While HDAC6 overexpression in diverse cell types leads to MT deacetylation, its inhibition induces MT hyperacetylation, which is considered to enhance their balance23. distribution of megakaryocyte (MK) organelles. CTTN silencing in individual MKs phenocopies HDAC6 knockdown and inactivation potential clients to a solid PPF defect. That is rescued by compelled expression of the deacetylated CTTN mimetic. Unexpectedly, unlike human-derived MKs, HDAC6 and CTTN are been shown to be dispensable for mouse PPF in platelet and vitro creation in vivo. Our results high light an urgent function of HDAC6CCTTN axis being a positive regulator of human but not mouse MK maturation. Introduction Megakaryocytes (MKs) are highly specialized bone marrow cells that give rise to anucleated blood cells known as platelets1. MK progenitor proliferation occurs by classical mitosis, which, during MK differentiation, subsequently switches to an endomitotic mode2C4. At the end of the endomitotic process, MK cytoplasm matures, leading to increased organelle biosynthesis5 and the development of the demarcation membrane system (DMS)6. Once a MK matures, the DMS extends to form long pseudopods called proplatelets PF-06424439 (PPTs)6 that fragment, leading to platelet release in marrow sinusoids or lung circulation.1 Platelet production is due to MK fragmentation by a dynamic regulation of cytoplasmic extension, which mainly depends on microtubules (MTs) and actin cytoskeleton. While MT sliding powers PPT elongation, actin cytoskeleleton dynamics is critical for early stages of PPF by regulating DMS formation and actomyosin by controlling cortical contractile forces7C9. In addition, actin cytoskeleleton is also important for PPT branching and platelet release amplification10,11. A new class of anticancer agents targeting histone deacetylases (HDACs) induce profound thrombocytopenia12,13 by several mechanisms, including a toxic effect on hematopoietic progenitors through reactive oxygen species (ROS) and DNA damage and an alteration in late MK differentiation leading to a defect in PPF14. The focus has been on the changes in the MK cytoskeleton, especially on tubulin hyperacetylation and MT dynamics alteration as the mechanism of HDACi-induced thrombocytopenia. However, there is no direct evidence that tubulin hyperacetylation is involved in the defect of MK maturation15. In humans, 18 HDACs are grouped into four classes, but the precise role of these proteins in hemostasis is not well defined. HDAC6 belongs to the class IIb of HDACs that shuttles between the cytoplasm and the nucleus16. In contrast to class I HDACs, the role of HDAC6 has not yet been described during human megakaryopoiesis. HDAC6 is well expressed in platelets and may be involved in C13orf15 platelet functions17,18. Predominantly cytoplasmic19,20, HDAC6 possesses two catalytically active domains that deacetylate nonhistone proteins such as tubulin, HSP90, and cortactin PF-06424439 (CTTN)21C23. While HDAC6 overexpression in diverse cell types results in MT deacetylation, its inhibition induces MT hyperacetylation, which is thought to enhance their stability23. Recently, it was shown that knockout mice are viable and develop normally24. Our present work shows that human HDAC6 is a positive regulator of MK terminal differentiation and consequently of PPT generation. Our findings demonstrate that HDAC6 inhibition induces a defect in the development of DMS and -granules and actin disorganization, thus impairing PPF. This defect is mediated by CTTN hyperacetylation. We also show that HDAC6 inhibition in humans and the mouse displays divergent effects on PF-06424439 MK differentiation due to a differential role of CTTN. Altogether, our results highlight the role of HDAC6CCTTN axis in human MK maturation and point to a previously unknown mechanism underlying the HDACi-induced thrombocytopenia14. Results Expression of HDAC6 increases during MK differentiation In order to study the function of HDAC6 during megakaryopoiesis, we determined PF-06424439 its expression pattern. CD34+ cells were differentiated to MKs and sorted on expression of CD34 and CD41 at day 7 of culture. A fraction of the CD41+ cells were grown for 2 and 5 additional days allowing MK maturation. We studied the expression of 1C11 transcripts. was the HDAC expressed at the highest level all along the MK differentiation and then HDAC2, 1, and 7 (Supplementary Fig.?1). messenger RNA (mRNA) PF-06424439 level was also detected.