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Abstract

  1. Top of page
  2. Abstract
  3. PATIENTS AND METHODS
  4. RESULTS
  5. DISCUSSION
  6. AUTHOR CONTRIBUTIONS
  7. ADDITIONAL DISCLOSURES
  8. Acknowledgments
  9. REFERENCES

Objective

To examine the prevalence of isolated IgA anti–β2-glycoprotein I (anti-β2GPI) positivity and the association of these antibodies, and a subgroup that bind specifically to domain IV/V of β2GPI, with clinical manifestations of the antiphospholipid syndrome (APS) in 3 patient groups and to evaluate the pathogenicity of IgA anti-β2GPI in a mouse model of thrombosis.

Methods

Patients with systemic lupus erythematosus (SLE) from a multiethnic, multicenter cohort (LUpus in MInorities, NAture versus nurture [LUMINA]) (n = 558), patients with SLE from the Hopkins Lupus Cohort (n = 215), and serum samples referred to the Antiphospholipid Standardization Laboratory (APLS) (n = 5,098) were evaluated. IgA anti-β2GPI titers and binding to domain IV/V of β2GPI were examined by enzyme-linked immunosorbent assay (ELISA). CD1 mice were inoculated with purified IgA anti-β2GPI antibodies, and surgical procedures and ELISAs were performed to evaluate thrombus development and tissue factor (TF) activity.

Results

A total of 198 patients were found to be positive for IgA anti-β2GPI isotype, and 57 patients were positive exclusively for IgA anti-β2GPI antibodies. Of these, 13 of 23 patients (56.5%) in the LUMINA cohort, 17 of 17 patients (100%) in the Hopkins cohort, and 10 of 17 patients (58.9%) referred to APLS had at least one APS-related clinical manifestation. Fifty-four percent of all the IgA anti-β2GPI–positive serum samples reacted with domain IV/V of anti-β2GPI, and 77% of those had clinical features of APS. Isolated IgA anti-β2GPI positivity was associated with an increased risk of arterial thrombosis (P < 0.001), venous thrombosis (P = 0.015), and all thrombosis (P < 0.001). The association between isolated IgA anti-β2GPI and arterial thrombosis (P = 0.0003) and all thrombosis (P = 0.0003) remained significant after adjusting for other risk factors for thrombosis. In vivo mouse studies demonstrated that IgA anti-β2GPI antibodies induced significantly larger thrombi and higher TF levels compared to controls.

Conclusion

Isolated IgA anti-β2GPI–positive titers may identify additional patients with clinical features of APS. Testing for these antibodies when other antiphospholipid tests are negative and APS is suspected is recommended. IgA anti-β2GPI antibodies directed to domain IV/V of β2GPI represent an important subgroup of clinically relevant antiphospholipids.

The current classification criteria for the antiphospholipid syndrome (APS) do not include determination of the presence of IgA anticardiolipin (aCL) or anti–β2-glycoprotein I (anti-β2GPI) antibodies ([1]). IgA aCL antibodies are more frequently found in African Caribbean populations, usually in association with other IgG and/or IgM aCL antibodies. IgA aCL antibodies have been shown to be pathogenic in animal models, but their clinical significance has remained elusive ([2, 3]). Previous studies have highlighted the association of IgA anti-β2GPI positivity with clinical manifestations of APS and have shown that systemic lupus erythematosus (SLE) patients with APS appear to be more prone to being positive for the IgA isotype ([4-6]). Of particular interest is the study conducted by Fanopoulos et al, which demonstrated that IgA positivity occurred more frequently and at higher titers in SLE patients with APS manifestations ([7]). Recently, Mehrani et al reported that IgA anti-β2GPI antibodies were more strongly associated with deep venous thrombosis (DVT) and stroke than the IgM isotype ([8]). In addition, it has been suggested that IgA anti-β2GPI antibodies may recognize epitopes in domain IV/V of β2GPI, and these antibodies appear to be associated with certain manifestations of APS ([9, 10]). The majority of these studies, however, describe patients who were also positive for other isotypes of antiphospholipid antibodies, limiting conclusions that can be drawn with respect to the clinical associations of IgA anti-β2GPI and aCL antibodies.

Recently, our group reported 5 isolated cases of individuals who were exclusively positive for IgA anti-β2GPI and had concomitant clinical manifestations of APS ([11]). Subsequently, Sweiss et al found that the presence of isolated IgA anti-β2GPI positivity was associated with an increased occurrence of thromboembolic events in a small group of patients, especially among patients with SLE ([12]). Isolated IgA anti-β2GPI positivity has also been reported in scleroderma and in autoimmune hepatitis and has been shown to correlate with disease severity and endothelial damage ([13, 14]). However, the clinical importance of isolated IgA anti-β2GPI positivity is largely unknown.

Our aim was therefore to determine the prevalence of isolated IgA anti-β2GPI antibody and to correlate its presence with APS-related clinical manifestations in 3 large groups of patients. In addition, we further examined the clinical relevance of IgA anti-β2GPI antibodies binding to domain IV/V of β2GPI and the pathogenicity of IgA anti-β2GPI antibodies in a mouse model of thrombosis.

PATIENTS AND METHODS

  1. Top of page
  2. Abstract
  3. PATIENTS AND METHODS
  4. RESULTS
  5. DISCUSSION
  6. AUTHOR CONTRIBUTIONS
  7. ADDITIONAL DISCLOSURES
  8. Acknowledgments
  9. REFERENCES

Patients and demographics

Patient serum samples were obtained from 3 independent sources: 588 from the LUpus in MInorities, NAture versus nurture (LUMINA) cohort, 215 from the Hopkins Lupus Cohort (Johns Hopkins University), and 5,098 sent to the Antiphospholipid Standardization Laboratory (APLS; University of Texas Medical Branch) between January 2008 and March 2010 for antiphospholipid antibody evaluation. Of these 5,098 APLS samples, 35 were found to be positive for IgA anti-β2GPI. We obtained APS-related clinical information about this subset of patients by medical chart review. LUMINA is a longitudinal study of outcomes in multiethnic SLE patients (Hispanic [Mexican/Central American and Puerto Rican], African American, and white) enrolled within 5 years of fulfillment of the American College of Rheumatology criteria at participating institutions in Alabama, Houston, Galveston, and Puerto Rico ([15, 16]). The Hopkins Lupus Cohort is a longitudinal study of lupus activity, organ damage, and quality of life in SLE patients. The demographic composition is balanced between white and African American patients with SLE. The APLS Laboratory is a reference laboratory that routinely evaluates serum and plasma samples from patients referred for APS evaluation.

We evaluated all patients for classic manifestations of APS as per the revised Sapporo criteria ([1]). We also explored nontraditional APS manifestations, such as seizures, valvular heart disease, pulmonary arterial hypertension, thrombocytopenia, livedo reticularis, skin ulcers, osteonecrosis, transverse myelitis, and headache. Other information, such as age at time of enrollment and history of smoking, and the presence of variables such as obesity (body mass index ≥30 kg/m2), pregnancy, end-stage renal disease (serum creatinine level of >3.0 mg/dl), and hydroxychloroquine, nonsteroidal antiinflammatory drug (NSAID), and/or oral contraceptive use, at the time of the visit during which the blood sample used in this study was drawn, were also available for the 3 groups of patients.

The institutional review boards of the respective institutions approved the use of samples and clinical data from all patients. The study was conducted according to the guidelines of the Declaration of Helsinki for the inclusion of human subjects in research, and informed consent was obtained from all subjects.

Antiphospholipid antibody testing

Anticardiolipin antibodies (IgG, IgM, and IgA) were evaluated using an in-house enzyme-linked immunosorbent assay (ELISA) method as previously described ([17]). The IgG, IgM, and IgA anti-β2GPI antibodies were determined using at least 1 of 2 commercial ELISA kits (kit A [TheraTest] and kit B [Inova Diagnostics]). A number of samples were tested for IgA anti-β2GPI activity in both kits to assess the correlation of positivity between the two. To better characterize the antigenic target recognized by those IgA anti-β2GPI antibodies, we performed an ELISA to detect IgA antibodies specific to domain IV/V of β2GPI (Research Use Only; Inova) on a selected number of positive samples (n = 126).

All assays were performed according to the manufacturer's instructions and were considered positive when titers were above the manufacturer's pre-established cutoff points. (For aCL assays, ≥10 IgG phospholipid units or IgM phospholipid units and ≥15 IgA phospholipid units; for Inova IgA anti-β2GPI assays, >20 standard IgA anti-β2GPI antibody units (SAU); for TheraTest IgA, IgG, and IgM anti-β2GPI assays, >4 anti-β2GPI units; and for domain IV/V, >20 units.)

IgA anti-β2GPI antibodies in a mouse model of thrombosis

IgA from the pooled sera of 4 patients with IgA anti-β2GPI titers ≥80 SAU (APS IgA) (2 had strokes, 1 had a confirmed DVT, and 2 had pregnancy losses) and IgA from the serum of a healthy subject with no evidence of inflammatory or thrombotic disease (control IgA) were purified by affinity chromatography using Immobilized Jacalin columns according to the recommendations of the manufacturer (Pierce). Endotoxin was removed from solutions of purified IgA antibodies using Detoxi-Gel Endotoxin Removing Columns (Thermo Fisher Scientific) and confirmed to be endotoxin free by Limulus amebocyte cell lysate assay (Sigma-Aldrich). The concentration of purified IgA was determined by the Bradford method, and the total IgA anti-β2GPI activity and the specific IgA binding activity to domain I and domain IV/V of β2GPI were determined by ELISA using commercial assays (Inova Diagnostics). Lupus anticoagulant (LAC) activity was measured using a modified silica clotting time assay (HemosIL; Beckman Coulter).

CD1 mice (The Jackson Laboratory) were used for animal experiments. The mice (n = 5 per group) were injected intraperitoneally with either APS IgA (500 μg) or control IgA (500 μg) on 2 occasions at 48-hour intervals. Surgical procedures were performed to study thrombus dynamics 72 hours after the first injection was administered, as previously described ([3]). Tissue factor (TF) activity was subsequently determined in mouse peritoneal macrophages and carotid homogenates using a chromogenic assay (Assaypro).

Statistical analysis

The strengths of the associations between IgA anti-β2GPI positivity and thromboses were measured by calculating the odds ratio (OR) and 95% confidence interval (95% CI) using univariate and multivariate logistic regression (SAS version 9.2 software, SAS Institute). The correlation of IgA anti-β2GPI titers between kits was evaluated using Spearman's rho (PASW version 18.0 software, SPSS). Student's t-test was used to determine differences in mean thrombus size and TF activity between mice treated with APS IgA and those treated with control IgA.

RESULTS

  1. Top of page
  2. Abstract
  3. PATIENTS AND METHODS
  4. RESULTS
  5. DISCUSSION
  6. AUTHOR CONTRIBUTIONS
  7. ADDITIONAL DISCLOSURES
  8. Acknowledgments
  9. REFERENCES

Prevalence of IgA anti-β2GPI antibodies

The demographic characteristics of the patients from the Hopkins and LUMINA cohorts are summarized in Table 1. Of the 35 patients in the APLS group found to be positive for IgA anti-β2GPI, 25 (71.4%) were women. Most were either white (12 of 35 [34.3%]) or of African descent (10 of 35 [28.5%]); other ethnic groups accounted for the rest (13 of 35 [37.1%]). The mean age of these patients was 42.6 years (range 23–70 years) and 14 of 35 (40.0%) had SLE. In addition to the 35 patients from the APLS group, 129 of the 588 patients from the LUMINA cohort (21.9%) and 34 of the 215 patients from the Hopkins cohort (15.8%) were positive for IgA anti-β2GPI. A total of 57 of these patients were positive for IgA anti-β2GPI exclusively (negative for aCL and IgG and IgM anti-β2GPI; data not shown), including 23 from the LUMINA cohort, 17 from the Hopkins Lupus Cohort, and 17 from the APLS group.

Table 1. Demographic characteristics of the 2 groups of patients with SLE*
 LUMINA (n = 588)Hopkins Lupus Cohort (n = 215)P
  1. SLE = systemic lupus erythematosus; LUMINA = LUpus in MInorities, NAture versus nurture.

Sex, % female/male86/1490/100.117
Ethnicity, %  <0.001
African American43.039.1 
White22.052.1 
Other35.08.8 
Age, mean (range) years36.8 (19–66)45.9 (19–81)<0.001

In order to confirm that these results were not kit specific, the IgA anti-β2GPI activity in 126 positive samples identified by kit A was re-evaluated with kit B, and 97 of 126 (77.0%) were also positive when tested using kit B. Despite both assays expressing results in different arbitrary units, there was good correlation between the kits (Spearman's ρ = 0.78, P < 0.001) (Figure 1).

image

Figure 1. Correlation of IgA anti–β2-glycoprotein I (anti-β2GPI) titers in two assays. A scatterplot of IgA anti-β2GPI titers of 126 samples tested using kit A and kit B, as described in Patients and Methods, is shown. Results were expressed as units/ml in kit A and as standard IgA anti-β2GPI antibody units (SAU) in kit B.

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Prevalence of IgA aCL

Of the 198 cases of individuals who were positive for IgA anti-β2GPI, 12 were also positive for IgA aCL. IgA aCL positivity occurred in complete isolation in only 2 cases.

Binding of IgA to anti-β2GPI domain IV/V

Of the 115 IgA anti–β2GPI-positive samples from the LUMINA and APLS cohorts (80 from LUMINA and 35 from APLS) that were evaluated for binding to domain IV/V of β2GPI, 62 (54%) were found to be positive in this assay. The correlation of IgA anti-β2GPI domain IV/V titers and IgA anti-β2GPI titers was 0.81 (P < 0.0001).

Clinical manifestations in patients with isolated IgA anti-β2GPI positivity and IgA anti-β2GPI domain IV/V positivity

A considerable number of the subjects with isolated IgA anti-β2GPI positivity in the 3 groups had at least one APS-related clinical manifestation (Table 2). These included classic features such as DVT and arterial thrombosis (stroke, myocardial infarction, and transient ischemic attacks) and miscarriages. Nontraditional APS manifestations included seizures, valvular heart disease, avascular necrosis, pulmonary hypertension, thrombocytopenia, superficial thrombophlebitis, livedo reticularis, skin ulcers, transverse myelitis, and headache. Of the 62 patients whose samples were positive for binding of IgA to anti-β2GPI domain IV/V, 77% had clinical manifestations of APS that included DVT, strokes, myocardial infarction, pulmonary arterial hypertension, seizures, pregnancy losses, skin ulcers, and livedo reticularis.

Table 2. Clinical manifestations in patients with isolated IgA positivity*
Clinical manifestationsaLUMINA (n = 23)Hopkins Lupus Cohort (n = 17)APLS (n = 17)
  1. Except where indicated otherwise, values are the number (%) of patients. LUMINA = LUpus in MInorities, NAture versus nurture; APLS = Antiphospholipid Standardization Laboratory; NA = not available; ESRD = end-stage renal disease; SLAM-R = Systemic Lupus Activity Measure, Revised; SELENA–SLEDAI = Safety of Estrogens in Lupus Erythematosus National Assessment version of the Systemic Lupus Erythematosus Disease Activity Index.

  2. a

    Includes traditional and nontraditional antiphospholipid syndrome–associated manifestations.

All thrombosis (arterial and/or venous)6 (26.1)12 (70.6)6 (35.3)
Pregnancy loss/complications3 (13.0)9 (52.9)1 (5.8)
Stroke1 (4.3)4 (23.5)2 (11.7)
Seizure1 (4.3)6 (35.3)0 (0)
Myocardial infarction2 (8.7)4 (23.5)0 (0)
Valvular heart disease1 (4.3)1 (5.8)0 (0)
Pulmonary arterial hypertension0 (0)5 (29.4)0 (0)
Skin ulcers1 (4.3)0 (0)0 (0)
Livedo reticularisNA3 (17.6)0 (0)
Thrombocytopenia5 (21.7)7 (41.2)2 (11.7)
ESRD0 (0)3 (17.6)1 (5.8)
SLAM-R, mean ± SD7.30 ± 5.49NANA
SELENA-SLEDAI, mean ± SDNA4.04 ± 3.53NA

Association of IgA anti-β2GPI with thrombosis in SLE patients

Of the 817 patients with SLE from the LUMINA and Hopkins cohorts, 202 (24.7%) had some form of thrombosis. Arterial thrombosis was seen in 95 patients (11.6%) and venous thrombosis in 132 (16.2%). Patients with overall IgA anti-β2GPI positivity (IgA positivity in the presence or absence of other isotypes) were significantly more likely to have arterial thrombosis than patients who were negative for this antibody (17.0% versus 10.4%; OR 1.8 [95% CI 1.1–2.8], P = 0.018). Overall IgA anti-β2GPI positivity was also significantly associated with all forms of thrombosis (31.6% of positive patients versus 23.3% of negative patients; OR 1.5 [1.1–2.2], P = 0.027). Although venous thrombosis occurred more frequently in patients who were positive for this antibody than in those who were not (20.5% versus 15.3%), the difference was not statistically significant (P = 0.103). After adjusting for age, obesity, pregnancy, use of oral contraceptives, end-stage renal disease, and use of hydroxychloroquine or NSAIDs at the time when the blood samples were obtained, as well as for ever smoking, IgA anti-β2GPI positivity remained significantly associated with both arterial thrombosis (adjusted P = 0.021) and all thrombosis (adjusted P = 0.016) but still had no significant association with venous thrombosis (adjusted P = 0.12) (Table 3).

Table 3. Association of anti-β2GPI IgA positivity with thrombosis*
Antiphospholipid antibodyArterial thrombosisVenous thrombosisAll thrombosis
  1. P values represent comparisons of autoantibody-positive versus autoantibody-negative patients. Anti-β2GPI = anti–β2-glycoprotein I; OR = odds ratio; 95% CI = 95% confidence interval.

  2. a

    Adjusted for age at visit, smoking, obesity (body mass index), pregnancy at visit, oral contraceptives, end-stage renal disease (measured by serum creatinine level), hydroxychloroquine treatment, and nonsteroidal antiinflammatory drug treatment.

IgA anti-β2GPI   
Positive, %17.020.531.6
Negative, %10.415.323.3
OR (95% CI)1.8 (1.1–2.8)1.4 (0.9–2.2)1.5 (1.1–2.2)
P0.0180.1030.027
Adjusted OR (95% CI)a2.2 (1.1–4.4)1.6 (0.9–2.8)1.9 (1.1–3.3)
P0.0210.1200.016
Isolated IgA anti-β2GPI   
Positive, %31.829.550.0
Negative, %10.615.623.6
OR (95% CI)3.9 (2.0–7.7)2.3 (1.2–4.5)3.2 (1.8–6.0)
P<0.0010.015<0.001
Adjusted OR (95% CI)a5.8 (2.3–15.2)2.3 (1.0–5.4)5.1 (2.2–12.4)
P0.00030.0610.0003

Isolated IgA anti-β2GPI positivity was associated with all forms of thrombosis: 31.8% versus 10.6% for arterial thrombosis (OR 3.9 [95% CI 2.0–7.7], P < 0.001), 29.5% versus 15.6% for venous thrombosis (OR 2.3 [95% CI 1.2–4.5], P = 0.015), and 50.0% versus 23.6% for all thrombosis (OR 3.2 [1.8–6.0], P < 0.001) in the 3 cohorts (Table 3). After adjusting for age, obesity, pregnancy, use of oral contraceptives, end-stage renal disease, use of hydroxychloroquine or NSAIDs at the time of visit when the blood samples were obtained, and for ever smoking, isolated IgA anti-β2GPI positivity remained significantly associated with arterial and all thrombosis, and the strength of association increased (OR 5.8, adjusted P = 0.0003 and OR 5.1, P = 0.0003, respectively). However, the association with venous thrombosis did not reach statistical significance (P = 0.061) (Table 3).

Pathogenicity of IgA anti-β2GPI antibodies in a mouse model of thrombosis

The final concentration of total IgA in the fraction obtained from APS patients (APS IgA) was 40.4 mg/dl, while the concentrations of IgG and IgM were below the lower limits of the assay. The APS IgA preparation had high levels of total IgA anti-β2GPI (103.7 SAU) but was negative for IgG (8.7 standard IgG anti-β2GPI antibody units) and IgM (6.6 standard IgM anti-β2GPI antibody units) anti-β2GPI antibodies. The APS IgA preparation also had high IgA binding activity for domain I and domain IV/V of β2GPI at several dilutions, as shown in Table 4. The control IgA had a total IgA concentration of 67.6 mg/dl but was negative for IgA, IgG, and IgM anti-β2GPI antibodies. The LAC activity of the APS IgA preparation was greater than that of the control IgA preparation (silica clotting time ratio of APS IgA to control IgA = 2; normal <1.2). The mean area of the thrombi induced in mice inoculated with APS IgA was significantly higher than that in mice inoculated with control IgA. Similarly, TF activity in the peritoneal macrophages and carotid homogenates of mice treated with APS IgA was significantly greater than in mice treated with control IgA (Figure 2).

Table 4. Specific IgA binding activity of purified whole IgA from APS patients to domains I and IV/V at several dilutions*
MaterialIgA domain IIgA domain IV/V
ODUnitsODUnits
  1. APS = antiphospholipid syndrome; OD = optical density.

APS IgA dilution    
Neat3.624163.73.655268.8
1:200.65829.70.80259.0
1:400.35716.20.45933.8
1:800.1968.90.27920.5
1:1000.1767.90.19514.3
Negative control0.0170.70.0392.9
image

Figure 2. Thrombus formation and tissue factor (TF) activity in mice inoculated with IgA anti–β2-glycoprotein I (anti-β2GPI) antibodies. Thrombus size (1.7-fold increase), TF activity in carotid homogenates (2.9-fold increase), and TF activity in peritoneal macrophages (3.5-fold increase) were significantly elevated in IgA anti-β2GPI–inoculated mice compared to controls inoculated with IgA antibodies from normal human serum (NHS). ∗ = P = 0.02; ¶ = P < 0.01 versus mice inoculated with antiphospholipid syndrome (APS) IgA.

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DISCUSSION

  1. Top of page
  2. Abstract
  3. PATIENTS AND METHODS
  4. RESULTS
  5. DISCUSSION
  6. AUTHOR CONTRIBUTIONS
  7. ADDITIONAL DISCLOSURES
  8. Acknowledgments
  9. REFERENCES

In this study, we demonstrated a fair prevalence of IgA anti-β2GPI positivity in the LUMINA and Hopkins cohorts of SLE patients. Our prevalence rates were similar to those of some previous studies ([5, 8]), including one of the Hopkins Lupus Cohort that reported 20.2% positivity of the IgA isotype in 796 patients, but lower than reported rates in other studies ([7, 18]). Importantly, we demonstrated for the first time that IgA anti-β2GPI was the sole antiphospholipid antibody present in 18% and 50% of cases in the LUMINA cohort and in the Hopkins Lupus Cohort, respectively. Additionally, we showed that a large number of the IgA anti-β2GPI antibodies present in our patients reacted preferentially against domain IV and V of β2GPI, confirming previous observations ([9, 10]). We have also shown quite clearly, and for the first time, that IgA anti-β2GPI antibodies, particularly in the absence of other antiphospholipid antibodies, are associated with clinically significant thrombosis, especially arterial thrombosis. Furthermore, we have demonstrated the pathogenicity of purified IgA anti-β2GPI antibodies, with binding activity for domains I and IV/V, in a mouse model of thrombosis.

Our study used two different commercial kits that express IgA anti-β2GPI titers in different units of measurements and have different cutoff points and reference intervals, and yet showed very good correlation, confirming that IgA anti-β2GPI positivity was not kit specific. Of note, kit A identified more positive samples when compared to kit B (127 versus 96 positive samples). The differences observed cannot be attributed to cross-reactivity with similar antibodies of the IgG or IgM isotype, since both kits utilize α-chain–specific affinity-purified anti-human IgA antibodies as secondary antibody, but rather to increased sensitivity of one kit versus the other. Other kit-related characteristics, such as the type and/or amount of β2GPI used to coat the ELISA plates or other features of the assays not disclosed by the manufacturer, may account for these apparent discrepancies.

Prior studies have shown an association of IgA anti-β2GPI with clinical manifestations of APS, but these findings are still a subject of controversy. Several studies have demonstrated that SLE patients with APS are more prone to being positive for the IgA isotype ([4, 5]). Fanopoulos et al found that in a cohort of 48 patients with SLE, the most prevalent isotype was IgA anti-β2GPI antibody (58%). Importantly, significantly higher frequencies (P < 0.001) and titers (P < 0.05) of IgA anti-β2GPI were observed in patients with APS than in those without APS manifestations ([7]). Lakos et al found that the prevalence of IgA anti-β2GPI antibody in their cohort of 70 patients was 59.3% and that this assay had the highest specificity (83%) for the diagnosis of APS ([19]). They also reported an association of this isotype with venous thrombosis (P = 0.007), thrombocytopenia (P = 0.02), livedo reticularis (P = 0.01), epilepsy (P = 0.01), and valvular heart disease (P = 0.02) ([18, 20, 21]).

Furthermore, it seems that IgA anti-β2GPI antibodies are independent risk factors for acute myocardial infarction and atherosclerosis in populations without APS ([18, 20, 21]), and the same positive association was found for acute cerebral ischemia ([22]). Cucurull et al studied both IgA aCL and anti-β2GPI antibodies in African American patients with SLE and found an association between thrombotic events and elevated levels of both of these autoantibodies ([2]). Recently, Mehrani et al found that the IgA isotype was strongly associated with DVT and stroke as compared to the IgM isotype ([8]). Other researchers have demonstrated the association of IgA anti-β2GPI antibodies with obstetric complications in APS patients ([23, 24]).

It is clear that IgA anti-β2GPI antibodies have an association with many clinical manifestations in APS. What remains uncertain is the significance of these antibodies as sole entities in different patient populations and the pathogenic effects most attributed to their presence. We recently published a case series, reporting 5 isolated cases of individuals who were exclusively positive for IgA anti-β2GPI and had concomitant clinical manifestations of APS ([11]). Another study showed that IgA anti-β2GPI positivity was associated with an increase in thromboembolic events in a small subset of patients with SLE (n = 31) ([12]). In addition, Shen et al ([25]) retrospectively examined 472 patients for whom clinical information on thrombotic events and complete laboratory evaluation for antiphospholipid antibodies were available. Univariate analysis showed a statistically significant risk of thrombosis in patients with elevated titers of IgA of any ELISA-based antiphospholipid antibodies (aCL, antiphosphatidylserine, and anti-β2GPI; OR 1.77). Stepwise logistic regression (multivariate) analysis identified elevated titers of any ELISA-based IgA antibody as an independent risk factor for thrombosis (OR 1.6) in the entire cohort and in a subgroup of patients without concurrent LAC positivity (OR 1.8) ([25]). Our analysis of the association of isolated IgA anti-β2GPI positivity with the increased risk of thromboembolic events included a total of 817 subjects from 3 independent groups of patients, using multivariate analysis and adjusting for possible confounding factors.

In the present study, we demonstrated an association of IgA anti-β2GPI antibodies with thrombosis, specifically arterial thrombosis and an association of isolated IgA anti-β2GPI positivity with both venous and arterial thrombosis. After adjusting for confounding factors, isolated IgA anti-β2GPI positivity remained significantly associated with arterial thrombosis, but not with venous thrombosis. Perhaps this indicates that IgA anti-β2GPI antibodies in certain subgroups of patients are more thrombogenic than other isotypes. Further subset analysis will be needed to identify possible markers that would identify these groups of patients and possibly identify predisposing clinical and ethnic components.

Thrombogenic effects of IgG and IgM antiphospholipid antibodies using in vitro and in vivo animal models have been shown ([3, 26, 27]). However, similar evidence demonstrating the pathogenic effects of IgA antiphospholipid antibodies has been comparatively limited. In a mouse model designed to study thrombus formation, injected IgA immunoglobulins with aCL activity from 2 patients with APS were shown to cause thrombosis ([28]). We have now also demonstrated, for the first time, that IgA anti-β2GPI antibodies induce increased thrombus formation and the up-regulation of TF activity using the same mouse model.

IgA anti-β2GPI antibody positivity was not included in the revised classification criteria for APS in 2006, due to lack of supporting evidence at that time ([1]). The question of whether IgA anti-β2GPI may have diagnostic value for APS was subsequently addressed by the “noncriteria” antiphospholipid task force during the 13th International Congress on Antiphospholipid Antibodies held in April 2010 in Galveston, Texas ([29]). The task force concluded that the IgA anti-β2GPI antibodies should be tested in the presence of clinical signs and symptoms of SLE and/or APS, particularly when other antiphospholipid tests are negative. Importantly, the Systemic Lupus International Collaborating Clinics group has recently proposed that anti-β2GPI antibodies be included as serologic markers of SLE as part of the revised classification criteria for SLE, including the IgA isotype ([30]). Our study provides enough data to indicate that isolated IgA anti-β2GPI positivity should be considered for APS diagnosis and classification in the presence of clinical signs and symptoms. There is still a need for larger longitudinal studies in order to confirm the relative importance of this laboratory marker in the diagnosis of APS, either primary or secondary. Such studies would help to define its value as a marker for disease activity and risk for thrombosis.

AUTHOR CONTRIBUTIONS

  1. Top of page
  2. Abstract
  3. PATIENTS AND METHODS
  4. RESULTS
  5. DISCUSSION
  6. AUTHOR CONTRIBUTIONS
  7. ADDITIONAL DISCLOSURES
  8. Acknowledgments
  9. REFERENCES

All authors were involved in drafting the article or revising it critically for important intellectual content, and all authors approved the final version to be published. Dr. Pierangeli had full access to all of the data in the study and takes responsibility for the integrity of the data and the accuracy of the data analysis.

Study conception and design. Willis, Jatwani, Alarcón, Sunkureddi, Tarantino, Akhter, Gonzalez, Teodorescu, Reveille, Petri, Pierangeli.

Acquisition of data. Murthy, Willis, Romay-Penabad, Ruiz-Limón, Martínez-Martínez, Jatwani, Jajoria, Seif, Alarcón, Papalardo, Liu, Vilá, McGwin, Maganti, Parekh, Tarantino, Gonzalez, Binder, Norman, Shums, Pierangeli.

Analysis and interpretation of data. Murthy, Willis, Ruiz-Limón, Martínez-Martínez, Alarcón, McGwin, McNearney, Fang, Gonzalez, Binder, Norman, Petri, Pierangeli.

ADDITIONAL DISCLOSURES

  1. Top of page
  2. Abstract
  3. PATIENTS AND METHODS
  4. RESULTS
  5. DISCUSSION
  6. AUTHOR CONTRIBUTIONS
  7. ADDITIONAL DISCLOSURES
  8. Acknowledgments
  9. REFERENCES

Author McNearney is currently an employee of Eli Lilly, but was employed by University of Texas Medical Branch, Galveston, during the time the study was conducted. Authors Binder, Norman, and Shums are employees of Inova Diagnostics. Author Teodorescu is an employee of TheraTest Laboratories.

REFERENCES

  1. Top of page
  2. Abstract
  3. PATIENTS AND METHODS
  4. RESULTS
  5. DISCUSSION
  6. AUTHOR CONTRIBUTIONS
  7. ADDITIONAL DISCLOSURES
  8. Acknowledgments
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