A modified assay method for determining serotonin uptake in human platelets

Abstract

Effects of various experimental conditions on serotonin (5-HT) uptake in human platelets were examined. The experimental design allowed the evaluation of the effect of diffusion and other non-saturable processes on the affinity and maximum activity of the membrane pump for 5-HT uptake. Total 5-HT uptake was determined by incubating platelet-rich plasma (PRP) with increasing concentrations of serotonin at 37 degrees C for 4 min. The passive uptake was measured by the addition of various 5-HT concentrations to PRP in buffer at 37 degrees C, followed by immediate transfer to an ice-cold water bath. The difference between the total and passive uptake was linear for 6 min. The affinity (Km) for active platelet serotonin uptake was 0.45 +/- 0.09 mumol/l and maximal rate of uptake (V) was 10.7 +/- 2.1 pmol/10(7) platelets/min. The described method provides a convenient and reliable measure of active 5-HT uptake suitable for clinical investigation. The effect of passive diffusion on kinetic parameters is discussed.