Correspondence

Antiphospholipid Antibodies, Annexin V, and Pregnancy Loss

N Engl J Med 1997; 337:1630-1631November 27, 1997

Article

To the Editor:

Although the antiphospholipid-antibody syndrome is defined clinically as thrombosis, thrombocytopenia, or pregnancy loss, with the presence of a positive test for antiphospholipid antibodies, the incidence of pregnancy disorders greatly exceeds the incidence of the other symptoms. Rand et al. (July 17 issue)1 report that in their study, IgG fractions containing antiphospholipid antibodies prevented the formation of annexin V complexes on the surfaces of choriocarcinoma (BeWo) cells and primary trophoblast cells and increased the activation of coagulation by these cells. This mechanism may lead to thrombosis at the maternal–fetal interface, with damage to the placenta.

A common problem with many such studies is the authors' assumption that antiphospholipid antibodies are the active component in crude IgG preparations. These preparations frequently contain other autoantibodies, and it is therefore very difficult to attribute biologic activity to a particular IgG specificity.

In addition, the procedure used by Rand et al. may not specifically measure annexin V on the cell surface, but instead may measure annexin V released from dying or antibody-damaged cells. BeWo cells are undifferentiated and have cytoplasmic, but not surface, annexin V.2 The cells express surface annexin V in relation to induced differentiation and the resultant externalization of phosphatidylserine. As with most cells, phosphatidylserine is sequestered on the inner leaflet of the trophoblast plasma membrane and is externalized only during differentiation.3 Trophoblasts externalize phosphatidylserine in relation to differentiation-associated intercellular fusion, which is a necessary event for normal placental growth. In order to prevent the assemblage of the prothrombinase complex on the phosphatidylserine-rich surface of the fusing trophoblast, annexin V is also secreted. Therefore, until the trophoblasts differentiate, the externalization of annexin V is probably unnecessary.

Despite these points, I agree with, and would expand on, the conclusions of the study. In a study that my colleagues and I conducted,2 a monoclonal antiphospholipid antibody removed annexin V from the surface of BeWo cells, resulting in increased thrombin binding. Rand et al. suggest that antiphospholipid antibodies prevent annexin V from clustering. In our experiments, annexin V was allowed to form natural clusters and was then displaced with antiphospholipid antibodies.

I agree that trophoblasts appear to be a principal target of antiphospholipid antibodies, which remove annexin V and induce a procoagulant trophoblast surface. Our work has demonstrated additional mechanisms by which trophoblasts are affected, including decreased syncytium formation, hormone production, and decidual invasion.4,5 These effects may lead to well-recognized pregnancy disorders associated with antiphospholipid antibodies, such as pregnancy loss, preeclampsia, and intrauterine growth restriction.

Neal S. Rote, Ph.D.
Wright State University, Dayton, OH 45435

5 References

1. 1

   Rand JH, Wu X-X, Andree HAM, et al. Pregnancy loss in the antiphospholipid-antibody syndrome -- a possible thrombogenic mechanism. N Engl J Med 1997;337:154-160
   Full Text | Web of Science | Medline

2. 2

   Vogt E, Ng AK, Rote NS. Anti-phosphatidylserine antibody removes annexin V and facilitates the binding of prothrombin at the surface of a choriocarcinoma model of trophoblast differentiation. Am J Obstet Gynecol (in press).
   

3. 3

   Obringer AR, Dean KW, Channel S, Rote NS. Membrane phospholipid translocase activity in JEG-3, a choriocarcinoma model of cytotrophoblast differentiation. Placenta (in press).
   

4. 4

   Adler RR, Ng AK, Rote NS. Monoclonal antiphosphatidylserine antibody inhibits intercellular fusion of the choriocarcinoma line, JAR. Biol Reprod 1995;53:905-910
   CrossRef | Web of Science | Medline

5. 5

   Katsuragawa H, Kanzaki H, Inoue T, Hirano T, Mori T, Rote NS. Monoclonal antibody against phosphatidylserine inhibits in vitro human trophoblastic hormone production and invasion. Biol Reprod 1997;56:50-58
   CrossRef | Web of Science | Medline

To the Editor:

Rand et al. report data suggesting that IgG antibodies from patients with the antiphospholipid-antibody syndrome can cause a reduction in cell-surface annexin V on placental villi. This finding was taken to suggest that antiphospholipid antibodies can promote a greater procoagulant potential of these cells by means of a decrease in the anticoagulant activity of annexin V.

I wish to propose an alternative mechanism. The reduction in surface annexin V as detected in vitro by the enzyme-linked immunosorbent assay may only be apparent. The annexin may be bound and thus blocked by specific antibodies; such anti–annexin V autoantibodies have been described in association with thrombotic events in pregnancy failure and systemic lupus erythematosus.1,2 Since annexin V may be involved in cellular processes such as exocytosis and membrane fusion (e.g., the syncytiotrophoblast),3 antibodies to annexin V may interfere with its function and result in the expression of anionic phospholipids from the inner layer to the outer layer of the plasma membrane.

In addition to possessing antiphospholipid and lupus-anticoagulant properties, anti–annexin V antibodies have been shown to induce apoptosis in human umbilical-vein endothelial cells.4 This may be another activating factor for the selective externalization, in apoptotic membranes, of the procoagulant phospholipid, phosphatidylserine.

Hwee-Ming Cheng, Ph.D.
Universiti Malaya, 50603 Kuala Lumpur, Malaysia

4 References

1. 1

   Matsuda J, Gotoh M, Saitoh N, Gohchi K, Tsukamoto M, Yamamoto T. Anti-annexin antibody in the sera of patients with habitual fetal loss or preeclampsia. Thromb Res 1994;75:105-106
   CrossRef | Web of Science | Medline

2. 2

   Kaburaki J, Kuwana M, Yamamoto M, Kawai S, Ikeda Y. Clinical significance of anti-annexin V antibodies in patients with systemic lupus erythematosus. Am J Hematol 1997;54:209-213
   CrossRef | Web of Science | Medline

3. 3

   Burgoyne RD, Clague MJ. Annexins in the endocytic pathway. Trends Biochem Sci 1994;19:231-232
   CrossRef | Web of Science | Medline

4. 4

   Nakamura N, Kuragaki C, Shidara Y, Yamaji K, Wada Y. Antibody to annexin V has anti-phospholipid and lupus anticoagulant properties. Am J Hematol 1995;49:347-348
   CrossRef | Web of Science | Medline

Author/Editor Response

The authors reply:

To the Editor: We are pleased to hear that Dr. Rote and colleagues have reached conclusions similar to ours using monoclonal antiphospholipid antibody and BeWo trophoblasts. Regarding his question about the antibody preparations, we used IgG fractions from clinically affected patients for the culture studies, because the specificities of the antiphospholipid-antibody subtypes, with respect to their relevance to the pathogenesis of the antiphospholipid-antibody syndrome, have not yet been defined. Indeed, as we noted in the Discussion section of our article,1 there is evidence that the serum glycoprotein β₂-glycoprotein I and other phospholipid-binding proteins may by themselves or in a complex with anionic phospholipids constitute antigenic sites for the antibodies. At this point, one would therefore not know whether findings with any particular antibody specificity (such as the monoclonal antiphosphatidylserine antibody referred to by Dr. Rote and his colleagues in the title of their paper) are relevant to the pathophysiology of the antiphospholipid-antibody syndrome. For this reason, we chose to work with IgG fractions from affected patients.

The design of our study was based on our previous study, which showed the reduction of immunohistochemically detectable annexin V on placental villi from patients with antiphospholipid antibody and duplicated those findings in cultured placental villi with the use of IgG fractions.2 The results of our work with IgG fractions and placental villi, BeWo trophoblasts, cultured primary trophoblasts, and human umbilical-vein endothelial cells establish a foundation for further studies using defined antibody subgroups in model systems.

Regarding the question of whether the measured annexin V may be released from dying or antibody-injured cells, Trypan-blue exclusion studies showed that the cells were intact, as we reported.1 We look forward to reading Dr. Rote's evidence that undifferentiated BeWo cells do not express any annexin V, since we were able to quantify this protein on the cells. Also, because BeWo is a choriocarcinoma-derived trophoblast cell line, we performed experiments with cultured primary placental trophoblasts and found the same reduction of annexin V by antiphospholipid-antibody IgG.

In response to Dr. Cheng's suggestion that we may have been observing the effects of anti–annexin V antibodies, none of the IgG fractions had any detectable anti–annexin V activity in immunoblot screening tests. Also, as we stated in our article,1 the IgG did not interfere with the ability of the polyclonal rabbit IgG used for the immunoassay to detect known quantities of purified annexin V. We therefore think that human antiphospholipid-antibody IgG causes a reduction in the actual quantity of annexin V.

Jacob H. Rand, M.D.
Peter Harpel, M.D.
Mount Sinai School of Medicine, New York, NY 10029

Charles Lockwood, M.D.
New York University School of Medicine, New York, NY 10016

2 References

1. 1

   Rand JH, Wu X-X, Andree HAM, et al. Pregnancy loss in the antiphospholipid-antibody syndrome -- a possible thrombogenic mechanism. N Engl J Med 1997;337:154-160
   Full Text | Web of Science | Medline

2. 2

   Rand JH, Wu X-X, Guller S, et al. Reduction of annexin-V (placental anticoagulant protein-I) on placental villi of women with antiphospholipid antibodies and recurrent spontaneous abortion. Am J Obstet Gynecol 1994;171:1566-1572
   Web of Science | Medline

Citing Articles (5)

Citing Articles

1. 1

   Marta P. Baleva, Maria H. Hristova, Krasimir V. Nikolov. (2010) Diagnostic significance of anti-annexin-A5 antibody determination. Central European Journal of Medicine 5:1, 6-11
   CrossRef

2. 2

   Hidehiko Matsubayashi. (2009) Autoantibodies and coagulation in reproductive medicine. Reproductive Medicine and Biology 8:4, 131-140
   CrossRef

3. 3

   Olivier Vittecoq, Fabienne Jouen-Beades, François Tron, Xavier Le Loët. (2001) Antibodies and vascular involvement in inflammatory joint disease: clinical relevance. Joint Bone Spine 68:6, 466-476
   CrossRef

4. 4

   Hidehiko Matsubayashi, Tadashi Arai, Shun-ichiro Izumi, Toshitaka Sugi, John A McIntyre, Tsunehisa Makino. (2001) Anti-annexin V antibodies in patients with early pregnancy loss or implantation failures. Fertility and Sterility 76:4, 694-699
   CrossRef

5. 5

   Azzudin E. Gharavi, Silvia S. Pierangeli, Roger A. Levy, E. Nigel Harris. (2001) Mechanisms of Pregnancy Loss in Antiphospholipid Syndrome. Clinical Obstetrics and Gynecology 44:1, 11-19
   CrossRef