Posttranslational processing of the thrombopoietin receptor is impaired inpolycythemia vera

Recently, we demonstrated a marked reduction in the expression of the throm bopoietin receptor, Mpl, in polycythemia vera (PV) platelets and megakaryoc ytes using an antiserum against the Mpl extracellular domain. To further ex amine this abnormality, we raised an antibody to the Mpl C-terminus. Immuno logic analysis of PV platelets with this antiserum confirmed the reduction in Mpl expression. However, the C-terminal antiserum detected 2 forms of Mp l in PV platelets in contrast to normal platelets, in which a single form o f Mpl was detected by both the extracellular domain and C-terminal antisera . Two-dimensional gel electrophoresis studies with isoelectric focusing in the first dimension identified normal platelet Mpl as an 85 to 92 kD protei n with an isoelectric point (pl) of 5.5. PV platelets contained an addition al 80 to 82 kD Mpl Mpl isoform with a pl of 6.5. Analysis of Mpl expressed by the human megakaryocytic cell line, Dami, showed 2 isoforms similar to t hose found in PV platelets suggesting a precursor-product relationship, Dig estion of Dami cell and normal platelet lysates with neuraminidase converte d the more acidic Mpl isoform to the more basic one, indicating that the 2 isoforms differed with respect to posttranslational glycosylation, Futhermo re, in contrast to normal platelet Mpl, PV platelet Mpl was susceptible to endoglycosidase H digestion, indicating defective Mpl processing by PV mega karyocytes, The glycosylation defect was specific for Mpl, as 2 other plate let membrane glycoproteins, glycoprotein IIb and multimerin, were processed normally. Importantly, the extent of the PV platelet Mpl glycosylation def ect correlated with disease duration and extramedullary hematopoiesis. (C) 1999 by The American Society of Hematology.