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Store-operated Ca2+ influx represents a major route by which cytosolic Ca2+ can be elevated during platelet activation, yet its molecular identity in this cell type remains highly controversial. Using quantitative RT-PCR analysis of candidate receptor-operated cation entry pathways in human platelets, we show a >30-fold higher expression of message for the recently discovered Orai1 store-operated Ca2+ channel, and also the store Ca2+ sensor STIM1, when compared to the non-selective cation channels TRPC1, TRPC6 and TRPM2. Orai1 and STIM1 gene transcripts were also detected at higher levels than TRPC1, TRPC6 and TRPM2 in primary murine megakaryocytes and human megakaryocytic cell lines. In direct electrophysiological recordings from murine megakaryocytes, Ca2+ ionophore-induced store depletion stimulated CRAC currents, which are known to require Orai1, and these overlapped with TRPC6-like currents following P2Y receptor activation. Together with recent transgenic studies, these data provide evidence for STIM1:Orai1 as a primary pathway for agonist-evoked Ca2+ influx in the platelet and megakaryocyte. [ABSTRACT FROM AUTHOR]

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Megakaryocytes constitute less than 1% of all marrow cells, therefore purification of these giant platelet precursor cells represents a challenge. We describe two methods to ultra-purify mature megakaryocytes from murine marrow for the purpose of extracting RNA suitable for studies of gene expression. In the first approach, unit velocity gradients are used to enrich for megakaryocytes, which are then selected by fluorescence-activated cell sorting based upon size and high surface expression of CD41. In the second method, individual megakaryocytes, identified by their distinct morphology, are extracted using glass suction pipettes. Despite the small numbers of cells that can be isolated via the latter technique, recent studies have demonstrated how this pure population can be used to detect mRNA transcripts encoding ion channels and other proteins in the native megakaryocyte.