Inhibition of Ca2+ entry by Ca2+ overloading of intracellular Ca2+ stores in human platelets.

1. This study examined the effect of overloading human platelet intracellular Ca2+ stores on the rate of agonist‐evoked external Ca2+ entry. To overload the Ca2+ stores (presumably dense tubules), external Na(+)‐dependent Ca2+ efflux via Na(+)‐Ca2+ exchange was inhibited by pretreating the cells with ouabain or Na(+)‐free medium. Ca2+ regulation was then examined after exposure to thrombin, ADP and thapsigargin. Cytosolic free Ca2+ levels were monitored using the fluorescent probe fura‐2 and external Ca2+ influx was assessed by the rates of extracellular Mn2+ or 45Ca2+ uptake. 2. Both ouabain and Na(+)‐free pretreatments caused a slight increase in the resting cytosolic free Ca2+. 3. In 1 mM Ca(2+)‐containing medium, Ca(2+)‐overloaded platelets showed similar thrombin‐evoked cytosolic free Ca2+ responses to those of control platelets. However, in Ca(2+)‐free medium, they showed substantially greater thrombin‐evoked cytosolic free Ca2+ responses than control platelets. Moreover, increased thrombin‐evoked Ca2+ mobilization from Ca2+ storage sites was accompanied by a diminished rate of thrombin‐evoked external Ca2+ entry. 4. Similar reductions in the rate of external Ca2+ entry were observed after treatment with ADP and thapsigargin. 5. Protein kinase C inhibitors (calphostin C and staurosporine) failed to reverse the effect of ouabain pretreatment on thrombin‐induced changes in the cytosolic free Ca2+ response. 6. Inositol 1,4,5‐trisphosphate profiles in ouabain‐treated and non‐treated platelets were not significantly different. 7. These data indicate that increased Ca2+ in the dense tubules is associated with diminished agonist‐evoked external Ca2+ influx.