Methodological aspects of studies on the 5-HT uptake mechanism in normal platelets

The 5-HT uptake mechanism in human platelets was studied in undiluted platelet-rich plasma (PRP). The kinetic data obtained, which were based on 5-HT concentrations ranging from 0.17 to 1.7 mumol/l, were analyzed according to Lineweaver-Burk, Eadie-Hofstee, Scatchard and Sips. In undiluted PRP all the analyses revealed a strict linearity between 5-HT concentrations and initial uptake velocities. Only one site for active transport seemed to be involved and no passive diffusion was present. The apparent Km and Vmax in a 95 per cent confidence interval (n = 25) were 1.20 +/- 0.14 mumol/l and 1.40 +/- 0.15 pmol/10(6) platelets and minutes, respectively. Dilution of PRP with autologous platelet-poor plasma or Ringer's solution markedly influenced the active 5-HT transport mechanism. The addition of minute amounts of supernatant from lysed autologous platelets to PRP totally impaired the active 5-HT uptake. It is concluded that relevant studies on the active 5-HT transport can be performed only in fresh undiluted PRP. The commonly used Lineweaver-Burk analysis does not discriminate sufficiently between fully saturable and non-saturable transport mechanism and therefore cannot be used for the detection of irregularities in the 5-HT transport.