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Thromb Haemost 1992; 68(03): 352-356
DOI: 10.1055/s-0038-1656379
Original Article
Schattauer GmbH Stuttgart

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

R Malmgren
1   The Department of Experimental Surgery, Karolinska Institute, Stockholm, Sweden
,
S Grunfelt
2   The Department of Neurology, Henry Fords Hospital, Detroit, Ml, USA
,
Rajiv Joseph
2   The Department of Neurology, Henry Fords Hospital, Detroit, Ml, USA
› Author Affiliations
Further Information

Publication History

Received 06 November 1991

Accepted after revision 16 April 1992

Publication Date:
04 July 2018 (online)

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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.

 

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