Phosphoproteomics and Morphology of Stored Human Red Blood Cells Treated by Protein-Tyrosine-Phosphatases Inhibitor.

The process of protein phosphorylation is involved in numerous cell functions. In particular, phosphotyrosine (pY) has been reported to play a role in red blood cell (RBC) functions, including the cytoskeleton organization. During their storage before transfusion, RBCs suffer from storage lesions that affect their energy metabolism and morphology. The present study investigated the relationship between the pY and the storage lesions. To do so, RBCs were treated (in the absence of calcium) with a protein tyrosine phosphatase inhibitor (orthovanadate, OV) to stimulate phosphorylation, and with three selective kinase inhibitors (KIs). Erythrocyte membrane proteins were studied by Western blot analyses and phosphoproteomics (data are available via ProteomeXchange with identifier PXD039914), and cell morphology by digital holographic microscopy. The increase of pY triggered by OV treatment (inducing a global downregulation of pS and pT) disappeared during the storage. Phosphoproteomic analysis identified 609 phosphoproteins containing 1'752 phosphosites, of which 41 pY were upregulated and 2 downregulated by OV. Following these phosphorylation processes, the shape of RBCs shifted from discocytes to spherocytes, and the addition of KIs partially inhibited this transition. The KIs modulated either pY, or pS and pT via diverse mechanisms related to cell shape, therefore all an impact on the RBC morphology. The capacity of RBCs to maintain their function is central in transfusion medicine, and the presented results contribute to a better understanding of RBC biology.

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