Methodological Aspects of Studies on the 5‐HT Uptake Mechanism in Normal Platelets

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Abstract
The 5-HT uptake mechanism in human platelets [used as a functional model of the presynaptic nerve terminal] was studied in undiluted and diluted platelet-rich plasma (PRP). The kinetic data obtained, based on 5-HT concentrations from 0.17-1.7 .mu.mol/l, were analyzed according to Lineweaver-Burk, Eadie-Hofstee and Scatchard and Sips. In undiluted PRP all the analyses revealed a strict linearity between 5-HT concentrations and initial uptake velocities. Only 1 site for active transport seemed to be involved and no passive diffusion was present. The apparent Km and Vmax in a 95% confidence interval (n = 25) were 1.20 .+-. 0.14 .mu.mol/l and 1.40 .+-. 0.15 pmol/106 platelets and min, 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. Evidently relevant studies on 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.