Platelet generation under shear force modulated by site-specific phosphorylation of myosin-IIA heavy chain

Megakaryocytes (MKs) are rare cells that every day give rise to 10
¹⁰
platelets, which are necessary for vascular homeostasis via clot formation and contraction. Understanding MK differentiation is of broad clinical importance and includes a need to augment platelet numbers in patients (e.g. post-surgery). Our group has demonstrated that reversible but sustained inhibition of NMM-II (non-muscle myosin-II) increases MK polyploidization, proplatelet formation and membrane flexibility, thereby increasing platelet generation [1]. NMM-II mutants have been implicated in platelet diseases. Furthermore, while physiological inhibition of NMM-II by phosphorylation regulates the early stage MK polyploidization, it is unknown whether phosphorylation is also important in the late stage fragmentation of platelets from MKs. We have demonstrated that applying controlled shear force in vitro increases fragmentation of platelet-like particles (PLPs) from an established human MK cell line system (MEG-01), and that pharmacological inhibition of NMM-II facilitates fragmentation even further under shear. Our current efforts our focused on introducing NMM-IIA mutants into MEG-01 cells at sites implicated in phosphorylation and May-Hegglin disorder.