Reduced Membrane Fluidity Research Articles

The interaction of beta-amyloid protein with cellular membranes stimulates its own production

Gradual changes in steady-state levels of beta amyloid peptides (Aβ) in brain are considered an initial step in the amyloid cascade hypothesis of Alzheimer's disease. Aβ is a product of the secretase cleavage of amyloid precursor protein (APP). There is evidence that the membrane lipid environment may modulate secretase activity and alters its function. Cleavage of APP strongly depends on membrane properties. Since Aβ perturbs cell membrane fluidity, the cell membrane may be the location where the neurotoxic cascade of Aβ is initiated. Therefore, we tested effects of oligomeric Aβ on membrane fluidity of whole living cells, the impact of exogenous and cellular Aβ on the processing of APP and the role of GM-1 ganglioside. We present evidence that oligoAβ (1–40) stimulates the amyloidogenic processing of APP by reducing membrane fluidity and complexing with GM-1 ganglioside. This dynamic action of Aβ may start a vicious circle, where endogenous Aβ stimulates its own production. Based on our novel findings, we propose that oligoAβ (1–40) accelerates the proteolytic cleavage of APP by decreasing membrane fluidity.

ENOS T-786C polymorphism affects atorvastatin-induced changes in erythrocyte membrane fluidity

Statins have pleiotropic effects, including endothelial nitric oxide synthase (eNOS) upregulation and increased nitric oxide formation, which can be modulated by a genetic polymorphism in the promoter region of the eNOS gene (T-786C). Here, we report our investigation of whether this polymorphism modulates the effects of atorvastatin on the fluidity of erythrocyte membranes. We genotyped 200 healthy subjects (males, 18-60 years of age) and then randomly selected 15 of these with the TT genotype and 15 with the CC genotype to receive placebo or atorvastatin (10 mg/day oral administration) for 14 days. Cell membrane fluidity was evaluated by electron paramagnetic resonance (EPR) and spin-labeling method. The EPR spectra were registered on a VARIAN-E4 spectrometer. Thiobarbituric acid-reactive species (TBA-RS) and plasma membrane cholesterol were determined in the erythrocytes. Atorvastatin reduced membrane fluidity in CC subjects (P < 0.05) but not in those with the TT genotype (P > 0.05). While no significant differences were found in plasma membrane cholesterol concentrations, higher TBA-RS concentrations were found in the CC subjects than in the TT subjects (P < 0.05). These findings suggest that a short treatment with atorvastatin is disadvantageous to subjects with the CC genotype for the T-786C polymorphism compared to those with TT genotype, at least in terms of the hemorheological properties of erythrocytes.

Different mechanisms of saturated versus polyunsaturated FFA-induced apoptosis in human endothelial cells

Apoptosis and underlying mechanisms were evaluated in human umbilical vein endothelial cells (HUVECs), in target tissues of late diabetic vascular complications [human aortic endothelial cells (HAECs) and human retinal endothelial cells (HRECs)], and in endothelial progenitor cells (EPCs) exposed to FFAs, which are elevated in obesity and diabetes. Saturated stearic acid concentration dependently induced apoptosis that could be mediated via reduced membrane fluidity, because both apoptosis and membrane rigidity are counteracted by eicosapentaenoic acid. PUFAs triggered apoptosis at a concentration of 300 micromol/l in HUVECs, HAECs, and EPCs, but not HRECs, and, in contrast to stearic acid, involved caspase-8 activation. PUFA-induced apoptosis, but not stearic acid-induced apoptosis, strictly correlated (P < 0.01) with protein expression of E2F-1 (r = 0.878) and c-myc (r = 0.966). Lack of c-myc expression and activity owing to quiescence or transfection with dominant negative In373-Myc, respectively, renders HUVECs resistant to PUFA-induced apoptosis. Because c-myc is abundant in growing cells only, apoptosis triggered by PUFAs, but not by saturated stearic acid, obviously depends on the growth/proliferation status of the cells. Finally, this study shows that FFA-induced apoptosis depends on the vascular origin and growth/proliferation status of endothelial cells, and that saturated stearic acid-induced apoptosis and PUFA-induced apoptosis are mediated via different mechanisms.

Membrane Regulation of the Stress Response from Prokaryotic Models to Mammalian Cells

"Membrane regulation" of stress responses in various systems is widely studied. In poikilotherms, membrane rigidification could be the first reaction to cold perception: reducing membrane fluidity of membranes at physiological temperatures is coupled with enhanced cold inducibility of a number of genes, including desaturases (see J.L. Harwood's article in this Proceedings volume). A similar role of changes in membrane physical state in heat (oxidative stress, etc.) sensing- and signaling gained support recently from prokaryotes to mammalian cells. Stress-induced remodeling of membrane lipids could influence generation, transduction, and deactivation of stress signals, either through global effects on the fluidity of the membrane matrix, or by specific interactions of boundary (or raft) lipids with receptor proteins, lipases, ion channels, etc. Our data point to membranes not only as targets of stress, but also as sensors in activating a stress response.

Alpha-tocopheryl phosphate is a novel apoptotic agent

Alpha-tocopheryl succinate (TOS) is a well-known potent and selective apoptotic agent. This apoptotic activity has been ascribed to its detergent-like property which is also shared by the structurally related compound, alpha-tocopheryl phosphate (TOP). TOP meets the structural requirements that have been described for the apoptotic activity of TO esters, i.e. the combination of three structural, one functional, one signalling and one hydrophobic domain. In this study, we have investigated the effect of TOP on the osteosarcoma cell line MG-63 using TOS as a reference compound. As compared with TOS, TOP showed a higher proliferative and apoptosis inducing activity on the MG-63 cancer cell line. The cytotoxic effect of TOP and TOS seems to be due to the effect of the intact compounds, since only a minor conversion into alpha-tocopheryl (TO) could be detected. EPR experiments showed that TOS and TOP reduced membrane fluidity, whereas TO had no effect. In addition, induction of erythrocyte hemolysis by TOP depended on the pH. These results suggest that the detergent-like activity of these compounds might be involved in their biological effect. Due to the potent biological activities, TOP might be clinically useful.

An Association Between Plasma Asymmetric Dimethylarginine and Membrane Fluidity of Erythrocytes in Hypertensive and Normotensive Men: An Electron Paramagnetic Resonance Investigation

Evidence indicates that increased levels of asymmetric dimethylarginine (ADMA), an endogenous inhibitor of nitric oxide (NO) synthase, are associated with increased risk of vascular dysfunction. The present study was performed to assess the possible relationship between plasma ADMA levels and membrane fluidity in hypertensive and normotensive men. Membrane fluidity (a reciprocal value of membrane microviscosity) of erythrocytes in hypertensive (n = 38) and normotensive (n = 35) men were measured using an electron paramagnetic resonance (EPR) and spin-labeling method. The order parameter (S) for the spin-label agent (5-nitroxide stearate) in the EPR spectra of erythrocyte membranes was significantly higher in hypertensive men than in normotensive men (mean +/- SEM: hypertensive subjects, 0.725 +/- 0.001,n = 38; normotensive subjects, 0.713 +/- 0.001, n = 35, P < .001), indicating that membrane fluidity of erythrocytes was decreased in hypertensive men compared with normotensive men. Plasma NO metabolite (nitrite and nitrate) concentration was lower in hypertensive than in normotensive men. In contrast, plasma ADMA levels were higher in hypertensive than in normotensive men. In the overall analysis of hypertensive and normotensive men, the order parameter (S) of erythrocytes was inversely correlated with plasma NO metabolites (r = -0.36, n = 73, P < .01) and positively correlated with plasma ADMA levels (r = 0.44, n = 73, P < .001), indicating that the reduced membrane fluidity of erythrocytes was associated with decreased NO and increased ADMA levels in plasma. When correlation analysis was performed separately for hypertensive and normotensive men, there was a positive correlation between plasma ADMA levels and the order parameter (S) of erythrocytes in both groups. The results of the present study showed that the plasma ADMA levels were elevated in hypertensive men and associated with lower membrane fluidity of erythrocytes, suggesting that ADMA might have a close correlation with the rheologic behavior of erythrocytes and the microcirculation in hypertension.

Inhibition of phospholipase A2 activity reduces membrane fluidity in rat hippocampus

Phospholipase A2 (PLA2) is a family of key enzymes in membrane phospholipid metabolism. In rats, the inhibition of PLA2 activity in the hippocampus was found to impair memory formation. Because memory function is largely dependent on the fluidity of brain membranes, we performed the present study to investigate the effects of in vivo PLA2 inhibition (with PACOCF3) on the fluidity of hippocampal membranes from rats trained in a learning task. Hippocampal tissue from rats injected with 100 microM PACOCF3 showed reduced membrane fluidity as compared to vehicle (p < 0.01), and the reduction of membrane fluidity was highly correlated with PLA2 inhibition (r = .76, p < 0.03). This finding is of interest because reduction of brain membrane fluidity impairs memory formation and both decreased PLA2 activity and reduced membrane fluidity have been reported in the brain from patients with Alzheimer's disease.

Membrane Toxicity Accounts for Apoptosis Induced by Realgar Nanoparticles in Promyelocytic Leukemia HL-60 Cells

Previous studies have demonstrated that realgar nanoparticles might provide a less toxic agent for antineoplasia by suppressing angiogenesis. Here, we addressed the question of whether the size effects on apoptosis induction mainly contribute to the comparably higher concentration of easily soluble As2O3 present in realgar nanoparticles. Results revealed that treatment with realgar nanoparticles resulted in considerably low cell viability and produced characteristic apoptotic events in HL-60 cells, including morphological changes, DNA ladder formation, and increased number of cells with sub-G1-phase, whereas raw realgar particles with the same As2O3 concentration failed to induce apoptosis. On the other hand, the effects of realgar nanoparticles and raw realgar particles on cell membrane were examined. Realgar nanoparticles had acute toxicity to cell membrane, potentiating lipid peroxidation, increasing lactate dehydrogenase (LDH) release, and reducing membrane fluidity, whereas raw realgar particles had little effect on cell membrane besides a similar reduction of membrane fluidity. These results suggest that the promotion of lipid peroxidation and membrane permeability might play an important role in the process of apoptotic induction by realgar nanoparticles. However, raw realgar particles are not sufficient to elicit apoptosis, although they can reduce membrane fluidity in HL-60 cells.

Emerging insights into hypertension and dyslipidaemia synergies

Hypertension frequently occurs in conjunction with metabolic disturbances, most notably hypercholesterolaemia. If there is an association between hypertension and hypercholesterolaemia, it is important to understand the mechanisms responsible and investigate whether elevated blood pressure (BP) increases the atherogenicity of cholesterol-rich lipoproteins. Evidence from studies suggests that hypercholesterolaemia contributes to the progression of hypertension via a number of possible mechanisms: low nitric oxide bioavailability, enhanced activity of the renin–angiotensin–aldosterone system, enhanced endothelin levels and receptor expression, and endothelial dysfunction. Other possible mechanisms include salt sensitivity (which is worsened by endothelial dysfunction), the secretion of vasoconstrictive molecules and an enrichment of cholesterol in cellular membranes, all of which reduce membrane fluidity and ion channel transporter activity. In response to cholesterol-enriched cellular membranes, the fractional clearance rate of sodium is reduced, which, in turn, favours sodium retention. Enriched cellular membrane cholesterol can also affect the transport of calcium in vascular smooth muscle cells, allowing an influx of calcium into these cells, which in turn increases conductivity and leads to microvessel constriction. Conversely, it is also evident that arterial distension and increased intra-luminal pressure induced by hypertension can increase the accumulation of atherogenic lipoproteins in the arterial wall. To optimize the management of cardiovascular disease, future treatment strategies should be directed at controlling both BP and cholesterol, particularly in high-risk patients.

The extraordinary antioxidant activity of vitamin E phosphate

The antioxidant activities of RRR-vitamin E (VE), all- rac-vitamin E (all- rac-VE), trolox, RRR-vitamin E acetate (VEA), all- rac-vitamin E phosphate (VEP) and RRR-vitamin E succinate (VES) were compared. In this study, the rank order in the inhibition of lipid peroxidation (LPO) of VE and its derivatives was trolox>VE≈all- rac-VE>VEA>VES. VE and trolox inhibited LPO in non-heated and heated rat liver microsomes. It has generally been accepted that this is due to scavenging of free radicals by these antioxidants, and during this protection the antioxidants are oxidized. VEA and VES have to be converted into VE by esterases to obtain antioxidant activity against LPO. VEP, however, had a potent antioxidant effect of its own without conversion to VE. In contrast to VE, VEP is not consumed during this protection. Of the compounds tested, VEP is the most potent in induction of hemolysis of erythrocytes. EPR experiments using the spin label 16-doxylstearic acid showed that VEP reduces membrane fluidity, in contrast to VE. This indicates that VEP acts as a detergent and forms a barrier that might inhibit the transfer of radicals from one polyunsaturated fatty acid to another. This new mechanism may form the basis for a new class of antioxidants.

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Structural basis of urothelial permeability barrier function as revealed by Cryo-EM studies of the 16 nm uroplakin particle.

The apical surface of terminally differentiated mammalian urothelial umbrella cells is covered by numerous plaques consisting of two-dimensional (2D) crystals of hexagonally packed 16 nm uroplakin particles, and functions as a remarkable permeability barrier. To determine the structural basis of this barrier function, we generated, by electron cryo microscopy, a projection map of the isolated mouse urothelial plaques at 7 A and a 3D structure at 10 A resolution. Our results indicate that each 16 nm particle has a central 6 nm lipid-filled 'hole' surrounded by 6 inverted U-shaped subunits, each consisting of an inner and an outer subdomain connected via a distal joint. The transmembrane portion of each subdomain can fit about 5 helices. This finding, coupled with our STEM and EM data, suggests that uroplakin pairs Ia/II and Ib/III are associated with the inner and outer subdomains, respectively. Since the inner subdomains interconnect to form a ring, which can potentially segregate the lipids of the central hole from those outside, the 2D crystalline uroplakin network may impose an organized state and a severely restricted freedom of movement on the lipid components, thus reducing membrane fluidity and contributing to the barrier function of urothelial plaques. Our finding that distinct uroplakin substructures are in contact with the cytoplasmic and exoplasmic leaflets of the plaque suggests that the two leaflets may have different lipid composition and contribute asymmetrically to the barrier function. We propose that the crystalline lattice structure of uroplakin, through its interactions with specialized lipids, plays a major role in the remarkable permeability barrier function of urothelial apical surface. Our results also have implications for the transmembrane signal transduction in urothelial cells as induced by the binding of uropathogenic E. coli to its uroplakin receptor.

Membrane fluidity adjustments in ethanol-stressed Oenococcus oeni cells.

The effect of ethanol on the cytoplasmic membrane of Oenococcus oeni cells and the role of membrane changes in the acquired tolerance to ethanol were investigated. Membrane tolerance to ethanol was defined as the resistance to ethanol-induced leakage of preloaded carboxyfluorescein (cF) from cells. To probe the fluidity of the cytoplasmic membrane, intact cells were labeled with doxyl-stearic acids and analyzed by electron spin resonance spectroscopy. Although the effect of ethanol was noticeable across the width of the membrane, we focused on fluidity changes at the lipid-water interface. Fluidity increased with increasing concentrations of ethanol. Cells responded to growth in the presence of 8% (vol/vol) ethanol by decreasing fluidity. Upon exposure to a range of ethanol concentrations, these adapted cells had reduced fluidity and cF leakage compared with cells grown in the absence of ethanol. Analysis of the membrane composition revealed an increase in the degree of fatty acid unsaturation and a decrease in the total amount of lipids in the cells grown in the presence of 8% (vol/vol) ethanol. Preexposure for 2 h to 12% (vol/vol) ethanol also reduced membrane fluidity and cF leakage. This short-term adaptation was not prevented in the presence of chloramphenicol, suggesting that de novo protein synthesis was not involved. We found a strong correlation between fluidity and cF leakage for all treatments and alcohol concentrations tested. We propose that the protective effect of growth in the presence of ethanol is, to a large extent, based on modification of the physicochemical state of the membrane, i.e., cells adjust their membrane permeability by decreasing fluidity at the lipid-water interface.

Melatonin protects against lipid peroxidation and membrane rigidity in erythrocytes from patients undergoing cardiopulmonary bypass surgery.

The first aim of this study was to test whether there are changes in erythrocyte lipid peroxidation products and membrane fluidity during cardiac surgery involving cardiopulmonary bypass. Secondly, in vitro tests were performed to examine whether melatonin alters induced lipid peroxidation and reduced membrane fluidity in erythrocytes from these patients. Fifteen patients undergoing cardiac surgery involving cardiopulmonary bypass (CPB) were selected. Five blood samples were taken at different times during surgery for analysis of thiobarbituric acid reactive substances (TBARS) content and membrane fluidity of the erythrocytes. TBARS are an index of the level of lipid peroxidation. The results revealed an increase in TBARS levels and a parallel decrease in erythrocyte membrane fluidity (increased membrane rigidity) after the onset of CPB with respect to the reference sample. Likewise, in vitro induction of lipid peroxidation in the erythrocyte samples from CPB patients followed a similar pattern. The changes in TBARS levels and membrane fluidity were suppressed by pre-incubation of erythrocytes with melatonin prior to the induction (by 70 microm Fe2+ + 250 microm ascorbate) of lipid peroxidation in a concentration-dependent manner. The results constitute a persuasive argument for the use of melatonin for preventive and therapeutic purposes during CPB.

HDL derived from the different phases of conjugated diene formation reduces membrane fluidity and contributes to a decrease in free cholesterol efflux from human THP-1 macrophages

Oxidized HDL (ox-HDL) has been reported to reduce free cholesterol efflux from cells. In this study we investigate the effect of different stages of ox-HDL on macrophage membrane fluidity and its effect on free cholesterol efflux from macrophages as a cell function influenced by ox-HDL. HDL was oxidized by means of conjugated diene production using copper as a prooxidant. Fluidity of HDL and human THP-1 macrophage membranes was evaluated by changes in fluorescence anisotropy ( r) by DPH probe where lower ( r) values give higher fluidity. We found that ox-HDL derived from the propagation phase (PP-HDL) and the decomposition phase (DP-HDL) became less fluid (( r): 0.263±0.001, 0.279±0.002, respectively) than HDL from the lag phase (LP-HDL) and native HDL (nat-HDL) (( r): 0.206±0.001) ( P<0.05). Macrophages incubated with PP-HDL and DP-HDL had less fluid membranes (( r): 0.231±0.001, 0.243±0.002, respectively) than those incubated with LP-HDL and nat-HDL (( r): 0.223±0.001) ( P<0.05). Consequently, fluidity was reduced not only in ox-HDL but also in the cell membranes exposed to ox-HDL. A significant negative correlation was observed between macrophage membrane fluorescence anisotropy ( r) and free cholesterol efflux from these cells (−0.876; P<0.05). Thus, lower membrane fluidity was associated with lower free cholesterol efflux from cells. In conclusion, the increase in the HDL oxidation process leads to a lost of macrophage membrane fluidity that could contribute to an explanation of the reduction of free cholesterol efflux from cells by ox-HDL.

Unique reciprocal changes of hepatocellular membrane transporter expression and fluidity in rats with selective biliary obstruction

Background and aims: The localized occlusion of bile ducts by intrahepaticlithiasis and intrahepatic neoplasms dose not cause severe jaundice, although the reason is unclear. The aim of this study was to clarify the molecular change of the liver in partial cholestasis, especially with respect to transporter expression (Mrp2, Mrp3, Bsep, P-gps) and membrane fluidity. Methods and results: After 3 days of selective bile duct ligation in Sprague–Dawley rats, the obstructed lobe (OL) and non-OL were analyzed. Canalicular and sinusoidal membrane fluidity were decreased in the OL compared with the non-OL. Mrp2 protein expression was significantly lower in the OL (by 62.9±6.9%) when compared with the non-OL. Mdr1b and Mdr2 mRNA were up-regulated in the OL when compared with the non-OL. Interestingly, Mrp3 protein and mRNA were induced in both the non-OL and OL of rats without hyperbilirubinemia. Conclusions: It seems that the reduction of membrane fluidity in the OL is a local response to localized cholestasis, while the induction of Mrp3 observed in both the OL and non-OL may be a response of the whole liver to localized impairment of bile secretion, although the regulatory mechanism is yet to be established.

Increased membrane sphingomyelin and arachidonic acid in stroke-prone spontaneously hypertensive rats

Background:Cell membrane composition and fluidity are altered in hypertension. Previous reports suggest arachidonic acid, a metabolically active fatty acid, is increased in the membranes of hypertensive animals compared to control. This increase in unsaturated fatty acids does not explain the observed reduction in fluidity in hypertensive rats, suggesting some other factors affecting fluidity may be present. It has been suggested that the metabolism of sphingomyelin is altered in genetic hypertension. We hypothesized that membrane sphingomyelin content is increased in hypertensive animals. Procedures:Stroke-prone spontaneously hypertensive rats (n = 8) were compared with Wistar-Kyoto rats (n = 8). Erythrocyte membranes were prepared and the lipids extracted and separated. Fatty acid methyl esters were produced, identified, and quantified by gas chromatography–mass spectrometry; membrane lipid content was also assessed. Results:The concentration of sphingomyelin was higher in the membrane of the hypertensive rats (45.7 ± 6 v 22.4 ± 2 μg/mg of protein) compared to control. The previously observed increase in membrane arachidonic acid content was observed in hypertensive animals when compared to control (130 ± 32 v 40 ± 3 μg/mg of protein). However, this difference was confined to the phosphatidylinositol (18 ± 4 v 6.5 ± 1.5 μg/mg of protein) and free fatty acid (2.1 ± 0.4 v 0.6 ± 0.1 μg/mg of protein) fractions. Conclusion:We hypothesize that reports of reduced membrane fluidity observed in hypertension may be due to an increase in the proportion of sphingomyelin in the cell membrane.

Profile of changes in lipid bilayer structure caused by beta-amyloid peptide.

beta-Amyloid peptide (A beta) is the primary constituent of senile plaques, a defining feature of Alzheimer's disease. Aggregated A beta is toxic to neurons, but the mechanism of toxicity is uncertain. One hypothesis is that interactions between A beta aggregates and cell membranes mediate A beta toxicity. Previously, we described a positive correlation between the A beta aggregation state and surface hydrophobicity, and the ability of the peptide to decrease fluidity in the center of the membrane bilayer [Kremer, J. J., et al. (2000) Biochemistry 39, 10309--10318]. In this work, we report that A beta aggregates increased the steady-state anisotropy of 1,6-diphenyl-1,3,5-hexatriene (DPH) embedded in the hydrophobic center of the membrane in phospholipids with anionic, cationic, and zwitterionic headgroups, suggesting that specific charge--charge interactions are not required for A beta--membrane interactions. A beta did not affect the fluorescence lifetime of DPH, indicating that the increase in anisotropy is due to increased ordering of the phospholipid acyl chains rather than changes in water penetration into the bilayer interior. A beta aggregates affected membrane fluidity above, but not below, the lipid phase-transition temperature and did not alter the temperature or enthalpy of the phospholipid phase transition. A beta induced little to no change in membrane structure or water penetration near the bilayer surface. Overall, these results suggest that exposed hydrophobic patches on the A beta aggregates interact with the hydrophobic core of the lipid bilayer, leading to a reduction in membrane fluidity. Decreases in membrane fluidity could hamper functioning of cell surface receptors and ion channel proteins; such decreases have been associated with cellular toxicity.

Fluorescence spectroscopic analysis of circulating platelet activation during coronary angioplasty

Background and Objective Platelet activation during percutaneous transluminal coronary angioplasty (PTCA) initiates thrombus formation and plaque regrowth at sites of arterial injury, limiting procedure efficacy. We have developed a simple assay for circulating platelet activation based on fluorescence analysis of membrane fluidity and intracellular calcium concentration and light scattering analysis of platelet aggregation. Study Design/Materials and Methods Platelet activation state was measured in 45 patients undergoing angioplasty, before and after treatment with platelet inhibitors. Results PTCA alone produced a decrease in pyrene dimer formation (P0.0083) and an increase in light scattering at 650 nm (P0.0128). Treatment with ADP and GPIIb/IIIa receptor antagonists reduced PTCA induced changes in pyrene dimer formation. An unexpected decrease in pyrene dimer formation (P0.05) was detected when the GPIIb/IIIa receptor antagonist was given together with an ADP receptor antagonist. Conclusions 1) Analysis of membrane fluidity provides a sensitive marker for platelet activation state. 2) Reduced membrane fluidity after combined platelet inhibitor treatments suggests reduced antiplatelet efficacy. Lasers Surg. Med. 39:414–426, 2001. © 2001 Wiley-Liss, Inc.

Fluorescence spectroscopic analysis of circulating platelet activation during coronary angioplasty.

Platelet activation during percutaneous transluminal coronary angioplasty (PTCA) initiates thrombus formation and plaque regrowth at sites of arterial injury, limiting procedure efficacy. We have developed a simple assay for circulating platelet activation based on fluorescence analysis of membrane fluidity and intracellular calcium concentration and light scattering analysis of platelet aggregation. Platelet activation state was measured in 45 patients undergoing angioplasty, before and after treatment with platelet inhibitors. PTCA alone produced a decrease in pyrene dimer formation (P0.0083) and an increase in light scattering at 650 nm (P0.0128). Treatment with ADP and GPIIb/IIIa receptor antagonists reduced PTCA induced changes in pyrene dimer formation. An unexpected decrease in pyrene dimer formation (P0.05) was detected when the GPIIb/IIIa receptor antagonist was given together with an ADP receptor antagonist. 1) Analysis of membrane fluidity provides a sensitive marker for platelet activation state. 2) Reduced membrane fluidity after combined platelet inhibitor treatments suggests reduced antiplatelet efficacy.