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Human platelets regulate agonist-evoked Ca2+ signalling through Ca2+ release from and sequestration into acidic organelles. Previous studies have pharmacologically characterised the presence of a Ca2+-H+ exchanger in these organelles. This exchanger appears to regulate a secondary plateau phase in agonist-evoked cytosolic Ca2+ signals in fura-2-loaded human platelets. Here we demonstrate that cytochalasin D treatment removes the secondary plateau in ADP-evoked Ca2+ signals elicited in the absence of external Ca2+. This effect was reversed by pretreatment with nigericin, a K+/H+ exchanger that short-circuits the Ca2+-H+ exchanger. Using Fluo-5N- or Lysosensor Green-loaded cells, cytochalasin D was found to enhance Ca2+ sequestration into acidic organelles by preventing their alkalinisation. Additional experiments demonstrated that ADP-evoked alkalinisation of acidic organelles and subsequent slowing of acidic organellar Ca2+ sequestration was mediated by autocrine 5-HT signalling. Enhancing this 5-HT signalling using fluoxetine overcame the inhibitory effect of cytochalasin D on ADP-evoked Ca2+ signals, indicating that cytochalasin D interferes with 5-HT autocrine signalling. The ability of Cytochalasin D to interfere with autocrine 5-HT signalling was downstream of the 5-HT2A receptor as secretion of [3H]-5-HT from ADP-stimulated human platelets was not reduced. These data provide the first evidence that the pH gradient across acidic organelles is dynamically regulated upon human platelet activation, and that this can play a significant role in controlling human platelet function by modulating Ca2+-H+ exchange and so [Ca2+]i.

Rises in cytosolic Ca(2+) concentration ([Ca(2+)]cyt) are central in platelet activation, yet many aspects of the underlying mechanisms are poorly understood. Most studies examine how experimental manipulations affect agonist-evoked rises in [Ca(2+)]cyt, but these only monitor the net effect of manipulations on the processes controlling [Ca(2+)]cyt (Ca(2+) buffering, sequestration, release, entry and removal), and cannot resolve the source of the Ca(2+) or the transporters or channels affected. To investigate the effects of protein kinase C (PKC) on platelet Ca(2+) signalling, we here monitor Ca(2+) flux around the platelet by measuring net Ca(2+) fluxes to or from the extracellular space and the intracellular Ca(2+) stores, which act as the major sources and sinks for Ca(2+) influx into and efflux from the cytosol, as well as monitoring the cytosolic Na(+) concentration ([Na(+)]cyt), which influences platelet Ca(2+) fluxes via Na(+)/Ca(2+) exchange. The intracellular store Ca(2+) concentration ([Ca(2+)]st) was monitored using Fluo-5N, the extracellular Ca(2+) concentration ([Ca(2+)]ext) was monitored using Fluo-4 whilst [Ca(2+)]cyt and [Na(+)]cyt were monitored using Fura-2 and SFBI, respectively. PKC inhibition using Ro-31-8220 or bisindolylmaleimide I potentiated ADP- and thrombin-evoked rises in [Ca(2+)]cyt in the absence of extracellular Ca(2+). PKC inhibition potentiated ADP-evoked but reduced thrombin-evoked intracellular Ca(2+) release and Ca(2+) removal into the extracellular medium. SERCA inhibition using thapsigargin and 2,5-di(tert-butyl) l,4-benzohydroquinone abolished the effect of PKC inhibitors on ADP-evoked changes in [Ca(2+)]cyt but only reduced the effect on thrombin-evoked responses. Thrombin evokes substantial rises in [Na(+)]cyt which would be expected to reduce Ca(2+) removal via the Na(+)/Ca(2+) exchanger (NCX). Thrombin-evoked rises in [Na(+)]cyt were potentiated by PKC inhibition, an effect which was not due to altered changes in non-selective cation permeability of the plasma membrane as assessed by Mn(2+) quench of Fura-2 fluorescence. PKC inhibition was without effect on thrombin-evoked rises in [Ca(2+)]cyt following SERCA inhibition and either removal of extracellular Na(+) or inhibition of Na(+)/K(+)-ATPase activity by removal of extracellular K(+) or treatment with digoxin. These data suggest that PKC limits ADP-evoked rises in [Ca(2+)]cyt by acceleration of SERCA activity, whilst rises in [Ca(2+)]cyt evoked by the stronger platelet activator thrombin are limited by PKC through acceleration of both SERCA and Na(+)/K(+)-ATPase activity, with the latter limiting the effect of thrombin on rises in [Na(+)]cyt and so forward mode NCX activity. The use of selective PKC inhibitors indicated that conventional and not novel PKC isoforms are responsible for the inhibition of agonist-evoked Ca(2+) signalling.