Physical properties of membrane fractions isolated from human platelets: implications for chilling induced platelet activation.

In previous studies, it has been suggested that chilling induced activation of human platelets is related to a lipid phase transition seen in membrane lipids. Those studies showed a single, surprisingly cooperative transition in human platelets, as determined by Fourier transform infrared (FTIR) spectroscopy, findings that are confirmed here with calorimetric measurements. Such transitions have now been studied in membrane fractions obtained from the platelets and it is reported that all fractions and purified phospholipids show similar transitions. In order to obtain these data it was necessary to develop means for separating these fractions. Therefore, a novel method for isolation and separation of dense tubular system (DTS) and plasma membranes in human platelets is described here. Lipid analysis showed that phosphatidylcholine (PC) and phosphatidylethanolamine (PE) were the dominant phospholipids in both fractions, whereas cholesterol and sphingomyelin (SM) were predominantly located in the plasma membranes. Thermotropic phase transitions in the two membrane fractions, determined by differential scanning calorimetry (DSC) and FTIR spectroscopy were found to occur at about 15 degrees C, similar to the Tm of intact human platelets. These data are discussed in relation to the role of the DTS and plasma membranes in the cold-induced activation of human platelets.     

Lipid phase separation correlates with activation in platelets during chilling

When human platelets are chilled below 22 degrees 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 that cold-induced 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 (Dil-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 of the 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.     

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.     

Evidence for a physiological role for membrane rafts in human platelets.

We have investigated raft formation in human platelets in response to cell activation. Lipid phase separation and domain formation were detected using the fluorescent dye 1,1'-dioctadecyl-3,3,3',3'-tetramethyl-indocarbocyanine perchlorate (diI-C(18)) that preferentially partitions into gel-like lipid domains. We showed that when human platelets are activated by cold and physiological agonists, rafts coalesce into visible aggregates. These events were disrupted by depletion of membrane cholesterol. Using Fourier transform infrared spectroscopy (FTIR), we measured a thermal phase transition at around 30 degrees C in intact platelets, which we have assigned as the liquid-ordered to the liquid-disordered phase transition of rafts. Phase separation of the phospholipid and the sphingomyelin-enriched rafts could be observed as two phase transitions at around 15 and 30 degrees C, respectively. The higher transition, assigned to the rafts, was greatly enhanced with removal of membrane cholesterol. Detergent-resistant membranes (DRMs) were enriched in cholesterol (50%) and sphingomyelin (20%). The multi-functional platelet receptor CD36 selectively partitioned into DRMs, whereas the GPI-linked protein CD55 and the major platelet integrin alpha(IIb)beta(3a) did not, which suggests that the clustering of proteins within rafts is a regulated process dependent on specific lipid protein interactions. We suggest that raft aggregation is a dynamic, reversible physiological event triggered by cell activation.     

Membrane and protein properties of freeze-dried mouse platelets

Membrane properties and the overall protein secondary structure of freeze-dried trehalose-loaded mouse platelets were studied using steady state fluorescence anisotropy and Fourier transform infrared spectroscopy (FTIR). FTIR results showed that fresh control mouse platelets have a main phase transition at ~14°C, whereas, freeze-dried platelets exhibited a main phase transition ~12°C. However, the cooperativity of the transition of the rehydrated platelets was greatly enhanced compared to that of control platelets. Anisotropy experiments performed with 1,6 diphenyl-1,3,5 hexatriene (DPH) complemented FTIR results and showed that the lipid order in the core of the membrane was affected by freeze-drying procedures. Similar experiments with trimethyl ammonium 1,6 diphenyl-1,3,5 hexatriene (TMA-DPH), a membrane surface probe, indicated that membrane properties at the membrane/water interface were less affected by freeze-drying procedures than the core of the membrane. Lyophilization did not result in massive protein denaturation, but the overall protein secondary structure was altered, based on in situ assessment of the amide-I and amide-II band profiles. Lyophilization-induced changes to endogenous platelet proteins were further investigated by studying the protein's heat stability. In fresh control platelets, proteins denatured at 42°C, whereas proteins in the rehydrated platelets denatured at 48°C.     

Temperature dependence of fluid phase endocytosis coincides with membrane properties of pig platelets

In previous studies we have shown that platelets take up low molecular weight molecules from the medium by fluid phase endocytosis, a phenomenon that we previously have used to load trehalose into human platelets, after which we have successfully freeze-dried them. We now extend those findings to a species to be used in animal trials of freeze-dried platelets:pigs. Further, we report results of studies aimed at elucidating the mechanism of the uptake. Temperature dependence of fluid-phase endocytosis was determined in pig platelets, using lucifer yellow carbohydrazide (LY) as a marker. A biphasic curve of marker uptake versus temperature was obtained. The activation energy was significantly higher above 22 degrees C (18.7+/-1.8 kcal/mol) than below that critical temperature (7.5+/-1.5 kcal/mol). The activation energy of fluid phase endocytosis in human platelets was 24.1+/-1.6 kcal/mol above 15 degrees C. In order to establish a correlation between the effect of temperature on fluid phase endocytosis and the membrane physical state, Fourier transform infrared spectroscopy (FTIR) and fluorescence anisotropy experiments were conducted. FTIR studies showed that pig platelets exhibit a main membrane phase transition at approximately 12 degrees C, and two smaller transitions at 26 and 37 degrees C. Anisotropy experiments performed with 1,6 diphenyl-1,3,5 hexatriene (DPH) complemented FTIR results and showed a major transition at 8 degrees C and smaller transitions at 26 and 35 degrees C. In order to investigate the relative roles of known participants in fluid phase endocytosis, the effects of several chemical inhibitors were investigated. LY uptake was unaffected by colchicine, methylamine, and amiloride. However, disruption of specific microdomains in the membrane (rafts) by methyl-beta-cyclodextrin reduced uptake of LY by 35%. Treatment with cytochalasin B, which inhibits actin polymerization, reduced the uptake by 25%. We conclude that the inflection point in the LY uptake versus temperature plot at around 22 degrees C is correlated with changes in membrane physical state, and that optimal LY internalization requires an intact cytoskeleton and intact membrane rafts.     

Temperature dependence of fluid phase endocytosis coincides with membrane properties of pig platelets

In previous studies we have shown that platelets take up low molecular weight molecules from the medium by fluid phase endocytosis, a phenomenon that we previously have used to load trehalose into human platelets, after which we have successfully freeze-dried them. We now extend those findings to a species to be used in animal trials of freeze-dried platelets:pigs. Further, we report results of studies aimed at elucidating the mechanism of the uptake. Temperature dependence of fluid-phase endocytosis was determined in pig platelets, using lucifer yellow carbohydrazide (LY) as a marker. A biphasic curve of marker uptake versus temperature was obtained. The activation energy was significantly higher above 22 °C (18.7±1.8 kcal/mol) than below that critical temperature (7.5±1.5 kcal/mol). The activation energy of fluid phase endocytosis in human platelets was 24.1±1.6 kcal/mol above 15 °C. In order to establish a correlation between the effect of temperature on fluid phase endocytosis and the membrane physical state, Fourier transform infrared spectroscopy (FTIR) and fluorescence anisotropy experiments were conducted. FTIR studies showed that pig platelets exhibit a main membrane phase transition at approximately 12 °C, and two smaller transitions at 26 and 37 °C. Anisotropy experiments performed with 1,6 diphenyl-1,3,5 hexatriene (DPH) complemented FTIR results and showed a major transition at 8 °C and smaller transitions at 26 and 35 °C. In order to investigate the relative roles of known participants in fluid phase endocytosis, the effects of several chemical inhibitors were investigated. LY uptake was unaffected by colchicine, methylamine, and amiloride. However, disruption of specific microdomains in the membrane (rafts) by methyl-β-cyclodextrin reduced uptake of LY by 35%. Treatment with cytochalasin B, which inhibits actin polymerization, reduced the uptake by 25%. We conclude that the inflection point in the LY uptake versus temperature plot at around 22 °C is correlated with changes in membrane physical state, and that optimal LY internalization requires an intact cytoskeleton and intact membrane rafts.     

In situ assessment of erythrocyte membrane properties during cold storage

Membrane fluidity and overall protein secondary structure of human erythrocytes were studied in situ using Fourier transform infrared spectroscopy (FTIR). Erythrocyte membranes were found to have weakly cooperative phase transitions at 14 degrees C and at 34 degrees C, which were tentatively assigned to the melting of the inner membrane leaflet and the sphingolipid rich outer leaflet, respectively. Cholesterol depletion by methyl-beta-cyclodextrin (MbetaCD) resulted in a large increase in the cooperativity of these transitions, and led to the appearance of another phospholipid transition at 25 degrees C. Multiple, sharp membrane phase transitions were observed after 5 days cold storage (4 degrees C ), which indicated phase separation of the membrane lipids. Using fluorescence microscopy, it was determined that the lipid probe 1,1'-dioctadecyl-3,3,3',3-tetramethyl-indocarbocyanine perchlorate (dil-C18) remained homogeneously distributed in the erythrocyte membrane during cold storage, suggesting that lipid domains were below the resolution limit of the microscope. Using thin layer chromatography, changes in the membrane lipid composition were detected during cold storage. By contrast, assessment of the amide-II band with FTIR showed that the overall protein secondary structure of haemoglobin was stable during cold storage.     

Thermotropic behavior of retinal rod membranes and dispersions of extracted phospholipids

Summary High sensitivity, differential scanning calorimetry studies of vovine retinal rod outer segment (ROS) disk membranes and aqueous dispersions of the extracted ROS phospholipids have been performed. ROS disk membranes were found to exhibit a broad peak of excess heat capacity with a maximum at less than about 3°C, ascribable to a gel-to-liquid crystalline phase transition of traction of the phospholipids. A similar thermotropic transition was observed for aqueous dispersions of the total extracted and purified ROS phospholipids. Comparison of the results obtained for the dispersion of total ROS phospholipids to those of the purified head group fractions. suggests that the thermotropic behavior reffects a gel-to-liquid crystalline transition, leading to lateral phase separation, involving those phosphatidylcholine (PC) molecules containing saturated fatty acylchains, possibley together with the highest melting ROS phosphatidylethanolamine (PE) and phosphatidylserine (PS) components. The interpretation of the thermal behavior of the ROS disk membranes depends on whether the transition is assumed to derive from the ROS PC and/or PE/PS fractions, and whether the transbilayer arrangement of the ROS phospholipids is assumed to be symmetric or asymmetric. The calorimetric data can be simply explained in terms of an asymmetric distribution of the major ROS disk membrane phospholipids (G.P. Miljanich et al.,J. Membrane Biol. 60:249–255, 1981). In this case, the transition would arise from the PE/PS fractions in the outer ROS disk membrane monolyer, and the anticipated transition from the PC in the inner monolayer would be broadened due to interaction with cholesterol. For the ROS membranes at higher temperatures, two additional, irreversible transitions are observed at 57 and 72°C, corresponding to the thermal denauturation of opsin and rhodopsin, respectively.     

Lipid phase transitions measured in intact cells with Fourier transform infrared spectroscopy

Lipid phase transitions in membranes are thought to be a major damaging event during cooling of cells prior to cryopreservation or during warming after freeze-thaw has been completed. Although there is abundant evidence that such transitions occur in isolated phospholipids, the evidence that they are found in membranes in intact cells is less clear, due largely to technical difficulties in detecting such transitions in the complex mixtures of lipids and proteins found in natural membranes. We show here that Fourier transform infrared spectroscopy provides a rapid, convenient method for detecting these transitions in intact cells. We have used intact pollen grains of cattail (Typha latifolia) as a primary experimental subject. Spectra taken of the intact pollen grains show most of the features commonly seen in natural membrane vesicles or pure phospholipids. Shifts in the vibrational frequency and width of the CH2 bands with temperature can be used to detect lipid phase transitions. Biochemical analysis, coupled with the spectroscopy, was used to assign transitions to nonpolar and polar lipids. Finally, although assignment of the melting lipid unambiguously in other cells has not yet been made, we show that the transitions can nevertheless be detected in other intact cells, including those of four plant species and sperm of three animals.     

The effects of cotyledon senescence on the composition and physical properties of membrane lipid

The phospholipid content of rough and smooth microsomal fractions from cotyledons of germinating bean declines as the tissue becomes senescent. Both types of membrane contain comparable proportions of three major phospholipids, phosphatidylcholine, phosphatidylethanolamine and phosphatidylinositol, which collectively comprise about 90% of the total. This proportionality does not change appreciably during senescence. Only small quantities of lysophosphatides were noted at all stages of senescence. The unsaturated:saturated fatty acid ratio for total extracted lipid declined only slightly in both membrane systems, but pronounced differences in this ratio were observed among the major phospholipids of the membranes. The most striking alteration in lipid composition with advancing senescence was an increase in the sterol:phospholipid ratio; this rose by about 50% for rough microsomes and 400% for smooth microsomes. For both types of membrane the patterns of change in this ratio correlated with previously reported changes in bulk lipid transition temperature, suggesting that the increase in sterol level may contribute to changes in phase behaviour of the membranes during senescence. Arrhenius plots of rotational correlation times for the electron spin label 2,2-dimethyl-5-dodecyl-5-methyloxazolidine-N-oxide (2N14) partitioned into the membrane lipid showed an increase in viscosity with advancing senescence and a corresponding increase in activation energy for both types of membrane. These changes in activation energy and viscosity correlated closely with the increase in sterol:phospholipid ratio. However, no phase transitions were detectable between temperatures of 2 and 55 degrees C despite the fact that transitions from a lipid-crystalline to gel state are detectable within this temperature range by wide angle X-ray diffraction.     

Phase transitions as a function of osmotic pressure in Saccharomyces cerevisiae whole cells, membrane extracts and phospholipid mixtures

Fourier Transform Infrared spectroscopy (FTIR) was used to determine the phase transition temperature of whole Saccharomyces cerevisiae W303-1 A cells as a function of Aw in binary water-glycerol media. A phase transition occurred at 12 degrees C in water, at 16.5 degrees C at Aw=0.75, and at 19.5 degrees C at Aw=0.65. The temperature ranges over which transition occurred increased with decreasing Aw. A total lipid extract of the plasma membranes isolated from S. cerevisiae cells was also studied, with a phase transition temperature determined at 20 degrees C in pure water and at 27 degrees C in binary water-glycerol solutions for both Aw levels tested. The pure phospholipids dimyristoylphosphatidylcholine (DMPC) and dimyristoylphosphatidylethanolamine (DMPE) and three binary mixtures of these phospholipids (percentage molar mixtures of DMPC/DMPE of 90.5/9.5, 74.8/25.2, and 39.7/60.3) were studied. For DMPC, there was no influence of Aw on the phase transition temperature (always 23 degrees C). On the other hand, the phase transition temperature of DMPE increased with decreasing Aw for the three aqueous solutions tested (glycerol, sorbitol and sucrose), from 48 degrees C in water, to 64 degrees C for a solution at Aw=0.67. For the DMPC/DMPE mixtures, transitions were found intermediate between those of the two phospholipids, and a cooperative state was observed between species at the gel and at the fluid phases.     

Anomalous change in the specific conductivity in lipoproteins at around physiologic temperatures

Studying the temperature dependence of conductivity sigma of rat and human lipoproteins and apoprotein A-I fractions revealed an anomalous region in the range of temperatures (35-38) +/- 0.5 degree C. The activation energy delta H and temperature coefficient sigma (delta sigma/delta T) on both sides of Tc and the heat of transition (delta H of transition) were calculated. In high-density human lipoproteins and apoA-I, the delta H value was found to be very low. Some mechanisms of interaction of hydrocortizone with high-density lipoproteins and apoA-I were studied by using IR-spectroscopy and conductometry were studied. It was found that the hormone considerably increases the portion of alpha-helices and beta-structures in these proteins (coil<-->alpha-helix and coil<-->beta-structure transitions). In this case, delta H value of the transition increases 13-fold; in addition, the abnormal region in apoA-I shifts 1-2 degrees C downwards. The anomalous changes in conductivity in the range of physiological temperatures in all lipoprotein fractions including apoA-I are probably related to structural phase transitions both in proteins and in phospholipids. Since the delta H value of the transition in human high-density lipoproteins is small, it is assumed that, in phospholipids of these particles, an orientation transition of the A<-->C smectic type takes place, which is assigned to the second-order phase transition. The structural transition in apoA-I can probably also be assigned to the second-order phase transition since the enthalpia of the transition is very small; presumably, this transition is related to changes in symmetry due to changes in the secondary structure (coil<-->beta-tructure transition).     

The effects of phospholipids on the properties of hepatic 5'-nucleotidase

Arrhenius plots of 5'-nucleotidase activity in microsomes or plasma membranes from rat liver exhibited transitions at approximately 35 degrees C. The enzyme was purified from homogenates after solubilization in 2% Triton X-100 and 1% sodium deoxycholate. After the initial steps of the purification, the enzyme was recovered in membranes, as judged by both thin section and freeze-fracture electron microscopy, which contained sphingomyelin, phosphatidylcholine, and phosphatidylethanolamine. The purest fractions of 5'-nucleotidase were enriched approximate 3,000-fold, consisted of similar membranes, but only contained sphingomyelin. Thermal transitions were detected in Arrhenius plots of 5'-nucleotidase after detergent solubilization, in the membranes which contained the three phospholipids, but not in the purified fraction which contained only sphingomyelin; transitions were also detected after reassociation of the purified enzyme with microsomal or plasma membrane lipids and phosphatidylcholine but not with phosphatidylethanolamine. Phosphatidylcholines containing specific fatty acids all affected the energy of activation of 5'-nucleotidase, and the detergent Sarkosyl, which has been shown to dissociate phospholipids from 5'-nucleotidase (Evans, W. H., and Gurd, J. W. (1973) Biochem. J. 133, 189-199), caused a marked decrease in the stability of the enzyme to heating. Inhibition of 5'-nucleotidase by concanavalin A followed by reactivation with alpha-methyl-D-mannoside resulted in linear Arrhenius plots of 5'-nucleotidase activity in membrane fractions, and in lower transition temperatures for the detergent, solubilized enzyme. It is concluded that in situ, 5'-nucleotidase interacts with both sphingomyelin and phosphatidylcholine; the first apparently influences the stability of the enzyme and the second, the energy of activation. In addition, the lipid environment of the enzyme seems to be altered as a result of lectin binding.     

Phase transitions as a function of osmotic pressure in Saccharomyces cerevisiae whole cells, membrane extracts and phospholipid mixtures

Fourier Transform Infrared spectroscopy (FTIR) was used to determine the phase transition temperature of whole Saccharomyces cerevisiae W303-1 A cells as a function of Aw in binary water-glycerol media. A phase transition occurred at 12 °C in water, at 16.5 °C at Aw=0.75, and at 19.5 °C at Aw=0.65. The temperature ranges over which transition occurred increased with decreasing Aw. A total lipid extract of the plasma membranes isolated from S. cerevisiae cells was also studied, with a phase transition temperature determined at 20 °C in pure water and at 27 °C in binary water-glycerol solutions for both Aw levels tested. The pure phospholipids dimyristoylphosphatidylcholine (DMPC) and dimyristoylphosphatidylethanolamine (DMPE) and three binary mixtures of these phospholipids (percentage molar mixtures of DMPC/DMPE of 90.5/9.5, 74.8/25.2, and 39.7/60.3) were studied. For DMPC, there was no influence of Aw on the phase transition temperature (always 23 °C). On the other hand, the phase transition temperature of DMPE increased with decreasing Aw for the three aqueous solutions tested (glycerol, sorbitol and sucrose), from 48 °C in water, to 64 °C for a solution at Aw=0.67. For the DMPC/DMPE mixtures, transitions were found intermediate between those of the two phospholipids, and a cooperative state was observed between species at the gel and at the fluid phases.     

Membrane and protein properties of freeze-dried mouse platelets

Membrane properties and the overall protein secondary structure of freeze-dried trehalose-loaded mouse platelets were studied using steady state fluorescence anisotropy and Fourier transform infrared spectroscopy (FTIR). FTIR results showed that fresh control mouse platelets have a main phase transition at approximately 14 degrees C, whereas, freeze-dried platelets exhibited a main phase transition approximately 12 degrees C. However, the cooperativity of the transition of the rehydrated platelets was greatly enhanced compared to that of control platelets. Anisotropy experiments performed with 1,6 diphenyl-1,3,5 hexatriene (DPH) complemented FTIR results and showed that the lipid order in the core of the membrane was affected by freeze-drying procedures. Similar experiments with trimethyl ammonium 1,6 diphenyl-1,3,5 hexatriene (TMA-DPH), a membrane surface probe, indicated that membrane properties at the membrane/water interface were less affected by freeze-drying procedures than the core of the membrane. Lyophilization did not result in massive protein denaturation, but the overall protein secondary structure was altered, based on in situ assessment of the amide-I and amide-II band profiles. Lyophilization-induced changes to endogenous platelet proteins were further investigated by studying the protein's heat stability. In fresh control platelets, proteins denatured at 42 degrees C, whereas proteins in the rehydrated platelets denatured at 48 degrees C.     

Phosphatidylcholine-cholesterol interactions: bilayers of heteroacid lipids containing linoleate lose calorimetric transitions at low cholesterol concentration

Model membranes composed of cholesterol plus one of two phosphatidylcholines (PC), each containing a saturated and a dienoic acyl chain, have been studied by differential scanning calorimetry. The gel to liquid-crystalline phase transition temperature of 1-palmitoyl-2-linoleoyl PC was -19.5 degrees C and that of 1-stearoyl-2-linoleoyl PC was -13.7 degrees C. The addition of cholesterol to the phosphatidylcholines in aqueous dispersion resulted in the progressive removal of the phase transition as observed by differential scanning calorimetry. Per mole of sterol in the membrane, cholesterol was more effective at reducing the enthalpy change of the phase transitions of these bilayers containing dienoic phosphatidylcholines than it is in eliminating the transition of membranes made with other phospholipids that contain more saturated chains. No transitions in membranes made with palmitoyl-linoleoyl PC or stearoyl-linoleoyl PC could be detected calorimetrically when 17 mol% cholesterol was present.     

Are lipid phase transitions responsible for chilling damage in human platelets?

In previous studies we have proposed that the well-known chilling-induced activation of human blood platelets can be ascribed at least in part to a thermotropic phase transition in membrane lipids. The evidence that this is the case is reviewed and amplified in this review, followed by an examination of the available physical data concerning phase transitions in lipid mixtures that mimic the mixture found in platelet membranes. Assuming complete mixing at all temperatures and equal contributions of the members of the mixture to the phase transition, the lipid mixture found in platelets should give values for Tm ranging from about 1 degrees C to about 16 degrees C, depending on the isomers present in the mixture. (The former value is not in agreement with the observed Tm, but the latter is in excellent agreement.) However, examination of the phase diagram for a binary pair of lipids found in platelet membranes shows that ideal mixing almost certainly does not occur; instead of a linear phase diagram, a convex one was obtained. This shape for the phase diagram, which would displace Tm to an unexpectedly elevated temperature, is in agreement with previously published phase diagrams for mixtures of this type. The prediction, based on thermodynamic properties of lipids found in the platelets, is that Tm will be displaced upward in more complex mixtures of the composition found in platelets, a prediction that requires experimental testing.     

Biochemical characterization of plasma membranes and intracellular membranes isolated from human platelets using Percoll gradients

Two kinds of membranes (plasma membranes and intracellular membranes) have been separated from human platelets by fractionation on Percoll gradients (successively at pH 7.4 and pH 9.6). On alkaline Percoll gradient, plasma membranes floated at low density, as shown with specific markers such as [3H]concanavalin A and monoacylglycerol lipase, whereas intracellular membranes sedimented in the higher densities and displayed a 5.6-12.4-fold enrichment in NADH diaphorase, antimycin insensitive NADH-cytochrome-c oxidoreductase and Ca2+-ATPase. Another criterion allowing differentiation of two membrane populations of human platelets was their lipid composition, which showed a cholesterol/phospholipid molar ratio of 0.5 in plasma membranes against 0.2 in intracellular membranes. Phospholipid analysis of the two kinds of membranes displayed also quite different profiles, since phosphatidylcholine increased from 30-32% in the plasma membrane to 52-66% in the intracellular membranes. This was at the expense of sphingomyelin (20-23% in plasma membrane, against 6.8-7.7% in intracellular membranes) and of phosphatidylserine (12-13% in plasma membrane, against 2-6% in intracellular membranes). Other striking differences between plasma membranes and intracellular membranes were obtained by SDS-polyacrylamide gel electrophoresis, which revealed the absence of actin and myosin in the intracellular membrane, whereas both proteins were present in significant amounts in plasma membranes. Finally, intracellular membranes but not plasma membranes were able to incorporate calcium. These results suggest that intracellular membrane fractions are derived from the dense tubular system and plasma membranes should correspond to the whole surface membrane of human platelets.     

An assessment of phase transitions in soybean membranes

Phase transitions were measured in vesicles of phospholipids, alone and in various combinations, and in pelleted mitochondrial membranes, using thermal (DSC) and optical methods. The objective was to consider their possible involvement in chilling injury of soybeans (Glycine max [L.] Merr. cv Wayne 1977). Saturated phospholipids showed clear transitions in the temperature range of 50°C to near 0°C. When mixtures of two phospholipids were examined, there was a marked lowering and broadening of the transition peaks, and a shift in the transition temperatures to intermediate temperatures. The unsaturated phospholipids that occur naturally in soybeans showed no detectable phase transitions in this temperature range, alone or in combinations. Examination of the polar lipids from soybean asolectin revealed no transitions in the biological temperature range; the additions of cations such as Ca²⁺ and La³⁺ did not evoke a detectable phase transition in them. Mitochondrial membrane pellets likewise showed no transitions. The application of these two direct methods of examination of membrane components without the addition of foreign agents did not support the suggested occurrence of a bulk phase transition which could be related to chilling injury in soybeans.