BIDIRECTIONAL TRANSBILAYER LIPID MOVEMENT IN HUMAN PLATELETS AS VISUALIZED BY THE FLUORESCENT MEMBRANE PROBE 1-[4-(TRIMETHYLAMMONIO)PHENYL]-6-PHENYL-1,3,5-HEXATRIENE

Transbilayer movement of the fluorescent membrane probe TMA-DPH [l-[4-(trimethylammonio)phenyl]-6-phenyl-1,3,5-hexatriene] in the plasma membrane of human platelets was investigated by measuring fluorescence intensity and fluorescence decay. Labeling of unstimulated platelets by TMA-DPH results in a rapid increase in fluorescence intensity, leveling off within 1 min. Dilution of platelets into buffer without TMA-DPH leads to an almost complete rapid efflux of TMA-DPH, indicating that TMA-DPH labels only the outer leaflet of the plasma membrane. Transbilayer movement of the fluorescent probe in unstimulated platelets could be observed upon prolonged incubation and occurs with a t1/2 of 60–90 min. Stimulation of platelets with thrombin directly after the initial rapid uptake of TMA-DPH results in a fast increase in membrane-bound TMA-DPH, fully explained by the increase in plasma membrane caused by secretion of intracellular storage organelles. No indications for increased transbilayer movement of the probe were found, since dilution of thrombin-stimulated TMA-DPH-labeled platelets into buffer without TMA-DPH indicated no uptake of TMA-DPH by intracellular membranes. In contrast to thrombin, stimulation of TMA-DPH-labeled platelets with the Ca2+-ionophore ionomycin results in a much larger increase in fluorescence intensity. This process is accompanied by labeling of intracellular membranes as indicated by incomplete efflux of TMA-DPH after dilution of the stimulated platelets. Thus, stimulation of platelets by ionomycin gives rise to rapid and massive inward movement of TMA-DPH (t1/2 ~ 10–12 s). Prolonged incubation of platelets in the absence of any stimulus allows labeling of the total lipid pool, including intracellular membranes. Dilution experiments with these platelets showed that rapid outward movement of TMA-DPH only occurs upon stimulation with ionomycin. The high rate of transbilayer movement of TMA-DPHboth inward and outward-during stimulation by ionomycin corresponds to the randomization of phospholipids that has been described previously for platelets stimulated by Ca2+-ionophore A23187 [Bevers et al. (1983) Biochim. Biophys. Acta 736, 57–66]. These results suggest that stimulation by ionophore causes the formation of local defects (flip sites) in the plasma membrane along which both endogenous phospholipid and exogenous added lipidlike compounds can cross the bilayer in both directions. Further examination of the “flip sites” during platelet stimulation by ionomycin showed that they were only transient, disappearing within 1 min following addition of the stimulus. © 1990 American Chemical Society. All rights reserved.