IgG antibodies that recognize epitope Gly40-Arg43 in domain I of 2–glycoprotein I cause LAC, and their presence correlates strongly with thr
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《血液学杂志》
the Departments of Haematology and Rheumatology and Clinical Immunology, University Medical Centre, Utrecht, the Netherlands.
Abstract
Anti–2–glycoprotein I antibodies are known to have a heterogeneous reactivity against 2–glycoprotein I. We performed this study to characterize the epitope on 2–glycoprotein I to which pathologic anti–2–glycoprotein I antibodies are directed. Plasma samples from 198 patients with various systemic autoimmune diseases were tested for the presence of lupus anticoagulant and anti–2–glycoprotein I immunoglobulin G (IgG) antibodies. The reactivity of the anti–2–glycoprotein I–positive samples was further tested by coating recombinant full-length 2–glycoprotein I and 8 deletion mutants of 2–glycoprotein I onto hydrophilic and hydrophobic enzyme-linked immunosorbent assay (ELISA) plates. Full-length 2–glycoprotein I with point mutations in domain I at positions 8, 40, and 43 were used in inhibition experiments. Fifty-two patients with anti–2–glycoprotein I IgG antibodies could be divided into 2 patterns. Type A antibodies only recognize domain I when coated onto hydrophobic plates; they do not recognize domain I coated onto hydrophilic plates. Type B antibodies have heterogeneous reactivity for all domains. Type A antibodies recognize the epitope around amino acids Gly40-Arg43 and cause lupus anticoagulant activity. In contrast to type B antibodies, those of type A strongly correlated with thrombosis. In conclusion, antibodies directed at domain I (epitope comprising Gly40 and Arg43) have lupus anticoagulant activity and strongly associate with thrombosis.
Introduction
The antiphospholipid syndrome (APS) is an autoimmune disease characterized by the presence of circulating antiphospholipid antibodies (aPLs), notably anticardiolipin antibodies (aCLs) and antibodies that prolong phospholipid coagulation tests (lupus anticoagulant [LAC]) in patients with thrombosis and/or well-defined pregnancy morbidity.1-3 Initially it was thought that aPLs bind directly to negatively charged phospholipids. However, it is now generally accepted that the clinically relevant aPLs bind to proteins with affinity for phospholipids.4-7 Probably the most important epitope of APS-related aPL resides on 2–glycoprotein I (2GPI).4 Most authorities do agree that anti-2GPI antibodies of the immunoglobulin G (IgG) class are more related to clinical symptoms like thrombosis than IgM-class antibodies.
2GPI is a 50-kDa glycoprotein that is present in plasma at a concentration of approximately 200 μg/mL. The protein is a member of the complement control proteins (CCPs).8 2GPI consists of 5 CCP repeats in which the first 4 domains are regular repeats consisting of 60 amino acids. The fifth domain is different from the other 4 domains as it consists of 82 amino acids, 4 conserved hydrophobic amino acids (313-316), 3 disulfide bonds instead of 2, and a cluster of positively charged amino acids (282-287), which is responsible for binding of 2GPI to phospholipids.9-12
To understand the pathophysiology of APS, it is necessary to know the domains involved in anti-2GPI antibody binding and to know whether pathologic antibodies are all directed toward the same epitope. There is substantial controversy on the specificity of anti-2GPI antibodies. Some groups claim that the epitope for binding of anti-2GPI antibodies is located on domain IV.13,14 Other studies showed that binding of anti-2GPI antibodies to 2GPI was abrogated when there was a clip at 316-317 in domain V, suggesting that domain V is involved.15 Arvieux et al16 found that anti-2GPI antibodies inhibited the binding of 2GPI to cardiolipin, suggesting again that domain V harbors the epitope for binding of anti-2GPI antibodies. Blank et al17 found that anti-2GPI antibodies react with peptides that cover sequences present in domains I, II, III, and IV. Most of these groups used murine monoclonal antibodies or a restricted number of affinity-purified patient anti-2GPI antibodies for their studies. Interestingly, studies using only anti-2GPI antibodies purified from patients concluded that domain I is the major domain of 2GPI involved in the binding of anti-2GPI antibodies.18-20
Recently it was suggested that the positively charged epitope R39-R43 on domain I is important in binding of anti-2GPI antibodies.21 Here we further investigate that suggestion by testing the specificity of anti-2GPI antibodies in patients with systemic lupus erythematosus, lupuslike disease, and primary antiphospholipid syndrome by using deletion mutants of 2GPI, recombinant full-length 2GPI, point-mutated recombinant full-length 2GPI, and both hydrophilic and hydrophobic enzyme-linked immunosorbent assay (ELISA) plates. Findings were related to the presence or absence of a history of thrombosis and anti-2GPI–dependent LAC activity.
Patients, materials, and methods
Patients
Included in this study were 198 patients with a diagnosis of systemic lupus erythematosus (SLE; n = 176), lupuslike disease (LLD; n = 16), and primary antiphospholipid syndrome (PAPS; n = 6) that were seen consecutively at the lupus clinic of the University Medical Center, Utrecht, the Netherlands. Patients with SLE meet at least 4 American College of Rheumatology (ACR) criteria for the classification of SLE, and patients with LLD meet at least 1 to 3 of these criteria. Patients with PAPS have aPLs and a history of thrombosis in absence of other signs for a systemic autoimmune disease. By chart review, the number of objectively verified thromboembolic events was recorded for each patient. Computed tomographic scanning or magnetic resonance imaging were used to diagnose a patient having thrombosis of intracerebral vessels. Typical electrocardiographic features and an elevated-fraction creatine kinase muscle and brain (MB) diagnosed myocardial infarction. Peripheral arterial thrombosis and thrombosis of the distal aorta was diagnosed by arteriography or thrombectomy at surgery. Retinal thrombosis was documented by fundoscopy and fluorescence angiography. Deep vein thrombosis was diagnosed by ultrasonography or venography, pulmonary embolism by radionuclide lung scanning, and portal vein thrombosis by angiography. The Institutional Review Board of the University Medical Center Utrecht approved this study, and informed consent was obtained from all patients.
Blood samples
Blood samples were taken at an arbitrary visit of the patients to the outpatient department and were collected by venipuncture using plastic tubes containing 3.8% trisodium citrate (0.129 M) as the anticoagulant (9:1; vol/vol). To obtain platelet-poor plasma, the samples were centrifuged twice at 2000g for 10 minutes and subsequently stored at –50°C until use.
Serologic assays to measure antiphospholipid antibodies
All patient samples were measured with 2 LAC assays: an activated partial thromboplastin time (APTT)–like assay (PTT-LA) and a dilute Russell's Viper Venom time (dRVVT). The APTT (PTT-LA; Diagnostica Stago, Gennevilliers, France) was performed as follows: 50 μL patient plasma was diluted 1:1 with normal pool plasma of 40 healthy volunteers and incubated with 50 μL APTT reagent (Diagnostica Stago). Coagulation was initiated by the addition of 50 μL CaCl2 (25 mM). As a control, samples were tested in an APTT with actin-FS (Dade-Behring, Marburg, Germany) as activator, a LAC-insensitive assay.22 Patients were considered positive when the ratioAPTT-LA/APTT-FS was greater than 1.20. The dRVVT was performed according to the instructions of the manufacturer (Gradipore, North Ryde, Australia) and considered positive when LAC screen/LAC confirm was greater than 1.20. A patient was considered LAC positive if 1 of the 2 LAC assays was positive.
Anticardiolipin antibodies were measured in an ELISA as described before.23 Nine IgG/IgM calibrators were used to report aCL levels as GPL or MPL units. Levels above 10 GPL or GPM units were considered positive.
IgG-class antibody binding to 2GPI and 2GPI mutants
Anti–2GPI IgG antibody ELISA. IgG-class antibodies against 2GPI were measured in an ELISA.24 In short, plasma-purified 2GPI (10 μg/mL in Tris-based solution [TBS]: 50 mM Tris in 100 mM NaCl) was coated onto in a high-binding ELISA plate (9102; Costar, New York, NY) by incubation for 1 hour. Then the plates were washed with 0.1% Tween in TBS (washing solution) and incubated with 4% bovine serum albumin (BSA) in TBS for 1 hour (blocking buffer) to prevent aspecific binding. The plates were washed again, followed by incubation with patient plasma (1:50 diluted in blocking buffer) for 1 hour. Then the plates were washed and incubated with a goat antihuman alkaline phosphatase–labeled antibody (diluted 1:1000; Biosource, Camarillo, CA) followed by staining with para-nitrophenyl phosphatase (PnPP; 0.4 mg/mL). A sample was considered positive if the optical density (OD) was greater than 3SD above the mean OD obtained with plasma from 40 healthy volunteers.
Mutants of 2GPI. By a generous gift of Dr Iverson of La Jolla Pharmaceutical Company, we obtained recombinant full-length 2GPI (DI-V); 8 deletion mutants of 2GPI (comprising domain I [DI]; domains I and II [DI-II]; domains I, II, and III [DI-III]; domains I, II, III, and IV [DI-IV]; domains II, III, IV, and V [DII-V]; domains III, IV, and V [III-V]; domains IV and V [DIV-V]; and domain V [DV]); and 3 recombinant 2GPI molecules with different point mutations in domain I (aspartate-alanine mutation at position 8 [D8A], glycine-glutamate mutation at position 40 [G40E], and an arginine-glycine mutation at position 43 [R43G]).21
Anti–2GPI IgG ELISA with 2GPI deletion mutants. To determine the IgG reactivity against 2GPI deletion mutants we used both 96-well hydrophilic high-binding ELISA plates (9102; Costar) and hydrophobic ELISA plates (2595; Costar). The plates were coated with deletion mutants in a concentration (10 μg/mL) that allows the use of the maximum binding capacity of the wells (1 hour at 37°C). The plates were washed 4 times with TBS/0.1% Tween and subsequently blocked with a 4% BSA/TBS solution. Patient plasma (diluted 1:50 in blocking solution) was added to the wells (50 μL/well) and incubated for 1 hour at 37°C. The plates were washed 4 times (0.1% Tween/TBS) and incubated with alkalic-phosphatase–labeled goat antihuman IgG antibodies (Abs; diluted 1:1000; Biosource) for 1 hour at 37°C. Staining was performed by using PnPP (Sigma, St Louis, MO) at a concentration of 0.6 mg/mL diluted in diethynolamine (DEA) buffer. Adding 2.4 M/L NaOH to the wells after 10 minutes at room temperature stopped the reaction. Absorption was measured at 405 nm. Extinctions obtained with normal pooled plasma were subtracted from those with patient plasma.
Inhibition experiments with point-mutated 2GPI coupled to Sepharose beads. Recombinant 2GPI and 3 different point-mutated forms of 2GPI were covalently coupled to chelating Sepharose beads by the following procedure. Chelating Sepharose beads (Pharmacia, Uppsala, Sweden) were centrifuged for 15 seconds at 10 000g followed by removal of ethanol and washing of the beads with 4 volumes 50 mM EDTA (ethylenediaminetetraacetic acid)/H2O. All washing procedures were done at room temperature by spinning down the beads at 57g. Subsequently beads were washed with 4 volumes H2O, 4 volumes 50 mM CoCl2/HO2, 4 volumes H2O, and 4 volumes PBS, followed by incubation for 15 minutes with point-mutated 2GPI or full-length recombinant 2GPI (2 volumes, 20 μM/mL) at room temperature during rotation. Then the beads were washed (with 6 volumes PBS) and incubated with 2 volumes 0.03% H2O2/PBS for 2 hours at room temperature during rotation, followed by washing with 4 volumes PBS, 1 volume 50 mM EDTA, 1 volume PBS, 1 volume 0.5 M imidazole/PBS, and 1 volume PBS. Blocking of aspecific binding places was done by incubation of beads with 4% BSA/PBS for 1 hour at room temperature during rotation.
Purified patient IgG was incubated for 1 hour at room temperature during rotation with Sepharose beads alone and Sepharose beads to which recombinant 2GPI or mutated 2GPI–Sepharose beads was coupled. Then the mixture was centrifuged for 1 minute at 57g. The supernatant was added to ELISA plates (Costar; high absorbing plates) coated with full-length recombinant 2GPI and incubated for 1 hour at 37°C. After washing with 0.1% Tween-20/TBS five times, the plates were incubated with 50 μL/well alkalic-phosphatase–labeled goat antihuman IgG diluted 1:1000 in 4% BSA/PBS for 1 hour at 37°C. Then plates were washed 5 times with 0.1% Tween/TBS, and color development was performed by adding 100 μL/well PNPP at a concentration of 6 mg/10 mL DEA buffer. After 20 minutes, addition of 50 μL/well 2.4 M/L NaOH stopped the coloring reaction. Absorption was measured at 405 nm. From extinctions with purified patient IgGs, extinctions obtained with normal pool IgG were subtracted.
Anti-2GPI–specific LAC assay
To discriminate between a 2GPI-dependent LAC and a 2GPI-independent LAC we made use of an aPTT-based clotting test (PTT-LA; Diagnostica Stago), as described before.25 In short, 25 μL PTT-LA reagent, 50 μL patient plasma that was mixed 1:1 with normal pool plasma (of 40 healthy volunteers), and 25 μL of different concentrations (0, 25, 50, 100, 200 μM) of cardiolipin vesicles diluted in TBS were mixed and added to a KC-10 microcoagulometer (Amelung, Lemgo, Germany). The cardiolipin vesicles were made as described before.26 After 3 minutes of incubation at 37°C, coagulation was initiated by the addition of 50 μL CaCl2 and clotting time was measured. A LAC was considered 2GPI dependent when the ratio of coagulation times of patient plasma and normal pool plasma was decreased by at least 0.05 with addition of cardiolipin vesicles at a concentration of 25 μM cardiolipin vesicles.22 An example of a patient with a 2GPI-dependent LAC and a patient with a 2GPI-independent LAC is given in Table 1.
LAC assay with point-mutated 2GPI
First, 2GPI-deficient plasma was obtained by adding normal pool plasma (mixture of 40 healthy volunteers) to a Sepharose column coupled with 21B2 (a monoclonal murine anti-2GPI antibody, generous gift of Prof J. Arnout). After this procedure, 2GPI could not be demonstrated anymore by chromatography. The 2GPI-deficient plasma was reconstituted with either recombinant 2GPI or mutated 2GPI (R43G). Of this mixture, 75 μL was added to the KC-10 (Amelung) and incubated at 37°C. After 3 minutes of incubation, a dRVVT solution (Gradipore, Frenchs Forrest, Australia) was added and the coagulation time was measured.
Statistical analysis
Chi-square statistics was used to compare the prevalence of thrombosis with serologic findings. Odds ratios and 95% confidence intervals were calculated by binary logistic regression. Student t test or Mann-Whitney test was used to calculate differences between 2 groups. For these calculations we used SPSS (Chicago, IL). P values less than .05 were considered significant.
Results
Patient population
The median age of the 198 patients (180 female) was 33 years. In 60 (30%) of 198 patients there was a history of objectively verified thrombosis. Thirty-two patients had a history of arterial thrombosis (stroke, n = 19; myocardial infarction, n = 5; peripheral artery, n = 7; distal aorta, n = 2; retinal artery, n = 3) and 38 patients had a history of venous thrombosis (deep venous thrombosis, n = 30; pulmonary embolism, n = 19; thrombophlebitis, n = 7; portal vein thrombosis, n = 1). Anticardiolipin antibodies were present in 112 (57%) of 198 patients, LAC in 63 (32%) of 198 patients, and anti–2GPI IgG antibodies in 52 (26%) of 198 patients (Table 2). Table 3 shows the interassay variability as an indication for the quality of the assays used in this study.
Domain specificity of anti–2GPI IgG antibodies
Fine specificity of anti–2GPI IgG antibodies
We hypothesized that patients of group A recognize a positive epitope on domain I that is shielded off from recognition when coated to hydrophilic plates. To demonstrate the involvement of this positively charged epitope on domain I in recognition by type A anti-2GPI antibodies, we performed inhibition experiments. We coated Sepharose beads with native full-length recombinant 2GPI or 1 of 3 point mutations (D8A, G40E, or R43G) of full-length recombinant 2GPI, blocked the beads with BSA, and incubated them with IgG fractions isolated from plasma from 3 patients from type A. The supernatants were added to a hydrophilic plate that was coated with full-length recombinant 2GPI. We found that in all 3 samples with type A anti-2GPI antibodies, the anti-2GPI activity could be fully absorbed by full-length recombinant 2GPI and with D8A and only partially with G40E or R43G (Figure 2).
When we tested the affinity for 2GPI in an anti–2GPI IgG-class ELISA in the presence of 1 M NaCl, 26 of 30 patients from group A recognized an epitope in the presence of 1 M NaCl (data not shown), indicating the recognition of a specific epitope rather than charge. Only 8 of 22 of the IgG of group B recognized 2GPI in the presence of 1 M NaCl, indicating that the binding of those antibodies was of lower affinity (data not shown).
Specific anti-2GPI reactivity and LAC
Out of the 30 samples from type A, 28 (93%) were LAC positive. In 23 of 28 samples the LAC activity was dependent on 2GPI (Table 4). To give support to the association between anti-2GPI reactivity and 2GPI-dependent LAC, we added anti–2GPI IgG isolated from plasma of 3 patients with type A reactivity to 2GPI-depleted plasma that was reconstituted with either recombinant full-length 2GPI or point-mutated 2GPI (R43G). We observed an increase in coagulation time from 104.0 ± 3.4 (mean ± SEM, n = 4) seconds to 175.5 ± 32.4 (mean ± SEM, n = 4) seconds when plasma was reconstituted with 150 μg/mL recombinant 2GPI and 100 μg/mL type A anti–2GPI IgG antibodies. However, little prolongation (104.0 ± 3.4 122.8 ± 17.3, mean ± SEM, n = 4) was seen when plasma was reconstituted with 150 μg/mL R43G and 100 μg/mL type A anti–2GPI IgG antibodies. This experiment is representative of 4 experiments performed in the same way.
Correlation between thrombosis and presence of type A or type B anti-2GPI antibodies
A history of thrombosis was present in 32 of 52 patients with IgG anti-2GPI antibodies, in 25 (83%) of 30 patients IgG anti-2GPI antibodies with type A reactivity, and in 7 (32%) of 22 patients with type B reactivity (Table 4). The odds ratios for thrombosis were 6.7 (95% confidence interval [95% CI]: 13.5-3.4) for IgG anti-2GPI antibodies, 18.9 (95% CI: 53.2-6.8) for type A reactivity, and 1.1 (95% CI: 2.8-0.4) for type B reactivity.
Anti–domain I ELISA for clinical practice
To enable differentiation between patients with IgG anti-2GPI antibodies of type A and type B reactivity, one can coat domain I of 2GPI onto a hydrophilic and a hydrophobic ELISA plate. Plasma from patients with type A IgG anti-2GPI antibodies do not recognize domain I on a hydrophilic plate but recognize domain I on a hydrophobic plate, whereas samples from patients with type B reactivity recognize domain I on both plates. A ratio of greater than 2 between the OD measured with the hydrophobic plate and the OD with the hydrophilic plate discriminates between type A and type B IgG anti-2GPI antibodies. This ratio is an indication for the relative amount of IgG anti-2GPI antibodies that recognize the positive charge on domain I. We determined a coefficient of variation of 3.7% (interassay variability) as an indication for the quality of this assay (Table 3). Figure 3 shows that within the group of patients with IgG anti-2GPI antibodies with type A reactivity, the ratio in those with a thrombotic history is significantly higher than in patients without thrombosis (P = .014). Within the group of patients with IgG anti-2GPI antibodies of type B reactivity, the ratios are low and there is no difference in ratio between patients with or without thrombosis.
Discussion
The identification of the domains of 2GPI that are involved in binding of anti-2GPI antibodies has been the subject of many studies.13-21 The cumulative results show that each domain of 2GPI has been indicated as the location where anti-2GPI antibodies may bind. A recent paper strongly suggests that for binding of anti-2GPI antibodies purified from patient plasma, a positively charged epitope, spanning amino acids 40-43 on domain I of 2GPI, is very important. We tested this suggestion by studying the reactivity of 52 samples (52 patients) with anti–2GPI IgG antibodies that were found within a group of 198 patients with SLE, LLD, or PAPS. The major conclusions from this study are as follows: (1) not all anti–2GPI IgG antibodies have the same epitope specificity; (2) a substantial number of antibodies found in patients recognize an epitope around G40-R43 on domain I; (3) these latter antibodies seem to be the pathologic ones because their presence was highly correlated with a history of thrombosis; (4) the anti–2GPI IgG antibodies against epitope G40-R43 in domain I are antibodies that cause LAC activity; and (5) anti–2GPI IgG antibodies that do not recognize G40-R43 in domain I are not related to a history of thrombosis. For anti-2GPI antibodies of the IgM class we did not find an increase in their association with thrombosis by using the anti–domain I ELISA. Based on these observations we have developed a simple assay to detect a subset of anti–2GPI IgG antibodies that highly correlates with thrombosis. Although the proportion of patients with primary APS was very small in our population, previous studies found similar clinical and laboratory features for primary and secondary APS.27 It seems likely that this also accounts for this anti–domain I ELISA.
By coating 8 domain-deleted mutants of 2GPI and full-length recombinant 2GPI on both hydrophilic and hydrophobic ELISA plates we discriminated 2 different reactivity patterns (designated types A and B) for the anti-2GPI antibodies. Samples with type A reactivity (present in 58% of anti-2GPI antibody–positive samples) recognize full-length 2GPI but not deletion mutants when hydrophilic ELISA plates are used and a strong reactivity for domain I (and extensions of this domain) with use of hydrophobic ELISA plates (Figure 1). Samples designated type B do not seem to have a preferred binding site on 2GPI. With hydrophilic ELISA plates these antibodies react both with full-length 2GPI, domain I and V, and extensions of these. When hydrophobic ELISA plates are used, the pattern of reactivity against 2GPI and domain-deleted mutants is similar, albeit the ODs are much lower.
We hypothesized that our observation of the reactivity of type A antibodies is dependent on the type of ELISA plate used and could indicate that type A anti-2GPI antibodies are directed against the G40-R43 epitope in domain I because it is conceivable that interactions between the positive charge of this epitope and the negative charge of a hydrophilic ELISA plate may hamper interaction of antibodies with that epitope.21 We showed that the reactivity of type A anti-2GPI antibodies against 2GPI can be fully absorbed by full-length recombinant 2GPI and full-length recombinant 2GPI mutated at position 8 (D8A). When we absorbed type A anti-2GPI antibodies with full-length recombinant 2GPI that had point mutations at 1 amino acid within the epitope G40-R43, namely R40E or R43G, 35% to 40% of the reactivity against 2GPI remained (Figure 2). This strongly suggests that these amino acids are part of the epitope to which type A anti-2GPI antibodies are directed. We previously reported that 2GPI-dependent LAC activity is a strong risk factor for thrombosis.22 We now demonstrated that 23 (92%) of 25 samples with 2GPI-dependent LAC activity contained type A anti-2GPI antibodies and that presence of type A antibodies is a strong risk factor for thrombosis (Table 4). The finding that reconstitution of 2GPI-depleted plasma with full-length 2GPI and type A anti-2GPI antibodies induces more prolongation of the clotting time than reconstitution with R43G and type A anti-2GPI antibodies suggested that type A anti-2GPI antibodies cause LAC activity. As a 2GPI-dependent LAC activity correlates very strongly with thrombosis (odds ratio, 42.3), it also supports the concept that pathologic antibodies are directed against the epitope G40-R43.
Collectively, type B antibodies directed to 2GPI are not correlated with a history of thrombotic complications. Whether this population contains clinical relevant subpopulations of antibodies needs further studies.
In conclusion, our study shows that anti-2GPI antibodies react with different epitopes on 2GPI. However, a substantial number of patient samples with anti-2GPI antibodies (in our series 58%) recognize an epitope on domain I, including positions 40 and 43 (type A antibodies). The strong correlation between presence of such type A antibodies with histories of thrombosis and presence of 2GPI-dependent LAC activity strongly suggests that type A antibodies constitute a pathologic subset. Our observations form the basis for a simple assay (using domain I coated onto hydrophilic and hydrophobic ELISA plates) for the detection of type A anti-2GPI antibodies.
Footnotes
Prepublished online as Blood First Edition Paper, October 26, 2004; DOI 10.1182/blood-2004-09-3387.
Supported by a grant of the Netherlands Organisation for Health Research and Development (ZonMw grant no. 902-26-290).
An Inside Blood analysis of this article appears in the front of this issue.
The publication costs of this article were defrayed in part by page charge payment. Therefore, and solely to indicate this fact, this article is hereby marked "advertisement" in accordance with 18 U.S.C. section 1734.
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Abstract
Anti–2–glycoprotein I antibodies are known to have a heterogeneous reactivity against 2–glycoprotein I. We performed this study to characterize the epitope on 2–glycoprotein I to which pathologic anti–2–glycoprotein I antibodies are directed. Plasma samples from 198 patients with various systemic autoimmune diseases were tested for the presence of lupus anticoagulant and anti–2–glycoprotein I immunoglobulin G (IgG) antibodies. The reactivity of the anti–2–glycoprotein I–positive samples was further tested by coating recombinant full-length 2–glycoprotein I and 8 deletion mutants of 2–glycoprotein I onto hydrophilic and hydrophobic enzyme-linked immunosorbent assay (ELISA) plates. Full-length 2–glycoprotein I with point mutations in domain I at positions 8, 40, and 43 were used in inhibition experiments. Fifty-two patients with anti–2–glycoprotein I IgG antibodies could be divided into 2 patterns. Type A antibodies only recognize domain I when coated onto hydrophobic plates; they do not recognize domain I coated onto hydrophilic plates. Type B antibodies have heterogeneous reactivity for all domains. Type A antibodies recognize the epitope around amino acids Gly40-Arg43 and cause lupus anticoagulant activity. In contrast to type B antibodies, those of type A strongly correlated with thrombosis. In conclusion, antibodies directed at domain I (epitope comprising Gly40 and Arg43) have lupus anticoagulant activity and strongly associate with thrombosis.
Introduction
The antiphospholipid syndrome (APS) is an autoimmune disease characterized by the presence of circulating antiphospholipid antibodies (aPLs), notably anticardiolipin antibodies (aCLs) and antibodies that prolong phospholipid coagulation tests (lupus anticoagulant [LAC]) in patients with thrombosis and/or well-defined pregnancy morbidity.1-3 Initially it was thought that aPLs bind directly to negatively charged phospholipids. However, it is now generally accepted that the clinically relevant aPLs bind to proteins with affinity for phospholipids.4-7 Probably the most important epitope of APS-related aPL resides on 2–glycoprotein I (2GPI).4 Most authorities do agree that anti-2GPI antibodies of the immunoglobulin G (IgG) class are more related to clinical symptoms like thrombosis than IgM-class antibodies.
2GPI is a 50-kDa glycoprotein that is present in plasma at a concentration of approximately 200 μg/mL. The protein is a member of the complement control proteins (CCPs).8 2GPI consists of 5 CCP repeats in which the first 4 domains are regular repeats consisting of 60 amino acids. The fifth domain is different from the other 4 domains as it consists of 82 amino acids, 4 conserved hydrophobic amino acids (313-316), 3 disulfide bonds instead of 2, and a cluster of positively charged amino acids (282-287), which is responsible for binding of 2GPI to phospholipids.9-12
To understand the pathophysiology of APS, it is necessary to know the domains involved in anti-2GPI antibody binding and to know whether pathologic antibodies are all directed toward the same epitope. There is substantial controversy on the specificity of anti-2GPI antibodies. Some groups claim that the epitope for binding of anti-2GPI antibodies is located on domain IV.13,14 Other studies showed that binding of anti-2GPI antibodies to 2GPI was abrogated when there was a clip at 316-317 in domain V, suggesting that domain V is involved.15 Arvieux et al16 found that anti-2GPI antibodies inhibited the binding of 2GPI to cardiolipin, suggesting again that domain V harbors the epitope for binding of anti-2GPI antibodies. Blank et al17 found that anti-2GPI antibodies react with peptides that cover sequences present in domains I, II, III, and IV. Most of these groups used murine monoclonal antibodies or a restricted number of affinity-purified patient anti-2GPI antibodies for their studies. Interestingly, studies using only anti-2GPI antibodies purified from patients concluded that domain I is the major domain of 2GPI involved in the binding of anti-2GPI antibodies.18-20
Recently it was suggested that the positively charged epitope R39-R43 on domain I is important in binding of anti-2GPI antibodies.21 Here we further investigate that suggestion by testing the specificity of anti-2GPI antibodies in patients with systemic lupus erythematosus, lupuslike disease, and primary antiphospholipid syndrome by using deletion mutants of 2GPI, recombinant full-length 2GPI, point-mutated recombinant full-length 2GPI, and both hydrophilic and hydrophobic enzyme-linked immunosorbent assay (ELISA) plates. Findings were related to the presence or absence of a history of thrombosis and anti-2GPI–dependent LAC activity.
Patients, materials, and methods
Patients
Included in this study were 198 patients with a diagnosis of systemic lupus erythematosus (SLE; n = 176), lupuslike disease (LLD; n = 16), and primary antiphospholipid syndrome (PAPS; n = 6) that were seen consecutively at the lupus clinic of the University Medical Center, Utrecht, the Netherlands. Patients with SLE meet at least 4 American College of Rheumatology (ACR) criteria for the classification of SLE, and patients with LLD meet at least 1 to 3 of these criteria. Patients with PAPS have aPLs and a history of thrombosis in absence of other signs for a systemic autoimmune disease. By chart review, the number of objectively verified thromboembolic events was recorded for each patient. Computed tomographic scanning or magnetic resonance imaging were used to diagnose a patient having thrombosis of intracerebral vessels. Typical electrocardiographic features and an elevated-fraction creatine kinase muscle and brain (MB) diagnosed myocardial infarction. Peripheral arterial thrombosis and thrombosis of the distal aorta was diagnosed by arteriography or thrombectomy at surgery. Retinal thrombosis was documented by fundoscopy and fluorescence angiography. Deep vein thrombosis was diagnosed by ultrasonography or venography, pulmonary embolism by radionuclide lung scanning, and portal vein thrombosis by angiography. The Institutional Review Board of the University Medical Center Utrecht approved this study, and informed consent was obtained from all patients.
Blood samples
Blood samples were taken at an arbitrary visit of the patients to the outpatient department and were collected by venipuncture using plastic tubes containing 3.8% trisodium citrate (0.129 M) as the anticoagulant (9:1; vol/vol). To obtain platelet-poor plasma, the samples were centrifuged twice at 2000g for 10 minutes and subsequently stored at –50°C until use.
Serologic assays to measure antiphospholipid antibodies
All patient samples were measured with 2 LAC assays: an activated partial thromboplastin time (APTT)–like assay (PTT-LA) and a dilute Russell's Viper Venom time (dRVVT). The APTT (PTT-LA; Diagnostica Stago, Gennevilliers, France) was performed as follows: 50 μL patient plasma was diluted 1:1 with normal pool plasma of 40 healthy volunteers and incubated with 50 μL APTT reagent (Diagnostica Stago). Coagulation was initiated by the addition of 50 μL CaCl2 (25 mM). As a control, samples were tested in an APTT with actin-FS (Dade-Behring, Marburg, Germany) as activator, a LAC-insensitive assay.22 Patients were considered positive when the ratioAPTT-LA/APTT-FS was greater than 1.20. The dRVVT was performed according to the instructions of the manufacturer (Gradipore, North Ryde, Australia) and considered positive when LAC screen/LAC confirm was greater than 1.20. A patient was considered LAC positive if 1 of the 2 LAC assays was positive.
Anticardiolipin antibodies were measured in an ELISA as described before.23 Nine IgG/IgM calibrators were used to report aCL levels as GPL or MPL units. Levels above 10 GPL or GPM units were considered positive.
IgG-class antibody binding to 2GPI and 2GPI mutants
Anti–2GPI IgG antibody ELISA. IgG-class antibodies against 2GPI were measured in an ELISA.24 In short, plasma-purified 2GPI (10 μg/mL in Tris-based solution [TBS]: 50 mM Tris in 100 mM NaCl) was coated onto in a high-binding ELISA plate (9102; Costar, New York, NY) by incubation for 1 hour. Then the plates were washed with 0.1% Tween in TBS (washing solution) and incubated with 4% bovine serum albumin (BSA) in TBS for 1 hour (blocking buffer) to prevent aspecific binding. The plates were washed again, followed by incubation with patient plasma (1:50 diluted in blocking buffer) for 1 hour. Then the plates were washed and incubated with a goat antihuman alkaline phosphatase–labeled antibody (diluted 1:1000; Biosource, Camarillo, CA) followed by staining with para-nitrophenyl phosphatase (PnPP; 0.4 mg/mL). A sample was considered positive if the optical density (OD) was greater than 3SD above the mean OD obtained with plasma from 40 healthy volunteers.
Mutants of 2GPI. By a generous gift of Dr Iverson of La Jolla Pharmaceutical Company, we obtained recombinant full-length 2GPI (DI-V); 8 deletion mutants of 2GPI (comprising domain I [DI]; domains I and II [DI-II]; domains I, II, and III [DI-III]; domains I, II, III, and IV [DI-IV]; domains II, III, IV, and V [DII-V]; domains III, IV, and V [III-V]; domains IV and V [DIV-V]; and domain V [DV]); and 3 recombinant 2GPI molecules with different point mutations in domain I (aspartate-alanine mutation at position 8 [D8A], glycine-glutamate mutation at position 40 [G40E], and an arginine-glycine mutation at position 43 [R43G]).21
Anti–2GPI IgG ELISA with 2GPI deletion mutants. To determine the IgG reactivity against 2GPI deletion mutants we used both 96-well hydrophilic high-binding ELISA plates (9102; Costar) and hydrophobic ELISA plates (2595; Costar). The plates were coated with deletion mutants in a concentration (10 μg/mL) that allows the use of the maximum binding capacity of the wells (1 hour at 37°C). The plates were washed 4 times with TBS/0.1% Tween and subsequently blocked with a 4% BSA/TBS solution. Patient plasma (diluted 1:50 in blocking solution) was added to the wells (50 μL/well) and incubated for 1 hour at 37°C. The plates were washed 4 times (0.1% Tween/TBS) and incubated with alkalic-phosphatase–labeled goat antihuman IgG antibodies (Abs; diluted 1:1000; Biosource) for 1 hour at 37°C. Staining was performed by using PnPP (Sigma, St Louis, MO) at a concentration of 0.6 mg/mL diluted in diethynolamine (DEA) buffer. Adding 2.4 M/L NaOH to the wells after 10 minutes at room temperature stopped the reaction. Absorption was measured at 405 nm. Extinctions obtained with normal pooled plasma were subtracted from those with patient plasma.
Inhibition experiments with point-mutated 2GPI coupled to Sepharose beads. Recombinant 2GPI and 3 different point-mutated forms of 2GPI were covalently coupled to chelating Sepharose beads by the following procedure. Chelating Sepharose beads (Pharmacia, Uppsala, Sweden) were centrifuged for 15 seconds at 10 000g followed by removal of ethanol and washing of the beads with 4 volumes 50 mM EDTA (ethylenediaminetetraacetic acid)/H2O. All washing procedures were done at room temperature by spinning down the beads at 57g. Subsequently beads were washed with 4 volumes H2O, 4 volumes 50 mM CoCl2/HO2, 4 volumes H2O, and 4 volumes PBS, followed by incubation for 15 minutes with point-mutated 2GPI or full-length recombinant 2GPI (2 volumes, 20 μM/mL) at room temperature during rotation. Then the beads were washed (with 6 volumes PBS) and incubated with 2 volumes 0.03% H2O2/PBS for 2 hours at room temperature during rotation, followed by washing with 4 volumes PBS, 1 volume 50 mM EDTA, 1 volume PBS, 1 volume 0.5 M imidazole/PBS, and 1 volume PBS. Blocking of aspecific binding places was done by incubation of beads with 4% BSA/PBS for 1 hour at room temperature during rotation.
Purified patient IgG was incubated for 1 hour at room temperature during rotation with Sepharose beads alone and Sepharose beads to which recombinant 2GPI or mutated 2GPI–Sepharose beads was coupled. Then the mixture was centrifuged for 1 minute at 57g. The supernatant was added to ELISA plates (Costar; high absorbing plates) coated with full-length recombinant 2GPI and incubated for 1 hour at 37°C. After washing with 0.1% Tween-20/TBS five times, the plates were incubated with 50 μL/well alkalic-phosphatase–labeled goat antihuman IgG diluted 1:1000 in 4% BSA/PBS for 1 hour at 37°C. Then plates were washed 5 times with 0.1% Tween/TBS, and color development was performed by adding 100 μL/well PNPP at a concentration of 6 mg/10 mL DEA buffer. After 20 minutes, addition of 50 μL/well 2.4 M/L NaOH stopped the coloring reaction. Absorption was measured at 405 nm. From extinctions with purified patient IgGs, extinctions obtained with normal pool IgG were subtracted.
Anti-2GPI–specific LAC assay
To discriminate between a 2GPI-dependent LAC and a 2GPI-independent LAC we made use of an aPTT-based clotting test (PTT-LA; Diagnostica Stago), as described before.25 In short, 25 μL PTT-LA reagent, 50 μL patient plasma that was mixed 1:1 with normal pool plasma (of 40 healthy volunteers), and 25 μL of different concentrations (0, 25, 50, 100, 200 μM) of cardiolipin vesicles diluted in TBS were mixed and added to a KC-10 microcoagulometer (Amelung, Lemgo, Germany). The cardiolipin vesicles were made as described before.26 After 3 minutes of incubation at 37°C, coagulation was initiated by the addition of 50 μL CaCl2 and clotting time was measured. A LAC was considered 2GPI dependent when the ratio of coagulation times of patient plasma and normal pool plasma was decreased by at least 0.05 with addition of cardiolipin vesicles at a concentration of 25 μM cardiolipin vesicles.22 An example of a patient with a 2GPI-dependent LAC and a patient with a 2GPI-independent LAC is given in Table 1.
LAC assay with point-mutated 2GPI
First, 2GPI-deficient plasma was obtained by adding normal pool plasma (mixture of 40 healthy volunteers) to a Sepharose column coupled with 21B2 (a monoclonal murine anti-2GPI antibody, generous gift of Prof J. Arnout). After this procedure, 2GPI could not be demonstrated anymore by chromatography. The 2GPI-deficient plasma was reconstituted with either recombinant 2GPI or mutated 2GPI (R43G). Of this mixture, 75 μL was added to the KC-10 (Amelung) and incubated at 37°C. After 3 minutes of incubation, a dRVVT solution (Gradipore, Frenchs Forrest, Australia) was added and the coagulation time was measured.
Statistical analysis
Chi-square statistics was used to compare the prevalence of thrombosis with serologic findings. Odds ratios and 95% confidence intervals were calculated by binary logistic regression. Student t test or Mann-Whitney test was used to calculate differences between 2 groups. For these calculations we used SPSS (Chicago, IL). P values less than .05 were considered significant.
Results
Patient population
The median age of the 198 patients (180 female) was 33 years. In 60 (30%) of 198 patients there was a history of objectively verified thrombosis. Thirty-two patients had a history of arterial thrombosis (stroke, n = 19; myocardial infarction, n = 5; peripheral artery, n = 7; distal aorta, n = 2; retinal artery, n = 3) and 38 patients had a history of venous thrombosis (deep venous thrombosis, n = 30; pulmonary embolism, n = 19; thrombophlebitis, n = 7; portal vein thrombosis, n = 1). Anticardiolipin antibodies were present in 112 (57%) of 198 patients, LAC in 63 (32%) of 198 patients, and anti–2GPI IgG antibodies in 52 (26%) of 198 patients (Table 2). Table 3 shows the interassay variability as an indication for the quality of the assays used in this study.
Domain specificity of anti–2GPI IgG antibodies
Fine specificity of anti–2GPI IgG antibodies
We hypothesized that patients of group A recognize a positive epitope on domain I that is shielded off from recognition when coated to hydrophilic plates. To demonstrate the involvement of this positively charged epitope on domain I in recognition by type A anti-2GPI antibodies, we performed inhibition experiments. We coated Sepharose beads with native full-length recombinant 2GPI or 1 of 3 point mutations (D8A, G40E, or R43G) of full-length recombinant 2GPI, blocked the beads with BSA, and incubated them with IgG fractions isolated from plasma from 3 patients from type A. The supernatants were added to a hydrophilic plate that was coated with full-length recombinant 2GPI. We found that in all 3 samples with type A anti-2GPI antibodies, the anti-2GPI activity could be fully absorbed by full-length recombinant 2GPI and with D8A and only partially with G40E or R43G (Figure 2).
When we tested the affinity for 2GPI in an anti–2GPI IgG-class ELISA in the presence of 1 M NaCl, 26 of 30 patients from group A recognized an epitope in the presence of 1 M NaCl (data not shown), indicating the recognition of a specific epitope rather than charge. Only 8 of 22 of the IgG of group B recognized 2GPI in the presence of 1 M NaCl, indicating that the binding of those antibodies was of lower affinity (data not shown).
Specific anti-2GPI reactivity and LAC
Out of the 30 samples from type A, 28 (93%) were LAC positive. In 23 of 28 samples the LAC activity was dependent on 2GPI (Table 4). To give support to the association between anti-2GPI reactivity and 2GPI-dependent LAC, we added anti–2GPI IgG isolated from plasma of 3 patients with type A reactivity to 2GPI-depleted plasma that was reconstituted with either recombinant full-length 2GPI or point-mutated 2GPI (R43G). We observed an increase in coagulation time from 104.0 ± 3.4 (mean ± SEM, n = 4) seconds to 175.5 ± 32.4 (mean ± SEM, n = 4) seconds when plasma was reconstituted with 150 μg/mL recombinant 2GPI and 100 μg/mL type A anti–2GPI IgG antibodies. However, little prolongation (104.0 ± 3.4 122.8 ± 17.3, mean ± SEM, n = 4) was seen when plasma was reconstituted with 150 μg/mL R43G and 100 μg/mL type A anti–2GPI IgG antibodies. This experiment is representative of 4 experiments performed in the same way.
Correlation between thrombosis and presence of type A or type B anti-2GPI antibodies
A history of thrombosis was present in 32 of 52 patients with IgG anti-2GPI antibodies, in 25 (83%) of 30 patients IgG anti-2GPI antibodies with type A reactivity, and in 7 (32%) of 22 patients with type B reactivity (Table 4). The odds ratios for thrombosis were 6.7 (95% confidence interval [95% CI]: 13.5-3.4) for IgG anti-2GPI antibodies, 18.9 (95% CI: 53.2-6.8) for type A reactivity, and 1.1 (95% CI: 2.8-0.4) for type B reactivity.
Anti–domain I ELISA for clinical practice
To enable differentiation between patients with IgG anti-2GPI antibodies of type A and type B reactivity, one can coat domain I of 2GPI onto a hydrophilic and a hydrophobic ELISA plate. Plasma from patients with type A IgG anti-2GPI antibodies do not recognize domain I on a hydrophilic plate but recognize domain I on a hydrophobic plate, whereas samples from patients with type B reactivity recognize domain I on both plates. A ratio of greater than 2 between the OD measured with the hydrophobic plate and the OD with the hydrophilic plate discriminates between type A and type B IgG anti-2GPI antibodies. This ratio is an indication for the relative amount of IgG anti-2GPI antibodies that recognize the positive charge on domain I. We determined a coefficient of variation of 3.7% (interassay variability) as an indication for the quality of this assay (Table 3). Figure 3 shows that within the group of patients with IgG anti-2GPI antibodies with type A reactivity, the ratio in those with a thrombotic history is significantly higher than in patients without thrombosis (P = .014). Within the group of patients with IgG anti-2GPI antibodies of type B reactivity, the ratios are low and there is no difference in ratio between patients with or without thrombosis.
Discussion
The identification of the domains of 2GPI that are involved in binding of anti-2GPI antibodies has been the subject of many studies.13-21 The cumulative results show that each domain of 2GPI has been indicated as the location where anti-2GPI antibodies may bind. A recent paper strongly suggests that for binding of anti-2GPI antibodies purified from patient plasma, a positively charged epitope, spanning amino acids 40-43 on domain I of 2GPI, is very important. We tested this suggestion by studying the reactivity of 52 samples (52 patients) with anti–2GPI IgG antibodies that were found within a group of 198 patients with SLE, LLD, or PAPS. The major conclusions from this study are as follows: (1) not all anti–2GPI IgG antibodies have the same epitope specificity; (2) a substantial number of antibodies found in patients recognize an epitope around G40-R43 on domain I; (3) these latter antibodies seem to be the pathologic ones because their presence was highly correlated with a history of thrombosis; (4) the anti–2GPI IgG antibodies against epitope G40-R43 in domain I are antibodies that cause LAC activity; and (5) anti–2GPI IgG antibodies that do not recognize G40-R43 in domain I are not related to a history of thrombosis. For anti-2GPI antibodies of the IgM class we did not find an increase in their association with thrombosis by using the anti–domain I ELISA. Based on these observations we have developed a simple assay to detect a subset of anti–2GPI IgG antibodies that highly correlates with thrombosis. Although the proportion of patients with primary APS was very small in our population, previous studies found similar clinical and laboratory features for primary and secondary APS.27 It seems likely that this also accounts for this anti–domain I ELISA.
By coating 8 domain-deleted mutants of 2GPI and full-length recombinant 2GPI on both hydrophilic and hydrophobic ELISA plates we discriminated 2 different reactivity patterns (designated types A and B) for the anti-2GPI antibodies. Samples with type A reactivity (present in 58% of anti-2GPI antibody–positive samples) recognize full-length 2GPI but not deletion mutants when hydrophilic ELISA plates are used and a strong reactivity for domain I (and extensions of this domain) with use of hydrophobic ELISA plates (Figure 1). Samples designated type B do not seem to have a preferred binding site on 2GPI. With hydrophilic ELISA plates these antibodies react both with full-length 2GPI, domain I and V, and extensions of these. When hydrophobic ELISA plates are used, the pattern of reactivity against 2GPI and domain-deleted mutants is similar, albeit the ODs are much lower.
We hypothesized that our observation of the reactivity of type A antibodies is dependent on the type of ELISA plate used and could indicate that type A anti-2GPI antibodies are directed against the G40-R43 epitope in domain I because it is conceivable that interactions between the positive charge of this epitope and the negative charge of a hydrophilic ELISA plate may hamper interaction of antibodies with that epitope.21 We showed that the reactivity of type A anti-2GPI antibodies against 2GPI can be fully absorbed by full-length recombinant 2GPI and full-length recombinant 2GPI mutated at position 8 (D8A). When we absorbed type A anti-2GPI antibodies with full-length recombinant 2GPI that had point mutations at 1 amino acid within the epitope G40-R43, namely R40E or R43G, 35% to 40% of the reactivity against 2GPI remained (Figure 2). This strongly suggests that these amino acids are part of the epitope to which type A anti-2GPI antibodies are directed. We previously reported that 2GPI-dependent LAC activity is a strong risk factor for thrombosis.22 We now demonstrated that 23 (92%) of 25 samples with 2GPI-dependent LAC activity contained type A anti-2GPI antibodies and that presence of type A antibodies is a strong risk factor for thrombosis (Table 4). The finding that reconstitution of 2GPI-depleted plasma with full-length 2GPI and type A anti-2GPI antibodies induces more prolongation of the clotting time than reconstitution with R43G and type A anti-2GPI antibodies suggested that type A anti-2GPI antibodies cause LAC activity. As a 2GPI-dependent LAC activity correlates very strongly with thrombosis (odds ratio, 42.3), it also supports the concept that pathologic antibodies are directed against the epitope G40-R43.
Collectively, type B antibodies directed to 2GPI are not correlated with a history of thrombotic complications. Whether this population contains clinical relevant subpopulations of antibodies needs further studies.
In conclusion, our study shows that anti-2GPI antibodies react with different epitopes on 2GPI. However, a substantial number of patient samples with anti-2GPI antibodies (in our series 58%) recognize an epitope on domain I, including positions 40 and 43 (type A antibodies). The strong correlation between presence of such type A antibodies with histories of thrombosis and presence of 2GPI-dependent LAC activity strongly suggests that type A antibodies constitute a pathologic subset. Our observations form the basis for a simple assay (using domain I coated onto hydrophilic and hydrophobic ELISA plates) for the detection of type A anti-2GPI antibodies.
Footnotes
Prepublished online as Blood First Edition Paper, October 26, 2004; DOI 10.1182/blood-2004-09-3387.
Supported by a grant of the Netherlands Organisation for Health Research and Development (ZonMw grant no. 902-26-290).
An Inside Blood analysis of this article appears in the front of this issue.
The publication costs of this article were defrayed in part by page charge payment. Therefore, and solely to indicate this fact, this article is hereby marked "advertisement" in accordance with 18 U.S.C. section 1734.
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