New insights into cytoskeletal remodeling during platelet production.

The past decade has brought unprecedented advances in our understanding of megakaryocyte (MK) biology and platelet production, processes that are strongly dependent on the cytoskeleton. Facilitated by technological innovations, such as new high-resolution imaging techniques (in vitro and in vivo) and lineage-specific gene knockout and reporter mouse strains, we are now able to visualize and characterize the molecular machinery required for MK development and proplatelet formation in live mice. Whole genome and RNA sequencing analysis of patients with rare platelet disorders, combined with targeted genetic interventions in mice, has led to the identification and characterization of numerous new genes important for MK development. Many of the genes important for proplatelet formation code for proteins that control cytoskeletal dynamics in cells, such as Rho GTPases and their downstream targets. In this review, we discuss how the final stages of MK development are controlled by the cellular cytoskeletons, and we compare changes in MK biology observed in patients and mice with mutations in cytoskeleton regulatory genes.

PRBC-derived plasma induces non-muscle myosin type IIA-mediated neutrophil migration and morphologic change.

CONTEXT: Neutrophils are the primary effector cells in the pathogenesis of transfusion-related acute lung injury or multiple organ failure after blood transfusion. OBJECTIVE: We aimed to investigate the effect of fresh (1 day preparation) and aged (42 day preparation) PRBC-derived plasma on neutrophil morphology, migration and phagocytosis. MATERIALS AND METHODS: We evaluated the production of reactive oxygen species (ROS) and the expression of non-muscle myosin heavy chain IIA (MYH9) in neutrophils treated with PRBC-derived plasma. We used western blots and antibody arrays to evaluate changes in signal transduction pathways in plasma-treated neutrophils. RESULTS: Aged PRBC-derived plasma elicited a stronger oxidative burst in neutrophils when compared with fresh PRBC-derived plasma (p < 0.05). Antibody arrays showed increased phosphorylation of NF-ĸB proteins (p105, p50 and Ikk) in aged PRBC-derived plasma-treated neutrophils. The expression of non-muscle myosin IIA (MYH9), a cytoskeleton protein involved in immune cell migration and morphological change, was also significantly upregulated in neutrophils treated with aged PRBC-derived plasma compared to fresh plasma (p < 0.05). Pretreatment of neutrophils with blebbistatin (a specific type II myosin inhibitor), ascorbic acid (an antioxidant), or staurosporine (a protein tyrosine kinase inhibitor), effectively abrogated the morphological changes, neutrophil migration, and phagocytosis induced by aged PRBC-derived plasma. CONCLUSION: Upregulation of MYH9 in neutrophils treated with aged PRBC-derived plasma and abrogation of neutrophil migration in blebbistatin-treated neutrophils suggested a functional role of MYH9 in the directional migration of immune cells. Our data help elucidate the cellular and molecular mechanisms of transfusion-related injury.

Myosin-II inhibition and soft 2D matrix maximize multinucleation and cellular projections typical of platelet-producing megakaryocytes.

Cell division, membrane rigidity, and strong adhesion to a rigid matrix are all promoted by myosin-II, and so multinucleated cells with distended membranes--typical of megakaryocytes (MKs)--seem predictable for low myosin activity in cells on soft matrices. Paradoxically, myosin mutations lead to defects in MKs and platelets. Here, reversible inhibition of myosin-II is sustained over several cell cycles to produce 3- to 10-fold increases in polyploid MK and a number of other cell types. Even brief inhibition generates highly distensible, proplatelet-like projections that fragment readily under shear, as seen in platelet generation from MKs in vivo. The effects are maximized with collagenous matrices that are soft and 2D, like the perivascular niches in marrow rather than 3D or rigid, like bone. Although multinucleation of other primary hematopoietic lineages helps to generalize a failure-to-fission mechanism, lineage-specific signaling with increased polyploidy proves possible and novel with phospho-regulation of myosin-II heavy chain. Label-free mass spectrometry quantitation of the MK proteome uses a unique proportional peak fingerprint (ProPF) analysis to also show upregulation of the cytoskeletal and adhesion machinery critical to platelet function. Myosin-inhibited MKs generate more platelets in vitro and also in vivo from the marrows of xenografted mice, while agonist stimulation activates platelet spreading and integrin αIIbß3. Myosin-II thus seems a central, matrix-regulated node for MK-poiesis and platelet generation.

The critical role of myosin IIA in platelet internal contraction.

BACKGROUND: Shape change and centralization of granules surrounded by a microtubular coil (internal contraction) are among the earliest morphologic changes observed following platelet activation. Myosin IIA contributes to initiation of platelet shape change, but its role in internal contraction has not been defined. OBJECTIVE: To define the contribution of myosin IIA to platelet internal contraction. METHODS: Aspirin-treated platelets suspended in calcium-free buffer were activated with a low concentration (25 nm) of the thromboxane A(2) analog U46619 which initiated shape change and internal contraction via a Rho kinase pathway. Shape change and internal contraction were assessed by aggregometry and transmission electron microscopy (TEM), and Rho activation and myosin regulatory light chain (MRLC) phosphorylation were studied concurrently. RESULTS AND CONCLUSIONS: Low-concentration blebbistatin (10 microm) inhibited internal contraction in the majority of platelets with minimal inhibition of shape change without significant suppression of MRLC phosphorylation. Higher blebbistatin concentrations (25-100 microm) produced concentration-dependent inhibition of aggregation, shape change, Rho activation, and MRLC phosphorylation. These data demonstrate: (i) direct platelet myosin IIA participation in internal contraction; and (ii) inhibition of Rho activation and MRLC phosphorylation by >10 microm blebbistatin.