Thrombosis leads to platelet activation and subsequent degradation; therefore, replenishment of platelets from hematopoietic stem/progenitor cells (HSPCs) is needed to maintain the physiological level of circulating platelets. Platelet-derived microparticles (PMPs) are protein- and RNA-containing vesicles released from activated platelets. We hypothesized that factors carried by PMPs might influence the production of platelets from HSPCs, in a positive feedback fashion. Here we show that, during mouse acute liver injury, the density of megakaryocyte in the bone marrow increases following an increase in circulating PMPs, but without thrombopoietin (TPO) upregulation. In vitro, PMPs are internalized by HSPCs and drive them toward a megakaryocytic fate. Mechanistically, miR-1915-3p, a miRNA highly enriched in PMPs, is transported to target cells and suppresses the expression levels of Rho GTPase family member B, thereby inducing megakaryopoiesis. In addition, direct injection of PMPs into irradiated mice increases the number of megakaryocytes and platelets without affecting TPO levels. In conclusion, our data reveal that PMPs have a role in promoting megakaryocytic differentiation and platelet production.

Platelets (PLTs) are produced by megakaryocytes (MKs) that completed differentiation and endomitosis. Endomitosis is an important process in which the cell replicates its DNA without cytokinesis and develops highly polyploid MK. In this study, to gain a better PLTs production, four small molecules (Rho-Rock inhibitor (RRI), nicotinamide (NIC), Src inhibitor (SI), and Aurora B inhibitor (ABI)) and their combinations were surveyed as MK culture supplements for promoting polyploidization. Three leukemia cell lines as well as primary mononuclear cells were chosen in the function and mechanism studies of the small molecules. In an optimal culture method, cells were treated with different small molecules and their combinations. The impact of the small molecules on megakaryocytic surface marker expression, polyploidy, proliferation, and apoptosis was examined for the best MK polyploidization supplement. The elaborate analysis confirmed that the combination of SI and RRI together with our MK induction system might result in efficient ploidy promotion. Our experiments demonstrated that, besides direct downregulation on the expression of cytoskeleton protein actin, SI and RRI could significantly enhance the level of cyclins through the suppression of p53 and p21. The verified small molecule combination might be further used in the in vitro PLT manufacture and clinical applications.

A better understanding of the mechanisms involved in megakaryocyte maturation will facilitate the generation of platelets in vitro and their clinical applications. A microRNA, miR-125b, has been suggested to have important roles in the self-renewal of megakaryocyte-erythroid progenitors and in platelet generation. However, miR-125b is also critical for hematopoietic stem cell self-renewal. Thus, the function of miR-125b and the complex signaling pathways regulating megakaryopoiesis remain to be elucidated. In this study, an attentive examination of the endogenous expression of miR-125b during megakaryocyte differentiation was performed. Accordingly, the differentiation of hematopoietic stem cells requires the downregulation of miR-125b, whereas megakaryocyte determination and maturation synchronize with miR-125b accumulation. The overexpression of miR-125b improves megakaryocytic differentiation of K562 and UT-7 cells. Furthermore, stage-specific overexpression of miR-125b in primary cells demonstrates that miR-125b mediates an enhancement of megakaryocytic differentiation after megakaryocyte determination, the stage at which megakaryocytes are negative for the expression of the hematopoietic progenitor marker CD34. The identification of miR-125b targets during megakaryopoiesis was focused on negative regulators of cell cycle because the transition of the G1/S phase has been associated with megakaryocyte polyploidization. Real-time PCR, western blot and luciferase reporter assay reveal that p19INK4D is a direct target of miR-125b. P19INK4D knockdown using small interfering RNA (siRNA) in megakaryocyte-induced K562 cells, UT-7 cells and CD61+ promegakaryocytes results in S-phase progression and increased polyploidy, as well as improved megakaryocyte differentiation, similarly to the effects of miR-125b overexpression. P19INK4D overexpression reverses these effects, as indicated by reduced expression of megakaryocyte markers, G1-phase arrest and polyploidy decrease. P19INK4D knockdown in miR-125b downregulated cells or p19INK4D overexpression in miR-125b upregulated cells rescued the effect of miR-125b. Taken together, these findings suggest that miR-125b expression positively regulates megakaryocyte development since the initial phases of megakaryocyte determination, and p19INK4D is one of the key mediators of miR-125b activity during the onset of megakaryocyte polyploidization.