Skip Navigation


Rheumatology Advance Access originally published online on July 4, 2006
Rheumatology 2007 46(1):81-86; doi:10.1093/rheumatology/kel200
This Article
Right arrow Abstract Freely available
Right arrow FREE Full Text (PDF) Freely available
Right arrow All Versions of this Article:
46/1/81    most recent
kel200v1
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Services
Right arrow Email this article to a friend
Right arrow Similar articles in this journal
Right arrow Similar articles in ISI Web of Science
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Add to My Personal Archive
Right arrow Download to citation manager
Right arrow Search for citing articles in:
ISI Web of Science (2)
Right arrowRequest Permissions
Right arrow Disclaimer
Google Scholar
Right arrow Articles by Gaspersic, N.
Right arrow Articles by Rozman, B.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Gaspersic, N.
Right arrow Articles by Rozman, B.
Related Collections
Right arrow Systemic Lupus Erythematosus and Autoimmunity
Social Bookmarking
Add to CiteULike   Add to Connotea   Add to Del.icio.us  
What's this?

© The Author 2006. Published by Oxford University Press on behalf of the British Society for Rheumatology. All rights reserved. For Permissions, please email: journals.permissions@oxfordjournals.org

Annexin A5 binding to giant phospholipid vesicles is differentially affected by anti-ß2-glycoprotein I and anti-annexin A5 antibodies

N. Gaspersic, A. Ambrozic, B. Bozic, J. Majhenc1, S. Svetina1 and B. Rozman

Department of Rheumatology, University Medical Centre and 1Institute of Biophysics, School of Medicine, University of Ljubljana, Ljubljana, Slovenia

Correspondence to: N. Gaspersic, University Medical Centre, Department of Rheumatology, Vodnikova 62, SI-1000 Ljubljana, Slovenia. E-mail: ngaspersic{at}yahoo.com


   Abstract
Top
 Abstract
Introduction
 Methods
 Results
 Discussion
 Acknowledgement
 References
 
Objectives. Anti-phospholipid antibodies have been recognized to play a role in vascular thrombosis and pregnancy morbidity. They were first thought to be directed to phospholipids, but it is now known that the majority of pathogenic antibodies recognizes epitopes on phospholipid-binding plasma proteins such as ß2-glycoprotein I (ß2GPI) or possibly also annexin A5 (ANXA5). The mechanism of their prothrombotic action is still not completely understood. The aim of the present study was to observe the effect of antibodies against ANXA5 (aANXA5) and antibodies against ß2GPI (aß2GPI) on the binding of ANXA5 to the negatively charged phospholipid membrane.

Methods. Giant phospholipid vesicles (GPVs) were used as a simple model of the membrane surface. GPVs composed of phosphatidylserine and phosphatidylcholine were produced in an aqueous medium. A single GPV was transferred to the solution containing ANXA5 conjugated with Alexa Fluor 488 (FANXA5) and (i) aANXA5 or aß2GPI and (ii) different concentrations of aß2GPI together with ß2GPI. The emission of the fluorescent light from the GPV surface, as the result of FANXA5 binding, was measured.

Results. ß2GPI together with aß2GPI reduced the binding of FANXA5 to GPVs. On the contrary, aANXA5 enhanced the binding of ANXA5 to the GPV surface.

Conclusions. Our results point to the competition between FANXA5 and complexes of ß2GPI–aß2GPI for the same binding sites and therefore support the hypothesis of the disruption of the ANXA5 protective shield on procoagulant phospholipid surface. The influence of increased cell surface ANXA5 concentration in the presence of aANXA5 on coagulation needs to be further studied.

KEY WORDS: Annexin A5, Anti-annexin A5, Anti-ß2-glycoprotein I, Giant phospholipid vesicles


   Introduction
Top
 Abstract
 Introduction
Methods
 Results
 Discussion
 Acknowledgement
 References
 
Anti-phospholipid antibodies (aPL) are a heterogeneous group of antibodies associated with vascular thrombosis and pregnancy morbidity [1]. The majority of pathogenic aPL are not directed to the anionic phospholipids, but recognize epitopes on phospholipid-binding plasma proteins, most notably ß2-glycoprotein I (ß2GPI) and prothrombin [2]. Another possible protein cofactor of aPL is annexin A5 (ANXA5) [3].

ANXA5, a 36 kDa protein, has the ability to bind with high affinity to the negatively charged phospholipids in a calcium-dependent manner. The protein was isolated from different tissues and organs; it is mostly an intracellular protein although extracellular localization was reported as well [4]. It is most abundant in the placenta cells [5], where it is highly expressed also on the microvillus surface of the villous syncytiotrophoblasts [6]. ANXA5 is also found in other cells exposed to blood, such as platelets and endothelial cells [7, 8]. ANXA5 is a multifunctional protein; one of its proposed roles is the prevention of thrombosis. The anti-coagulant action of extracellularly localized ANXA5 was indirectly confirmed by several in vitro [5] and ex vivo studies or animal models [9–14].

Rand et al. [15] proposed the mechanism of the prothrombotic action of aPL: antibodies in the presence of the cofactor ß2GPI compete with ANXA5 for the same binding sites on phospholipids, causing the disruption of the protective ANXA5 shield on the procoagulant surfaces. The dysfunction of the protective shield results in enhanced activation of the coagulation factors and therefore leads to thrombosis. However, the pathogenic mechanism explaining how antibodies against ANXA5 (aANXA5) act is still unknown. It was already shown that aANXA5 (i) induce the placental thrombosis and fetal absorption in mice [16], (ii) shorten the coagulation time when plasma is added to the culture of trophoblast cells pre-incubated with aANXA5 [17], (iii) induce apoptosis [18] and (iv) decrease intercellular fusion of the differentiating trophoblast cells and thus induce miscarriage by a non-thrombogenic mechanism [19]. The exact role of aANXA5 has to be studied further.

To get some insight into the mechanisms of certain biological processes in vivo different models are used for in vitro studies. Planar lipid bilayers or small liposomes have already been used [20–23]. Recently, we reported a novel model of membrane surface—giant phospholipid vesicles (GPVs)—to study the interactions of ß2GPI and antibodies against ß2GPI (aß2GPI) [24]. The GPVs due to their size (diameter of 5–100 µm) mimic more closely the cell membrane and can be directly studied by light microscopy.

The aim of the present study was to reveal the influence of ß2GPI alone and in the presence of aß2GPI and aANXA5 on the binding of ANXA5 to the negatively charged phospholipids. Using fluorescent microscopy, we measured the accumulation of ANXA5 on the GPVs surface in the presence of purified immunoglobulin G (IgG) containing aß2GPI or aANXA5 and ß2GPI. The aANXA5 and aß2GPI were shown to have different influences on the binding of ANXA5 to negatively charged phospholipid membranes.


   Methods
Top
 Abstract
 Introduction
 Methods
Results
 Discussion
 Acknowledgement
 References
 
Patient sera and affinity purification of IgG
Patient sera were pre-tested by an in-house enzyme-linked immunosorbent assay for the presence of different subtypes of aPL as described before: anti-cardiolipin antibodies [25], aß2GPI [26], anti-prothrombin antibodies [27, 28] and aANXA5, directed to purified human placental ANXA5 (Sigma-Aldrich, Wien, Austria) [29]. For the present study we selected: serum from a patient with primary anti-phospholipid syndrome positive for IgG aß2GPI and sera from a patient with systemic sclerosis and an anonymous blood donor, positive only for IgG aANXA5. As a control, serum from another anonymous blood donor, negative for all tested aPL, was applied.

We purified total IgG from four selected sera by affinity purification on a protein G column, according to the manufacturer's protocol (ImmunoPure G, Pierce, Rockford, USA). After purification, the preparations were dialysed against Tris-buffered saline (25 mM Tris, 130 mM NaCl), pH 7.4. The concentrations of purified proteins were determined by the Bio-Rad method [30] using bovine serum albumin (BSA) (Sigma, St Louis, MO, USA) as a standard.

Preparation of giant phospholipid vesicles
Unilamellar GPVs were prepared out of 15 mol% 1-palmitoyl-2-oleoyl-phosphatidylserine (POPS) and 85 mol% 1-palmitoyl-2-oleoyl-phosphatidylcholine (POPC) lipid mixture (both from Avanti Polar-Lipids, Alabaster, AL, USA) according to a modified method of Angelova et al. [31]. The lipids, dissolved in chloroform–methanol, were spread over platinum electrodes and vacuum-dried for 2 h. The electrodes were then placed in an electroformation chamber, filled with 2 ml of 0.2 M sucrose solution intended to be encapsulated in the GPVs. An AC field (1 V/mm, 10 Hz) was applied for 2 h and afterwards consecutively reduced in two 15-min steps (0.75 V/mm, 5 Hz; 0.5 V/mm, 2 Hz) to a final 30-min application of 0.25 V/mm and 2 Hz. After the final step, the sample containing GPVs of different sizes (from a few micrometres to more than 100 µm) was washed out of the electroformation chamber with 2 ml of 0.2 M glucose solution and stored in a plastic beaker. The GPV suspension was stored in the dark at room temperature and was used for the measurements no longer than 2 days after the formation.

Binding of conjugated annexin A5 to the giant phospholipid vesicles
BSA was added to the GPV suspension to prevent interactions of vesicles with the glass surface. Because of Ca2+-dependent ANXA5 binding, Ca2+ was also added in all experiments unless indicated otherwise. The final external solution in the GPV suspension contained 0.09 M sucrose, 0.09 M glucose, 0.1% BSA and 3 mM CaCl2 in Tris-buffered saline, pH 7.4.

Micropipette-based transfer technique and observation of giant phospholipid vesicles
A single GPV of medium size (diameter about 40–60 µm) and without irregularities on the surface visible by light microscopy was aspirated by a glass micropipette from the compartment containing GPVs and transferred to a compartment containing the test solution. The basic composition of the test solution was the same as the solution with GPVs, additionally it contained ANXA5 conjugated with Alexa Fluor 488 (FANXA5) (Molecular Probes, Eugene, OR, USA) at a concentration of 1 µl FANXA5/40 µl solution (the half concentration as recommended by the manufacturer for detecting apoptosis [32]). In particular experiments, the following IgGs were also added: (i) affinity purified IgG from the selected sera containing aANXA5, aß2GPI or IgG of control serum separately where the final antibody concentration in the solution was always 35 mg/l, (ii) IgG aß2GPI at different concentrations together with ß2GPI (105 mg/l). ß2GPI was purified by the perchloric acid precipitation method from pooled human sera [33].

An IMT-2 Olympus microscope (objective: Plan40Pl, Na0.55J) was used for the fluorescent and phase-contrast microscopy. The emission of the fluorescent light at the circumference of each GPV was measured (Fig. 1). Each GPV was observed up to 30 min, but the exposure to the fluorescent light was pulsewise at the same regular intervals for all GPVs. The accumulation time of the exposure was <1 min in the time relevant for further analysis, when the bleaching does not exceed 10% (data not shown). It was assumed that the maximal emission of the fluorescent light (E0) is proportional to the amount of bound FANXA5. The images were acquired by a black and white CCD camera (Chilled CCD 5985, Hamamatsu Photonics k.k., Hamamatsu City, Japan) and digitized using the computer program Image Pals 2.0. Later on the data were translated by 8-bit frame grabber (DT 2851, Data Translation Inc.) and analysed with homemade software.


Figure 1
View larger version (31K):
[in this window]
[in a new window]
[Download PowerPoint slide]
  		FIG. 1. (A) Halo of the GPV in the fluorescent image due to the accumulation of FANXA5 on the surface of the vesicle. Bar indicates 10 µm. (B) Measurement of the brightness on the vesicle cross-section along the line indicated in (A). E0 is the maximal emission of the fluorescent light at the GPV surface at a particular time. The background brightness was subtracted.

 
The binding of FANXA5 was followed for each GPV for at least 30 min after the GPV was transferred into the test solution. By their slopes the best-fitting linear regression lines described the initial accumulation rate (single rate constant) of FANXA5 on each vesicle. The fit was done on the data for the first 14 min or shorter when (i) the maximum emission was reached before that time; (ii) the measuring capacity of the camera (255 levels) was reached before 14 min. A minimum 5 up to 18 GPVs were transferred into particular test solutions. The rate constants of binding FANXA5 were determined as the mean values of single rate constants for a particular test solution [mean rate constants, k (min–1)].

Statistical analysis
Mann–Whitney U-test was used for the comparison of the rate constants for two different independent experiments, and Kruskal–Wallis test when more than two independent experiments were compared. P-values <0.05 were considered statistically significant.

The study was approved by Slovenian ethical committee (Master's Degree thesis of the first author).


   Results
Top
 Abstract
 Introduction
 Methods
 Results
Discussion
 Acknowledgement
 References
 
To test the Ca2+-dependent binding of FANXA5 to the anionic phospholipids, GPVs were transferred into the test solutions containing FANXA5 with individual IgG preparations (IgG aANXA5 from the patient, IgG aANXA5 from the blood donor, IgG aß2GPI or IgG of the control). One at a time, each GPV was transferred into the test solution. For each GPV the rate constant from the best-fitting regression line was determined. Irrespective of the type of antibody, the binding of FANXA5 was significantly larger in the presence of Ca2+ (P < 0.05) (Fig. 2). In the absence of Ca2+, the binding of FANXA5 was similarly low for all tested antibodies, without any increase in binding during the time of the experiment. Further tests were performed in the presence of Ca2+ only.


Figure 2
View larger version (24K):
[in this window]
[in a new window]
[Download PowerPoint slide]
  		FIG. 2. The binding of FANXA5 to the GPVs when transferred to the test solution containing FANXA5 with or without Ca2+ and four different IgG preparations separately: IgG aANXA5 from the patient, IgG aANXA5 from blood donor, IgG aß2GPI and control IgG. Each line represents an average increase of fluorescence emission in time for all transferred GPVs. Bars indicate the maximum and minimum values. The binding of FANXA5 was significantly larger in the presence of Ca2+. (a.u., arbitrary units)

 
Next, we studied the time course of different types of IgG on the binding of ANXA5 to the GPVs by preparing test solutions containing FANXA5 and Ca2+ with different IgG preparations in the final concentration of 35 mg/l each. The intensity of the emission as a result of FANXA5 binding grew faster when IgG aANXA5 were present in the test solutions. The binding of FANXA5 was similar in the presence of either IgG aANXA5 from the patient (k = 11.01 min–1) or IgG aANXA5 from the blood donor (k = 10.76 min–1) (Fig. 2). In the case of the blood donor's IgG aANXA5, the binding was significantly faster when compared with the accumulation of FANXA5 at the GPV in the presence of either aß2GPI (k = 7.58 min–1) or control IgG (k = 7.49 min–1) (P < 0.05). Although observed, the difference between the binding of FANXA5 in the presence of the patients’ aANXA5 and the aß2GPI or control IgG did not reach statistical significance.

Further, we studied the binding of FANXA5 in the presence of aß2GPI and their cofactor ß2GPI. The GPVs were transferred to the test solutions, containing FANXA5 and Ca2+ with ß2GPI in the final concentration of 105 mg/l (close to physiological free ß2GPI plasma concentration [34, 35]) and different concentrations of aß2GPI (0, 30, 60 or 1440 mg/l). At higher concentrations of aß2GPI in the test solution, the accumulation of FANXA5 at the GPV surfaces was progressively reduced. In addition to a slower accumulation rate, the maximum fluorescent emission of FANXA5 on the GPV was also reduced by the increasing concentration of aß2GPI. At the highest aß2GPI concentration the fluorescent emission of FANXA5 was nearly completely abolished (Fig. 3).


Figure 3
View larger version (63K):
[in this window]
[in a new window]
[Download PowerPoint slide]
  		FIG. 3. (A) Images of GPV transferred into the test solution containing FANXA5, Ca2+ and ß2GPI in the final concentration of 105 mg/l and (a) no aß2GPI; (b) 60 mg/l aß2GPI and (c) 1440 mg/l aß2GPI. Time after the transfer is indicated below images. (B) The influence of aß2GPI at different concentrations on the binding of FANXA5 in the presence of ß2GPI (105 mg/l) and Ca2+. Each line represents the average growth of emission at a particular time for three GPVs; bars indicate the maximum and minimum values. (a.u., arbitrary units).

 

   Discussion
Top
 Abstract
 Introduction
 Methods
 Results
 Discussion
Acknowledgement
 References
 
Although there is a procoagulant state in the presence of aPL and there are different mechanisms of their prothrombotic activity proposed, it is still not completely understood how they act in vivo. Rand et al. [15] proposed the disruptive role of non-aANXA5 aPL on the ANXA5 anti-coagulant shield. Using different models such as cultures of endothelial and trophoblast cells, competition tests on the artificial phospholipid surfaces and coagulation tests [22] and recently by atomic force microscopy, representing the first morphological evidence [36], IgG aPL fractions were found to reduce the quantity of ANXA5 on the cell surfaces and also accelerate the coagulation of plasma that was incubated with the cells after their exposure to aPL. Similarly, Tomer [37] demonstrated the displacement of FANXA5 by aPL-positive sera from platelets by flow cytometry. On the contrary, Willems et al. [23] using ellipsometry showed the superior affinity of ANXA5 binding to the negative surfaces and, therefore, a competition between ANXA5 and the complexes of ß2GPI-aß2GPI for the same binding sites as less likely.

In our study, we used GPVs as the model for studying the interactions between aPL, their antigens and phospholipids. GPVs are 5–100 µm in diameter and can be directly studied by a light microscopy. They are also closer to the in vivo models than flat unilamellar surfaces. Furthermore, the micropipette manipulation technique enables the observation of a single giant vesicle after it is added to a solution containing different reagents [38]. The weakness of this model is that GPV may have different ratios of POPS/POPC, which could influence the interactions of GPV with proteins causing variability of the measured single rate constants. The model has been proven to be convenient in studying ß2GPI–aß2GPI interactions with membrane phospholipids [24]. However, even using only aß2GPI from a single patient and aANXA5 from two subjects, we have shown different influences on the binding of ANXA5 to negatively charged phospholipid membranes.

In the present work, we found slower accumulation and lowering of the total fluorescent emission of FANXA5 parallel to the increasing amount of aß2GPI in the presence of ß2GPI. This can be interpreted as the competition between FANXA5 and complexes of ß2GPI–aß2GPI for the same binding sites, suggesting higher affinity of complexes for negatively charged phospholipids in comparison with ANXA5. The displacement of prebound ANXA5 by the complexes is therefore most likely, as previously reported, irrespective of the order of protein additions [36, 37]. Willems et al. [23] did not show such a competition. Their phospholipid layers were prepared in the absence of BSA, and they suggested that BSA incorporated in the phospholipid layer could be the reason for the reduced binding of ANXA5 in the presence of aPL. It should be stressed that our GPVs were also prepared in the absence of BSA; therefore its possible influence was excluded. However, in our experiments Ca2+ was added to the test solutions that enabled the high-affinity binding of ANXA5. In such circumstances, the dose-dependent effect of aß2GPI was observed, which suggests the competition of aPL with ANXA5 and the disruption of ANXA5 shield as a more likely mechanism of aPL action.

Studying the role of aANXA5, a possible subgroup of aPL, we found IgG aANXA5 to enhance the binding of ANXA5 to the GPV surfaces in comparison with either IgG aß2GPI or control IgG. That difference was statistically significant for IgG aANXA5 from the blood donor. But in the case of IgG aANXA5 from the patient, although evident, it was not statistically significant (probably due to the previously mentioned variability between single rate constants measured in the same test conditions).

The adsorption of ANXA5 is in vivo limited by the transport of the molecules to the membrane, where the stabilization occurs [39]. The enhanced accumulation of ANXA5 at the GPV surfaces in the presence of aANXA5, found in our study, might be the result of the bivalent binding of aANXA5 with two molecules of ANXA5, similarly as observed in ß2GPI–aß2GPI binding [40]. Enhanced binding of ANXA5 in the presence of aANXA5 could diminish the binding of other negatively charged phospholipids-dependent proteins and therefore their activity. As already suggested, the proapoptotic activity of aANXA5–ANXA5 complexes [18] through immunogenicity could result in the procoagulant state. In addition, our findings support the clinical relevance of aANXA5 in pregnancy morbidity found in different studies [41]. Inadequate ANXA5 concentration on the surface of trophoblast cells associated with aANXA5 could interfere with the normal function as endometrial invasion.

In conclusion, we found a distinct difference in the influence of aß2GPI compared with aANXA5 on the ANXA5 binding to the negatively charged phospholipids on GPVs, which suggests different mechanisms of their pathogenic action: aß2GPI acting as a procoagulant and aANXA5 interfering with ANXA5 concentration on the involved cells. Our results support the proposed destructive role of aß2GPI on the ANXA5-protective shield on the negatively charged phospholipids. Further, whether or not aANXA5 have a procoagulant effect, the mechanism of their action is different from that of the competitive/destructive action of aß2GPI-ß2GPI complexes.

Formula


   Acknowledgement
Top
 Abstract
 Introduction
 Methods
 Results
 Discussion
 Acknowledgement
References
 
The authors thank Ms Vesna Arrigler for her precise preparation of the phospholipid vesicles and her practical help in laboratory work.

The authors have declared no conflicts of interest.


   References
Top
 Abstract
 Introduction
 Methods
 Results
 Discussion
 Acknowledgement
 References
 

  1. Wilson WA, Gharavi AE, Koike T, et al. (1999) International consensus statement on preliminary classification criteria for definite antiphospholipid syndrome: report of an international workshop. Arthritis Rheum 42:1309–11.[CrossRef][Web of Science][Medline]
  2. Roubey RAS. (1994) Autoantibodies to phospholipid-binding plasma proteins: a new view of lupus anticoagulants and other »antiphospholipid« autoantibodies. Blood 84:2854–67.[Free Full Text]
  3. Galli M. (1996) Non ß2-glycoprotein I cofactors for antiphospholipid antibodies. Lupus 5:388–92.[Web of Science][Medline]
  4. Reutelingsperger CPM and van Heerde WL. (1997) Annexin V, the regulator of phosphatidylserine-catalyzed inflammation and coagulation during apoptosis. CMLS 53:527–32.
  5. Van Heerde WL, de Groot PG, Reutelingsperger CPM. (1995) The complexity of the phospholipid binding protein annexin V. Thromb Haemost 73:172–9.[Web of Science][Medline]
  6. Krikun G, Lockwood CJ, Wu XX, et al. (1994) The expression of the placental anticoagulant protein, annexin V, by villous trophoblasts: immunolocalisation and in vitro regulation. Placenta 15:601–12.[Web of Science][Medline]
  7. Kaetzel MA, Hazarika P, Dedman JR. (1989) Differential tissue expression of three 35-kDa annexin calcium-dependent phospholipid-binding proteins. J Biol Chem 264:14463–70.[Abstract/Free Full Text]
  8. Flaherty MJ, West S, Heimark RL. (1990) Placental anticoagulant protein I: measurement in extracellular fluids and cells of the hemostatic system. J Lab Clin Med 115:174–9.[Web of Science][Medline]
  9. Chollet P, Malecaze F, Hullin F, et al. (1992) Inhibition of intraocular fibrin formation with annexin V. Br J Ophthalmol 76:450–2.[Abstract/Free Full Text]
  10. Van Ryn-McKenna J, Merk H, Müller TH, Buchanan MR, Eisert WG. (1993) The effects of heparin and annexin V on fibrin accretion after injury in the jugular veins of rabbits. Thromb Haemost 69:227–30.[Web of Science][Medline]
  11. Van Heerde WL, Sakariassen KS, Hemker HC, Sixma JJ, Reutelingsperger CP, de Groot PG. (1994) Annexin V inhibits the procoagulant activity of matrices of TNF-stimulated endothelium under blood flow conditions. Arterioscler Thromb 14:824–30.[Abstract/Free Full Text]
  12. Rand JH, Wu XX, Guller S, et al. (1994) Reduction of annexin V (placental anticoagulant protein-I) on placental villi of women with antiphospholipid antibodies and recurrent spontaneous abortion. Am J Obstet Gynecol 171:1566–72.[Web of Science][Medline]
  13. Thiagarajan P and Benedict CR. (1997) Inhibition of arterial thrombosis by recombinant annexin V in a rabbit carotid artery injury model. Circulation 96:2339–47.
  14. Ravassa S, Bennaghmouch A, Kenis H, et al. (2005) Annexin A5 downregulates surface expression of tissue factor: a novel mechanism of regulating the membrane receptor repertoire. J Biol Chem 280:6028–35.[Abstract/Free Full Text]
  15. Rand JH and Wu XX. (1999) Antibody-mediated disruption of the annexin-V antithrombotic shield: a new mechanism for thrombosis in the antiphospholipid syndrome. Thromb Haemost 82:649–55.[Web of Science][Medline]
  16. Wang X, Campos B, Kaetzel MA, Dedman JR. (1999) Annexin V is critical in the maintenance of murine placental integrity. Am J Obstet Gynecol 180:1008–16.[CrossRef][Web of Science][Medline]
  17. Rand JH, Wu XX, Andree HAM, et al. (1997) Pregnancy loss in the antiphospholipid-antibody syndrome – a possible thrombogenic mechanism. N Engl J Med 337:154–60.[Abstract/Free Full Text]
  18. Nakamura N, Ban T, Yamaji K, Yoneda Y, Wada Y. (1998) Localization of the apoptosis-inducing activity of lupus anticoagulant in an annexin V-binding antibody subset. J Clin Invest 101:1951–9.[Web of Science][Medline]
  19. Rote NS, Kumar N, Chang SH, Liln L, Xu B, Kent MN. (2002) Monoclonal antibody against annexin V prevents intertrophoblast fusion. Lupus 11:564.
  20. Sammaritano LR, Gharavi AE, Soberano C, Levy RA, Lockshin MD. (1992) Phospholipid binding of antiphospholipid antibodies and placental anticoagulant protein. J Clin Immunol 12:27–35.[CrossRef][Web of Science][Medline]
  21. Andree HAM, Stuart MCA, Hermens WT, et al. (1992) Clustering of lipid-bound annexin V may explain its anticoagulant effect. J Biol Chem 267:17907–12.[Abstract/Free Full Text]
  22. Rand JH. (2000) Antiphospholipid antibody-mediated disruption of the annexin-V antithrombotic shield: a thrombogenic mechanism for the antiphospholipid syndrome. J Autoimmun 15:107–11.[CrossRef][Web of Science][Medline]
  23. Willems GM, Janssen MP, Comfurius P, Galli M, Zwaal RFA, Bevers EM. (2000) Competition of annexin V and anticardiolipin antibodies for binding to phosphatidylserine containing membranes. Biochemistry 39:1982–89.[CrossRef][Medline]
  24. Ambrozic A, Bozic B, Kveder T, et al. (2005) Budding, vesiculation and permeabilization of phospholipid membranes – evidence for a feasible physiologic role of ß2-glycoprotein I and pathogenic actions of anti-ß2-glycoprotein I antibodies. Biochim Biophys Acta 1740:38–44.[Medline]
  25. Bozic B, Kveder T, Stegnar M, et al. (1997) Influence of degraded phosphatidylserine on binding of antiphospholipid antibodies. Int Arch Allerg Immunol 112:19–26.[CrossRef][Web of Science][Medline]
  26. Avcin T, Ambrozic A, Kuhar M, Kveder T, Rozman B. (2001) Anticardiolipin and anti-ß2 glycoprotein I antibodies in sera of 61 apparently healthy children at regular preventive visits. Rheumatol 40:565–73.[Abstract/Free Full Text]
  27. Atsumi T, Ieko M, Bertolaccini ML, Matsuura E, Koike T. (2000) Association of autoantibodies against the phosphatidylserine-prothrombin complex with manifestations of the antiphospholipid syndrome and with the presence of lupus anticoagulant. Arthritis Rheum 43:1982–93.[CrossRef][Web of Science][Medline]
  28. Ambrozic A, Kveder T, Zigon P, Bozic B. (2002) A modified ELISA for the detection of phosphatidylserine dependent anti-prothrombin antibodies with increased sensitivity and specificity. Ann Rheum Dis 61:Suppl 1, 218.
  29. Matsubayashi H, Arai T, Izumi S, Sugi T, McIntyre JA, Makino T. (2001) Anti-annexin V antibodies in patients with early pregnancy loss or implantations failures. Fertil Steril 76:694–9.[CrossRef][Web of Science][Medline]
  30. Bio-Rad protein assay. (1989) For research use only(Bio-Rad Laboratories, München, West Germany) pp. 9–10.
  31. Angelova MI, Soleau S, Méléard P, Faucon JF, Bothorel P. (1992) Preparation of giant vesicles by external AC electric fields. Kinetics and applications. Prog Colloid Polym Sci 89:127–31.[CrossRef][Web of Science]
  32. Product information sheet. (2001) Annexin V conjugates for apoptosis detection. Molecular Probes.
  33. Cucnik S, Ambrozic A, Bozic B, Skitek M, Kveder T. (2000) Anti-ß2-glycoprotein I ELISA: methodology, determination of cut-off values in 434 healthy Caucasians and evaluation of monoclonal antibodies as possible international standards. Clin Chem Lab Med 38:777–83.[CrossRef][Web of Science][Medline]
  34. McNally TIJ, MacKie DA, Isenberg SJ. (1993) Machin, immunoelectrophoresis and ELISA techniques for assay of plasma-beta(2) glycoprotein-I and the influence of plasma lipids. Thromb Res 72:275–86.[CrossRef][Web of Science][Medline]
  35. Polz E and Kostner GM. (1979) Binding of beta 2-glycoprotein-I to human serum lipoproteins-distribution among density fractions. FEBS Lett 102:183–6.[CrossRef][Web of Science][Medline]
  36. Rand JH, Wu XX, Quinn AS, et al. (2003) Human monoclonal antiphospholipid antibodies disrupt the annexin A5 anticoagulant crystal shield on phospholipid bilayers. Evidence from atomic force microscopy and functional assay. Am J Path 163:1193–200.[Abstract/Free Full Text]
  37. Tomer A. (2002) Antiphospholipid antibody syndrome: rapid, sensitive, and specific flow cytometric assay for determination of anti-platelet phospholipids autoantibodies. J Lab Clin Med 139:147–54.[CrossRef][Web of Science][Medline]
  38. Mally M, Majhenc J, Svetina S, Zeks B. (2002) Mechanisms of equinatoxin II-induced transport through the membrane of a giant phospholipids vesicle. Biophys J 83:944–53.
  39. Andree HA, Reutelingsperger CP, Hauptmann R, Hemker HC, Hermens WT, Willems GM. (1990) Binding of vascular anticoagulant {alpha} (VAC {alpha}) to planar phospholipid bilayers. J Biol Chem 265:4923–8.[Abstract/Free Full Text]
  40. Reddel SW, Wang YX, Krilis SA. (2003) Anti-ß2-glycoprotein I autoantibodies require an antigen density threshold, consistent with bivalent binding. Lupus 12:37–45.[Abstract/Free Full Text]
  41. Bozic B, Irman S, Gaspersic N, Kveder T, Rozman B. (2005) Antibodies against annexin A5: detection pitfalls and clinical associations. Autoimmunity 38:425–30.[Medline]
Submitted 25 January 2006; revised version accepted 9 May 2006.
Add to CiteULike CiteULike   Add to Connotea Connotea   Add to Del.icio.us Del.icio.us    What's this?


This article has been cited by other articles:


Home page
 The Journal of Rheumatology SupplementHome page
E. SOSCIA, R. SCARPA, M. A. CIMMINO, M. ATTENO, R. PELUSO, C. SIRIGNANO, L. COSTA, S. IERVOLINO, F. CASO, A. DEL PUENTE, et al.
Magnetic Resonance Imaging of Nail Unit in Psoriatic Arthritis
J Rheumatol Suppl, August 1, 2009; 83(0): 42 - 45.
[Abstract] [Full Text] [PDF]

This Article
Right arrow Abstract Freely available
Right arrow FREE Full Text (PDF) Freely available
Right arrow All Versions of this Article:
46/1/81    most recent
kel200v1
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Services
Right arrow Email this article to a friend
Right arrow Similar articles in this journal
Right arrow Similar articles in ISI Web of Science
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Add to My Personal Archive
Right arrow Download to citation manager
Right arrow Search for citing articles in:
ISI Web of Science (2)
Right arrowRequest Permissions
Right arrow Disclaimer
Google Scholar
Right arrow Articles by Gaspersic, N.
Right arrow Articles by Rozman, B.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Gaspersic, N.
Right arrow Articles by Rozman, B.
Related Collections
Right arrow Systemic Lupus Erythematosus and Autoimmunity
Social Bookmarking
Add to CiteULike   Add to Connotea   Add to Del.icio.us  
What's this?