Molecular order and fluidity of the plasma membrane of human platelets from time-resolved fluorescence depolarization

The ability of seven fluorescence polarization probes (1,6-diphenyl-1,3,5-hexatriene, 1-(4-trimethylamino)phenyl]-6-phenyl-1,3,5-hexatriene, (2-carboxyethyl)-1,6-diphenyl-1,3,5-hexatriene, 16(9-anthroyloxy)palmitic acid, CIS-parinaric acid, trans-parinaric acid and perylene) to report changes induced by temperature and Ca²⁺ in the plasma membrane of human platelets has been examined. The steady-state fluorescence anisotropy of the probes was compared after being incorporated into whole resting platelets, fragments of platelet plasma membrane and multilayers of lipids extracted from these membranes. In addition, we have investigated the molecular order and dynamics of the three preparations by time-resolved fluorescence depolarization of DPH and CE-DPH as a function of temperature and Ca²⁺ concentration. The high values of the order parameters found in intact platelets (S_{DPH, 36.c}=0.70) were almost identical to those in membrane fragments and lipid vesicles, suggesting that lipid-lipid interactions and, therefore, the lipid composition are the main factors influencing the probe order parameter. Other lipid interactions such as those with membrane proteins and intracellular components have little effect on the SDP, in platelets. These measurements also showed that the stationary fluorescence anisotropy of DPH and CE-DPH in platelets is largely determined (80%) by the structural order of the lipid bilayer. Therefore, the previous microviscosity values based on stationary anisotropy data reflect the alignment and packing rather than the mobility of the bilayer components. The dynamic component of the anisotropy decay of these probes was analyzed in terms of the wobbling-in-cone model, allowing an estimation of the apparent viscosity of platelet plasma membrane (DPH, 36°C =–0–5 P) that is similar to that of the erythrocyte membrane. This value decreased substantially in multilayers of native lipids, indicating a large effect of the lipidprotein interactions on the probe dynamics within the bilayer. When the temperature was raised from 25° to 36°C a pronounced decrease was observed in the order parameter and apparent viscosity, followed by a tendency to level-off in the 36°-40°C interval. This may be related to the end-point of the lipid phase separation reported by Gordon et al. (1983). Finally, the rigidifying (lipid ordering) effect of Ca²⁺ on the platelet plasma membrane could also be observed by the fluorescence anisotropy measurements, in the form of an increase (2%) of the order parameter of CE-DPH for Ca²⁺ concentrations in the millimolar range.Abbreviations DPH 1,6-diphenyl-1,3,5-hexatriene - TMA-DPH 1-(4-trimethyl-amino)phenyl]-6-phenyl-1,3,5-hexatriene - CE-DPH (2-carboxyethyl)-1,6-diphenyl-1,3,5-hexatriene - 16AP 16-(9-anthroyloxy)-palmitic acid; c-PnA, CIS-parinaric acid; t-PnA, trans-parinaric acid - PER perylene - POPOP p-bis2(5-phenyl-oxazolyl)benzene] - ESR electron spin resonance Offprint requests to: A. U. Acuña