Megakaryocytes from MPNs express increased B4GALT1 under the control of JAK/STAT pathway. Megakaryocytes were differentiated from human peripheral blood progenitors from HCs and MPN patients. (A) Representative immunofluorescence staining of GpIIIa (green, GpIIIa; blue, nuclei; scale bar, 25 µm). (B) Percentage of GpIIb⁺GpIbα⁺ megakaryocytes (MKs) at the end of the culture. Data are presented as mean ± SD (n = 8; P, not significant). (C) B4GALT1 expression differentiated from HCs and patients. Data are presented as mean ± SD (n = 3; *P < .05). (D) Immunofluorescence of megakaryocytes differentiated from HC and patients with MPN using ECL (red, ECL; blue, nuclei; scale bar, 20 μm). The quantification of ECL fluorescence in megakaryocytes is reported. Data are presented as mean ± SD (n = 8; *P < .001). (E) MPN megakaryocytes were cytokine starved and treated or not with ruxolitinib (i). HC megakaryocytes were cultured with a high dose of TPO and treated or not with ruxolitinib (ii). Two MPNs (JAK2 and MPL) and 3 HCs were tested with comparable results. Here, we present the representative western blot analysis of total lysates from the JAK2-mutated patient and 1 HC, probed with ECL in order to evaluate the galactosylation status. GpIbα, GpIIIa, and β-actin were used as loading controls. (F) Diagram of how B4GALT1 regulates megakaryocyte and platelet galactosylation in physiology and disease. In HCs (left panel), B4GALT1 is expressed under control of TPO. Megakaryocytes produce and release platelets into the bloodstream that expose galactose becoming ligands for the AMRs to promote TPO production by the liver. In MPNs (right panel), constitutive activation of JAK/STAT signaling determines an increased expression of B4GALT1 resulting in the production of platelets that are highly galactosylated. This can stimulate hepatocytes to release high amounts of TPO into bloodstream.