Adenine nucleotide storage and secretion in platelets as studied by 31P nuclear magnetic resonance.

Abstract

Suspensions of human and pig blood platelets were studied by 31P NMR at 145.7 MHz and by chemical and radiochemcial determination of nucleotide levels. In both types of platelets the cytoplamic nucleotide pool, which was prelabeled by incubation with [14C]adenine, was selectively reduced by addition of H2O2/NaN3 or 2-deoxyglucose/antimycin A. After the reduction of cytoplasmic ATP in human platelets, the 31P NMR spectra showed an almost complete loss of the nucleoside di- and triphosphate resonances at temperatures examined (4-50.degree. C), indicating that only the cytoplasmic nucleotides were observed, with no detectable contributions from the granular ATP, ADP and pyrophosphate. Slow tumbling of the granular nucleotides, due to aggregation, is the probable explanation of their undetectability at 145.7 MHz. Similar experiments showed that, in pig platelets granular ATP and ADP were not detected by 31P NMR at 4.degree. C but were observed at higher temperatures, indicating that aggregation may be occurring at the lower temperatures. Upon thrombin stimulation of human platelets, the NMR spectra and the chemical and radioactivity analyses showed that the granular adenylates and pyrophosphate were secreted, and that cytoplasmic ATP levels were appreciably reduced.