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See detailThrombopoietin dose-response curves reflect the number of doublings undergone by human megakaryocyte progenitors
Paulus, Jean-Michel ULg; Debili, N.; Larbret, F. et al

Poster (2004, July)

To assess the variation of thrombopoietin (TPO) responsiveness associated with MK progenitor amplification, TPO dose-response curves were obtained for MK clones derived from normal human CD34+CD41+ cells ... [more ▼]

To assess the variation of thrombopoietin (TPO) responsiveness associated with MK progenitor amplification, TPO dose-response curves were obtained for MK clones derived from normal human CD34+CD41+ cells which had been single-cell plated in serum-deprived medium with TPO concentrations of 0-1000 pg/mL per well. On day 5, the number of MK cells per well was determined in situ and expressed as number of doublings (NbD). Dose-response curves of the means of the frequency of clones of each size vs logTPO concentration showed highly significant differences in the TPO concentration needed for half-maximum generation of clones of different sizes (TPO50). Based on 3 independent experiments, TPO50, expressed in pg/mL, was 1.89 ±SD 0.87 for 1 cell MK clone; 7.75 ±1.40 for 2-3 cell MK clone; 38.5 ±8.73 for 4-7 cell MK clone and 93.6 ±27.7 for 8-15 cell MK clone (p<0.001). Compared to the CD42- fraction of CD34+CD41+ progenitors, the corresponding CD42+ fraction had a lower TPO50 (p<0.001), underwent lesser NbD (p<0.001) and expressed a 10-fold greater mean mpl receptor density. In order to interpret the conclusion that the most responsive progenitors were those which proliferated less, we tested a prediction of the generation-age model, namely that the percentage of progenitors undergoing a given number of residual doublings in vitro (NbDr) depends on the number of previous doublings they had undergone in vivo (NbDp). It was demonstrated that logTPO50 had a positive correlation with NbDr and negative correlation with NbDp (p<0.001). The reciprocal relationship between NbDp and NbDr was evidence for a generation-age model in which TPO responsiveness increases by a factor of approximately 4 per doubling. This model suggests that progenitors which have completed their proliferative program have maximum factor responsiveness and are preferentially induced to terminal differentiation. [less ▲]

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See detailMegakaryocyte (MK) clone size is inversely related to CFU-MK responsiveness to thrombopoietin (TPO)
Paulus, Jean-Michel ULg; Vainchenker, W.; Debili, N.

in Blood (2001, November 16), 98(11, Part 2), 119-119

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See detailExpression and function of the collagen receptor GPVI during megakaryocyte maturation.
Lagrue-Lak-Hal, A. H.; Debili, N.; Kingbury, G. et al

in Journal of Biological Chemistry (2001), 276(18), 15316-25

In this report, the expression and function of the platelet collagen receptor glycoprotein VI (GPVI) were studied in human megakaryocytes during differentiation and maturation of mobilized blood and cord ... [more ▼]

In this report, the expression and function of the platelet collagen receptor glycoprotein VI (GPVI) were studied in human megakaryocytes during differentiation and maturation of mobilized blood and cord blood derived CD34(+) cells. By flow cytometry, using an anti-GPVI monoclonal antibody or convulxin, a GPVI-specific ligand, GPVI was detected only on CD41(+) cells including some CD41(+)/CD34(+) cells, suggesting expression at a stage of differentiation similar to CD41. These results were confirmed at the mRNA level using reverse transcription-polymerase chain reaction. GPVI expression was low during megakaryocytic differentiation but increased in the more mature megakaryocytes (CD41(high)). As in platelets, megakaryocyte GPVI associates with the Fc receptor gamma chain (FcRgamma). The FcR gamma chain was detected at the RNA and protein level at all stages of megakaryocyte maturation preceding the expression of GPVI. The other collagen receptor, alpha(2)beta(1) integrin (CD49b/CD29), had a pattern of expression similar to GPVI. Megakaryocytic GPVI was recognized as a 55-kDa protein by immunoblotting and ligand blotting, and thus it presented a slightly lower apparent molecular mass than platelet GPVI (58 kDa). Megakaryocytes began to adhere to immobilized convulxin via GPVI after only 8-10 days of culture, at a time when megakaryocytes were maturing. At this stage of maturation, they also adhered to immobilized collagen by alpha(2)beta(1) integrin-dependent and -independent mechanisms. Convulxin induced a very similar pattern of protein tyrosine phosphorylation in megakaryocytes and platelets including Syk, FcRgamma, and PLC(gamma)2. Our results showed that GPVI is expressed early during megakaryocytic differentiation but functionally allows megakaryocyte adherence to collagen only at late stages of differentiation when its expression increases. [less ▲]

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