The importance of calcium in the regulation of megakaryocyte function

Abstract

© F e r r a t a S t o r t i F o u n d a t i o ndepletion and activates Orai1 (also known as calciumrelease-activated calcium-modulator, CRACM), the poreforming subunit of store-operated Ca 2+ channels. 19 The role of the additional STIM1 and Orai1 paralogs, i.e. STIM2 and Orai2-3, is far from being fully understood in naïve cell systems, albeit they may recapitulate SOCE when ectopically expressed. 19 Moreover, several evidences demonstrated that members of the canonical transient receptor potential (TRPC) family of cation channels may represent additional candidates for SOCE. 20 Agonistinduced elevation of intracellular Ca 2+ levels is essential for platelet activation. In this regard, STIM1 and Orai1 proteins have been shown to be key players in human platelet SOCE during aggregation, 21,22 whereas TRPC involvement has been questioned. 23,24 Importantly, it has been demonstrated that SOCE plays a major role in mediating adhesion and motility onto ECM components of different cell types, including hematopoietic stem cells.9,25 Thus we hypothesized that purinergic signaling and SOCE may be responsible for Mk interaction with the ECM environment and consequent regulation of platelet production. Our results demonstrate that ADP induces both intracellular Ca 2+ mobilization and extracellular Ca 2+ inflow in human Mks, which in turn support the cytoskeletal reorganization responsible for cellular adhesion and migration and final proplatelet formation on ECM components that promote such a dynamic process, such as fibrinogen and fibronectin. These findings provide the first evidence that Ca 2+ signaling is a fundamental regulator of human Mk functions. MethodsHuman cord blood was collected from the local blood bank following normal pregnancies and deliveries with informed consent of the parents, in accordance with the ethical committee of the IRCCS Policlinico San Matteo Foundation, Pavia, Italy, and the principles of the Declaration of Helsinki. CD34 + cells from cord blood samples were separated by immunomagnetic bead selection (Miltenyi Biotec, Bologna, Italy) and differentiated, as previously described. 4 At the end of cell culture, Mks were harvested and plated onto glass cover-slips previously coated with different ECM components in order to evaluate cell adhesion, migration and proplatelet formation. All images were acquired by Olympus BX51 microscope (Olympus, Deutschland GmbH, Hamburg, Germany). In some experiments, before being seeded, cells were pre-incubated with the following substances, at the indicated final concentrations: apyrase 1 U/mL, ADP 25 μM, 2-APB 20 µM, U-73122 10 μM, BTP-2 20 μM.We employed Ca 2+ imaging to investigate the expression and functionality of SOCE on the same extracellular matrix components. Specifically, Mks were loaded with 4 μM fura-2 acetoxymethyl ester (AM) or 5 mM FLUO-3 AM and observed using an upright epifluorescence Axiolab microscope (Carl Zeiss) equipped with a Zeiss X63 Achroplan objective or a laser-scanning confocal microscope (Nikon, Eclipse TE300).For all ...

N‐methyl‐d‐aspartate receptor mediated calcium influx supports in vitro differentiation of normal mouse megakaryocytes but proliferation of leukemic cell lines

“…Previous work showed that extracellular Ca 2+ entry regulates actin polymerization,23 so we stained cells with phalloidin to visualize actin fibers (Figures 5D and S9). Control megakaryocytes displayed obvious signs of actin reorganization, including peripheral enhancement of staining, focal complex formation, and peripherally extending filaments (Figure 5D.i).…”

AMPA Receptors Are Involved in Store-Operated Calcium Entry and Interact with STIM Proteins in Rat Primary Cortical Neurons

“…Refilling these stores occurs through store-operated calcium entry (SOCE; Putney, 1986). This is the major mechanism that triggers Ca 2+ influx in non-excitable cells (Parekh and Putney, 2005; Abdullaev et al, 2008; Sánchez-Hernández et al, 2010; Di Buduo et al, 2014). The key proteins that are involved in this process are Ca 2+ sensors that are located in the ER (stromal interaction molecule 1 [STIM1] and STIM2) and Ca 2+ channel-forming proteins (Orai1–3) that are present in the plasma membrane.…”

Pathophysiological Significance of Store-Operated Calcium Entry in Megakaryocyte Function: Opening New Paths for Understanding the Role of Calcium in Thrombopoiesis

“…Further, increasing evidences have recently identified the contribution of SOCE in thrombopoiesis, the process that ensures the differentiation of megakaryocytes, the platelet precursors, and final platelet production [28]. …”