Ragged spiking of free calcium in ADP-stimulated human platelets:: regulation of puff-like calcium signals in vitro and ex vivo

Human platelets respond to agonists of G protein (G(q))-coupled receptors by generating an irregular pattern of spiking changes in cytosolic Ca2+ ([Ca2+](i)). We have investigated the ADP-induced Ca2+ responses of single, Fluo-3-loaded platelets in the presence or absence of autologous plasma or whole blood under flow conditions. In plasma-free platelets, incubated in buffer medium, baseline separated [Ca2+](i) peaks always consisted of a rapid rising phase (median time 0.8 s) which was abruptly followed by a slower, mono-exponential decay phase. The decay constant differed from platelet to platelet, ranging from 0.23 +/- 0.02 to 0.63 +/- 0.03 s(-1) (mean +/- S.E.M., n = 3-5), and was used to identify individual Ca2+ release events and to determine the Ca2+ fluxes of the events. Confocal, high-frequency measurements of adherent, spread platelets (diameter 3-5 mum) indicated that different optical regions had simultaneous patterns of both low- and high-amplitude Ca2+ release events. With or without plasma or flowing blood, the ADP-induced Ca2+ signals in platelets had the characteristics of irregular Ca2+ puffs as well as more regular Ca2+ oscillations. Individual [Ca2+]i peaks varied in amplitude and peak-to-peak interval, as observed for separated Ca2+ Puffs within larger cells. On the other hand, the peaks appeared to group into periods of ragged, C, shorter-interval Ca2+ release events with little integration, which were alternated with longer interval events. We conclude that the spiking Ca2+ signal generated in these small cells has the characteristics of a 'poor' oscillator with an irregular frequency being reactivated from period to period. This platelet signal appears to be similar in an environment of non-physiological buffer medium and in flowing, whole blood.