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Annals of Clinical & Laboratory Science 31:85-90 (2001)
© 2001 Association of Clinical Scientists

The Influence of Lipid Composition and Divalent Cations on Annexin V Binding to Phospholipid Mixtures

Karen G. Maffey, Lynn B. Keil and Vincent A. DeBari
Laboratory of Experimental Medicine, St. Joseph’s Hospital and Medical Center, Paterson, New Jersey

Correspondence to Vincent A. DeBari, PhD, St. Joseph’s Hospital Medical Center, 703 Main Street, Paterson, NJ 07503, USA; tel 973 754 3561; fax 973 754 3555; e-mail debariv{at}sjhmc.org


   Abstract
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 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
Annexin V is a 36-kDa protein which, it has been suggested, is a factor in protecting the vascular endothelium from attack by antibodies to other phospholipid-binding proteins. Competition between annexin V and ß2-glycoprotein I (ß2GPI) for phospholipid surfaces is complicated by empirical observations regarding alterations in binding to anionic phospholipid, primarily phosphatidylserine. In order to elucidate the effect of phospholipid composition and divalent cations (Ca+2 and Mg+2) on annexin V binding to phospholipid, we used biotinylated annexin V and peroxidase-conjugated avidin D to probe the binding of annexin V to phospholipid-coated wells of polystyrene microtiter plates. Binding of annexin V to anionic phospholipid is Ca+2-dependent and, in its absence, annexin V was found to bind most avidly to 100% phosphatidylcholine in a saturable manner, followed by decreasing percentages of phosphatidylcholine. Ca+2 was found to inhibit phosphatidylcholine binding and promote the binding of phospholipid mixtures containing phosphatidylserine. Phosphatidylserine (100%) did not bind annexin V as strongly as mixtures of 50% and 75% phosphatidylserine. The effect with Ca+2 suggests saturation of Ca+2-binding sites on annexin V, reached under our experimental conditions at approximately 1 mM. Under the same conditions, Mg+2 slightly enhanced the binding of all of the phospholipid compositions studied. Ca+2 -dependent binding of annexin V was competitively inhibited by Mg+2; 5 mM Mg+2 reduced binding significantly (p < 0.0001 by ANOVA, p < 0.05 for post hoc test of 5 mM vs 0 mM). These data suggest that the translocation of membrane phospholipid under the dynamics of ion transport in vascular endothelium may alter annexin V binding.

(received 7 July 2000; accepted 25 September 2000)

Keywords: Annexin V, phospholipids, ß2-glycoprotein I, antiphospholipid syndrome


   Introduction
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 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
Annexin V (placental anticoagulant protein) is one of a group of about 20 homologous proteins in the annexin family [1]. Several members of this family, notably annexin II and annexin V, have recently been implicated as potential intermediates in the regulation of thrombogenesis on endothelial surfaces [2]. Annexin V is a potent anticoagulant and, due to its affinity for anionic phospholipid, is capable of displacing various coagulation factors from the surface of vascular endothelium [3]. Annexin V evidently functions as an antithrombotic agent by protecting vascular endothelium from procoagulant proteins [4]. Abnormality in this "annexin shield" may be responsible for the thrombogenic effect of antiphos-pholipid antibodies in patients with an autoimmune disorder, antiphospholipid syndrome (APS) [5,6].

Ca+2 is required for annexin V to bind anionic phospholipid [7]. In addition to its putative role as an anticoagulant, annexin V participates in calcium channel activity [8]. Cation-induced conformational changes in annexin V may contribute to its ability to regulate transmembrane Ca+2 flux and, thus, may alter surface annexin V. This study explored the effects of divalent cations and phospholipid composition on annexin V binding to phospholipid-coated surfaces.


   Materials and Methods
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 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Materials.  Biotinylated annexin V, phosphate buffered saline with potassium (0.01 M phosphate buffered saline, 0.138 M NaCl, 0.0027 M KCl, pH 7.4), tris-buffered saline (50 mM Tris-HCl, 150 mM NaCl, pH 7.6), and spectrophotometric grade methanol were obtained from Sigma (St. Louis, MO). Avanti Polar Lipids Co. (Alabaster, AL) supplied phosphatidyl-choline (1,2-dimyristoyl-sn-glycero-3-phosphocholine mono-sodium salt) and phosphatidylserine (1,2,dimyr-istoyl-sn-glycero-3-phosphoserine mono-sodium salt). Other materials included microtiter wells (untreated polystyrene, Fisher Scientific, Morris Plains, NJ), bovine serum albumin (fraction V, Boehringer Mannheim, Indianapolis, IN), RTU horseradish peroxidase avidin D (Vector, Burlingame, CA), and chromogenic substrate (3% hydrogen peroxide and tetramethylbenzidine in a kit from Kirkegaard and Perry Laboratories, Gaithersburg, MD).

Experimental Methods.  Microtiter plates were coated by adding 100 µl of solutions of varying phosphatidylserine /phosphatidylcholine ratios (PS/PC = 100/0, 75/ 25, 50/50, 25/75, 0/100) to untreated wells and allowing the methanol to evaporate in a vacuum desiccator for 24 hr at 4–6°C, as previously described [9]. The total mass of phospholipid was 2.5 µg/well. The microtiter wells were blocked with a solution of 3% (w/v) bovine serum albumin in tris-buffered saline. Annexin V binding was determined by incubating 100 µl of a solution of biotinylated annexin V (0.005 µg/ 100 µl tris-buffered saline) in the phospholipid-coated wells for 2 hr at 37°C, then washing 3 times with bovine serum albumin/tris-buffered saline. Bovine serum albumin/tris-buffered saline was used as the diluent and wash solution in all steps of the assay. Bound annexin V was detected using horse radish peroxidase-conjugated avidin D. Briefly, the wells were incubated with 100 µl of avidin, as supplied, for 30 min at room temperature (21–25°C), washed 3 times, and then incubated with 100 µl of chromogenic substrate solution for 10 min at room temperature. The reaction was stopped with 100 µl of 3 N sulfuric acid and the absorbance was read at 450 nm using a microtiter plate reader (model Elx800, BioTek Instruments, Winooski, VT).

Statistical Methods.  Descriptive and inferential statistical tests were performed using Prism programs (GraphPad Software, San Diego, CA). Analysis of variance (ANOVA) was used to assess the significance of variations among groups (at {alpha} = 0.05). Groups that were significant by ANOVA (two-tailed, p < 0.05) were subjected to the Newman-Keuls test, set at p < 0.05.


   Results
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 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Initial experiments focused on the determination of baseline binding of annexin V to phospholipid mixtures. Adsorption isotherms (37°C) of annexin V (0–0.20 µg/well) on phospholipid mixtures from 100% phosphatidylserine /0% phosphatidylcholine to 0% phosphatidylserine /100% phosphatidylcholine were generated in phosphate-buffered saline (Fig. 1AGo) or tris-buffered saline (Fig. 1BGo). A surprising aspect of this study was the avidity of annexin V binding to phosphatidylcholine, a neutral phospholipid, in the absence of Ca+2. In phosphate-buffered saline, there was a substantial difference between the strong binding to 100% phosphatidylcholine and modest binding to 100% phosphatidylserine. The likelihood of phosphate contributing to this effect is indicated by the data in Fig. 1BGo, where various mixtures containing phosphatidylserine show little difference in annexin V binding. However, in tris-buffered saline as well as phosphate-buffered saline, substantial increase of annexin V binding is evident in the 100% phosphatidylcholine isotherm.



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  		Fig. 1. Binding isotherms of annexin V. Isotherms were run in (A) phosphate-buffered saline and (B) tris-buffered saline on PL mixtures containing various concentrations of phosphatidylserine (PS) and phosphatidylcholine (PC). Solid square = 100% PS; solid normal triangle = 75% PS/ 25% PC; solid inverted triangle = 50% PS/50% PC; solid diamond = 25% PS/75% PC; solid circle = 100% PC. The error bars show ± 1 SD; data are means of duplicates. The absorbance axis is broken at 3 (emphasized by the dotted line), which is the maximum absorbance value given by the microtiter plate reader.

 
Fig. 2Go shows the effect of varying divalent cation (Ca+2 and Mg+2) concentration on annexin V binding to phospholipid mixtures. The expected strong binding of phospholipid mixtures enriched in phosphatidylserine is seen in Fig. 2AGo (varying Ca+2). Interestingly, Ca+2 is observed to inhibit binding to phosphatidylcholine, so that the difference between anionic phospholipid binding of annexin V and the binding of annexin V to phosphatidylcholine alone is even greater in the presence of Ca+2. On the other hand, Mg+2 promotes minor enhancement of binding of annexin V to the phospholipid mixtures (Fig. 2BGo).



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  		Fig. 2. Effect of varying concentrations of (A) Ca+2 and (B) Mg+2 on annexin V binding. Data are means of duplicate values ± SD. The absorbance axis is broken at 3 (emphasized by the dotted line), the maximum absorbance value given by the microtiter plate reader. See legend to Fig. 1Go for symbols.

 
To relate potential translocations of annexin V to ion fluxes in cells, the binding of annexin V in 1 mM Ca+2 to phospholipid mixtures was studied with Mg+2 in the ranges expected in the intracellular cytosolic compartment. Fig. 3Go demonstrates the inhibition of binding of annexin V to 25% phosphatidylserine /75% phosphatidylcholine over the range from 0 to 5 mM Mg+2. The insert shows 0, 5, 10 mM Mg+2 inhibition of binding to anionic mixtures and promotion of binding to phosphatidylcholine, clearly demonstrating the competitive inhibition of Ca+2 effect by Mg+2. This was explored in greater detail by multiple trials of the Mg+2 experiments, as shown in Table 1Go. With the exception of 100% phosphatidylcholine, strong inhibition of annexin V binding was seen at Mg+2 concentrations as low as 5 mM.



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  		Fig. 3. Mg+2 inhibition of Ca+2 (1 mM)-dependent binding of annexin V on 25% phosphatidylserine/75% phos-phatidylcholine. Data are means ± SD for 3 experiments. Insert shows the effect for a single experiment (replicate values) for all 5 PL mixtures at 0, 5, and 10 mM Mg+2. See legend to Fig. 1Go for symbols.

 

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  		Table 1. The effect of Mg+2 on the inhibition of Ca+2 (1 mM)-dependent binding of annexin V on phosphatidylserine/ phosphatidylcholine mixtures. Data are means ± SD for absorbance at 450 nm (n in parentheses). Groups that were significantly different by ANOVA were subjected to groupwise comparisons by Newman-Keul’s post hoc test.
 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
The phospholipid-binding proteins appear to be an important group of autoantigens in APS. Antibodies from patients with APS bind ß2GPI on phospholipid and suitably-treated polymer surfaces [10–13]. Moreover, the anti-ß2GPI are more closely associated with APS than are antibodies directed against phospholipid [14]. Besides anti-ß2GPI, autoantibodies to other phospholipid-binding proteins have been proposed, eg, prothrombin, phospholipase A2, protein C, and protein S [15–17].

The mechanisms by which antibodies to phospholipid-binding proteins lead to the thrombotic state associated with APS have been intensely studied. Since ß2GPI inhibits the intrinsic pathway of coagulation, it has been suggested that anti-ß2GPI may act as an antagonist of this anticoagulant activity [18]. Others have implicated prothrombinase inhibition [19] and the prothrombin activation system [20–22]. The indication that annexin V may have an "anti-procoagulant" effect [2–6] provides an elaboration upon earlier work, suggesting a role for annexin V as a protective agent against the thrombotic activity of antibodies to phospholipid-binding proteins in APS [23,24].

Annexin V is primarily localized on the intra-cellular aspect of the plasma membrane [25], where it acts as a Ca+2 ion channel [1,7,8]. It requires Ca+2 for binding to anionic phospholipid [26] and is used as a measure of phosphatidylserine on the outer leaflet of the plasma membrane of apoptotic cells [27]. Based on these facts, cell surface phospholipid composition and the extracellular and intracellular concentrations of divalent cations impact the localization of annexin V and, by extension, its displacement by procoagulant autoimmune complexes. Accordingly, this study was undertaken to elucidate the interactions of annexin V with phospholipid mixtures having different compositions (ratios of phosphatidylserine to phosphatidylcholine) and at different concentrations of divalent cations. A system that has been well standardized was used. This is important, since in some earlier studies [28–33], phospholipid vesicles or micelles of different types were used to investigate these interactions. Previous work from our laboratory demonstrated the correspondence of the microtiter well-coated PL binding system and a system using large multilamellar vesicles [34]. Our data generally parallel those obtained with suspension-based systems (vesicles or micelles) with one exception: Meers and Mealy [28] observed a Ca+2-dependent binding to pure phosphatidylcholine. They suggested that subtleties of acyl chain structure-dependent binding might be responsible. We concur, and we suggest that such interactions may also account, in part, for the discrepant effect we observed regarding phosphatidylcholine binding by annexin V in the presence of Ca+2. A clear picture of annexin V binding under these conditions is emerging.

In the absence of Ca+2, annexin V binds phosphatidylcholine avidly and to a greater extent than anionic phospholipid mixtures. However, in the presence of Ca+2, annexin V binding to anionic phospholipid mixtures not only increases dramatically, but binding of the protein to phosphatidylcholine diminishes until it becomes negligible. Mg+2, on the other hand, has little effect on annexinV-phospholipid binding; it does, however inhibit Ca+2-dependent binding of annexin V to anionic phospholipid mixtures while restoring the Ca+2-independent binding of annexin V to phosphatidylcholine. Funakoshi et al [31] found that lanthanide ions promoted stronger annexin V-phospholipid binding than Ca+2. Likely, stable Mg+2-phosphate head group complexes neutralize potential ionic interactions between annexin V and anionic phospholipid.

These data demonstrate that annexin V binding to phospholipid is sensitive to phospholipid composition and the concentrations of divalent cations. Based on these observations and the dynamics of transmembrane ion movements, a mechanism is suggested whereby annexin V may translocate to the apical surface of the vascular endothelium under the appropriate conditions of cation flux.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

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