The metabolic pool of adenine nucleotides in platelets can be labeled by incubating platelets for 1 h in vitro with [14C]adenosine or [32P]orthophosphate. When these platelets are treated with thrombin, the adenine nucleotides released are not labeled. Because of this, Holmsen's suggestion of a metabolically inert pool of granule nucleotides has been generally accepted. We have found that upon incubation of labeled rabbit platelets for longer times (up to 6 h) in vitro, or upon reinjection and reharvesting at times up to 66 h, the releasable pool of adenine nucleotides becomes labeled. Because the rates of 32p and 14C incorporation into this releasable pool are similar, it seems likely that these labels enter the granules as ATP. Equilibrium between the metabolic and granule pools is complete by 18 h. When rabbit platelets are labeled in vivo by intravenous injection of [32P]orthophosphate, peak labeling occurs between 60 and 70 h; this corresponds to their maturation time. The platelets probably incorporate 32P during their production in the megakaryocytes. The specific radioactivity of the adenine nucleotides in the releasable (granule) pool of these platelets is the same as the specific radioactivity in the nonreleasable (metabolic) pool. Since inorganic phosphate in platelets (and undoubtedly in the megakaryocytes) exchanges with inorganic phosphate in plasma, and since the radioactivity of the latter decreases rapidly, the adenine nucleotides in the two pools must exchange to maintain the same specific radioactivity. Transfer of adenine nucleotides into storage granules may represent a general phenomenon because it has been observed in the chromaffin cells of the adrenal medulla also.
Repeated thrombin treatment of washed platelets prepared from rabbits can decrease the serotonin content of the platelets by about 80%. When these platelets are deaggregated they reaccumulate serotonin but their storage capacity for serotonin is reduced by about 60%. If thrombin-pretreated platelets are allowed to equilibrate with a high concentration of serotonin (123 mu M), they release a smaller percentage of their total serotonin upon further thrombin treatment, in comparison with the percentage of serotonin released from control platelets equilibrated with the same concentration of serotonin calculations indicate that in thrombin-treated platelets reequilibrated with serotonin, two-thirds of the serotonin is in the granule compartment and one-third is in the extragranular compartment, presumably the cytoplasm. Analysis of the exchange of serotonin between the suspending fluid and the platelets showed that thrombin treatment does not alter the transport rate of serotonin across the platelet membrane and does not cause increased diffusion of serotonin from the platelets into the suspending fluid. The primary reason for the reduced serotonin accumulation by the thrombin-treated platelets appears to be loss of amine storage granules or of the storage capacity within the granules.