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 Ca 2+ ([Ca 2+ ] i ). We have investigated the ADP-induced Ca 2+ 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 [Ca 2+ ] 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 Ca 2+ release events and to determine the Ca 2+ fluxes of the events. Confocal, high-frequency measurements of adherent, spread platelets (diameter 3-5 μm) indicated that different optical regions had simultaneous patterns of both low- and high-amplitude Ca 2+ release events. With or without plasma or flowing blood, the ADP-induced Ca 2+ signals in platelets had the characteristics of irregular Ca 2+ puffs as well as more regular Ca 2+ oscillations. Individual [Ca 2+ ] i peaks varied in amplitude and peak-to-peak interval, as observed for separated Ca 2+ puffs within larger cells. On the other hand, the peaks appeared to group into periods of ragged, shorter-interval Ca 2+ release events with little integration, which were alternated with longer-interval events. We conclude that the spiking Ca 2+ 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.