 on re ticulocytes often leads to blood circulation complications in some diseases . Moreover, the presence of alpha 4 beta 1 on the exosome surface, by allow ing binding to endothelial cells through vascular cell adhesion molecule 1 (VCAM-1), might confer another physiological function to the secreted vesic les.