Platelet-Like-Particles Sheared From Myosin-II-Inhibited Megakaryocytes Highlights The Elevated Thrombocrit Of May-Hegglin Anomaly

Megakaryocytes (MKs) in the marrow extend projections into blood flow and generate platelets under shear. Understanding MK differentiation and platelet production is of broad clinical importance and extends a need to augment platelet numbers in patients. Reversible but sustained inhibition of non-muscle myosin-II (NMM-II) with the drug blebbistatin increases MK polyploidization, proplatelet formation, and membrane flexibility, thereby increasing platelet generation under shear. Using a cone and plate rheometer to apply fluid shear to drug-treated MKs in bulk, platelet-like-particles (PLPs) that are collagen-I responsive can be generated with intermediate shear. The MKs naturally down-regulate NMM-IIA activity through phosphorylation of S1943, but this site proves shear sensitive, consistent with results for human platelets. Using micropipette aspiration of MKs, inhibition of NMM-IIA is found necessary to generate CD41⁺ fragments that approximate the size of human platelets. Localization of NMM-IIA to the fragments is modulated by S1943 as seen by unique distribution patterns resulting from specific S1943 mutations that can be abrogated by addition of blebbistatin. The approach is extended to clinically relevant mutations associated with May-Hegglin anomaly (MHA) co-expressed with wild type protein to mimic heterozygotes. As with blebbistatin inhibition of myosin, May-Hegglin mutants result in a higher frequency of fragmentation during micropipette aspiration, indicating a dominant negative effect. Immunofluorescence documents abnormal myosin aggregation in cells transfected with May-Hegglin myosin mutations compared to wild type constructs. Finally, peripheral blood from a patient with a D1414N May-Hegglin mutation is cultured to produce megakaryocytes used to support both the micropipette and immunofluorescence results. These findings reveal a phospho-switch in NMM-II, from inactive to active in the terminal stages of platelet-poiesis, and that proper myosin activity is critical to fragment size and number. Disruption of normal activity enhances fragment generation suggesting a novel mechanism in MHA: in particular, MHA thrombocytopenia results in an increased thrombocrit due to abnormally large platelets, which overcompensates for the reduction in platelet number.