Detection and determination of adenosine diphosphate and related substances in plasma

Abstract

A method is described for detecting and determining the products of metabolism of ADP added to plasma at initial concentrations of about 1 [mu]M-ADP. ATP, ADP, AMP, adenosine, inosine and hypoxanthine were detected in human platelet-rich plasma after incubation with ADP and in the presence of either heparin or heparin-citrate. The products of incubation of ADP with human platelet-poor plasma in the presence of heparin were the same as with platelet-rich plasma, except that, when the initial concentration of ADP was 1.5 [mu][image], little or no ATP was detected. The ATP detected in platelet-rich plasma when 1.5 [mu][image] ADP was initially incubated was present in the platelets and not in the plasma. The time for 50% decay of ADP in either platelet-rich or platelet-poor plasma in the presence of heparin was about 20 min. when the initial concentration of ADP was 200 [mu][image], but was 6-9 min. when the initial ADP concentration was 1.5-2.5[mu][image]. The corresponding values in the presence of heparin-citrate were about 45 min. and about 9-12 min. respectively. Hypoxanthine accumulated to a greater extent in platelet-rich than in platelet-poor plasma after the addition of ADP. After incubation for 15-20 min. of either platelet-rich plasma or suspensions of washed platelets in saline with adenosine at an initial concentration of about 3-4 [mu][image], ATP, ADP and AMP were detected in the platelets. Similar incubations of washed platelets with inosine also showed the formation of these substances, but to a much less extent. After the addition of adenosine to suspensions of washed platelets in saline, inosine and hypoxanthine were detected in the incubation mixture. After the addition of inosine, hypoxanthine was detected. When ADP at an initial concentration of 1.5 [mu][image] was added to platelet-rich plasma containing adenosine deaminase, no adenosine was detected in the incubation mixture. There was no difference in the rate of decay of ADP in the presence or absence of the deaminase, but ATP formation was decreased in its presence.