Effect of common agonists on cytoplasmic ionized calcium concentration in platelets. Measurement with 2-methyl-6-methoxy 8-nitroquinoline (quin2) and aequorin.

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Abstract

Because of controversy regarding the relationship of cytoplasmic ionized calcium concentration ([Cai2+]) to platelet activation, we studied the correlation of platelet aggregation and ATP secretion with [Cai2+] as determined by 2-methyl-6-methoxy 8-nitroquinoline (quin2) and aequorin in response to ADP, epinephrine, collagen, the Ca2+ ionophore A23187, and thrombin. Both indicators showed a concentration-dependent increase in [Cai2+] in response to all agonists except epinephrine when gel-filtered platelets were suspended in media containing 1 mM Ca2+. With epinephrine, a rise in [Cai2+] was indicated by aequorin, but not by quin2; [Cai2+] signals, aggregation, and secretion were suppressed by EGTA. ADP [0.5 microM] produced a rise in [Cai2+] that was registered by both aequorin and quin2 in platelets in Ca2+-containing media; addition of EGTA to the medium raised the threshold concentration of ADP to 5.0 microM for both indicators. Collagen produced progressive concentration-related increases in [Cai2+] and aggregation in aspirin-treated aequorin-loaded platelets. Quin2 failed to indicate a rise in [Cai2+]at lower collagen concentrations with EGTA or aspirin. [Cai2+] response to A23187 and thrombin was reduced by addition of EGTA to platelets loaded with either aequorin or quin2. With all five agonists in all conditions tested, aequorin [Cai2+] signals occurred at the same agonist concentration as that or lower than that which produced platelet shape change, aggregation, or secretion. Platelet activation was better correlated with changes in [Cai2+] indicated by aequorin than with the response of quin2, possibly because aequorin is more sensitive to local zones of [Cai2+] elevation.

Authors

J A Ware, P C Johnson, M Smith, E W Salzman