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

Abstract

Because of controversy regarding the relationship of cytoplasmic ionized calcium concentration ([Ca2+(i)]) to platelet activation, we studied the correlation of platelet aggregation and ATP secretion with [Ca2+(i)] 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 [Ca2+(i)] in response to all agonists except epinephrine when gel-filtered platelets were suspended in media containing 1 mM Ca2+. With epinephrine, a rise in [Ca2+(i)] was indicated by aequorin, but not by quin2; [Ca2+(i)] signals, aggregation, and secretion were suppressed by EGTA. ADP [0.5 μM] produced a rise in [Ca2+(i)] 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 μM for both indicators. Collagen produced progressive concentration-related increases in [Ca2+(i)] and aggregation in aspirin-treated aequorin-loaded platelets. Quin2 failed to indicate a rise in [Ca2+(i)] at lower collagen concentrations with EGTA or aspirin. [Ca2+(i)] 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 [Ca2+(i)] 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 [Ca2+(i)] indicated by aequorin than with the response of quin2, possibly because aequorin is more sensitive to local zones of [Ca2+(i)] elevation.