Subsecond calcium dynamics in ADP- and thrombin-stimulated platelets: A continuous-flow approach using indo-1

Abstract

The regulation and kinetics (<5 seconds) of cytosolic calcium changes ([Ca2+](i)) in stimulated blood platelets have been investigated under physiological blood flow conditions. Using a newly-developed continuous-flow approach with indo-1-loaded human platelets, adenosine diphosphate (ADP, 10 µmol/L) and thrombin (5 U/mL) were equally effective in significantly increasing [Ca2+](i) by 0.5 seconds. ADP induced a transient [Ca2+](i) peak of 1 to 2 µmol/L near 2 seconds, whereas thrombin caused a sustained and larger response. The first phase (<2 seconds) was not influenced by a lack of extracellular Ca2+, in contrast to the subsequent [Ca2+](i) increase that only reached about 0.7 µmol/L for either ADP or thrombin. The shear rates used in our continuous-flow apparatus were physiological (<1,258 sec-1) and only slightly increased the basal [Ca2+](i) of 0.1 µmol/L. Platelet aggregation (<5 seconds), assessed by single-particle counting, was not altered in platelets loaded with indo-1/AM (2.5 µmol/L).