Asymmetry of membrane fluidity in the lipid bilayer of blood platelets: Fluorescence study with diphenylhexatriene and analogs

Summary Membrane fluidity of bovine platelets was examined with diphenylhexatriene (DPH), its cationic trimethylammonium derivative (TMA-DPH) and anionic propionic acid derivative (DPH-PA). After addition of these probes to platelet suspensions at 37°C, the fluorescence intensity of DPH-PA reached equilibrium within 2 min, whereas those of DPH and TMA-DPH increased gradually. With increase in the fluorescence intensity of TMA-DPH, its fluorescence anisotropy decreased significantly, but the fluorescence anisotropies of DPH-PA and DPH did not change during incubation. The gradual increase of fluorescence intensity of TMA-DPH was due to its penetration into the cytoplasmic side of the platelet membrane, as shown quantitatively by monitoring decrease in its extractability with albumin. Transbilayer movement of TMA-DPH was markedly temperature-dependent, and was scarcely observed at 15°C. The fluorescence intensity of TMA-DPH was much higher in platelet membranes and vesicles of extracted membrane lipids than the initial intensity in intact platelets. Moreover, the fluorescence anisotropy of TMA-DPH was much lower in the former preparations than the initial value in intact platelets. These results suggest that binding sites for TMA-DPH in the cytoplasmic side of the platelet membrane are more fluid than those in the outer leaflet of the plasma membrane. Platelet activation by ionomycin induced specific change in the fluorescence properties of TMA-DPH without causing transbilayer incorporation of the probe.     

Influence of dietary fatty acids on membrane fluidity and activation of rat platelets

The apparent steady-state fluorescence anisotropy of DPH- or TMA-DPH-labeled washed rat platelets is strongly affected by factors that also influence the turbidity by these platelet suspensions. Sonicated preparations from platelet lipids have a low turbidity and give anisotropy values which are hardly affected by the experimental conditions. We studied the effect of four high-fat diets on membrane fluidity, lipid composition and activation tendency of washed platelets. The diets contained 50 energy% of oils with different levels of saturated and (poly)unsaturated fatty acids. Only small diet-induced differences in DPH fluorescence anisotropy were found, which were comparable for intact platelets and platelet lipids. These differences were unrelated to the degree of saturation of the dietary fatty acids. Platelets from rats fed mainly saturated fatty acids differed significantly from other diet groups in a higher unsaturation degree of phospholipids and a lower cholesterol/phospholipid ratio, but this was not detected by DPH in terms of decreased anisotropy. These platelets aggregated less than other platelets in response to thrombin or collagen. The lower response to collagen persisted in indomethacin-treated platelets activated with the thromboxane A2 mimetic U46619, indicating a different sensitivity of these platelets for thromboxane A2. We conclude that in rat platelets: (a) the overall membrane fluidity and phospholipid unsaturation degree are subject to strong homeostatic control; (b) steady-state anisotropy with DPH or TMA-DPH label is inadequate to reveal subtile changes in lipid profile; (c) changes in platelet responsiveness to thrombin and thromboxane A2, rather than (plasma) membrane fluidity, determine the effect of dietary fatty acids on platelet aggregation.     

Effects of alcohols on fluorescence anisotropies of diphenylhexatriene and its derivatives in bovine blood platelets: relationships of the depth-dependent change in membrane fluidity by alcohols with their effects on platelet aggregation and adenylate cyclase activity.

The effects of three short-chain alkyl alcohols and benzyl alcohol on the membrane fluidity of bovine blood platelets were investigated by studies on the fluorescence anisotropies of diphenylhexatriene (DPH), its cationic trimethylammonium derivative (TMA-DPH) and its anionic propionic acid derivative (DPH-PA). These alcohols decreased the fluorescence anisotropy of DPH, which is thought to be located within the hydrophobic core of the membrane, in concentration ranges that inhibited platelet aggregation. On the other hand, they had little or no effects on the fluorescence anisotropy of DPH-PA which is thought to be located in the interfacial region of the lipid bilayer. Likewise, they had little or no effects on the fluorescence anisotropy of TMA-DPH, which is also thought to be located in the interfacial region of the lipid bilayer, either when the probe was located in the outer layer of the plasma membrane or when the probe was located in the inner membrane compartment. These results suggest that alcohols mainly increase the fluidity in the central region of the lipid bilayer. Consistent with their effects on the fluorescence anisotropy of DPH, these alcohols increased the intracellular cyclic AMP concentration. Thus alcohols may inhibit platelet function due to stimulation of adenylate cyclase, which is mediated by perturbation of the central region of the membrane lipid bilayer.     

A study of the Interaction Between Cetirizine and Plasma Membrane of Eosinophils, Neutrophils, Platelets and Lymphocytes using A fluorescence Technique

The effect of cetirizine on plasma membrane fluidity and heterogeneity of human eosinophils, neutrophils, platelets and lymphocytes was investigated using a fluorescence technique. Membrane fluidity and heterogeneity were studied by measuring the steady-state fluorescence anisotropy and fluorescence decay of 1-(4- trimethylammonium-phenyl)-6-phenyl-1, 3, 5-hexatriene (TMA-DPH) incorporated in the membrane. The results demonstrate that cetirizine (1 mug/ml) induced a significant increase in the Hpid order in the exterior part of the membrane and a decrease in membrane heterogeneity in eosinophils, neutrophils and platelets. Moreover, cetirizine blocked the PAF induced changes in membrane fluidity in these cells. Cetirizine did not influence significantly the plasma membrane of lymphocytes. These data may partially explain the effect ofcetirizine on inflammatory cell activities.     

Membrane fluidity aspects in endocytosis; a study with the fluorescent probe trimethylamino-diphenylhexatriene in L929 cells

The fluorescent hydrophobic plasma membrane probe, trimethylamino-diphenylhexatriene (TMA-DPH) was previously shown to follow the plasma membrane throughout its internalization and recycling process and thus to behave as a marker for endo- and exocytosis in living cell systems. In this paper, we made use of these properties to investigate membrane fluidity effects associated with endocytosis in L929 cells. For that purpose we performed TMA-DPH fluorescence anisotrophy measurements which showed that endocytosis starts from particularly rigid regions of the plasma membrane (probably coated pits). The fluorescence anisotropy then continuously decreases to a lower limit corresponding to the membrane fluidity of the probe in the lysosomial membrane. Strikingly, the value of this limit is identical to the average anisotropy value in the peripheral membrane, which suggests that lysosomes and plasma membrane may have a similar phospholipidic composition and a possible common origin.     

Acute sodium-dependent changes in membrane dynamic properties.

Na+ ions, which can play a pathogenic role in the development of high blood pressure, have been reported to regulate membrane enzymatic activities, receptor-ligand interaction and coupling of G-protein receptors to their effectors. This study was designed to investigate the in vitro effects of Na+ ions on membrane dynamic properties. The fluorescence anisotropy values of TMA-DPH (trimethylamino-diphenylhexatriene, probe selectively incorporated into the outer leaflet of the plasma membrane) was evaluated in platelets and erythrocytes of sodium-dependent hypertension-prone and -resistant rats of the Sabra Strain. Whereas no difference was observed between the 2 strains, TMA-DPH anisotropy was found to be strongly influenced in platelets by external Na+ ions. In the absence of external Na+, TMA-DPH anisotropy increased in human and rat platelets. In contrast, Na+ ions did not affect the anisotropy when the probe was inserted into erythrocyte ghosts. This indicates that Na+ ions can acutely regulate order parameter and microviscosity of platelet plasma membrane in the regions explored by the probe.     

Membrane microenvironmental changes during activation of human blood platelets by thrombin. A study with a fluorescent probe.

Membrane microenvironmental changes associated with thrombin-induced platelet activation were followed by fluorescence intensity and polarization studies of 1,6-diphenyl-1,3,5-hexatriene (DPH)-labeled human platelets. The labeling of washed platelets with DPH did not alter platelet intactness and morphology. In response to thrombin, DPH-labeled platelets exhibited reduced serotonin release, yet aggregation was barely inhibited. Shape change induced by thrombin or ADP was indistinguishable in control and in DPH-labeled platelets. During platelet aggregation induced by thrombin, fluorescence intensity increased by about 14%, which may indicate a more hydrophobic exposure of the probe. However, no change in fluorescence was detected during platelet shape change, induced either by thrombin in presence of EDTA or by ADP. Thrombin-activated platelets exhibited an increase in values of fluorescence polarization (P) during the stages of shape change and secretion, which further increased during aggregation. A similar pattern of increase in P values characterized platelet shape changes, caused either by thrombin in the presence of EDTA or by ADP. Changes in individual platelets are discernible from the alterations of the aggregating cells. These results may indicate that platelet activation is accompanied by an increase in rigidity of the membrane lipids. Functionally, the elevated "microviscosity" may reflect a primary role of membrane lipids in modulating the process of platelet activation or secondary transitions in lipids due to membrane events mediated by proteins.     

Membrane oxidative damage induced by ionizing radiation detected by fluorescence polarization

Effects of ionizing radiation on biological membranes include alterations in membrane proteins, peroxidation of unsaturated lipids accompanied by perturbations of the lipid bilayer polarity. We have measured radiation-induced membrane modifications using two fluorescent lipophilic membrane probes (TMA-DPH and DPH) by the technique of fluorescence polarization on two different cell lines (Chinese hamster ovary CHO-K1 and lymphoblastic RPMI 1788 cell lines). γ-Irradiation was performed using a ⁶⁰Co source with dose rates of 0.1 and 1 Gy/min for final doses of 4 and 8 Gy. Irradiation induced a decrease of fluorescence intensity and anisotropy of DPH and TMA-DPH in both cell lines, which was dose-dependent but varied inversely with the dose rate. Moreover, the fluorescence anisotropy measured in lymphoblastic cells using TMA-DPH was found to decrease as early as 1 h after irradiation, and remained significantly lower 24 h after irradiation. This study indicates that some alterations of membrane fluidity are observed after low irradiation doses and for some time thereafter. The changes in membrane fluidity might reflect oxidative damage, thus confirming a radiation-induced fluidization of biological membranes. The use of membrane fluidity changes as a potential biological indicator of radiation injury is discussed. Received: 14 May 1996 / Accepted in revised form: 30 September 1996     

Fluorescence studies of the blood platelet membranes associated with fibrinogen

To follow microviscosity changes in membranes associated with fibrinogen binding to human platelets, specific fluorescent probes were used and their fluorescence anisotropy was analysed. The degree of fluorescence anisotropy of diphenylhexatriene, anilinonaphthalene sulfonate (ANS) and fluorescamine increased significantly when fibrinogen reacted with its membrane receptors. Fluorescence polarization analyses showed that fibrinogen binding to platelet membranes is accompanied by an increase in the membrane lipid rigidity. On the other hand, changes in the fluorescence anisotropy of membrane tryptophans and N-(3-pyrene)maleimide suggest augmented mobility of the membrane proteins. The binding of fibrinogen to the membrane receptors is not accompanied by any change in the fluorescence intensity of ANS attached to the membranes. This may suggest that covering of platelets with fibrinogen molecules does not influence the surface membrane charge.     

Cultured human skin fibroblasts modify their plasma membrane lipid composition and fluidity according to growth temperature suggesting homeoviscous adaptation at hypothermic (30 degrees C) but not at hyperthermic (40 degrees C) temperatures.

Mammalian cell metabolism is responding to changes in temperature. Body temperature is regulated around 37 degrees C, but temperatures of exposed skin areas may vary between 20 degrees C and 40 degrees C for extended periods of time without apparent disturbance of adequate cellular functions. Cellular membrane functions are depending from temperatures but also from their lipid environment, which is a major component of membrane fluidity. Temperature-induced changes of membrane fluidity may be counterbalanced by adaptive modification of membrane lipids. Temperature-dependent changes of whole cell- and of purified membrane lipids and possible homeoviscous adaptation of membrane fluidity have been studied in human skin fibroblasts cultured at 30 degrees C, 37 degrees C, and 40 degrees C for ten days. Membrane anisotropy was measured by polarized fluorescence spectroscopy using TMA-DPH for superficial and DPH for deeper membrane layers. Human fibroblasts were able to adapt themselves to hypothermic temperatures (30 degrees C) by modifying the fluidity of the deeper apolar regions of the plasma membranes as reported by changes of fluorescence anisotropy due to appropriate changes of their plasma membrane lipid composition. This could not be shown for the whole cells. At 40 degrees C growth temperature, adaptive changes of the membrane lipid composition, except for some changes in fatty acid compositions, were not seen. Independent from the changes of the membrane lipid composition, the fluorescence anisotropy of the more superficial membrane layers (TMA-DPH) increased in cells growing at 30 degrees C and decreased in cells growing at 40 degrees C.     

Multidimensional stopped-flow photometry monitoring initial processes of platelet activation

A group of initial processes in platelet activation, consisting of a platelet shape change, an intracellular calcium mobilization, a calcium efflux, and a membrane fluidity (mobility) change, has been examined in rabbit platelets by a multidimensional stopped-flow method with light scattering, light transmission, and fluorescence measurements. It was found that a 90 degrees light scattering change and internal calcium release (monitored in terms of chlortetracycline fluorescence) take place after a short lag (5 s at 25 degrees C and 2 s at 37 degrees C) following activation by thrombin. The duration of the lag was the same in both cases. During the initial lag period, a rapid increase in platelet membrane fluidity (mobility) was observed by the use of pyrene excimer fluorescence. These results suggest that the intracellular calcium mobilization and the shape change are triggered by the same rate-determining step, and increase in membrane mobility may play some role in the initial stage of platelet activation before intracellular calcium mobilization occurs.     

TMA-DPH a fluorescent probe of membrane dynamics in living cells. How to use it in phagocytosis

Trimethylammonium-diphenylhexatriene (TMA-DPH), a hydrophobic fluorescent probe, has been shown in earlier studies to possess a variety of particular properties in interaction with intact living cells--specific and rapid incorporation into the plasma membrane and partition equilibrium between the membranes and the buffer. These properties offer promising applications in membrane fluidity studies and in monitoring exocytosis kinetics. Furthermore, these properties offer a method described here for quantitative monitoring of phagocytosis kinetics, by means of simple fluorescence intensity measurements. This method is original in that it evaluates only the particles which have actually been internalized by phagocytosis, and not those adsorbed on the cell surface, and that it gives quantitative information on the amount of plasma membrane involved in the process. It has been tested on mouse bone marrow macrophages.     

Comparative study on fluorescent probes distributed in human erythrocytes and platelets

Important cellular functions, such as rheological properties of cells are presumably related to the membrane lipid fluidity which may be approached by the use of fluorescence polarization method. However, biological membranes represent very heterogeneous media and the knowledge of the fluidity of membrane compartments requires the use of different probes. Two fluorescent probes, DPH and its cationic derivative, TMA-DPH, have been employed to probe the lipid fluidity of human platelets and red cell membranes. The results show that the informations given by DPH and TMA-DPH can present important differences, suggesting that DPH and TMA-DPH are localized in different regions of cell membranes. In an attempt to investigate relations between lipid fluidity and rheological properties of red cells, the behavior of probes was studied in a "Couette" viscometer with a device for studying the emissive properties of probes when red cell membranes are under shear conditions.     

Cell membrane fluidity in the intact kidney proximal tubule measured by orientation-independent fluorescence anisotropy imaging.

Membrane fluidity was measured in the isolated perfused proximal tubule from rabbit kidney. The apical and basolateral plasma membranes of tubule cells were stained separately with the fluidity-sensitive fluorophore trimethylammonium-diphenyl-hexatriene (TMA-DPH) by luminal or bath perfusion. Fluorescence anisotropy (r) of TMA-DPH was mapped with spatial resolution using an epifluorescence microscope (excitation 380 nm, emission greater than 410 nm) equipped with rotatable polarizers and a quantitative imaging system. To measure r without the confounding effects of fluorophore orientation, images were recorded with emission polarizer parallel and perpendicular to a continuum of orientations of the excitation polarizer. The theoretical basis of this approach was developed and its limitations were evaluated by mathematical modeling. The tubule inner surface (brush border) was brightly stained when the lumen was perfused with 1 microM TMA-DPH for 5 min; apical membrane r was 0.281 +/- 0.006 (23 degrees C). Staining of the tubule basolateral membrane by addition of TMA-DPH to the bath gave a significantly lower r of 0.242 +/- 0.010 (P less than 0.005); there was no staining of the brush border membrane. To interpret anisotropy images quantitatively, effects of tubule geometry, TMA-DPH lifetime, fluorescence anisotropy decay, and objective-depolarization were evaluated. Steady-state and time-resolved r and lifetimes in the intact tubule, measured by a nanosecond pulsed microscopy method, were compared with results in isolated apical and basolateral membrane vesicles from rabbit proximal tubule measured by cuvette fluorometry; r was 0.281 (apical membrane) and 0.276 (basolateral membrane) (23 degrees C). These results establish a methodology to quantitate membrane fluidity in the intact proximal tubule, and demonstrate a significantly higher fluidity in the basolateral membrane than in the apical membrane.     

TMA-DPH A fluorescent probe of membrane dynamics in living cells

Trimethylammonium-diphenylhexatriene (TMA-DPH), a hydrophobic fluorescent probe, has been shown in earlier studies to possess a variety of particular properties in interaction with intact living cells —specific and rapid incorporation into the plasma membrane and partition equilibrium between the membranes and the buffer. These properties offer promising applications in membrane fluidity studies and in monitoring exocytosis kinetics. Furthermore, these properties offer a method described here for quantitative monitoring of phago-cytosis kinetics, by means of simple fluorescence intensity measurements. This method is original in that it evaluates only the particles which have actually been internalized by phagocytosis, and not those adsorbed on the cell surface, and that it gives quantitative information on the amount of plasma membrane involved in the process. It has been tested on mouse bone marrow macrophages.     

Bidirectional transbilayer lipid movement in human platelets as vizualized by the fluorescent membrane probe 1-[4-(trimethylammonio)phenyl]-6-phenyl-1,3,5-hexatriene

Transbilayer movement of the fluorescent membrane probe TMA-DPH [1-[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 approximately 10-12 s). Prolonged incubation of platelets in the absence of any stimulus allows labeling of the total lipid pool, including intracellular membranes.(ABSTRACT TRUNCATED AT 250 WORDS)     

Alterations in erythrocyte membrane fluidity in children with trisomy 21: a fluorescence study.

Membrane fluidity of erythrocytes obtained from 15 children with trisomy 21 and 20 healthy controls were studied by measuring steady-state fluorescence anisotropy and fluorescence lifetime of 1,6-diphenyl-1,3,5-hexatriene (DPH) and 1-(4-trimethylammoniumphenyl)-6-phenyl-1,3,5-hexatriene (TMA-DPH) incorporated in hemoglobin-free erythrocyte membranes. Our results demonstrate a significant decrease in DPH fluorescence anisotropy and a significant increase in TMA-DPH fluorescence anistropy in erythrocytes from subjects with trisomy 21. No significant differences between the two groups were observed in the fluorescence lifetime of DPH and TMA-DPH. These data suggest an increase in membrane fluidity in the interior part of the membrane and a decrease in fluidity at the lipid-water interface region. This could be in part attributed to an increased oxidative damage in trisomy 21.     

Microenvironment changes of human blood platelet membranes associated with fibrinogen binding

Alterations in the membrane organization caused by fibrinogen binding to human blood platelets and their isolated membranes were analyzed by fluorescence and electron spin resonance measurements. The degree of fluorescent anisotropy of DPH, ANS and fluorescamine increased significantly when fibrinogen reacted with its membrane receptors. Both fluorescence and ESR analyses showed that fibrinogen binding to platelet membranes is accompanied by an increase of the membrane lipid rigidity. This effect seems to be indirect in nature and is mediated by altered membrane protein interactions. As it has been shown that an increased membrane lipid rigidity leads to a greater exposure of membrane proteins, including fibrinogen receptors, this might facilitate a formation of molecular linkages between neighboring platelets. On the other hand, changes of fluorescence anisotropy of membrane tryptophans and N-(3-pyrene) maleimide suggest the augmented mobility of the membrane proteins. Evidence is presented which indicated that the binding of fibrinogen to the membrane receptors is not accompanied by any changes in the fluorescence intensity of ANS attached to the membranes. It may suggest that the covering of platelets with fibrinogen does not influence the surface membrane charge. In contrast to fibrinogen, calcium ions caused an increase of the fluorescence intensity resulting from the more efficient binding of ANS to the platelet membranes.     

Oxidative response and membrane modification of diabetic platelets challenged with PAF

Alterations in the functional activities of platelets (PLT) in type I diabetes have been widely observed. These changes play a key role in the development of cardiovascular complications in diabetes. Various functional activities of PLT are the result of the interaction of numerous stimuli with PLT plasma membrane. This study was designed to evaluate the oxidative response and membrane modifications of diabetic PLT stimulated by platelet activating factor (PAF). The oxidative response was assessed by employing luminol- and lucigenin-amplified chemiluminescence. Luminol-amplified chemiluminescence is sensitive to the release of hydrogen peroxide whereas lucigenin-amplified chemiluminescence is sensitive to the production of superoxide anion. Membrane fluidity and polarity were studied using fluorescence spectroscopy. Membrane fluidity was investigated by measuring steady-state fluorescence anisotropy of 1-[4-trimethylammonium-phenyl]-6-phenyl-1,3,5-hexatriene (TMA-DPH) and membrane polarity was studied by measuring the steady-state fluorescence emission and excitation spectra of 2-dimethylamino[6-lauroyl]-naphthalene (Laurdan). The diabetic group consisted of 20 type I diabetic children with good metabolic control. Our results show a significant decrease in the luminol- and lucigenin-amplified chemiluminescence of PAF stimulated PLT in the diabetic group with respect to controls. These data indicate a decrement in the release of reactive oxygen species by diabetic PLT. We observed a significant increase in steady-state fluorescence anisotropy of diabetic PLT membrane that reflects a decrease in membrane fluidity. Laurdan showed a blue shift of the fluorescence emission and excitation spectra in diabetic PLT with respect to the control group, indicating a decrease in membrane polarity. The addition of PAF to PLT induced a red shift of Laurdan spectra in both groups, indicating an increase in membrane polarity. Our study [table: see text] demonstrates an altered oxidative response to PAF stimulation of diabetic PLT, probably due to altered generation or handling of reactive oxygen species, and alterations in the physico-chemical properties of the plasma membrane which could influence various functional activities of PLT.     

Lipid phase separation correlates with activation in platelets during chilling

When human platelets are chilled below 22°C, they spontaneously activate, a phenomenon that severely limits their storage life. It has previously been proposed that there is a correlation between cold-induced platelet activation and passage of the membranes through a liquid-crystalline to gel phase transition. Because animal models are essential for developing methods for cold storage of platelets, it is necessary to investigate such a correlation in animal platelets. In this work, horse platelets were used as a model, and it was found thatcoldinduced morphological activation is related to the lipid phase transition. Using fluorescence microscopy with the lipophilic fluorescent dye 1,1'-dioctadecyl-3,3,3',3'-tetramethyl-indocarbocyanine perchlorate (DiI-C₁₈), and Fourier transform infrared spectroscopy (FTIR), it was found that lipid phase separation occurs during cooling and low temperature storage. Furthermore, removal of cholesterol from the plasma membrane also induced a phase separation, possibly between specific phospholipid classes. Steady-state fluorescence anisotropy of 1,6-diphenyl-1,3,5-hexatriene (DPH) and trimethylammonium-DPH (TMA-DPH) were compared in cells and multilamellar vesicles (MLV)composed of platelet lipids. Cholesterol depletion led to a decrease in the fluorescence anisotropy of the two probes, which can be explained by changes in the order of the phospholipid molecules. In addition, the lipid composition and fatty acid profile of the cellular phospholipids were determined. Based ofthe similarities between horse and human platelets, it is suggested that horse platelets may be used as a model for studying cold-stored platelets. The results are discussed in relation to the possible role of phase separation during cell signalling.