Anomalous mixing of zwitterionic and anionic phospholipids with double-chain cationic amphiphiles in lipid bilayers.

High-sensitivity scanning calorimetry has been used to examine the thermotropic behavior of mixtures combining dipalmitoylphosphatidylcholine (DPPC), phosphatidylethanolamine (DPPE) and O-methylphosphatidic acid (DPPA-OMe) with the double-chain cationic amphiphiles N,N-dihexadecyl-N,N- dimethylammonium chloride (DHDAC), 1,2-dipalmitoyloxy-3-(trimethylammonio)propane (DPTAP) and the corresponding monomethylated tertiary amino compounds (DHMMA-H+ and DPDAP-H+). At physiological ionic strength, mixtures of these cationic amphiphiles with the anionic phospholipid DPPA-OMe can show gel-to-liquid-crystalline phase transitions at considerably higher temperatures than do either of the pure components. Surprisingly, binary mixtures of DPPC and these cationic amphiphiles also show strongly nonideal mixing, with phase diagrams exhibiting pronounced maxima in their solidus and liquidus curves. Similar behavior is not observed for mixtures of DPPC with DPPA-OMe, which closely resembles DPTAP and DPDAP-H+ in backbone configuration and polar headgroup size. The present results suggest that perturbation of the orientation of the phosphatidylcholine headgroup by cationic amphiphiles, as demonstrated previously by Seelig and co-workers (Biochemistry 28 [1989], 7720-7728), can significantly affect the thermotropic behavior of phospholipids such as DPPC. Such effects may exert a generally important (though not always easily recognizable) influence on the organization and thermotropic behavior of systems where zwitterionic phospholipids are combined with charged bilayer-associated molecules.     

Landau theory of two-component phospholipid bilayers. I. Phosphatidylcholine/phosphatidylethanolamine mixtures

Priest's phenomenological model (Mol. Cryst. Liq. Cryst. 60 (1980) 167.) on one- and two-component PC bilayers is extended here. We constructed a new excess free energy term in the state function to describe the thermodynamic properties of the two-component phospholipid bilayers where the chain lengths and the polar heads of the components can be different simultaneously. By means of this generalized state function, we can calculate the phase diagrams of DPPC/DPPE, DMPC/DMPE, DMPC/DPPE, DPPC/DMPE and DSPC/DMPE mixtures. We obtained complete miscibility both in the liquid crystalline and in the gel phase if the chain lengths of the components were the same. If the chain length of the PE component was longer than that of the PC component, we obtained a peritectic system. A eutectic system was obtained in the reverse case. The results of the model were compared with the experimental data available. Applying the quasichemical approximation, we determined the molecular meaning of the phenomenological model parameters. Namely, sigma and gamma are proportional to the sublimation heat of the CH2 group in the long-chain alkanes and to the hydrogen-bonding energy between the polar heads of the ethanolamines; otherwise the model resulted in--1.94 kcal/mol per CH2 for the sublimation heat and --1.4 kcal/mol for the hydrogen-bond energy.     

Gene transfer efficacies of novel cationic amphiphiles with alanine, beta-alanine, and serine headgroups: a structure-activity investigation

Herein, we report on the relative in vitro efficacies of nine novel non-glycerol based cationic amphiphiles with increasing hydrophobic tails and the amino acids serine, alanine and beta-alanine as the headgroup functionalities (lipids 1-9, Scheme 1) in transfecting multiple cultured cells including CHO, COS-1, MCF-7, and HepG2. The gene transfer efficiencies of lipids 1-9 were evaluated using the reporter gene assays in all the four cell lines and the whole cell histochemical X-gal staining assays in representative CHO cells. In CHO, HepG2, and MCF-7 cells, cationic lipids with alanine (4-6) and beta-alanine (7-9) headgroups were found to be remarkably more transfection efficient than their serine headgroup counterparts (1-3). Most notably, in CHO, HepG2, and MCF-7 cells, in combination with cholesterol as auxiliary lipid, the transfection efficiencies of the cationic lipids with alanine and beta-alanine headgroups and myristyl and palmityl tails (lipids 4, 5, 7 and 8) were significantly higher (2-3-fold) than that of LipofectAmine-2000, a widely used commercially available liposomal tranfection vectors. Surprisingly, in COS-1 cells, although cationic lipids with beta-alanine headgroups (7-9) were strikingly transfection efficient (3-4-fold more efficacious than LipofectAmine-2000), the gene transfer properties of both their structural isomers (4-6) and their serine headgroup counterparts (1-3) were adversely affected. In summary, the present structure-activity investigation demonstrate that high gene delivery efficacies of cationic amphiphiles containing alanine or beta-alanine headgroups can get seriously compromised by substituting the alanine or beta-alanine with serine presumably due to the enhanced sensitivity of DNA associated with such serine-head-containing cationic lipids.     

Space-filling effects in membrane disruption by cationic amphiphiles

We studied the hemolytic activity towards bovine erythrocytes of novel synthetic steroid-polyamine conjugates consisting of a rigid hydrophobic steroid unit, and a flexible hydrophilic polyamine unit connected by a linker. The steroid structure, polyamine chain length, and the presence of a hydrophobic substituent on the steroid, all influenced the activity. Analysis of the time dependence of hemolysis suggested that these structurally related cationic amphiphiles have different mechanisms of membrane perturbation.     

Gene transfer effects on various cationic amphiphiles in CHO cells

Gene transfer is an important tool to explore genomic, cell biologic, or gene therapeutic research. In this paper we report that several cationic amphiphiles have the potential to efficiently deliver DNA into CHO cells, which is one of the cell lines considered to be important for production of proteins including therapeutic proteins. We have found that O,O′-ditetradecanoyl-N-(trimethylammonio acetyl) diethanolamine chloride (14Dea2), among 29 types of cationic amphiphiles tested, shows a transfection efficiency of more than 40% in CHO cells. In addition, the results from a series of hydrocarbon chains of varying lengths bound to a connector have shown that an optimal chain length is important for the efficient delivery of DNA into cells. Moreover, flow cytometer analysis has shown that 14Dea2 transfection leads to high levels of expression of the reporter gene (green fluorescent protein) in individual cells. These findings have suggested that 14Dea2 is able to effectively deliver a number of plasmids into a cell nucleus. Thus, our system might be a powerful tool for high efficiency gene transfer and production of high levels of recombinant protein.     

Tunable pH-sensitive liposomes composed of mixtures of cationic and anionic lipids

The pH-dependent fusion properties of large unilamellar vesicles (LUVs) composed of binary mixtures of anionic and cationic lipids have been investigated. It is shown that stable LUVs can be prepared from the ionizable anionic lipid cholesteryl hemisuccinate (CHEMS) and the permanently charged cationic lipid N,N-dioleoyl-N, N-dimethylammonium chloride (DODAC) at neutral pH values and that these LUVs undergo fusion as the pH is reduced. The critical pH at which fusion was observed (pH(f)) was dependent on the cationic lipid-to-anionic lipid ratio. LUVs prepared from DODAC/CHEMS mixtures at molar ratios of 0 to 0.85 resulted in vesicles with pH(f) values that ranged from pH 4.0 to 6.7, respectively. This behavior is consistent with a model in which fusion occurs at pH values such that the DODAC/CHEMS LUV surface charge is zero. Related behavior was observed for LUVs composed of the ionizable cationic lipid 3alpha-[N-(N',N'-dimethylaminoethane)-carbamoyl] cholesterol hydrochloride (DC-Chol) and the acidic lipid dioleoylphosphatidic acid (DOPA). Freeze-fracture and (31)P NMR evidence is presented which indicates that pH-dependent fusion results from a preference of mixtures of cationic and anionic lipid for "inverted" nonbilayer lipid phases under conditions where the surface charge is zero. It is concluded that tunable pH-sensitive LUVs composed of cationic and anionic lipids may be of utility for drug delivery applications. It is also suggested that the ability of cationic lipids to adopt inverted nonbilayer structures in combination with anionic lipids may be related to the ability of cationic lipids to facilitate the intracellular delivery of macromolecules.     

Interactions of anionic and cationic fluorescent probes with proteins: The effect of charge

A family of fluorescent probes, consisting of 2-p-toluidinylnapththalene-6-sulfonate (TNS) and neutral and cationic sulfonamido derivatives has been utilized to study the influence of electrostatic forces in protein-amphiphile interactions. 2-p-Toluidinylnaphthalene-6-[N--ethylammonium chloride] sulfonamide (III) binds to a lower number of discrete sites in bovine serum albumin and sperm whale apomyoglobin than does TNS, and is also bound less efficiently by -lactoglobulin. The fluorescence characteristics of the bound probes indicate that their environments are hydrophobic, but thatpH and ionic strength influence the binding. The initial binding of III to discrete sites on both apomyoglobin and bovine serum albumin induces the cooperative binding of additional probe molecules. TNS, but not III, fluoresces in -chymotrypsin solutions. An [N--ethyltrimethyl ammonium] sulfonamido derivative (IV), but not TNS, fluoresces in bovine trypsin solutions. Fluorescence-enhancing interactions were detected between TNS, III, and polyvinylpyrrolidone, but not between these probes and ribonuclease A, -chymotrypsinogen, lysozyme, or -globulins. The wheat prolamin A-gliadin binds more TNS than III. The accessibility of all binding sites of gliadin is lower atpH 5.0 than atpH 3.1. It is suggested that, in general, charge effects are more likely to enhance the binding of anionic than cationic amphibiles by proteins.Dedicated to my teacher, Prof. Robert E. Feeney, on the occasion of his 70th Birthday. Presented in part at the Symposium on Chemical Modification and Structure-Function Relationships of Macromolecules, Division of Agricultural and Food Chemistry, 186th National Meeting of the American Chemical Society, 30 August 1983, Washington, D.C.     

Restoring the charge and surface activity of bovine lung extract surfactants with cationic and anionic polysaccharides

Chitosan, a cationic polysaccharide, has been found to improve the surface activity of lung surfactant extracts in the presence of various inhibitors. It has been proposed that chitosan binds to anionic lipids (e.g. phosphatidyl glycerols) in lung surfactants, producing stable lipid films at the air-water interface. This binding also reverses the net charge of the surfactant aggregates, from negative to positive. Unfortunately, positively charged aggregates may adsorb or interact with the negatively charged epithelial tissue, leading to poor surfactant performance. To address this issue an anionic polysaccharide, dextran sulfate (dexS), was used as a secondary coating to reverse the charge of chitosan-lung surfactant extracts without affecting the surface activity of the preparation. The dynamic surface tension and zeta potential of bovine lipid extract surfactant (BLES) containing chitosan chloride (chiCl) and dexS were evaluated as a function of dexS concentration. These studies were conducted in the absence and presence of sodium bicarbonate buffer, and in the absence and presence of bovine serum used as model inhibitor. It was determined that using an appropriate concentration of dexS, especially at physiological pH, it is possible to restore the negative charge of the surfactant aggregates, and retain their surface activity, even in the presence of bovine serum. High concentrations of dexS affect the binding of chiCl to BLES, and the surface activity of the preparation.     

Response of the phosphatidylcholine headgroup to membrane surface charge in ternary mixtures of neutral, cationic, and anionic lipids: a deuterium NMR study.

Deuterium nuclear magnetic resonance (2H NMR) spectroscopy was used to investigate the response of the phosphatidylcholine headgroup of 1,2-dimyristoyl-sn-glycero-3-phosphocholine (DMPC) to changes in surface electrostatic charge in membranes consisting of ternary mixtures of lipids. DMPC was deuterated at the choline alpha- and beta-methylene segments. The membrane surface charge was manipulated by the simultaneous addition of cationic didodecyldimethylammonium bromide (DDAB) and anionic 1,2-dimyristoyl-sn-glycero-3-phosphoglycerol (DMPG) to neutral DMPC. Addition of increasing amounts of DDAB caused a progressive decrease (increase) in the 2H NMR quadrupole splitting from DMPC-alpha-d2 (DMPC-beta-d2). Addition of increasing amounts of DMPG caused a progressive increase (decrease) in the quadrupole splitting from DMPC-alpha-d2 (DMPC-beta-d2). Qualitatively, the 2H NMR quadrupole splitting charge response exhibited the same main features for ternary mixtures of DDAB/DMPG/DMPC and binary mixtures of DDAB/DMPC or DMPG/DMPC. Quantitatively, however, the 2H NMR quadrupole splittings obtained from ternary mixtures did not coincide with those obtained from binary mixtures of nominally identical surface charge densities. Hence, the quadrupole splitting did not respond directly to the net membrane surface charge. Instead, the quadrupole splitting measured for a given ternary lipid composition could be reproduced by summing the individual effects of the charged lipids in binary mixtures, weighted according to their appropriate mole fractions.     

Comparison of salt effects on the reactions of acetylcholinesterase with cationic and anionic inhibitors

The influence of inorganic salts on the inhibition of acetylcholinesterase by charged organophosphorous inhibitors has been studied. It has been shown that the salt effect on the reaction of acetylcholinesterase with anionic bis(p-nitrophenyl) phosphate is determined by the influence of added salts on the activity coefficient of the inhibitor. In contrast to the salt effects on the reaction of acetylcholinesterase with cationic compounds, it does not include contribution from the enzyme charges. The smaller salt effect in the case of anionic inhibitor can be explained assuming that the anionic inhibitor does not form a non-covalent complex with the enzyme before the phosphorylation step of the reaction. Comparison of salt effects on the substrate turnover showed that in the case of cholinesterases from natural sources they are larger than in the case of enzymes expressed in recombinant cell clones. The enhanced salt effects may result from post-translational modification of the enzyme.     

pH-dependent membrane fusion and vesiculation of phospholipid large unilamellar vesicles induced by amphiphilic anionic and cationic peptides.

We studied fusion induced by a 20-amino acid peptide derived from the amino-terminal segment of hemagglutinin of influenza virus A/PR/8/34 [Murata, M., Sugahara, Y., Takahashi, S., & Ohnishi, S. (1987) J. Biochem. (Tokyo) 102, 957-962]. To extend the study, we have prepared several water-soluble amphiphilic peptides derived from the HA peptide; the anionic peptides D4, E5, and E5L contain four and five acidic residues and the cationic peptide K5 has five Lys residues in place of the five Glu residues in E5. Fusion of egg phosphatidylcholine large unilamellar vesicles induced by these peptides is assayed by two different fluorescence methods, lipid mixing and internal content mixing. Fusion is rapid in the initial stage (12-15% within 20 s) and remains nearly the same or slightly increasing afterward. The anionic peptides cause fusion at acidic pH lower than 6.0-6.5, and the cationic peptide causes fusion at alkaline pH higher than 9.0. Leakage and vesiculation of vesicles are also measured. These peptides are bound and associated with vesicles as shown by Ficoll discontinuous gradients and by the blue shift of tryptophan fluorescence. They take an alpha-helical structure in the presence of vesicles. They become more hydrophobic in the pH regions for fusion. When the suspension is made acidic or alkaline, the vesicles aggregate, as shown by the increase in light scattering. The fusion mechanism suggests that the amphiphilic peptides become more hydrophobic by neutralization due to protonation of the carboxyl groups or deprotonation of the lysyl amino groups, aggregate the vesicles together, and interact strongly with lipid bilayers to cause fusion. At higher peptide concentrations, E5 and E5L cause fusion transiently at acidic pH followed by vesiculation.     

The neuroleptic chlorpromazine inhibits the cationic and stimulates the anionic phospholipid precursor synthesis in human lymphocytes

The widely used neuroleptic drug chlorpromazine (CPZ) influences membrane functions at the levels of ionic channels and receptors as shown. Here we show the effect of short term treatments by CPZ (30 microM), on the nucleotide-containing phospholipid precursors in human lymphocyte primary cultures. During 60 minutes incubation of the cells, the CDP-ethanolamine (CDP-EA) content was only slightly reduced (87 to 76 pmol/10(6) cells), the amount of CDP-choline (CDP-Ch) was inhibited totally (from 25 to 0 pmol) upon the treatment with 30 microM CPZ under the same conditions. It has been shown earlier, that dCTP can be used as well as CTP for biosynthesis of phospholipids. Thus, the separation of the corresponding ribo- and deoxyribo-liponucleotides was developed. CPZ almost completely inhibited the synthesis of both dCDP-EA and dCDP-Ch under the same conditions The synthesis of the activated liponucleotide precursors, can be measured by incorporation of extracellular 14C-dCyt into both dCDP-EA and dCDP-Ch, as shown earlier. While the cationic deoxyribo-liponucleotide content (dCDP-Ch, dCDP-EA) was decreased, the labelling of the anionic phospholipid precursor dCDP-diacylglycerol (dCDP-DAG) was enhanced several times, it could be labelled only in the presence of CPZ from 14C-dCyd. Thus, a principal disturbance of the membrane phospholipid synthesis is presented (i.e., inhibition of the cationic and enhancement of the anionic dCDP-DAG synthesis). This profound influence on the membrane phospholipids by chlorpromazine, might be the primary effect that contributes to the wide spectrum of CPZ effects on neuronal cells.     

A test of discreteness-of-charge effects in phospholipid vesicles: measurements using paramagnetic amphiphiles

A new series of negatively charged, paramagnetic alkylsulfonate probes was synthesized and can be used to measure both the internal and the external surface potentials of model membrane systems. We tested for discreteness-of-charge effects in lipid membranes by comparing the surface potentials, estimated by use of these negatively charged amphiphiles, with that of a series of positively charged alkylammonium nitroxides in charged membranes. From the partitioning of these probes, the membrane surface potential was estimated in phosphatidylcholine membranes containing either phosphatidylserine or didodecyldimethylammonium bromide. The surface potentials, estimated with either positive or negative probes, were identical, within experimental error, in either positive or negative membranes, and they were well accounted for by a simple Gouy-Chapman-Stern theory. This symmetry, with respect to the sign of the charge, indicates that discreteness-of-charge effects are not significant in determining the potential-sensitive phase partitioning of these probes in model membranes. Thus, despite the fact that charge on membranes is discrete, models that assume a uniform density of charge in the plane of the membrane adequately account for the potentials measured by these amphiphilic probes.     

Nuclear antisense effects of neutral, anionic and cationic oligonucleotide analogs

The antisense activity of oligomers with 2'-O-methyl (2'-O-Me) phosphorothioate, 2'-O-methoxyethyl (2'-O-MOE) phosphorothioate, morpholino and peptide nucleic acid (PNA) backbones was investigated using a splicing assay in which the modified oligonucleotides blocked aberrant and restored correct splicing of modified enhanced green fluorescent protein (EGFP) precursor to mRNA (pre-mRNA), generating properly translated EGFP. In this approach, antisense activity of each oligomer was directly proportional to up-regulation of the EGFP reporter. This provided a positive, quantitative readout for sequence-specific antisense effects of the oligomers in the nuclei of individual cells. Nuclear localization of fluorescent labeled oligomers confirmed validity of the functional assay. The results showed that the free uptake and the antisense efficacy of neutral morpholino derivatives and cationic PNA were much higher than that of negatively charged 2'-O-Me and 2'-O-MOE congeners. The effects of the PNA oligomers were observed to be dependent on the number of L-lysine (Lys) residues at the C-terminus. The experiments suggest that the PNA containing Lys was taken up by a mechanism similar to that of cell-penetrating homeodomain proteins and that the Lys tail enhanced intracellular accumulation of PNA oligomer without affecting its ability to reach and hybridize to the target sequence.     

Phase behavior of cationic amphiphiles and their mixtures with helper lipid influences lipoplex shape,DNA translocation,and transfection efficiency

Cationic lipids are widely used for gene transfection, but their mechanism of action is still poorly understood. To improve this knowledge, a structure-function study was carried out with two pyridinium-based lipid analogs with identical headgroups but differing in alkyl chain (un)saturation, i.e., SAINT-2 (diC18:1) and SAINT-5 (diC18:0). Although both amphiphiles display transfection activity per se, DOPE strongly promotes SAINT-2-mediated transfection, but not that of SAINT-5, despite the fact that DOPE effectively facilitates plasmid dissociation from either lipoplex. This difference appears to correlate with membrane stiffness, dictated by the cationic lipid packing in the donor liposomes, which governs the kinetics of lipid recruitment by the plasmid upon lipoplex assembly. Because of its interaction with the relatively rigid SAINT-5 membranes, the plasmid becomes inappropriately condensed, which results in formation of structurally deformed lipoplexes. This structural deformation does not affect its cellular uptake but, rather, hampers plasmid translocation across endosomal and/or nuclear membranes. This is inferred from the observation that both lipoplexes effectively translocate much smaller oligonucleotides into cells. In fact, SAINT-5/DOPE-mediated transfection is greatly improved when, before lipoplex assembly, the plasmid is stabilized by condensation with polylysine. The results emphasize a role of the structural shape of the plasmid in gaining cytosolic/nuclear access. Moreover, it has been proposed that such a translocation is promoted when the lipoplex adopts the hexagonal phase, and data are presented that demonstrate that the lamellar SAINT-5/DOPE lipoplex adopts such a phase after its interaction with acidic phospholipid-containing membranes.     

Two-dimensional diffusion of amphiphiles in phospholipid monolayers at the air-water interface.

Steady-state and time-resolved fluorescence spectroscopy has been used to examine lateral diffusion in dipalmitoyl-L-alpha-phosphatidylcholine (DPPC) and dimyristoyl-L-alpha-phosphatidylcholine (DMPC) monolayers at the air-water interface, by studying the fluorescence quenching of a pyrene-labeled phospholipid (pyrene-DPPE) by two amphiphilic quenchers. Steady-state fluorescence measurements revealed pyrene-DPPE to be homogeneously distributed in the DMPC lipid matrix for all measured surface pressures and only in the liquid-expanded (LE) phase of the DPPC monolayer. Time-resolved fluorescence decays for pyrene-DPPE in DMPC and DPPC (LE phase) in the absence of quencher were best described by a single-exponential function, also suggesting a homogeneous distribution of pyrene-DPPE within the monolayer films. Addition of quencher to the monolayer film produced nonexponential decay behavior, which is adequately described by the continuum theory of diffusion-controlled quenching in a two-dimensional environment. Steady-state fluorescence measurements yielded lateral diffusion coefficients significantly larger than those obtained from time-resolved data. The difference in these values was ascribed to the influence of static quenching in the case of the steady-state measurements. The lateral diffusion coefficients obtained in the DMPC monolayers were found to decrease with increasing surface pressure, reflecting a decrease in monolayer fluidity with compression.     

Unusual electrostatic effects on binding of C1q to anionic liposomes: role of anionic phospholipid domains and their line tension.

The binding of 125I-C1q to anionic liposomes was studied as a function of protein concentration, pH, ionic strength, and anionic lipid composition. The maximum amount of protein bound per micromole of lipid was very sensitive to electrostatic factors, increasing strongly with decreased pH and ionic strength or increased anionic lipid content. The apparent association constant was independent of these electrostatic factors, however, in marked contrast to studies on basic peptide binding to anionic lipid vesicles. Microscopic observations of large unilamellar liposomes containing fluorescently labeled C1q or phosphatidylglycerol demonstrated, under conditions causing strong electrostatic interactions, that C1q and anionic lipids colocalized into domains whose radii of curvature were higher than that of the surrounding lipid. These domains were observed to bud and pinch off into brightly fluorescent vesicles. We propose a model for all of these observations in which the line tension or edge energy at the boundary of the domain resists its increase in circumference as the domain grows by electrostatic effects on binding, eventually resulting in vesiculation. We propose that under favorable electrostatic conditions, as larger domains form the edge energy balances the increases in the electrostatic contribution to binding, resulting in a net binding energy independent of electrostatic factors.     

The effect of anionic amphiphiles on the recruitment of Rac in neutrophils

Anionic amphiphiles have been shown to influence the NADPH oxidase system. Although one target of the amphiphile action is p47(phox), the cell-free activation of the enzyme in the absence of p47(phox) is also influenced. In the present study, we examined the actions of sodium dodecyl sulfate (SDS) on the NADPH oxidase system in vivo. Treatment of guinea pig neutrophils with the amphiphile caused the translocation of Rac to a membrane fraction and its conversion to the GTP-bound form. Because SDS had little effect on p47(phox), it increased the superoxide production only when p47(phox) was otherwise activated. Inhibitors of phosphoinositide 3-kinases had no effect on the SDS-induced translocation of Rac to the membrane. However, the inhibitors prevented the conversion of Rac to its GTP-bound form, indicating that these two processes can be controlled separately. In a cell-free system, SDS induced the binding of p47(phox) and Rac to the membrane preparation. The SDS concentration inducing the Rac binding was lower than that inducing the p47(phox) binding. Thus we observed that Rac is more sensitive to SDS than p47(phox) both in vivo and in vitro. The results suggest a role of natural amphiphiles such as unsaturated fatty acids in regulation of Rac activation.     

Collagen-induced exposure of anionic phospholipid in platelets and platelet-derived microparticles.

We have shown recently that the calcium-dependent phospholipid-binding protein annexin V (placental anticoagulant protein I) can be used to study the exposure of anionic phospholipid after platelet activation. In this study we have further examined the mechanism of this process. Collagen-induced exposure of annexin V binding sites correlated directly with increased ability to support activity of the reconstituted prothrombinase complex. The potency of annexin V as an inhibitor of platelet prothrombinase was the same as its Kd for platelets. Prior incubation of platelets with 5'-p-fluorosulfonylbenzoyladenosine or p-chloromercuribenzenesulfonate had no significant effect on annexin V binding. Similarly, inhibition of platelet cyclic endoperoxide synthesis by acetylsalicylic acid or indomethacin did not inhibit annexin V binding. Staurosporine inhibited collagen-induced, but not A23187-induced, annexin V binding. Agents that increase intraplatelet cyclic nucleotides partially inhibited collagen-induced annexin V binding. Thus, collagen-induced exposure of anionic phospholipid appears to depend primarily on increases in intraplatelet free calcium and may be independent of ADP- or endoperoxide-mediated pathways. Binding sites for annexin V on microparticles derived from collagen-stimulated platelets were demonstrated by flow cytometry and gel filtration. In addition, prior incubation of platelets with 100 nM annexin V inhibited factor Va binding to both platelets and platelet-derived microparticles. These results support the concept that the procoagulant effect of platelets and platelet-derived microparticles is mediated by calcium-induced exposure of anionic phospholipids.     

Different effects of sphingosine, R59022 and anionic amphiphiles on two diacylglycerol kinase isozymes purified from porcine thymus cytosol

Porcine thymus cytosol contains two immunologically distinct forms of diacylglycerol kinase (DGK) [Yamada, K. and Kanoh, H. (1988) Biochem. J. 255, 601-608]. These 2 DGK species, having apparent molecular masses of 80 and 150 kDa, were purified from the thymus cytosol. Upon sodium dodecyl sulfate-polyacrylamide gel electrophoresis, the 150-kDa DGK gave 2 polypeptide bands of 50 and 75 kDa, whereas the 80-kDa DGK yielded a single protein band. The 80-kDa DGK was markedly activated by 10-20 microM sphingosine as well as by the known anionic activators such as phosphatidylserine and deoxycholate. In contrast, the 150-kDa DGK was fully active in the absence of the anionic activators and was strongly inhibited by sphingosine (IC50, 20 microM). The putative DGK inhibitor R59022 inhibited the 80-kDa DGK (IC50, 10 microM), but had little effect on the 150-kDa form. It is therefore clear that in the thymus cytosol there are at least 2 DGK isozymes operating under different control mechanisms.