Fig. 5

Proplatelet formation in the presence of plasma from myeloproliferative neoplasms patients. Cord blood-derived day 12 megakaryocyte (MK) were incubated with 10% plasma during 72 hs. (A) Example of a MK producing proplatelets (inverted microscope) in the presence of control, essential thrombocythemia (ET) and myelofibrosis (MF) samples. The arrows indicate MK producing proplatelets. (B) Proplatelet processes were counted in an inverted microscope and proplatelets-bearing MK (PPF) number was calculated and referred to total cell count (Neubauer chamber). Results are expressed as percentage of PPF (Control, n = 40; ET, n = 28; MF, n = 30). Red empty circles represent post-ET MF, pink empty circles represent post-PV MF, red filled circles represent primary MF. Kruskal–Wallis followed by Dunn’s multiple comparisons test. Bars and error bars indicate the median with interquartile range. The experiments were carried out in triplicate in independent umbilical cord blood samples. (C) Percentage of plasma-induced PPF, according to the JAK2V617F mutational status of the myeloproliferative neoplasms (MPN) patients (p = ns, Mann-Whitney test). (D) Correlation between plasma-induced PPF in culture and peripheral platelet count in MPN patients (Spearman correlation). (E) Proplatelet morphology in the presence of plasma samples. MK were seeded in fibrinogen-coated glass during 72 hs, incubated in the presence of control (n = 6), ET (n = 6) or MF (n = 6) plasma. Then, cells were labeled with Hoechst (nucleus, blue) and CD61-FITC antibody (green). Number of swelling and tips per proplatelet process were counted in at least 50 MK. Kruskal–Wallis followed by Dunn’s multiple comparisons test. Bars and error bars indicate the median with interquartile range. (F) Representative example of proplatelet-bearing MK incubated with control, ET and MF samples. Swelling and tip are pointed out. Scale bars, 20 μm.