Evidence that secreted dense granule
adenine nucleotides mediate part of the agonist-induced cytosolic
calcium ([Ca2+]i) responses in human platelets was obtained from comparisons of fura-2-loaded platelets from normal subjects and from patients with a form of
platelet storage pool deficiency (SPD) in which the secretory dense granules and their contents are virtually absent. SPD platelets had normal initial [Ca2+]i increases induced by
thrombin and the endoperoxide analog
U46619, but a significantly enhanced decay of elevated [Ca2+]i levels following the initial increases. With
thrombin, this enhanced [Ca2+]i decay was associated with decreased Ca2+ influx, as measured by Mn2+ quench of
fura-2 fluorescence. Addition of micromolar concentrations of
ADP, alone or together with
ATP, after stimulation reversed the enhanced [Ca2+]i decay and increased Mn2+ quench in SPD platelets, but had no effect on these responses in normal platelets, while addition of 100-fold higher concentrations of
ATP or
apyrase before stimulation increased [Ca2+]i decay and decreased Mn2+ quench in normal platelets, but had little effect in SPD platelets.
ATP and
alpha,beta-methylene ATP, a specific agonist for P2X1 receptors, at micromolar concentrations also increased Mn2+ quench, but to lesser extents than did
ADP, in SPD platelets isolated and loaded with
fura-2 in the presence of
apyrase. Similar effects of
ADP and excess
ATP were seen in U46619-stimulated platelets, but decreased Ca2+ influx could not be measured directly in SPD platelets, presumably due to the very transient influx response seen with
U46619. These results suggest that secreted dense granule
ADP and
ATP contribute to the maintenance of elevated [Ca2+]i levels, but not to the initial [Ca2+]i increases, in stimulated human platelets, most likely via a
nucleotide-specific component of Ca2+ influx which may be mediated by interactions with both P2X1 and
P2Y1 purinoceptors.