Effects of inhibitor of Src kinases, SU6656, on differentiation of megakaryocytic progenitors and activity of alpha1,6-fucosyltransferase.

Abstract

alpha1,6-fucosyltransferase (FUT8) attaches fucose residues via an alpha1,6 linkage to the innermost N-acetylglucosamine residue of N-linked glycans. Glycans with this type of structure are present in GpIIb/GpIIIa complex (CD41a) which is present on megakaryocytes (Mks) and platelets. CD41a is the earliest marker of megakaryocytopoiesis. The aim of this study was to analyse the morphology, phenotype, ploidy level and activity of FUT8 during induced differentiation/maturation of Mk progenitor cells in ex vivo culture. We used SU6656, a selective inhibitor of Src tyrosine kinases, as differentiation-inducing agent for Mks. The addition of SU6656 to the culture system of megakaryocytic progenitors from cord blood CD34(+) cells and Meg-01 cell line induced their maturation towards later stages of Mk differentiation with increased activity of FUT8. We suggest FUT8 as a candidate for an early marker of differentiation and possibly of the ploidy level of Mks. We confirm a special status of FUT8 in megakaryocytopoiesis.

Optimizing megakaryocyte polyploidization by targeting multiple pathways of cytokinesis

“…Differentiation of megakaryocytes from umbilical cord blood in a medium containing TPO and other cytokines, such as SCF, interleukin‐3, and Flt3‐l, has historically led to low ploidy megakaryocytes with a maximum DNA content of 32N at a low percentage of the cultured cells 2,5,9 . The introduction of reagents such as NIC, 17,18 SI, 19,20 and ABI 22,23 into the culture conditions has increased the maximum DNA content as well as the percentage of polyploid cells. Each of these reagents affects cytokinesis through different cellular targets and yet was able to increase the megakaryocyte ploidy.…”

“…The inhibition of Src family kinases has been shown to increase megakaryocyte polyploidization through the Lyn/Fyn pathway and inhibition of actin polymerization. 19,20 Addition of SI, Su6656, at a concentration of 2.5 mmol/L on Day 8 of megakaryocyte culture induced a moderate increase in ploidy ( Figs. 1A and 2E), a moderate fold expansion (Fig.…”

“…Much progress has been made toward understanding the mechanisms of megakaryocyte polyploidization 12,13 . Various reagents that inhibit specific steps of the cytokinesis process have been reported to increase megakaryocyte ploidy, for example, nicotinamide (NIC; vitamin B3), 17,18 Src inhibitor (SI; Su6656), 19‐21 and aurora B inhibitor (ABI; ZM447439) 22,23 . The role of aurora B inhibition remains controversial regarding its capacity to increase megakaryocyte polyploidization 24,25 .…”

Ex Vivo production of platelets from stem cells

“…Lastly, Src‐family kinases inhibit megakaryocyte proliferation and differentiation (Lannutti & Drachman, ). Accordingly, inhibition of Src‐family kinases increased megakaryocyte polyploidization and proplatelet formation in leukaemic cell lines through the Lyn/Fyn pathway and inhibition of actin polymerization (Lannutti et al , ; Kaminska et al , ). Each (or all) of these pathways could be manipulated to force ex vivo cultured megakaryocytes to undergo polyploidization.…”

Induction of differentiation of human stem cellsex vivo: Toward large-scale platelet production

“…Lyn-deficient mice produced more mature MKs above 8 N in the presence of the Src kinase inhibitor pyrrolopyrimidine 1. In addition, the Src kinase inhibitor SU6656 increased the proportion of polyploid MKs in the differentiation of cell lines UT-7, HEL, and Meg-01 and in patients with myeloid dysplasia syndrome[48,49].…”