The influence of storage time on the nucleotide concentration of canine platelets.

Summary Nucleotide concentrations of canine platelets during storage for 50 days were determined to find out the variation in the concentration of various nucleotides in canine platelets during storage at + 4 °C for 50 days. Platelets of 3 dogs were stored in whole blood in PVC bags containing CPDA-1 stabilizer. The bags were kept at 4 °C for 50 days. Changes in the energy metabolism of the platelets during storage were determined by successive measurements of the following nucleotides: adenosine-diphosphate (ADP), adenosine-tri-phosphate (ATP) and guanosine-tri-phosphate (GTP). In preparation for the measurement of their nucleotide content, the platelets were isolated from the blood at regular intervals and their membranes destroyed by deep freezing, thawing, double sonification in ice and protein precipitation with perchloric acid. The concentration of nucleotides within the platelets was determined by “High Pressure Liquid Chromatography” (HPLC). ADP, ATP and GTP were identified in the peak areas of the HPLC using 3 different techniques: comparison of retention time of standard nucleotide solutions as control samples, enzyme tests and thin layer chromatography. Adenosine-mono-phosphate (AMP), adenosine, hypoxanthine and inosine mono 5‘-phosphate (IMP) were not found in dog platelets. Platelet ATP concentration decreased significantly during 50 days of storage (p < 0.001), and was reduced to 24% of the concentration measured after 1 hour of storage on day 50. ADP concentration also dropped significantly during 50 days of storage (p < 0.001). On day 50 the average ADP concentration was reduced to about 34% of the concentration measured after 1 hour of storage. The greatest reduction was observed in GTP concentration (p < 0.001): after 50 days the GTP concentration was only 5% of the concentration measured after 1 hour of storage. In freshly drawn blood samples, the average ATP/ADP-ratio was 1.7 and the average ATP/GTP-ratio was 5.5. In all 3 dogs the ATP/ADP-ratios reached their lowest values between the 36th and 43rd day of storage, at which time the ratios were clearly below 1. After this time the ATP/ADP-ratios increased and were bigger than 1 after 50 days (an average of 75% of the values measured after 1 hour of storage). During storage the GTP concentration decreased much faster than the ATP concentration. On day 50 the ATP/GTP-ratio was 33.3. Our results indicate in concordance with the literature, that below a certain energy level, the platelets lose their functions and concentrate on surviving by saving energy for their metabolism.