On the significance of different aequorin loading techniques on intracellular aequorin discharge, baseline calcium, platelet aggregation and aequorin-indicated Ca(2+)-transients.

The study compares the decay of intracellular luminescence activity (Lmax), the levels of basal [Ca2+]i in resting platelets, and agonist-induced peak [Ca2+]i-signals in platelets loaded with aequorin using the EGTA-, DMSO- and hypoosmotic shock treatment (HOST)-techniques. The highest load of intracellular aequorin with almost unchanged luminescence activity during 4 h was achieved with HOST. Lmax decreased linearly in EGTA- and HOST-platelets, but the decay rate and the levels of basal [Ca2+]i were significantly lower in HOST-platelets. Platelet aggregation and aequorin-indicated [Ca2+]i-rise induced by thrombin and collagen were similar in EGTA- and HOST-platelets. In HOST-platelets, ADP-induced platelet aggregation was always accompanied by aequorin-signals, while at a similar time point, aequorin-signals were absent in 3 of 5 cases in EGTA-platelets. The initial aequorin loading was highest in DMSO-platelets, but Lmax described an exponential decay, which was most pronounced when DMSO-platelets were maintained in Ca(2+)-free buffer (R2 = 0.86). Agonist-induced platelet aggregation was significantly reduced in DMSO-platelets: thrombin-stimulation was accompanied by a significantly lower and delayed [Ca2+]i-rise and no aequorin-signal was obtained in response to ADP in 3 of 5 cases. The study shows that in addition of being a rapid loading-technique, the criteria of high intracellular aequorin load with low luminescence consumption, low basal [Ca2+]i and completely preserved platelet functions are most convincingly met by the HOST-method.