| Abstract: | The Ca2+-sensitive photoprotein aequorin (Mr = 20,000) was introduced into human blood platelets by incubation with 10 mM EGTA and 5 mM ATP. Platelet cytoplasmic and granule contents were retained during the loading procedure, and platelet morphology, aggregation, and secretion in response to agonists were normal after aequorin loading. Luminescence indicated an apparent resting cytoplasmic ionized calcium concentration ( Cai2+]) of 2-4 microM in media containing 1 mM Ca2+ and of 0.8-2 microM in 2-4 mM EGTA. The Ca2+ ionophore A23187 and the enzyme thrombin produced dose-related luminescent signals in both Ca2+-containing and EGTA-containing media. Peak Cai2+] after A23187 or thrombin stimulation of aequorin-loaded platelets was 2-10 microM, while peak Cai2+] determined using Quin 2 as the Cai2+] indicator was at least 1 log unit lower. In platelets loaded with both aequorin and Quin 2, the aequorin signal was delayed but not reduced in amplitude. Aequorin loading of Quin 2-loaded cells had no effect on the Quin 2 signal. Ca2+ buffering by Quin 2 (intracellular concentration greater than 1 mM) is also supported by a reciprocal relationship between Quin 2] and peak Cai2+] stimulated by A23187 in the presence of EGTA. Parallel experiments with Quin 2 and aequorin may identify inhomogeneous Cai2+] in platelets and give a more complete picture of platelet Ca2+ homeostasis than either indicator alone. |
