On the Significance of Different Aequorin Loading Techniques on Intracellular Aequorin Discharge, Baseline Calcium, Platelet Aggregation and Aequorin-Indicated Ca²⁺-Transients

Summary

The study compares the decay of intracellular luminescence activity (L _max), 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. L _max 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 L _max described an exponential decay, which was most pronounced when DMSO-platelets were maintained in Ca²⁺-free buffer (R ² = 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.

• References

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