Irregular spiking in free calcium concentration in single, human platelets - Regulation by modulation of the inositol trisphosphate receptors

Fluorescence ratio imaging indicates that immobilized, aspirin-treated platelets, loaded with Fura-2, respond to inositol 1,4,5-trisphosphate- (InsP(3))-generating agonists such as thrombin by high-frequency, irregular rises in cytosolic [Ca2+], with spikes that vary in peak level and peak-to-peak interval. This differs from the regular [Ca2+](i) oscillations observed in other, larger cells. We found that the thiol-reactive compounds thimerosal (10 muM) and U73122 (10 muM) evoked sinular irregular Ca2+ responses in platelets, but in this case in the absence of InsP3 generation. Thrombin-induced spiking was acutely abolished by inhibiting phospholipase C or elevating intracellular cAMP levels, while spiking with sulfhydryl reagents was only partially blocked by cAMP elevation. Confocal laser scanning microscopy using fluo-3-loaded platelets indicated that, with all agonists or conditions, the irregular spikes were almost instantaneously raised in various regions within a single platelet. When using saponin-permeabilized platelets, we found that InsSP(3)-induced Ca2+ release from stores was stimulated by modest Ca2+ concentrations, pointing to a mechanism of InsP(3)-dependent Ca2+-induced Ca2+ release (CICR). This process was completely inhibitable by heparin. The Ca2+ release by InsP(3), but not the CICR sensor, was negatively regulated by cAMP elevation. Thimerosal treatment did not release Ca2+ from intracellular stores, but markedly potentiated the stimulatory effect of InsP(3). In contrast, U73122 caused a heparin/CAMP-insensitive Ca2+ leak from stores that differed from those used by InsP3. Taken together, these results demonstrate that InsP(3) receptor channels play a crucial role in the irregular, spiking Ca2+ signal of intact platelets, even when induced by agents such as thimerosal or U73122 which do not stimulate InsP(3) formation. The irregular Ca2+ release events appear to be subjected to extensive regulation by: (a) InsP(3) level, (b) the potentiating effect of elevated Ca2+ on Ins(P)3 action via CICR, (c) InsP3 channel sensitization by sulfhydryl (thimerosal) modification, (d) InsP(3) channel-independent Ca2+ leak with U73122, and (e) down-regulation via cAMP elevation. The observation that individual Ca2+ peaks were generated in various parts of a platelet at similar intervals and amplitudes points to effective cooperation of the various stores in the Ca2+-release process.