Reversible and irreversible intracellular Ca2+ spiking in single isolated human platelets

1. We have developed conditions that permit long duration recordings of [Ca2+]i in single, isolated human platelets and studied the reversibility of Ca2+i spiking following activation by physiological and artificial stimuli. 2. Fura-2-loaded platelets were immobilized at the tip of a saline-filled glass pipette using gentle suction. 'Contact' activation of Ca2+i spiking was observed in a proportion (11 %) of platelets, which continued for the duration of each recording (range 8-45 min). 3. Platelets that displayed constant, resting Ca2+i levels were used to test the effects of agonists. ADP (10 microM) increased [Ca2+]i in the form of either one to two spikes followed by an elevated plateau level (60 % of cells) or multiple Ca2+ spikes of irregular amplitude (40 % of cells). ADP-induced Ca2+i mobilization was completely reversible and repeatable. 4. Thrombin (1 u ml-1) evoked Ca2+i spiking in the majority (88 %) of platelets tested, which was not inhibited by perfusion of agonist-free saline throughout the recording period (range 8-67 min). 5. The clear difference in the reversibility of activation by different stimuli may reflect the distinct roles of individual agonists in haemostasis and have important consequences in the design of treatments for thrombosis.