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Previous Volume 337:154-160 July 17, 1997 Number 3 Next

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ABSTRACT

Background The mechanisms of vascular thrombosis and pregnancy loss in the antiphospholipid-antibody syndrome are unknown. Levels of annexin V, a phospholipid-binding protein with potent anticoagulant activity, are markedly reduced on placental villi from women with this syndrome. Hypercoagulability in such women may therefore be due to the reduction of surface-bound annexin V by antiphospholipid antibodies. To test this idea, we studied how antiphospholipid antibodies affect levels of annexin V on cultured trophoblasts and human umbilical-vein endothelial cells and how they affect the procoagulant activity of these cells.

Methods We isolated IgG fractions from three patients with the antiphospholipid-antibody syndrome and from normal controls. These antibodies were incubated with cultured BeWo cells (a placental-trophoblast cell line), primary cultured trophoblasts, and human umbilical-vein endothelial cells. Annexin V on the cell surfaces was measured by an enzyme-linked immunosorbent assay. The coagulation times of plasma overlaid on the cells were also determined.

Results Trophoblasts and endothelial cells exposed to antiphospholipid-antibody IgG as compared with control IgG had reduced levels of annexin V (trophoblasts, 0.37±0.02 vs. 0.85±0.12 ng per well, P = 0.02; endothelial cells, 1.6±0.04 vs. 2.1±0.05 ng per well, P = 0.001). Also, trophoblasts and endothelial cells exposed to antiphospholipid-antibody IgG had faster mean (±SE) plasma coagulation times than cells exposed to control IgG (trophoblasts, 8.7±2.0 vs. 21.3±2.9 minutes, P = 0.02; endothelial cells, 9.8±0.8 vs. 14.2±1.2 minutes, P = 0.04).

Conclusions Antiphospholipid antibodies reduce the levels of annexin V and accelerate the coagulation of plasma on cultured trophoblasts and endothelial cells. The reduction of annexin V levels on vascular cells may be an important mechanism of thrombosis and pregnancy loss in the antiphospholipid-antibody syndrome.

Annexin V (previously known as placental anticoagulant protein I and vascular anticoagulant ) is found in placenta and vascular endothelium, among other tissues.⁷ This protein, whose physiologic function has not yet been established, has potent anticoagulant properties that are based on its high affinity for anionic phospholipids and its capacity to displace coagulation factors from phospholipid surfaces.⁸ We previously reported that annexin V is found on the apical surface of placental syncytiotrophoblasts and subsequently found that levels of this protein are markedly reduced on placental villi in patients with the antiphospholipid-antibody syndrome.^9,10 We hypothesized that annexin V has an antithrombotic role in vivo and that thrombosis in patients with the antiphospholipid-antibody syndrome may be due to reduced levels of the protein at the sites where circulating blood contacts cells lining the vasculature.

We therefore investigated the effects of antiphospholipid antibodies on levels of annexin V on placental trophoblast cells. We then studied how these antibodies affect the coagulation of plasma on cultured trophoblasts and whether they also affect annexin V and plasma coagulation on cultured human umbilical-vein endothelial cells. Finally, we studied how purified annexin V and antiannexin IgG affect the coagulation of plasma on umbilical-vein endothelial cells.

Methods

Isolation of IgG

IgG antibodies were isolated from the citrated plasma of three patients with severe antiphospholipid-antibody syndrome and three normal control subjects with a protein G column, as described by Sammaritano et al.¹¹ A preparation of antiphospholipid antibody from each of the three patients was studied and compared with a preparation from one of the controls. The three patients all had severe primary antiphospholipid-antibody syndrome — that is, there was no evidence of systemic lupus erythematosus or any other autoimmune disorder — and high titers of anticardiolipin IgG.

The first patient was a 33-year-old woman (previously described¹²) who had evidence of a previous cerebral infarct on a computed tomographic scan, previous deep-vein thrombosis and pulmonary embolism, and four consecutive losses of pregnancy. She presented with a fifth pregnancy loss at 18 weeks' gestation, placental infarction, and infarcts on the skin of her hands and face, with fibrin thrombi in the small vessels of the dermis. The second patient was a 47-year-old man with catastrophic antiphospholipid syndrome, manifested by deep-vein thrombosis, pulmonary emboli, and stroke. The third patient was a 63-year-old woman with stroke, pulmonary embolism, and infarcts on the skin of her hands. The antiphospholipid IgG from the first patient was used to determine whether primary placental trophoblasts are affected in the same way as the trophoblast cell line.

Effects of IgG on Trophoblast Annexin V

A human trophoblast cell line (BeWo) was obtained from the American Type Culture Collection (Rockville, Md.) and maintained as described elsewhere.^13,14 The BeWo cells were resuspended in a basal medium composed of a 1:1 mixture of phenol red–free Ham's F12 and Dulbecco's modified Eagle's medium plus 10 percent fetal-calf serum. They were then plated at densities of 60,000 cells per well in 96-well culture plates and grown to confluence (approximately 130,000 cells per well). Either antiphospholipid-antibody IgG or control IgG (2 mg per milliliter) in basal medium plus 10 percent fetal-calf serum was added, and the cells were incubated for two hours at 4°C to inhibit the recycling of membranes and vesicles. The cells were then washed once in HEPES buffer (pH 7.4) containing 5 mM calcium chloride, followed by a wash in HEPES buffer containing 1 mM ethylene glycol-bis(-aminoethyl ether)-N,N,N',N'-tetraacetic acid (EGTA) in place of calcium, to dissociate cell-surface annexin V. Levels of annexin V were determined by an enzyme-linked immunosorbent assay^10,15 that used a previously characterized, affinity-purified, monospecific, polyclonal rabbit anti–annexin V IgG antibody.⁹ In assays in which known quantities of purified annexin V were added, the presence of antiphospholipid or control IgG did not in itself reduce the levels of annexin V. All the studies were performed with quadruplicate culture wells. Trypan-blue exclusion studies showed that the treated cells were at least 95 percent viable.

Experiments with Cultured Primary Trophoblasts

To determine whether the effects observed with the trophoblast cell line also occurred with primary cultured trophoblasts (cytotrophoblasts), we obtained the latter from women undergoing elective cesarean sections at term. The cells were isolated by a modification of the procedure of Douglas and King¹⁶ in which anti-CD45 antibodies conjugated to magnetic microspheres (Advanced Magnetics, Cambridge, Mass.) were substituted for the anti-HLA antibodies used in the original procedure.¹⁷ The cells were washed, resuspended in basal medium supplemented with 2 percent charcoal-stripped calf serum and culture supplement (ITS+, Collaborative Biomedical Products, Bedford, Mass.), and seeded in 96-well culture plates at a density of 100,000 cells per well. The cultures were maintained at 37°C in a humidified atmosphere containing 5 percent carbon dioxide and 95 percent air, and the medium was changed at 48 hours.

The cells were allowed to form syncytia for 72 hours before the IgG was added, which was done in the manner described above for the trophoblast cell line. The final wash, with HEPES buffer containing 1 mM EGTA, was assayed for annexin V as described above. Since cultured primary trophoblasts do not proliferate, the results of these experiments were normalized for the DNA concentrations, which were determined by fluorimetry on the cells after their detachment, as described elsewhere.¹⁸ All these experiments were performed with quadruplicate culture wells.

Experiments with Cultured Umbilical-Vein Endothelial Cells

Umbilical-vein endothelial cells were harvested and cultured as previously described.¹⁹ They were plated at a density of 20,000 cells per well in 96-well culture plates, allowed to grow to confluence (approximately 140,000 cells per well), and treated in the same way as the BeWo trophoblasts. In addition to the short-term cultures at 4°C, the umbilical-vein endothelial cells were also cultured with the IgG fractions at 37°C for 20 hours, after which the cells were washed once in HEPES buffer containing 5 mM calcium chloride and then washed in HEPES buffer containing 1 mM EGTA in place of calcium, to dissociate cell-surface annexin V. The levels of annexin V were determined by an enzyme-linked immunosorbent assay as described above. In addition, for coagulation studies with plasma, parallel cultures of umbilical-vein endothelial cells were incubated with the IgG fractions at 37°C for 20 hours, washed once in HEPES buffer containing 5 mM calcium chloride, and then tested as described in the following section. Quadruplicate culture wells were used in all the studies.

Studies of Coagulation

After the cells were grown to confluence in the 96-well tissue-culture microtiter plates, studies of coagulation were performed as follows: the cells were first washed three times in HEPES buffer containing 5 mM calcium chloride and then incubated with either antiphospholipid or control IgG (5 mg per milliliter) in basal medium plus 10 percent fetal-calf serum for 90 minutes at 4°C. After a washing in HEPES buffer, the cells were overlaid with normal pooled plasma (100 µl per well) recalcified with 11 µl of 70 mM calcium chloride in the case of the BeWo cells. It was necessary to add the same volume of 200 mM calcium chloride in order to observe coagulation of plasma in the case of the umbilical-vein endothelial cells. Quadruplicate culture wells were used in all the studies.

The culture plates were then placed in a kinetic microtiter-plate reader, and the formation of fibrin was observed as an increase in the optical density to 0.100 at a wavelength of 405 nm. We confirmed that this assay indeed monitors the formation of fibrin by determining that adding porcine intestinal-mucosa heparin (0.5 U per milliliter) (Steris Laboratories, Phoenix, Ariz.) or recombinant hirudin (0.5 µg per milliliter) (kindly provided by Ciba–Geigy, Summit, N.J.) to the plasma completely inhibited any change in optical density. Furthermore, in the absence of heparin or hirudin the formation of fibrin gels could be observed with the unaided eye.

We also sought to determine whether reducing cell-surface annexin V without antiphospholipid antibodies might affect the coagulation of plasma. We therefore performed experiments in which umbilical-vein endothelial cells that were not incubated with human IgG fractions were washed in HEPES buffer containing 5 mM calcium chloride and EGTA, to preserve or dissociate surface annexin V. The umbilical-vein endothelial cells were then incubated with rabbit polyclonal anti–annexin V IgG antibodies (100 µg per milliliter) for 90 minutes at 4°C, after which they were overlaid with recalcified plasma and the time to coagulation measured. The controls included equivalent concentrations of polyclonal rabbit anti–annexin II IgG (kindly provided by Dr. Katherine Hajjar, Cornell University Medical College) and a polyclonal rabbit antimouse idiotype IgG (kindly provided by Dr. Thomas Moran, Mount Sinai School of Medicine).

In addition, umbilical-vein endothelial cells washed three times in HEPES buffer that contained 5 mM calcium chloride, to preserve cell-surface annexin V, were compared with cells that were washed three times in HEPES buffer containing 1 mM EGTA, to dissociate cell-surface annexin V. Each of these treatments was followed by a washing in buffer containing calcium chloride, after which the cells were overlaid with recalcified normal pooled plasma containing various concentrations of annexin V; the epithelial cells were then monitored for coagulation as described above. In addition, the coagulation times of the epithelial cells incubated with plasma containing recombinant annexin II at a concentration of 4 µg per milliliter (kindly provided by Dr. Hajjar) were compared with those of cells incubated with plasma containing annexin V in the same concentration and cells incubated with HEPES-buffer control.

Statistical Analysis

The statistical analyses were performed with the use of Student's two-tailed t-test (InStat program, Graphpad, San Diego, Calif.).

Results

Effects of Antiphospholipid Antibodies on Annexin V and Plasma Coagulation in Trophoblasts

We studied the effects of antiphospholipid IgG on levels of annexin V associated with the trophoblast cell surface, using the BeWo trophoblast cell line. With the three different antiphospholipid IgG antibodies we used, the amount of annexin V associated with the trophoblast cell surface was significantly lower than that associated with control IgG, and the reductions were similar (Figure 1A and Figure 1B). We then determined whether these reductions also occurred with primary cultured placental trophoblasts (cytotrophoblasts). When these trophoblasts were incubated with antiphospholipid IgG, there was a significantly lower amount of annexin V, approximately 20 percent of the amount found in trophoblasts incubated with control IgG (Figure 1B).

View larger version (7K): [in this window] [in a new window]   Figure 1. Effects of Antiphospholipid-Antibody IgG on Annexin V and Plasma Coagulation on Trophoblasts. Cultured trophoblasts (from the BeWo cell line) grown to confluence were exposed to IgG preparations (2 mg per milliliter) from three patients and their controls for two hours at 4°C to inhibit the recycling of membranes and vesicles. Annexin V was then dissociated with buffer containing EGTA and measured by immunoassay. (All tests were performed in quadruplicate.) Panel A shows that the mean (±SE) level of annexin V, indicated by the horizontal line and error bar, was significantly lower after exposure to antiphospholipid IgG than after exposure to control IgG (0.37±0.02 vs. 0.85±0.12 ng per well, P = 0.02). Panel B shows how antiphospholipid IgG affects annexin V levels on primary cultured trophoblasts and BeWo trophoblasts. (The data on the former were normalized for the DNA concentration, and both sets of data were normalized as percentages of the control values so that the two cell types could be shown together.) Annexin V levels on the surface of both types of trophoblasts were significantly reduced (P<0.001 for both). Panel C shows the coagulation time of plasma added to BeWo trophoblasts exposed to preparations of IgG from the three patients for two hours at 4°C, as compared with controls. In these experiments, annexin V was not dissociated from the cells. The mean (±SE) coagulation time was significantly shorter in the antiphospholipid-IgG–exposed trophoblasts than in the controls (8.7±2.0 vs. 21.3±2.9 minutes, P = 0.02).

 
We then tested whether the reduction in the amount of this anticoagulant protein was associated with a shortening in the coagulation time of plasma exposed to these cells. There was indeed a significant shortening in the clotting times of plasma on the trophoblasts exposed to antiphospholipid IgG, as compared with those exposed to control IgG (Figure 1C).

Effects of Antiphospholipid Antibodies on Annexin V and Plasma Coagulation in Umbilical-Vein Endothelial Cells

The antiphospholipid-antibody syndrome may lead to thrombosis in veins and arteries. In view of our findings with trophoblasts, we also studied the effects of antiphospholipid antibodies on levels of annexin V and plasma coagulation on the surfaces of umbilical-vein endothelial cells. As we found with trophoblasts, levels of annexin V were reduced on the surface of epithelial cells exposed to antiphospholipid antibody (Figure 2A). There was also a significant acceleration of coagulation on the surface of epithelial cells exposed to antiphospholipid IgG as compared with control IgG (Figure 2B). The results were similar with epithelial cells cultured at 37°C for 20 hours with the antibodies (Figure 2C and Figure 2D).

View larger version (8K): [in this window] [in a new window]   Figure 2. Effects of Antiphospholipid-Antibody IgG on Annexin V and Plasma Coagulation on Umbilical-Vein Endothelial Cells. Umbilical-vein endothelial cells were exposed to IgG preparations from three patients and their controls for two hours at 4°C to inhibit the recycling of membranes and vesicles. Panel A shows that the mean (±SE) level of annexin V, indicated by the horizontal line and error bar, was significantly lower after exposure to antiphospholipid IgG than after exposure to control IgG (1.6±0.04 vs. 2.1±0.05 ng per well, P = 0.001). Panel B shows the coagulation time of plasma added to these cultures of endothelial cells. Overall, the mean (±SE) coagulation time was significantly shorter in the three groups of antiphospholipid-antibody–exposed endothelial cells than in the controls (9.8±0.8 vs. 14.2±1.2 minutes, P = 0.04). Panel C shows the annexin V levels after endothelial cells were cultured with preparations of antiphospholipid IgG from the three patients and control IgG for 20 hours at 37°C, a temperature at which recycling of membranes and vesicles occurs. The mean (±SE) level of annexin V in the cells exposed to antiphospholipid IgG was significantly lower than the level in the control cells (1.3±0.2 vs. 2.1±0.1 ng per well, P = 0.02). Panel D shows the coagulation time of plasma added to endothelial cells cultured with IgG preparations from the three patients and the controls for 20 hours at 37°C. Again, the coagulation time was significantly shorter for the endothelial cells exposed to antiphospholipid antibody, with a lower mean value in those cells than in the control cells (17.2±0.2 vs. 23.5±1.2 minutes, P = 0.006).

 
When umbilical-vein endothelial cells not treated with antiphospholipid IgG were incubated with rabbit polyclonal anti–annexin V IgG, the coagulation time of plasma applied to the cells was significantly shorter than after incubation with antimouse IgG, and treating the epithelial cells with anti–annexin II IgG had no effect on the coagulation time (Figure 3A). This shorter coagulation time did not occur with cells from which the annexin V was first dissociated with EGTA (Figure 3A). Also, removing annexin V from the endothelial surface by preincubation with EGTA significantly reduced the coagulation time (Figure 3A and Figure 3B). Furthermore, adding exogenous annexin V resulted in dose-dependent prolongations of coagulation in both cells whose annexin V had been removed by EGTA treatment and controls whose annexin V had been preserved by treatment with calcium-containing buffer (Figure 3B). In contrast, there was no difference in the mean (±SE) coagulation time between the epithelial cells exposed to plasma containing 4 µg of annexin II per milliliter and the controls exposed to buffer alone (19.5±0.6 vs. 19.8±0.2 minutes).

View larger version (6K): [in this window] [in a new window]   Figure 3. Effects of Polyclonal Antiannexin Antibodies and Purified Annexin V on the Coagulation of Plasma Exposed to Umbilical-Vein Endothelial Cells. Panel A shows that the mean (±SE) coagulation time of plasma was significantly less after cells were incubated with rabbit polyclonal anti–annexin V as compared with equal concentrations of control rabbit polyclonal IgG (20.1±0.6 vs. 23.3±0.5 minutes, P = 0.006). Treatment with rabbit polyclonal anti–annexin II had no effect (coagulation time, 23.8±0.8). When cells were pretreated with EGTA, which dissociates cell-surface annexin V, there was no difference between the results obtained with the various antibodies. Panel B shows how dissociating and restoring annexin V affects the coagulation of plasma exposed to umbilical-vein endothelial cells. The plasma coagulation time was significantly shorter after annexin V was dissociated from the cell surface by treatment with EGTA alone as compared with calcium (mean of eight experiments, 14.6±0.9 vs. 22.5±0.5 minutes; P<0.001). When exogenous annexin V was added to the culture, dose-dependent prolongations of plasma coagulation were observed. The difference in the coagulation time between cells treated with EGTA and those treated with calcium was also significant when 1 µg of annexin V was added per milliliter (P<0.001).

 
Discussion

The mechanism of pregnancy loss and thrombosis in the antiphospholipid-antibody syndrome remains unclear.^1,2,3,4,5,6 In this report we provide evidence of a potentially important prothrombotic effect of these antibodies — a reduction in the quantity of the potent anticoagulant protein annexin V on the surface of placental trophoblasts and vascular endothelial cells. This reduction correlates with our previous immunohistochemical findings¹⁰ and is associated with an increase in the rate of coagulation at the cell surface. It stands in contrast to the "lupus anticoagulant" phenomenon observed with routine phospholipid-dependent coagulation assays. Also, our study offers evidence that endogenous annexin V, whose physiologic function has been unknown, has an antithrombotic role at the interface of trophoblasts and endothelial cells with circulating blood.

Several findings lead to these conclusions. First, the decrease in levels of annexin V induced by antiphospholipid IgG is accompanied by a shortening of the coagulation time of plasma. Second, incubating umbilical-vein endothelial cells with polyclonal rabbit anti–annexin V results in faster coagulation of plasma than that induced by the control polyclonal IgG and polyclonal antibodies against another endothelial-surface annexin, annexin II. Moreover, treating cells with the calcium chelator EGTA, which removes annexin V from the cell surface, significantly accelerates the coagulation of plasma. Finally, adding exogenous annexin V lengthens the coagulation time of plasma applied to the cells. These findings are consistent with the concept that annexin V has an antithrombotic function on the vascular surface, one that is blocked by antiphospholipid antibodies.

Recent data in animal models indicate a causal relation between antiphospholipid antibodies and both pregnancy loss and thrombosis.^20,21,22,23 Our findings of reduced levels of annexin V and accelerated coagulation of plasma on trophoblasts and endothelial cells suggest a mechanism for these processes. Among the members of the annexin family, annexin V has the highest affinity for phospholipid.²⁴ We therefore speculate that antiphospholipid antibodies may have an effect similar to that of annexin V in displacing the lower-affinity annexins, such as annexin II, which may also have antithrombotic properties.^25,26 Our experiments with the polyclonal antibody against annexin II and the protein itself showed that they had no effects on the coagulation of plasma exposed to umbilical-vein endothelial cells. Nevertheless, it remains possible that the proposed fibrinolytic function of this protein²⁶ may also be affected by antiphospholipid.

The 54-kd serum glycoprotein ₂-glycoprotein I (₂GPI, also known as apolipoprotein H) and other phospholipid-binding proteins appear to serve as cofactors in the recognition of their putative antigens by antiphospholipid antibodies.²⁷ Either by itself or in complex with anionic phospholipids, ₂GPI may form an antigenic site for the antibodies. Since ₂GPI is present in fetal-calf serum and on trophoblasts,²⁸ our studies did not determine whether the decrease in annexin V results from the formation of antiphospholipid-antibody–₂GPI complexes or from binding to phospholipids alone.

There is an apparent paradox in that antiphospholipid antibodies bind tightly enough to displace annexin V but do not inhibit the binding of coagulation factors to the same extent. We propose that the explanation is that the antibodies disrupt the ability of annexin V to cluster on the anionic phospholipid that is exposed at the apical membrane (Figure 4A, Figure 4B, and Figure 4C). There is evidence that the high affinity of annexin V for anionic phospholipid is due to the clustering of this protein on the phospholipid surface,²⁹ which makes the protein multimeric and polyvalent. This clustering forms a "carpet" of annexin V and exerts an anticoagulant effect in two ways — first, by shielding the phospholipid and inhibiting coagulation-factor complexes from binding to it, and second, by limiting the lateral diffusion of any coagulation factors bound to the phospholipids. We hypothesize that antiphospholipid antibodies disrupt the ability of annexin V to cluster, resulting in a lower affinity for this protein, which consequently favors both the binding of coagulation-factor complexes and their ability to diffuse laterally.

View larger version (29K): [in this window] [in a new window]   Figure 4. Mechanisms of the Reduction of Annexin V Levels and the Acceleration of Coagulation Associated with Antiphospholipid Antibodies. In Panel A, anionic phospholipids (minus signs) on the surface of the cell-membrane bilayer serve as potent cofactors for the assembly of three coagulation complexes: the tissue-factor–VIIa complex, the IXa–VIIIa complex, and the Xa–Va complex. The presence of such phospholipids thus accelerates blood coagulation. The tissue-factor–VIIa complex yields either factor IXa or factor Xa; the IXa–VIIIa complex yields factor Xa; and the Xa formed from both these reactions becomes the active enzyme in the prothrombinase complex (Xa–Va), which yields factor IIa (thrombin) and in turn cleaves fibrinogen to form fibrin. In Panel B, when antiphospholipid antibodies are absent, annexin V forms clusters that bind with high affinity to the surface of anionic phospholipids and block the assembly of the phospholipid-dependent coagulation complexes, thereby inhibiting coagulation. In Panel C, directly or through an interaction with protein–phospholipid cofactors, antiphospholipid antibodies disrupt the ability of annexin V to cluster on the phospholipid surface. This action reduces the binding affinity of annexin V and permits more anionic phospholipid to be available to form complexes with coagulation proteins. As a result, coagulation is accelerated and the development of thrombosis is promoted. TF denotes tissue factor.

 
In conclusion, we have elucidated a mechanism by which antiphospholipid antibodies may promote thrombosis at the sites where fetal cells are exposed to maternal blood and where vascular endothelial cells contact circulating blood. We hypothesize that annexin V has an antithrombotic function at the apical surface of trophoblasts and endothelial cells and that the antiphospholipid-antibody–induced reduction in the level of annexin V at these sites may account for the thrombosis that occurs in the antiphospholipid-antibody syndrome.

Supported in part by grants (HL-32200, HL-29019, and AI-24671) from the National Institutes of Health and by the Hematology Division of the Mount Sinai School of Medicine.

We are indebted to Drs. Sami David, Harry Spiera, and Yale Nemerson for stimulating these studies; to Dr. Cesare Calandri for initiating our research in this area; to Dr. Barry Potter for helpful advice; and to Nayana Patel and Mayra G. Lema, M.S., for technical assistance.

Source Information

From the Department of Medicine, Divisions of Hematology (J.H.R., X.-X.W., P.C.H.) and Thrombosis (H.A.M.A.), and the Department of Obstetrics, Gynecology, and Reproductive Science (J.S.), Mount Sinai School of Medicine; and the Department of Obstetrics and Gynecology (C.J.L., S.G.) New York University School of Medicine — all in New York.

Address reprint requests to Dr. Rand at the Hematology Division, Mount Sinai Medical Center, Box 1079, 5 E. 98th St., New York, NY 10029.

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Antiphospholipid Antibodies, Annexin V, and Pregnancy Loss
Rote N. S., Cheng H.-M., Rand J. H., Harpel P., Lockwood C.
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N Engl J Med 1997; 337:1630-1631, Nov 27, 1997. Correspondence

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