Fluorescence digital image analysis of thrombin and ADP induced rise in intracellular Ca 2+ concentration of single blood platelets

Abstract

Cytoplasmic free Ca 2+ concentration, [Ca 2+] i, was estimated in single rabbit blood platelets by digital imaging microscopy with the use of the specific Ca 2+-indicator dye Fura-2. Uneven distribution and low level of [Ca 2+] i was found in the resting platelet even in the presence of extracellular 1 mM Ca 2+. Thrombin at 1 unit/ml immediately caused a transient increase in [Ca 2+] i, which was followed by a secondary and sustained increase in [Ca 2+] i. The distribution of increased levels of [Ca 2+] i was also shown to be uneven within the cell. The presence of 1 mM EGTA in the medium only slightly decreased the initial rise in [Ca 2+] i, but completely inhibited the latter phase, a sustained rise in [Ca 2+] i. This result shows that the initial rise of [Ca 2+] i might not be caused by Ca 2+ influx, but might be induced by mobilization of Ca 2+ from intracellular Ca 2+ storage sites. This speculation is further supported by the fact that the elevated [Ca 2+] i induced by thrombin immediately decreased to the base line value when 3 mM EGTA was applied. Thus, thrombin induced elevation of [Ca 2+] i is suggested to consist of two different processes, namely the mobilization of Ca 2+ from the intracellular storage sites and the successive Ca 2+ influx through the receptor activated Ca 2+ channels. Stimulation with ADP also caused a rapid elevation of platelet [Ca 2+] i, but this effect of ADP was different from that of thrombin. Thus, the ADP induced rise in [Ca 2+] i was accompanied by oscillation and was inhibited by extracellular EGTA. Our present experiment is the first report that clearly and directly reveals the differences between the effects of thrombin and ADP on [Ca 2+] i of platelets.