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 b y generating an irregular pattern of spiking changes in cytosolic Ca2+ ([Ca 2+](i)). We have investigated the ADP-induced Ca2+ responses of single, Flu o-3-loaded platelets in the presence or absence of autologous plasma or who le blood under flow conditions. In plasma-free platelets, incubated in buffer medium, baseline separated [C a2+](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 ident ify individual Ca2+ release events and to determine the Ca2+ fluxes of the events. Confocal, high-frequency measurements of adherent, spread platelets (diamet er 3-5 mum) indicated that different optical regions had simultaneous patte rns of both low- and high-amplitude Ca2+ release events. With or without plasma or flowing blood, the ADP-induced Ca2+ signals in pl atelets had the characteristics of irregular Ca2+ puffs as well as more reg ular Ca2+ oscillations. Individual [Ca2+]i peaks varied in amplitude and pe ak-to-peak interval, as observed for separated Ca2+ Puffs within larger cel ls. 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 bei ng reactivated from period to period. This platelet signal appears to be si milar in an environment of non-physiological buffer medium and in flowing, whole blood.