Parallel investigation of exocytosis kinetics and membrane fluidity changes in human platelets with the fluorescent probe, trimethylammonio-diphenylhexatriene

Abstract

A simple, flexible and sensitive fluorescence method is described, which, from the same experiment, provides coupled quantitative informations on membrane fluidity changes and exocytosis, and reliable kinetic analyses of these effects, in intact cell suspensions. The method is based on the features peculiar to trimethylammonio-diphenylhexatriene (TMA-DPH), a fluorescent hydrophobic probe, which, in intact cells, is incorporated specifically into the plasma membranes, according to an instantaneous partition equilibrium. The method was tested on human platelets upon stimulation with various agents, such as human α-thrombin, adenosine diphosphate (ADP), adrenalin and ionomycin, which act through different types of mechanism. The experimental conditions were chosen to allow platelet shape change and exocytosis, but no aggregation. The kinetics and the dose-dependence of the changes in TMA-DPH fluorescence intensity and anisotropy were compared to the simultaneous physiological responses of platelets to the same stimuli, under the same conditions. Quantitative correlations were established between serotonin secretion and the increase in fluorescence intensity, whereas fluorescence anisotropy, which monitors membrane fluidity changes was associated with platelet shape change. The specificity of the effects was confirmed with appropriate antagonistic or modulating agents.