Calcium sequestration by human platelet acidic organelles is regulated by the actin cytoskeleton and autocrine...

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-HT_2A 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.