Measurement of receptor induced changes in intracellular free calcium in human platelets.

The intracellular free calcium concentration plays a key role as second messenger in the regulation of cellular reactions. Post-receptor events can be investigated by measurement of free calcium concentration in cells. Our experience in measuring the intracellular free calcium concentration in platelets with the use of the fluorescent indicator quin-2-tetraacetoxymethyl- ester is described. Possible pitfalls in the preparation procedures of the platelets are discussed as well as critical steps and the limitations of the quin-2-method. The methodological approach is demonstrated by the presentation of an investigation with the adenylate cyclase inhibitors adenosine-5'-diphosphate and epinephrine as well as their interrelationship. The potentiation effect of epinephrine to adenosine 5'-diphosphate on platelet function such as aggregation is accompanied by a potentiation of the effect of these two platelet activators in elevating the intracellular free calcium concentration. Adenosine 5'-diphosphate elevates intra-platelet free calcium alone, whereas epinephrine acting through stimulation of the alpha2-receptor needs another permissive factor to immediately elevate the intra-platelet free calcium.