Measurement of the affinity and cooperativity of annexin V-membrane binding under conditions of low membrane occupancy

We developed a method for measuring the binding affinity of annexin V for phospholipid vesicles and cells at very low levels of membrane occupancy. The annexin V-117 mutant was labeled with fluorescein iodoacetamide on its single N-terminal cysteine residue; binding to phospholipid vesicles containing phosphatidylserine (PS) and 2% rhodamine-phosphatidylethanolamine was measured by fluorescence quenching due to resonance energy transfer; binding to cells with exposed PS was measured by fluorometry after elution of bound protein. The equilibrium constant was calculated as a function of the midpoint of the calcium titration curve, the Hill coefficient, and the concentration of membrane binding sites. Calcium titrations at very low ratios of protein to membrane revealed Hill coefficients of approximately 8 for both vesicles and cells, far higher than previously measured, but as the protein-membrane ratio was increased above 3% of maximum membrane occupancy, the value of the Hill coefficient progressively decreased to a limiting value of about 2. High Hill coefficients were also observed for measurements performed at different ionic strengths and with membrane PS content varied over the range from 20 to 50%. This method allows the accurate determination of the affinity and cooperativity of annexin V-membrane binding and will be useful for the evaluation of modified annexin V derivatives intended for diagnostic and therapeutic applications.     

Clustering of lipid-bound annexin V may explain its anticoagulant effect.

In 1985 we isolated a new vascular anticoagulant protein VAC alpha, now called annexin V, with a high binding affinity (Kd less than 10(-10) M) for phospholipids. Its anticoagulant effect was attributed to displacement of coagulation factors from the phospholipid membrane. The present study demonstrates that the inhibition of prothrombinase activity by annexin V strongly depends on the curvature of the membrane surface and on the calcium concentration. Half-maximal inhibition of prothrombinase on and binding of annexin V to small vesicles, composed of 20% phosphatidylserine and 80% phosphatidylcholine, requires 2-3 mM calcium. With large vesicles and planar bilayers considerably less calcium is required for inhibition of prothrombinase and for lipid binding. Half-maximal binding of annexin V to large vesicles and to planar bilayers occurs at 0.7 and 0.2 mM calcium, respectively. This seemingly confirms the displacement model. The displacement of coagulation factors, however, proved to be incomplete, with residual surface concentrations of factors Xa, Va, and prothrombin sufficient for effective production of thrombin. Cryoelectron microscopy revealed that annexin V binding to large vesicles caused planar facets, indicating the formation of large sheets of clustered annexin V. Apparently, the formation of these two-dimensional arrays is promoted by calcium and hampered by high surface curvature. It is speculated that the complete inhibition (greater than 99%) of prothrombinase activity by annexin V is caused by the reduced lateral mobility of prothrombin and factor Xa in rigid sheets of annexin V covering the membrane.     

Suicidal erythrocyte death following cellular K+ loss.

Hallmarks of apoptosis include cell shrinkage, which is at least partially due to cellular K(+) loss. The decline of cellular K(+) concentration has been suggested to participate in the triggering of apoptosis. Suicidal erythrocyte death or eryptosis is triggered by increased cytosolic Ca(2+) activity leading to activation of Ca(2+)-sensitive K(+) channels with subsequent cellular K(+) loss and cell shrinkage, and to Ca(2+)-sensitive scambling of the cell membrane with subsequent phosphatidylserine (PS) exposure at the cell surface. Phosphatidylserine exposing erythrocytes are recognized by macrophages, engulfed, degraded and thus cleared from circulating blood. The present study explored whether cellular loss of K(+) and/or cell shrinkage actively participate in the triggering of cell membrane phospholipid scrambling. Cellular K(+) loss was achieved by treatment of human erythrocytes with the K(+) ionophore valinomycin (1 nM) at different extracellular K(+) concentrations (5-125 mM) and osmolarities (300-550 m Osm). Cell volume was estimated from forward scatter and PS exposure from annexin V binding in FACS analysis. Treatment with 1 nM valinomycin indeed decreased forward scatter and increased annexin V binding. The effect was significantly blunted in the presence of staurosporine (1 microM). Increase of extracellular K(+) concentration gradually blunted the decrease of forward scatter but inhibited annexin V binding only at extracellular K(+) concentrations >or=75 mM. An increase of extracellular osmolarity (+150 mM or 250 mM sucrose) reversed the protective effect of 75 mM KCl during valinomycin treatment. A correlation between forward scatter and annexin binding at different osmolarities and K(+) concentrations suggests that the cellular K(+) content determines the rate of suicidal erythrocyte death primarily through its influence on cell volume.     

The crystal structure of calcium-bound annexin Gh1 from Gossypium hirsutum and its implications for membrane binding mechanisms of plant annexins

Plant annexins show distinct differences in comparison with their animal orthologues. In particular, the endonexin sequence, which is responsible for coordination of calcium ions in type II binding sites, is only partially conserved in plant annexins. The crystal structure of calcium-bound cotton annexin Gh1 was solved at 2.5 A resolution and shows three metal ions coordinated in the first and fourth repeat in types II and III binding sites. Although the protein has no detectable affinity for calcium in solution, in the presence of phospholipid vesicles, we determined a stoichiometry of four calcium ions per protein molecule using isothermal titration calorimetry. Further analysis of the crystal structure showed that binding of a fourth calcium ion is structurally possible in the DE loop of the first repeat. Data from this study are in agreement with the canonical membrane binding of annexins, which is facilitated by the convex surface associating with the phospholipid bilayer by a calcium bridging mechanism. In annexin Gh1, this membrane-binding state is characterized by four calcium bridges in the I/IV module of the protein and by direct interactions of several surface-exposed basic and hydrophobic residues with the phospholipid membrane. Analysis of the protein fold stability revealed that the presence of calcium lowers the thermal stability of plant annexins. Furthermore, an additional unfolding step was detected at lower temperatures, which can be explained by the anchoring of the N-terminal domain to the C-terminal core by two conserved hydrogen bonds.     

Key role of the N‐terminus of chicken annexin A5 in vesicle aggregation

Annexins are calcium‐dependent phospholipid‐binding proteins involved in calcium signaling and intracellular membrane trafficking among other functions. Vesicle aggregation is a crucial event to make possible the membrane remodeling but this process is energetically unfavorable, and phospholipid membranes do not aggregate and fuse spontaneously. This issue can be circumvented by the presence of different agents such as divalent cations and/or proteins, among them some annexins. Although human annexin A5 lacks the ability to aggregate vesicles, here we demonstrate that its highly similar chicken ortholog induces aggregation of vesicles containing acidic phospholipids even at low protein and/or calcium concentration by establishment of protein dimers. Our experiments show that the ability to aggregate vesicles mainly resides in the N‐terminus as truncation of the N‐terminus of chicken annexin A5 significantly decreases this process and replacement of the N‐terminus of human annexin A5 by that of chicken switches on aggregation; in both cases, there are no changes in the overall protein structure and only minor changes in phospholipid binding. Electrostatic repulsions between negatively charged residues in the concave face of the molecule, mainly in the N‐terminus, seem to be responsible for the impairment of dimer formation in human annexin A5. Taking into account that chicken annexin A5 presents a high sequence and structural similarity with mammalian annexins absent in birds, as annexins A3 and A4, some of the physiological functions exerted by these proteins may be carried out by chicken annexin A5, even those that could require calcium‐dependent membrane aggregation.     

Interactions of annexins with membrane phospholipids.

The annexins are proteins that bind to membranes and can aggregate vesicles and modulate fusion rates in a Ca2(+)-dependent manner. In this study, experiments are presented that utilize a pyrene derivative of phosphatidylcholine to examine the Ca2(+)-dependent membrane binding of soluble human annexin V and other annexins. When annexin V and other annexins were bound to liposomes containing 5 mol % acyl chain labeled 3-palmitoyl-2-(1-pyrenedecanoyl)-L-alpha-phosphatidylcholine, a decrease in the excimer-to-monomer fluorescence ratio was observed, indicating that annexin binding may decrease the lateral mobility of membrane phospholipids without inducing phase separation. The observed increases of monomer fluorescence occurred only with annexins and not with other proteins such as parvalbumin or bovine serum albumin. The extent of the increase of monomer fluorescence was dependent on the protein concentration and was completely and rapidly reversible by EDTA. Annexin V binding to phosphatidylserine liposomes was consistent with a binding surface area of 59 phospholipid molecules per protein. Binding required Ca2+ concentrations ranging between approximately 10 and 100 microM, where there was no significant aggregation or fusion of liposomes on the time scale of the experiments. The polycation spermine also displaced bound annexins, suggesting that binding is largely ionic in nature under these conditions.     

Stoichiometric and electrostatic characterization of calcium binding to native and lipid-substituted adenosinetriphosphatase of sarcoplasmic reticulum

The stoichiometry of calcium binding to specific sites (i.e., those producing enzyme activation) was found to be 8-10 nmol/mg protein in native sarcoplasmic reticulum vesicles, and 13.9-15.4 nmol/mg of ATPase purified by non-ionic detergent solubilization and anion exchange chromatography. Parallel measurements of phosphoenzyme yielded levels of 4.0-4.9 and 6.0-7.7 nmol/mg of protein in the two preparations, respectively, demonstrating that each 115 kDa ATPase chain includes one catalytic site and two calcium binding sites. The apparent association constant, K = (6 +/- 2) X 10(5) M-1, and the binding cooperativity, nH = 1.9, were unchanged when measurements were carried out with native sarcoplasmic reticulum vesicles and when the membrane surface charge was altered by lipid substitution with phosphatidylcholine or phosphatidylserine, at neutral pH in the presence of 10 mM MgCl2 and 80 mM KCl. On the other hand, the apparent association constant was increased in the absence of Mg2+ or, to a lesser extent, in the absence of monovalent cations. It was also observed that the cooperative character of the calcium binding isotherms was reduced in low ionic-strength media. Analysis of the electrostatic effects indicates that the calcium-binding domain is shielded from the membrane phospholipid surface charge by virtue of its location within the ATPase protein. The effects of various electrolytes are attributed to monovalent-cation binding in the calcium-binding domain. The apparent loss of cooperativity of the calcium binding isotherms at low ionic strength is attributed to a progressive displacement of the titration curve which is minimal at low degrees of saturation and becomes larger at higher degrees of saturation. This behavior is described quantitatively by the progressive effect of calcium binding on an electrostatic potential generated by localized protein charge densities within, or near, the calcium-binding domain.     

Phosphorylation of annexin II tetramer by protein kinase C inhibits aggregation of lipid vesicles by the protein.

Annexin II tetramer (A-IIt) is a member of the annexin family of Ca2+ and phospholipid-binding proteins. The ability of this protein to aggregate both phospholipid vesicles and chromaffin granules has suggested a role for the protein in membrane trafficking events such as exocytosis. A-IIt is also a major intracellular substrate of both pp60src and protein kinase C; however, the effect of phosphorylation on the activity of this protein is unknown. In the current report we have examined the effect of phosphorylation on the lipid vesicle aggregation activity of the protein. Protein kinase C catalyzed the incorporation of 2.1 +/- 0.8 mol of phosphate/mol of A-IIt. Phosphorylation of A-IIt caused a dramatic decrease in the rate and extent of lipid vesicle aggregation without significantly effecting Ca(2+)-dependent lipid binding by the phosphorylated protein. Phosphorylation of A-IIt increased the A50%(Ca2+) of lipid vesicle aggregation from 0.18 microM to 0.65 mM. Activation of A-IIt phosphorylation, concomitant with activation of lipid vesicle aggregation, inhibited both the rate and extent of lipid vesicle aggregation but did not cause disassembly of the aggregated lipid vesicles. These results suggest that protein kinase C-dependent phosphorylation of A-IIt blocks the ability of the protein to aggregate phospholipid vesicles without affecting the lipid vesicle binding properties of the protein.     

Organization and dynamics of the proteolipid complexes formed by annexin V and lipids in planar supported lipid bilayers

The consequences of the binding of annexin V on its lateral mobility and that of lipids were investigated by means of experimental and simulated FRAP experiments. Experiments were carried out on planar supported bilayers (PC/PS 9:1 mol/mol mixtures) in the presence of 1 mM CaCl2 in the subphase. The probes C12-NBD-PS and fluorescein-labeled annexin V were used and the data compared with that previously obtained for C12-NBD-PC [Saurel, O., Cézanne, L., Milon, A., Tocanne, J. F., & Demange, P. (1998) Biochemistry 37, 1403-1410]. At complete coverage of the lipid bilayer by the protein (Cannexin = 80 nM), the lateral mobility of C12-NBD-PC was reduced by 40% while C12-NBD-PS and bound annexin V molecules were nearly immobilized (D < 10(-)11 cm2/s). At moderate protein concentration (20 nM < Cannexin < 80 nM), best fitting of the lipid and protein probe recoveries was achieved with one single diffusion coefficient and a mobile fraction close to 100%, indicating homogeneous lipid and protein populations. In contrast, at low protein concentration (Cannexin < 20 nM), C12-NBD-PS showed a two-component diffusion. The slow PS population at Cannexin < 20 nM and the single PS population at Cannexin > 20 nM moved at the same rate that bound annexin V (mobile fraction close to 100%), indicating strong PS/protein interactions. With the aid of computer simulations of the lateral motion of PC molecules, based on the 2-D crystalline networks formed by annexin V in contact with the lipid bilayer, these FRAP results may be accounted for by considering a rather simple model of a proteolipidic complex consisting of an extended 2-D crystalline protein network facing the lipid bilayer and stabilized by strong interactions between annexin V and PS molecules. In this model, immobilization of annexin V and PS molecules originates from their mutual interactions. The slowing down of PC molecules is due to various obstacles to their lateral diffusion which can be described as: the four PS molecules bound to the protein, the tryptophan 187 which presumably interacts with the lipids at the level of their polar headgroups and probably the three other hydrophobic amino acid residues located on the AB calcium-binding loops of the protein.     

Association of protein kinase C with phospholipid vesicles

The Ca2+- and phospholipid-dependent protein kinase, protein kinase C (PKC), was purified from bovine brain by a modified procedure that provided sufficient quantities of stable protein for analysis of physical properties of protein-membrane binding. The binding of PKC to phospholipid vesicles of various compositions was investigated by light-scattering and fluorescence energy transfer measurements. The binding properties for membranes of low phosphatidylserine (PS) content were consistent with a peripheral membrane association; PKC showed Ca2+ -dependent binding to phospholipid vesicles containing phosphatidylserine, phosphatidylinositol, or phosphatidylglycerol. Membranes containing 0-20% PS (the remainder of the phospholipid was phosphatidylcholine) bound less protein than membranes containing greater than 20% PS; the factor limiting protein binding to membranes containing low PS appeared to be the availability of acidic phospholipids. Increasing the PS content above 20% did not increase the amount of membrane-bound protein at saturation, and the limiting factor was probably steric packing of protein on the membrane surface. The membranes bound about 1 g of protein/g of phospholipid at steric saturation. Binding was of relatively high affinity (Kd less than 5 nM), and the association rate was rapid on the time scale of the experiments. Addition of ethylene glycol bis(beta-aminoethyl ether)-N,N,N',N'-tetraacetic acid to phospholipid-bound PKC caused dissociation of the complex, and the properties of this dissociation indicated an equilibrium binding of protein to membrane. However, only partial dissociation of PKC was achieved when the PS content of the vesicles exceeded 20%. A number of comparisons revealed that binding of protein to the membrane, even in the presence of phorbol esters, was insufficient for development of enzyme activity.(ABSTRACT TRUNCATED AT 250 WORDS)     

Calcium-induced changes in permeability of dioleoylphosphatidylcholine model membranes containing bovine heart cardiolipin

At calcium concentrations up to about 4 mM a selective permeability increase of cardiolipin/dioleoylphosphatidylcholine (50:50, mol%) membranes for calcium and its chelator arsenazo III is observed. Under these conditions calcium does not occupy all the binding sites of cardiolipin at the membrane interface and no vesicle-vesicle interactions are found. Lowering of the cardiolipin content of the vesicles to 20 mol% extends the calcium concentration range in which a selective permeability for calcium and arsenazo III is appearing up to about 12 mM. We suggest that the observed selective permeability increase is caused by transient formation of inverted micellar structures in the membrane with cardiolipin as translocating membrane component for calcium and arsenazo III. At calcium concentrations of 4 mM and higher for 50 mol% cardiolipin-containing vesicles a general permeability increase is found together with calcium-cardiolipin binding in a 1:1 stoichiometry, vesicles aggregation and, above 8 mM of calcium, vesicle fusion. The loss of barrier function of the membrane under these conditions is correlated with vesicle aggregation and may be explained by a transition from a bilayer into a hexagonal HII organization of the phospholipids.     

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.     

Phorbol ester binding and activation of protein kinase C on triton X-100 mixed micelles containing phosphatidylserine

A mixed micellar assay for the binding of phorbol-esters to protein kinase C was developed to investigate the specificity and stoichiometry of phospholipid cofactor dependence and oligomeric state of protein kinase C (Ca2+/phospholipid-dependent enzyme) required for phorbol ester binding. [3H]Phorbol dibutyrate was bound to protein kinase C in the presence of Triton X-100 mixed micelles containing 20 mol % phosphatidylserine (PS) in a calcium-dependent manner with a Kd of 5 X 10(-9) M. The [3H]phorbol dibutyrate X protein kinase C . Triton X-100 . PS mixed micellar complex eluted on a Sephacryl S-200 molecular sieve at an Mr of approximately 200,000; this demonstrates that monomeric protein kinase C binds phorbol dibutyrate. This conclusion was supported by molecular sieve chromatography of a similar complex where Triton X-100 was replaced with beta-octylglucoside. Phorbol dibutyrate activation of protein kinase C in Triton X-100/PS mixed micelles occurred and was dependent on calcium. The PS dependence of both phorbol ester activation and binding to protein kinase C lagged initially and then was highly cooperative. The minimal mole per cent PS required was strongly dependent on the concentration of phorbol dibutyrate or phorbol myristic acetate employed. Even at the highest concentration of phorbol ester tested, a minimum of 3 mol % PS was required; this indicates that approximately four molecules of PS are required. [3H]Phorbol dibutyrate binding was independent of micelle number at 20 mol % PS. The phospholipid dependencies of phorbol ester binding and activation were similar, with PS being the most effective; anionic phospholipids (cardiolipin, phosphatidic acid, and phosphatidylglycerol were less effective, whereas phosphatidylcholine, phosphatidylethanolamine, and sphingomyelin did not support binding or activation. sn-1,2-Dioleoylglycerol displaced [3H]phorbol dibutyrate quantitatively and competitively. The data are discussed in relation to a molecular model of protein kinase C activation.     

Activation of annexin 7 by protein kinase C in vitro and in vivo

Annexin 7, a Ca(2+)/GTP-activated membrane fusion protein, is preferentially phosphorylated in intact chromaffin cells, and the levels of annexin 7 phosphorylation increase quantitatively in proportion to the extent of catecholamine secretion. Consistently, various protein kinase C inhibitors proportionately reduce both secretion and phosphorylation of annexin 7 in these cells. In vitro, annexin 7 is quantitatively phosphorylated by protein kinase C to a mole ratio of 2.0, and phosphorylation is extraordinarily sensitive to variables such as pH, calcium, phospholipid, phorbol ester, and annexin 7 concentration. Phosphorylation of annexin 7 by protein kinase C significantly potentiates the ability of the protein to fuse phospholipid vesicles and lowers the half-maximal concentration of calcium needed for this fusion process. Furthermore, other protein kinases, including cAMP-dependent protein kinase, cGMP-dependent protein kinase, and protein-tyrosine kinase pp60(c-)(src), also label annexin 7 with high efficiency but do not have this effect on membrane fusion. In the case of pp60(c-)(src), we note that this kinase, if anything, modestly suppresses the membrane fusion activity of annexin 7. These results thus lead us to hypothesize that annexin 7 may be a positive mediator for protein kinase C action in the exocytotic membrane fusion reaction in chromaffin cells.     

The interaction of bone Gla protein (osteocalcin) with phospholipid vesicles

The binding interaction of bone Gla protein (BGP), or osteocalcin, to phospholipid vesicles in the presence of calcium has been investigated. Two separate indirect methodologies involving displacement of pyrene-modified Factor Va bound to phospholipid vesicles, and competition with several coagulation proteins in a prothrombin activation assay were performed. Titration of BGP into a cuvette containing phospholipid vesicles (75:25, L-alpha-phosphatidylcholine/L-alpha-phosphatidylserine (PCPS] saturated with pyrene-modified Factor Va resulted in a systematic decrease in steady-state anisotropy, suggesting competition for membrane binding sites with pyrene-modified Factor Va. BGP was also found to inhibit thrombin generation in the prothrombin activation assay. Approximately 50% inhibition was observed at 3 microM BGP under phospholipid-limiting (0.5 microM PCPS) concentrations. No inhibition was observed under phospholipid excess (30 microM PCPS) concentrations. Direct measurement of phospholipid binding was measured using equilibrium gel filtration. Elution profiles using fixed lipid (3.4 mumol of PCPS) and varying BGP concentrations (1-17 microM) in the presence of 3 mM CaCl2 showed a BGP-phospholipid association. Quantitation of determined isotherm yielded a dissociation constant of 6 +/- 1 microM with a stoichiometry of 102 +/- 9 BGP molecules/vesicle at saturation (35 PCPS lipids/BGP) in the presence of 3 mM CaCl2. These results support the hypothesis that protein gamma-carboxylation events are coincident with membrane binding potential.     

Inhibition of protein kinase C by annexin V.

Annexin V is a protein of unknown biological function that undergoes Ca(2+)-dependent binding to phospholipids located on the cytosolic face of the plasma membrane. Preliminary results presented herein suggest that a biological function of annexin V is the inhibition of protein kinase C (PKC). In vitro assays showed that annexin V was a specific high-affinity inhibitor of PKC-mediated phosphorylation of annexin I and myosin light chain kinase substrates, with half-maximal inhibition occurring at approximately 0.4 microM. Annexin V did not inhibit epidermal growth factor receptor/kinase phosphorylation of annexin I or cAMP-dependent protein kinase phosphorylation of the Kemptide peptide substrate. Since annexin V purified from both human placenta and recombinant bacteria inhibited protein kinase C activity, it is not likely that the inhibitor activity was associated with a minor contaminant of the preparations. The following results indicated that the mechanism of inhibition did not involve annexin V sequestration of phospholipid that was required for protein kinase C activation: similar inhibition curves were observed as phospholipid concentration was varied from 0 to 800 micrograms/mL; the extent of inhibition was not significantly affected by the order of addition of phospholipid, substrate, or PKC, and the core domain of annexin I was not a high-affinity inhibitor of PKC even though it had similar Ca2+ and phospholipid binding properties as annexin V. These data indirectly indicate that inhibition occurred by direct interaction between annexin V and PKC. Since the concentration of annexin V in many cell types exceeds the amounts required to achieve PKC inhibition in vitro, it is possible that annexin V inhibits PKC in a biologically significant manner in intact cells.     

The effect of phospholipids, calcium ions and protein S on rate constants of human factor Va inactivation by activated human protein C.

Rate constants for human factor Va inactivation by activated human protein C (APC) were determined in the absence and presence of Ca2+ ions, protein S and varying concentrations of phospholipid vesicles of different lipid composition. APC-catalyzed factor Va inactivation in free solution (in the presence of 2 mM Ca2+) was studied under first-order reaction conditions with respect to both APC and factor Va and was characterized by an apparent second-order rate constant of 6.1 x 10(5) M-1 s-1. Stimulation of APC-catalyzed factor Va inactivation by phospholipids was dependent on the concentration and composition of the phospholipid vesicles. Optimal acceleration (230-fold) of factor Va inactivation was observed with 10 microM phospholipid vesicles composed of 20 mol% dioleoylglycerophosphoserine (Ole2GroPSer) and 80 mol% dioleoylglycerophosphocholine (Ole2GroPCho). At higher vesicle concentrations and at higher molar fractions of Ole2GroPSer some inhibition of APC-catalyzed factor Va inactivation was observed. Membranes that contained anionic phospholipids other than phosphatidylserine also promoted factor Va inactivation. The ability of different anionic lipids to enhance factor Va inactivation increased in the order phosphatidylethanolamine less than oleic acid less than phosphatidic acid less than phosphatidylglycerol less than phosphatidylmethanol less than phosphatidylserine. APC-catalyzed factor Va inactivation in the presence of phospholipid vesicles could be saturated with respect to factor Va and the reaction obeyed Michaelis-Menten kinetics. Both the Km for factor Va and the Vmax of factor Va inactivation were a function of the phospholipid concentration. The Km increased from 1 nM at 2.5 microM phospholipid (Ole2GroPSer/Ole2GroPCho 20:80, mol/mol) to 65 nM at 250 microM phospholipid. The Vmax increased from 20 mol factor Va inactivated.min-1.mol APC-1 at 2.5 microM phospholipid to 62 mol factor Va inactivated.min-1.mol APC-1 at 10 microM phospholipid and remained constant at higher phospholipid concentrations. Protein S appeared to be a rather poor stimulator of APC-catalyzed factor Va inactivation. Protein-S-dependent rate enhancements were only observed in reaction mixtures that contained negatively charged phospholipid vesicles. Independent of the concentration and the lipid composition of the vesicles, protein S caused a twofold stimulation of APC-catalyzed factor Va inactivation. This suggests that, in the human system, enhancement of APC binding to phospholipid vesicles by protein S is of minor importance. Considering that protein S is a physiologically essential antithrombotic agent, it is likely that other factors or phenomena contribute to the in vivo antithrombotic action of protein S.     

Phospholipid binding properties of human placental anticoagulant protein-I, a member of the lipocortin family

Human placental anticoagulant protein-I (PAP-I) is a member of the lipocortin/calpactin/annexin family of Ca2+-dependent phospholipid binding proteins. PAP-I was labeled with fluorescein 5-isothiocyanate (1 mol/mol); this derivative had anticoagulant activity identical to the unlabeled protein and could be used to measure Ca2+-dependent binding to phospholipid vesicles through changes in fluorescence quenching. At 1.2 mM Ca2+, 0.50 M ionic strength, pH 7.4, 25 degrees C, fluorescein-labeled PAP-I bound to phospholipid vesicles containing 80% phosphatidylcholine, 20% phosphatidylserine with a Kd of 1.2 +/- 0.2 nM (mean +/- S.D.). At an ionic strength of 0.15 M, the Kd decreased to less than 0.1 nM. Prothrombin and factor Xa both competed with fluorescein-labeled PAP-I for binding to anionic phospholipid vesicles, but with affinities at least 1000-fold weaker than PAP-I. PAP-I bound only weakly (Kd greater than 2 x 10(-5) M) to neutral or anionic phospholipid monomers, and this binding was not calcium-dependent. These results show that the affinity of PAP-I for anionic phospholipid surfaces is sufficient to explain its potency as an in vitro anticoagulant.     

Conformational flexibility of domain III of annexin V at membrane/water interfaces

The conformational dynamics of domain III in annexin V bound to negatively charged phospholipid vesicles of 1-palmitoyl-2-oleoyl-sn-glycerophosphocholine and 1-palmitoyl-2-oleoyl-sn-glycerophosphoserine or incorporated into reverse micelles of water/sodium bis(2-ethylhexyl) sulfosuccinate in isooctane, used to mimic the phospholipid/water interface, was studied by steady-state and time-resolved fluorescence of its single tryptophan residue (W187). Upon interaction with sonicated phospholipid vesicles in the presence of calcium, or upon incorporation into reverse micelles without calcium, a progressive 12-14 nm red shift of the fluorescence emission spectrum of W187 is observed. The indole environment becomes therefore more polar than in the unbound protein. Three major lifetime populations describe the fluorescence intensity decays of W187 in both systems. A long-lived excited-state population characterizes the membrane-bound state of the protein. The existence of local conformers with different subnanosecond mobility is suggested by specific association between lifetimes and correlation times both for the protein in buffer and in interaction with the membrane surface. The interaction of the protein with the membrane surface preserves the existence of a rapid unhindered rotational motion, which is coupled with all three lifetimes. The longest lifetime is coupled to restricted motions in subnanosecond and nanosecond time scales. The overall amplitude of rotation of the indole ring is increased in the membrane-bound conformation of the protein. In reverse micelles, the local dynamics reported by W187 is also considerably increased whereas the overall folding of the protein remains unaffected. The same conformational change of domain III can therefore be provoked by different conditions: calcium binding at high concentration, mild acidic pH [Sopkova, J., Vincent, M., Takahashi, M., Lewit-Bentley, A. , and Gallay, J. (1998) Biochemistry 37, 11962-11970] and the interaction of the protein with the membrane surface. The high flexibility of domain III in the membrane-bound protein suggests that this domain may not be crucial for the interaction of the protein with the membrane, in contrast with previous models. Our data are compatible with atomic force microscopy results which suggest that domain III of annexin V does not interact strongly with the membrane surface [Reviakine, I., Bergma-Schutter, W., and Brisson, A. (1998) J. Struct. Biol. 121, 356-361].     

Binding of vascular anticoagulant alpha (VAC alpha) to planar phospholipid bilayers

Vascular anticoagulant alpha (VAC alpha, annexin V) is a member of the family of calcium and phospholipid binding proteins, the annexins. The binding properties of VAC alpha to phospholipid bilayers were studied by ellipsometry. Adsorption was calcium-dependent and completely reversible upon calcium depletion. Half-maximal adsorptions to phospholipid bilayers consisting of 100, 20, 5, and 1% dioleoyl-phosphatidylserine (DOPS) supplemented with dioleoyl-phosphatidylcholine (DOPC) were reached at Ca2+ concentrations of 0.04, 0.22, 1.5, and 8.6 mM. These surfaces all showed the same maximal adsorption of 0.22 +/- 0.01 micrograms of VAC alpha/cm2 (mean +/- S.D.). The adsorption to bilayers containing more than 10% DOPS was independent of VAC alpha concentrations in the range of 0.5-100 nM. Dissociation constants for VAC alpha binding to these surfaces were estimated to be below 2 x 10(-10) M. No adsorption was observed on pure DOPC bilayers at a Ca2+ concentration of 3 mM. The ability to mediate VAC alpha binding to 20% DOPS/80% DOPC bilayers was highly specific for Ca2+. The use of other divalent cations resulted in decreased binding in the order Cd2+ greater than Zn2+ greater than Mn2+ greater than Co2+ greater than Ba2+ greater than Mg2+. Zinc ions had a synergistic effect on Ca2(+)-dependent VAC alpha binding. The Ca2+ concentration needed for half-maximal binding to cardiolipin, dioleoyl-phosphatidylglycerol, DOPS, phosphatidylinositol, phosphatidic acid, dioleoyl-phosphatidylethanolamine, and sphingomyelin increased in that order. Adsorption was independent of the overall surface charge of the phospholipid membrane.