Study of Megakaryopoiesis and Proplatelet Formation In Myeloproliferative Disorders

Myeloproliferative neoplsms (MPNs) are clonal disorders that include primary myelofibrosis (PMF), polycythemia vera (PV) and essential thrombocytemia (ET). Several abnormalities that could explain the myeloproliferation have been reported, and most important is a striking involvement of the megakaryocyte (Mk) lineage, with hyperplasia and dysplasia resulting in an excessive production of several cytokines and chemokines. Whereas molecular defect(s) associated with the development of MPNs have been described, their pathogenesis is not thoroughly elucidated.

The general objective of this research was to study the process of megakaryopoiesis in MPNs in order to understand whether the pathological mechanisms underlying MPNs are intrinsic defects of Mk function or depend on an altered regulation by the bone marrow microenvironment.

We analyzed in vitro the Mk differentiation and proplatelet formation in 25 PMF patients, 13 ET/PV patients, and 19 healthy controls (HCs). Mks were differentiated from peripheral blood CD34⁺ or CD45⁺ cells in the presence of TPO for 14 days. Mature Mks were, then, grown in suspension or plated onto glass coverslips coated with collagen I or fibrinogen for additional 16 hours. Mk differentiation-maturation and proplatelet formation were evaluated by phase contrast and fluorescence microscopy upon cell staining with anti alpha-tubulin and CD41 antibodies. Measurement of Mk diameters was performed on acquired images, at least one hundred Mks were analyzed for each sample. Mk ploidy was evaluated by flow cytometry after staining with propidium iodide. Controls were analyzed in parallel with each patient sample.

Mk output was higher in patients with MPNs than HCs. Patients with PMF and PV had the highest Mk output, and in PMF the number of Mks was strictly associated with the V617F JAK2 genotype. PMF Mks presented smaller size and increased nuclear/cytoplasm ratio with respect to the other MPNs and HCs. Further, the measurement of Mk diameters revealed that PMF-derived Mks rarely reached the diameter of 40 μm, while HCs and ET/PV presented larger Mks. Finally, PMF produced decreased numbers of polyploid Mks (>8N) with respect to HCs. In order to explore if defects in Mk development were associated to altered Mk function, we investigated the competence to generate proplatelet by MPN-derived and HC-derived Mks. In HC samples, a 7.5% median of the total Mks formed proplatelets, while a significant defect in extending proplatelets was observed in PMF-derived Mks with respect to other MPNs and HCs. On the contrary, patients with PV or ET showed increased numbers of Mks forming proplatelets with respect to PMF and HCs. No differences were observed between pre-fibrotic and fibrotic PMF, and pre-fibrotic PMF-derived Mks formed a decreased number of proplatelet as compared to both ET/PV and HCs. Most importantly, platelet counts correlated with Mk ability of extending proplatelets of different MPN patients. Finally, we found that the proplatelets extended by patients with PMF presented peculiar morphological features with respect to the normal counterpart and patients with PV and ET. Their more evident alteration was a significantly reduced number of bifurcations in secondary processes despite a normal length of the individual proplatelet shafts, thus indicating a defect in proplatelet branching. On the opposite, ET- and PV-derived proplatelets presented an enormous increase of bifurcations with respect to PMF and HCs.

Mks from patients affected by MPNs presented high proliferative capacity and defects in proplatelet formation. Thus, platelet production in MPNs may not depend only on an increased number of Mks, but also on intrinsic defects in Mk function. Interestingly, our results also showed that Mk of pre-fibrotic and fully fibrotic PMF present similar biological characteristics and different from those of ET, providing a rationale for considering pre-fibrotic as a different entity from ET. In conclusion, this study proposes important new elements in the understanding of the biology of Mk and proplatelet formation in MPNs, and open a new perspective into the understanding of the pathophysiology of defects of platelet production in MPNs.

No relevant conflicts of interest to declare.