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An altered cellular membrane fluidity secondary to changes of cholesterol metabolism is a potentially important mechanism in the pathogenesis of atherosclerosis. Especially in blood platelets an increased sensitivity for stimulation dependent aggregation which is a risk factor for thrombosis has been experimentally linked to disorders of lipid and lipoprotein metabolism. The goal of this study was the development of a flow cytometric assay for the direct analysis of cellular membrane microviscosity in correlation to activation associated phenotypic changes of platelets in vitro. The analysis of fluorescence polarization following the staining of hydrophobic lipid regions of cell membranes with the fluorescent dye 1,6-diphenyl-1,3,5-hexatriene (DPH) is a well established method for the analysis of membrane fluidity. The extent of fluorescence anisotropy dependent on the rotational mobility of this fluorochrome is indirectly proportional to the microviscosity of the stained membrane subcompartment. In this study, an alternative and more simple method based on the diffusion dependent excimer formation of pyrenedecanoic acid (PDA) (J. Immunol. Methods 96:225-31, 1987) was characterized in comparison to the DPH method as a reference. Human platelets showed a rapid uptake of both DPH and PDA resulting in the staining primarily of the plasma membrane after up to 30 min of incubation. Staining analyzed at 351 nm excitation resulted in a saturation of the depolarization coefficient of DPH at 20 (mu) M but an increase of the excimer to monomer ratio of PDA with increasing dye concentration. A 'membrane fluidity coefficient' which saturated at 5 (mu) M PDA was calculated as the excimer fluorescence divided through the square of monomer fluorescence thereby correcting for the influence of dye concentration on excimer formation. The temperature dependent changes of membrane viscosity were further used as a model for the comparison of both methods. Cells analyzed at temperatures between 12 degrees Celsius and 33 degrees Celsius showed a linear increase of the microviscosity values which were derived from the method by a factor of 3.1. The microviscosity calculated from the DPH method, in contrast, decreased only 2.2-fold with relatively smaller changes occurring above 24 degrees Celsius. Cholesterol depletion of platelets using cholesterol-poor phosphatidylcholine-cholesterol liposomes resulted in significant changes of the PDA fluorescence coefficient similar to the DPH polarization coefficient indicating similar specificity of both methods. A high sensitivity of the PDA method was further confirmed through the analysis of patient blood samples where the membrane viscosity of platelets as determined with PDA showed a good correlation to serum HDL cholesterol. In conclusion, the analysis of the excimer fluorescence of PDA is a technically simple, sensitive, and highly reproducible method for the flow cytometric analysis of an altered membrane fluidity of platelets.
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