Asymptomatic aPL Carriers Research Articles

Association of anti-β2-glycoprotein I/HLA-DR complex antibody with arterial thrombosis in female patients with systemic rheumatic diseases

Backgroundβ2-glycoprotein I (β2GPI) complexed with human leukocyte antigen DR (β2GPI/HLA-DR) was found to be a major autoantibody target in antiphospholipid syndrome (APS). This study aimed to reveal the association between anti-β2GPI/HLA-DR antibodies and vascular thromboses in women with systemic rheumatic diseases.MethodsWe conducted a retrospective longitudinal study. We measured anti-β2GPI/HLA-DR antibodies and compared them with anti-phospholipid antibody (aPL) profiles and the adjusted global antiphospholipid syndrome score (aGAPSS). Using receiver operating characteristic (ROC) analysis, we determined the best cut-off value for arterial thrombosis. We also evaluated the validity of anti-β2GPI/HLA-DR antibodies by adding to conventional cardiovascular risk factors in multivariate logistic analysis.ResultsWe evaluated 704 patients, including 66 (obstetric or thrombotic) APS, 13 primary APS, and 78 asymptomatic aPL carriers. Seventy-seven patients had a history of arterial thrombosis, and 14 patients had both arterial and venous thrombosis. These 14 patients, as well as patients with aGAPSS > 10 or triple-positive aPL profiles, displayed high anti-β2GPI/HLA-DR antibody titers. The ROC curve showed a sensitivity, specificity, and area under the curve (AUC) for arterial thrombosis of 33.8%, 91.4%, and 0.6009, respectively, with a cut-off value of 172.359 U/mL. The anti-β2GPI/HLA-DR antibody positivity using this cut-off value yielded an odds ratio of 5.13 (95%CI: 2.85–9.24), significantly improving the AUC from 0.677 to 0.730.ConclusionAnti-β2GPI/HLA-DR antibodies are associated with arterial thrombosis in female patients with systemic rheumatic diseases.

Identifying high-risk profile in primary antiphospholipid syndrome through cluster analysis: French multicentric cohort study

IntroductionAntiphospholipid syndrome (APS) is an autoimmune disease characterised by thrombosis (arterial, venous or small vessel) or obstetrical events and persistent antiphospholipid antibodies (aPL), according to the Sydney classification criteria. Many...

Evaluation of a Gene Signature Related to Thrombotic Manifestations in Antiphospholipid Syndrome

Introduction: Whether different manifestations of thrombotic antiphospholipid syndrome (APS) share pathological mechanisms has not been established. Transcriptome analysis may constitute a new approach to evaluate the mechanisms behind thrombotic manifestations in APS. Aim: To determine in patients with primary thrombotic APS (t-PAPS) and aPL asymptomatic (individuals carrying the antibodies but without clinical manifestation) the expression of genes already related to venous and arterial thrombosis in general population compared with control. Method: mRNA was obtained from total leucocyte and gene expression was measured by qPCR and the results were analyzed by QuantStudio™ Software. Results: 100 t-PAPS, 17 asymptomatic aPL carriers and 100 control individuals were included. The group of t-PAPS were 63.73% female, and the median age was 40 years (IQR 31-53). Asymptomatic aPL carriers were mostly female (82.36%) and the median age was 36 years (IQR 24-59). Controls were mostly female (67%) and their median was 39 years (IQR 29-49). No significant demographic differences between the groups was observed. Cardiovascular risk factors were more prevalent among t-PAPS than among asymptomatic aPL carriers and controls (37.25%, 5.88% and 9% respectively). ANXA3 (P=0.0292) and TNFAIP6 (P=0.0006) mRNA were up-regulated while TXK (P=< 0.0001), BACH2 (P=0.0085) and SERPINB2 (P=0.0491) were down-regulated in t-PAPS when compared to controls. In aPL carriers, the relative expression levels of TNFAIP6 were similar to t-PAPS while that of BACH was similar to controls; as shown in Figure 1. In a subgroup analysis, ANXA3 and TNFAIP6 mRNA expression was more pronounced in t-PAPS with multiple thrombosis (P= 0.0027 and P=< 0.0001 respectively), while TXK mRNA expression was lower in all t-PAPS subgroups in comparison with controls, being particularly lower in patients who had first thrombotic event and non-triple positivity (P=< 0.0001 and P= < 0.0001 respectively), BACH2 mRNA expression was lower in t-PAPS subgroup with first thrombotic event (P= 0.0019) compared to the control group and SERPINB2 mRNA expression was only significantly lower in t-PAPS with first thrombotic event (P= 0.0071). Table 1 demonstrates fold changes by different manifestations of t-PAPS. Discussion: We observed a dysregulation of genes related to hemostasis (ANXA3 and SERPINB2) and inflammation (TNFAIP6, TXK and BACH2) in t-PAPS, when compared to controls. ANXA3 plays a role in cell growth regulation and signal transduction pathways, and has previously been linked to stroke in animal studies. TNFAIP6 is involved in cell-cell and cell-matrix interactions during inflammation and tumorigenesis. Among TXK related pathways are the transmigration of immune cells and plays a role in regulating the adaptive immune response. BACH2 is responsible for immune regulation, mainly related to the adaptive immune response and has been implicated in B cell. SERPINB2 regulates the production of the plasminogen activator inhibitor-2 (PAI-2), being directly related to the hemostasis pathways. Such mechanisms were not evident among asymptomatic aPL carriers. In subgroup analysis, the relative expression of genes related to innate immunity has also been associated with signs of disease severity, such as multiple thrombosis and triple positivity. Conclusions: Our results showed that innate immunity and hemostasis pathways are associated with t-PAPS at a transcriptome level and innate immunity may play a role in disease severity. Acknowledgements: São Paulo Research Foundation FAPESP (2019/20136-0) Figure 1View largeDownload PPTFigure 1View largeDownload PPT Close modal

EVALUATION OF A GENE SIGNATURE RELATED TO THROMBOTIC MANIFESTATIONS IN ANTIPHOSPHOLIPID SYNDROME

Whether different manifestations of thrombotic antiphospholipid syndrome (APS) share pathological mechanisms has not been established. Transcriptome analysis may constitute a new approach to evaluating the mechanisms behind thrombotic manifestations in APS. To determine in patients with primary thrombotic APS (t-PAPS) and aPL asymptomatic (individuals carrying the antibodies but without clinical manifestation) the expression of genes already related to venous and arterial thrombosis in the general population compared with control. Method: mRNA was obtained from total leucocytes, gene expression was measured by qPCR, and the results were analyzed by QuantStudio™Software. 100 t-PAPS, 17 asymptomatic aPL carriers, and 100 control individuals were included. The group of t-PAPS was 63.73% female, and the median age was 40 years (IQR 31-53). Asymptomatic aPL carriers were predominantly female (82.36%) and the median age was 36 years (IQR 24-59). Controls were mostly female (67%) and their median was 39 years (IQR 29-49). No significant demographic differences between the groups were observed. Cardiovascular risk factors were more prevalent among t-PAPS than among asymptomatic aPL carriers and controls (37.25%, 5.88%, and 9% respectively). ANXA3 (p = 0.0292) and TNFAIP6 (p = 0.0006) mRNA were up-regulated while TXK (p = < 0.0001), BACH2 (p = 0.0085) and SERPINB2 (p = 0.0491) were down-regulated in t-PAPS when compared to controls. In aPL carriers, the relative expression levels of TNFAIP6 were similar to t-PAPS while that of BACH was similar to controls; as shown in Figure 1. In a subgroup analysis, ANXA3 and TNFAIP6 mRNA expression were more pronounced in t-PAPS with multiple thrombosis (P = 0.0027 and P = < 0.0001 respectively), while TXK mRNA expression was lower in all t-PAPS subgroups in comparison with controls, being particularly lower in patients who had first thrombotic event and non-triple positivity (p = < 0.0001 and p = < 0.0001 respectively), BACH2 mRNA expression was lower in t-PAPS subgroup with first thrombotic event (P = 0.0019) compared to the control group and SERPINB2 mRNA expression was only significantly lower in t-PAPS with first thrombotic event (p = 0.0071). Table 1 demonstrates fold changes by different manifestations of t-PAPS. We observed dysregulation of genes related to hemostasis ( ANXA3 and SERPINB2 ) and inflammation ( TNFAIP6 , TXK, and BACH2 ) in t-PAPS when compared to controls. ANXA3 plays a role in cell growth regulation and signal transduction pathways, and has previously been linked to stroke in animal studies. TNFAIP6 is involved in cell-cell and cell-matrix interactions during inflammation and tumorigenesis. Among TXK-related pathways is the transmigration of immune cells which plays a role in regulating the adaptive immune response. BACH2 is responsible for immune regulation, mainly related to the adaptive immune response, and has been implicated in B cell. SERPINB2 regulates the production of the plasminogen activator inhibitor-2 (PAI-2), directly related to the hemostasis pathways. Such mechanisms were not evident among asymptomatic aPL carriers. In subgroup analysis, the relative expression of genes related to innate immunity has also been associated with signs of disease severity, such as multiple thrombosis and triple positivity. Our results showed that innate immunity and hemostasis pathways are associated with t-PAPS at a transcriptome level and innate immunity may play a role in disease severity.

Association between interferon-I producing plasmacytoid dendritic cells and thrombotic antiphospholipid syndrome.

Background: Thrombotic risk in antiphospholipid syndrome (APS) is conferred by the association of antiphospholipid (aPL) antibodies (first hit) with additional pro-coagulant stimulus (second hit), such as inflammation. Among inflammatory responses, the production of large amounts of interferon (IFN)-I by plasmacytoid dendritic cells (pDCs) is at the basis of the pathophysiology of systemic autoimmune disorders, which raises the hypothesis that this mechanism could also be associated with vascular manifestations of APS. Purpose: Here, we determined the association of pDCs and IFN-I production with thrombotic APS. Research design: Patients with thrombotic primary (t-PAPS) and secondary APS (t-SAPS), asymptomatic aPL carriers and individuals without thrombosis (controls) were included. Data collection and analysis: Circulating pDCs and IFN-α intracellular expression (in the presence or not of oligodeoxynucleotides (CP) stimulus) were quantified by flow cytometry. The expression of five IFN-I inducing genes: ISG15, OASL, Ly6E, MX1, and OAS1 in mononuclear cells was determined by qPCR. Between-group differences were evaluated using chi-square or Kruskal-Wallis tests. Results: A total of 50 patients with t-PAPS, 50 patients with t-SAPS, 20 aPL carriers, and 50 individuals without thrombosis (controls) were included. Intracellular expression of IFN-α was increased after CPG stimulation in both t-SAPS (1.56%; IQR 1.07-2.02) and t-PAPS (0.96%; IQR 0.55-1.24), when compared to aPL carriers (0.71%; IQR 0.42-0.93) and controls (0.48%; IQR 0.24-0.78; p < .0001). ISG15, OASL, Ly6E, MX1, and OAS1 mRNA expressions were higher in t-SAPS (but not in t-PAPS) than in aPL carriers and controls. The expression of proteins and mRNA related to IFN-I response was similar between the triple aPL-positive profile and other aPL profiles. Conclusion: Our results indicate an association of IFN-I response and t-APS. Since IFN-I expression was not increased in aPL carriers or associated with a higher-risk aPL profile, this mechanism does not appear to be related to the presence of aPL alone. IFN-I response could possibly constitute a complementary mechanism for triggering clinical manifestations in APS.

Soluble LILRA3 is aberrantly expressed in antiphospholipid syndrome (APS) and is a potential marker of thrombotic APS.

Leucocyte immunoglobulin-like receptor A3 (LILRA3) belongs to a family of leucocyte receptors. Our previous study reported LILRA3 transcripts were markedly upregulated in neutrophils from patients with APS. We undertook this study to investigate clinical implications of LILRA3 in APS and its potential role in APS-associated thrombosis. Two independent cohorts were studied. The first consisted of 294 APS patients, 48 asymptomatic aPL carriers and 150 healthy controls (HCs) from Peking University People's Hospital. The second included 99 APS patients, 25 aPL carriers and 40 HCs from United States APS centres. Serum or plasma concentrations of LILRA3 and MPO-DNA complexes were measured. Additionally, 35 patients with thrombotic APS (tAPS) were evaluated to determine potential effects of immunosuppressive therapy on serum concentrations of LILRA3 and MPO-DNA complexes. Both positivity and serum concentration of LILRA3 were significantly increased in APS patients, especially in those with tAPS. LILRA3-positive tAPS patients displayed more severe thrombotic manifestations. Serum LILRA3 was positively correlated with MPO-DNA complexes in LILRA3-positive tAPS. After immunosuppressive treatment, LILRA3 and MPO-DNA complexes were consistently decreased in tAPS patients. Key findings from the Peking cohort were confirmed in the United States cohort. Our study provides first evidence that LILRA3 is aberrantly expressed in APS, especially in patients with tAPS. Serum LILRA3 correlated with MPO-DNA complexes, and the two indices were consistently decreased in tAPS patients after treatment. LILRA3 may play a role in thrombosis of APS and may serve as a biomarker and/or therapeutic target in tAPS.

Additional risk factors associated with thrombosis and pregnancy morbidity in a unique cohort of antiphospholipid antibody-positive patients.

Background:Antiphospholipid syndrome (APS) is an autoimmune prothrombotic condition with significant morbidity. The objective of this study was to identify additional clinical and epidemiological risks of arterial thrombosis, venous thrombosis, and pregnancy morbidities in a large cohort of persistent antiphospholipid antibodies (aPLs)-positive carriers.Methods:This was a cross-sectional cohort study of 453 consecutive patients with a documented positive aPL who attended Peking University People's Hospital. Among 453 patients screened, 297 patients had persistent positive aPL. We compared asymptomatic aPL carriers with thrombotic and obstetric APS patients. And the univariate analysis and multivariable logistic regression were used to evaluate the association between different risk factors and APS clinical manifestations. The levels of circulating markers of neutrophil extracellular traps (NETs) (cell-free DNA and citrullinated histone H3 [Cit-H3]) were assessed and compared among aPL-positive carriers with or without autoimmune disease and APS patients.Results:Additional risk factors associated with arterial thrombosis among aPL-positive carriers included: smoking (odds ratio [OR] = 6.137, 95% confidence interval [CI] = 2.408–15.637, P = 0.0001), hypertension (OR = 2.368, 95% CI = 1.249–4.491, P = 0.008), and the presence of underlying autoimmune disease (OR = 4.401, 95% CI = 2.387–8.113, P < 0.001). Additional risks associated with venous thrombosis among aPL carriers included: smoking (OR = 4.594, 95% CI = 1.681–12.553, P = 0.029) and the presence of underlying autoimmune disease (OR = 6.330, 95% CI = 3.355–11.940, P < 0.001). The presence of underlying autoimmune disease (OR = 3.301, 95% CI = 1.407–7.744, P = 0.006) is the additional risk, which demonstrated a significant association with APS pregnancy morbidity. Higher circulating levels of cell-free DNA and Cit-H3 were observed among APS patients and aPL patients with autoimmune diseases compared with those aPL carriers without underlying autoimmune diseases. Furthermore, control neutrophils that are conditioned with APS patients’ sera have more pronounced NET release compared with those treated with aPL carriers’ sera without underlying autoimmune diseases.Conclusion:We identified several potential additional risk factors for APS clinical manifestations among a large cohort of Chinese aPL carriers. Our data may help physicians to risk stratify aPL-positive Asian patients.

The impact of various entities of antiphospholipid antibodies positivity on adverse pregnancy outcome. An epidemiological perspective

The aim of the study was to evaluate the rate of obstetric complications and the burden of obstetric outcomes in antiphospholipid syndrome (APS), non-criteria APS and asymptomatic antiphospholipid antibodies (aPL) carriers. From 2013–2018, 163 pregnant subjects with aPL antibodies and 785 controls were enrolled. Penalized logistic regression was used to compare obstetric complications.Cases included 62 complete APS (38 %), 48 non-criteria APS (29.4 %) and 53 (32.5 %) asymptomatic aPL-carriers. Connective tissue diseases (CTDs) were diagnosed in 31.3 % of cases. The rate of high-risk aPL profile was higher (p < .01) in APS (67.7 %) compared to non-criteria (14.6 %) and aPL-carriers (9.4 %). Double/triple positivity was 33.9 % (p < .05 compared to non-criteria and aPL-carriers) in APS, 10.4 % in non-criteria and 9.4 % in aPL-carriers. The rate of adverse pregnancy outcomes were 5.6 % in controls, 41.9 % (adj.OR = 6.95 %CI = 2.7−13.5) in APS, 25 % (adj.OR = 4.4,95 %CI = 2−9.4) in non-criteria and 28.3 % (OR = 4.95 %CI = 1.8−8.8) in aPL-carriers. CTDs were independently associated with an increased risk of adverse obstetric outcomes (OR = 2.8,95 %CI = 1.36–5.89). The attributable fraction (AF) of adverse obstetric events was higher among low-risk antibodies compared to high-risk (AF = 0.27,95 %CI = 0.22−0.31 vs AF = 0.16,95 %CI = 0.16−0.2,p < .01) and among single positivity compared to double/triple positivity (AF = 0.32,95 %CI = 0.26−0.37 vs AF = 0.11,95 %CI = 0.09−0.13,p < .01) suggesting that low-risk subjects are responsible for a high burden of obstetric complications.

AB0408 DOES THE PRESENCE IN THE SERUM OF ANTIPHOSPHOLIPID ANTIBODIES CORRELATE WITH SPECIFIC/NON SPECIFIC CAPILLAROSCOPIC ABNORMALITIES?

Background:Antiphospholipid syndrome (APS) is a systemic autoimmune disease characterized by specific vascular and obstetric manifestations and by antiphospholipid antibodies (aPL) positivity [1].To date, little is known regarding nailfold videocapillaroscopy (NVC) alterations in APS patients and in asymptomatic aPL-carriers, non-specific abnormalities being the most frequently reported [2,3,4].Objectives:To retrospectively analyze NVC alterations in APS patients and in asymptomatic aPL-carriers and to correlate NCV alterations with both clinical manifestations and serum aPL profile.Methods:Thirty-five aPL positive patients having received at least one NCV investigation (mean age 47 years, range 16-81, 31 female and 4 male) were retrospectively included in the study. For each patient complete medical history was collected with a particular attention to past vascular thrombosis and pregnancy morbidity. Patients were classified as affected by APS according to the updated Sapporo classification criteria [5]. Lupus anticoagulant (LAC), IgM and IgG anti-cardiolipin antibodies (ACL) and IgM and IgG anti-Beta2 Glycoprotein 1 (anti-B2GP1) were assessed in each patient according to the recommended procedures [5]. NCV parameters were analyzed in each patient, with a particular interest to hemorrhages or nailfold bed-parallel hemosiderin deposits (“comb-like”hemorrhages) presence [2,6]. Statistical analysis was performed by parametric and non-parametric tests.Results:Seventeen patients (mean age 49 years, range 16-81 years) were asymptomatic aPL-carriers and 18 (mean age 46 years, range 26-71 years) were affected by APS. Within APS patients, 16 had a history of vascular thrombosis and 2 had pregnancy morbidity; in 6 patients APS was secondary to other autoimmune rheumatologic conditions (3 to Systemic Lupus Erythematosus, 2 to vasculitides and 1 to Mixed Connective Tissue Disease).Among the total number of aPL-carriers and APS patients six patients showed a normal NVC pattern, 24 patients had non-specific NVC abmormalities and 5 patients had a “scleroderma-like” pattern. Interestingly, NCV microhemorrhages were significantly more frequent in APS patients than in asymptomatic aPL-carriers, both in score and in absolute (p=0.05 andp=0.04, respectively). Particularly, in APS patients “comb-like”hemorrhages had a statistically significant higher prevalence than isolated hemorrhages (p=0.03). Dilated capillaries score was significantly higher in APS patients than in asymptomatic aPL-carriers (p=0.01).Not any statistically significant difference was observed regarding other capillary parameters (score of giant capillaries, loss of capillaries, or anormal shpaes, i.e. angiogenesis). Not any statistical correlation was observed between NVC parameters and different aPL profile.Conclusion:The study shows that the total number of microhemorrhages and in particular the“comb-like”subtype, are significantly the most frequent specific abnormalities in APL patients when compared to asymptomatic aPL carriers. The presence of the “scleroderma like” NVC pattern may suggest a concomitant overlap syndrome. Not any correlation was found between aPL profile and other NVC parameters. Further studies need to develop a more specific APS NVC pattern for APS patients.

Anti-phospholipid antibodies and reproductive failures.

Anti-phospholipid syndrome (APS) recapitulates the link between autoimmunity and pregnancy failure: Acquired anti-phospholipid antibodies (aPL) play a pathogenic role in pregnancy complications. The diagnosis of obstetric APS can easily be pursued when women present with laboratory and clinical features fulfilling the international classification criteria. Standard therapeutic approach to obstetric APS consists in the association of anti-platelet agents and anticoagulants. Most patients achieve a live birth thanks to conventional treatment; however, approximately 20% fail to respond and are managed with additional therapeutic tools added on the top of conventional treatment. Surely, a refinement of risk stratification tools would allow early identification of high-risk pregnancies that warrant tailored treatment. In real life, obstetricians and rheumatologists face complex diagnostic scenarios including women with pregnancy morbidities other than those mentioned in classification criteria such as one or two early losses and premature birth after 34weeks due to preeclampsia or placental insufficiency, women with low-titer aPL not fulfilling criteria laboratory requirements, women with positive non-criteria aPL, asymptomatic aPL carriers, and infertile women found to be aPL-positive. This review focuses on some of the several unanswered questions related to diagnostic, prognostic, and therapeutic aspects in obstetric APS.

Antiphospholipid Antibodies and Fertility: No Impact on Ovarian Reserve in Premenopausal Women

Objective: To investigate possible differences in levels of ovarian reserve between antiphospholipid antibodies (aPL) asymptomatic carriers and antiphospholipid syndrome (APS) patients, by measuring the levels of anti-Müllerian hormone (AMH). Methods: We enrolled 69 premenopausal women divided in 2 groups: a) patients with APS, either primary (PAPS) or secondary (SAPS), according to the Sydney classification criteria; b) asymptomatic aPL carriers. Aged-matched premenopausal healthy donors (HDs) were also recruited. Complete aPL testing was performed and AMH levels were measured using enzyme-linked immunosorbent assay. Results: Among the 69 patients included in the study, 22 were diagnosed with PAPS, 13 with SAPS, and 14 patients were asymptomatic aPL carriers. No differences in AMH levels were observed among the three groups [mean AMH: PAPS 3.09 ng/ml ± 1.9 (range 1.02 − 7.1); SAPS 3.1 ng/ml ± 2.2 (range 1.1 − 7.6); aPL carriers 2.2 ng/ml ± 5.4 (range 1 − 6.3)] and between patients/aPL carriers and HDs [mean AMH 2.82 ng/ml ± 2.9 (range 1 − 6.9)]. Any correlation between the global APS score (GAPSS) and AMH levels failed to be found (rho = 0.31; p = 0.073). Conclusion: With the limitations of the current study, as observed in women with APS, we confirm that ovarian reserve, assessed with AMH levels, is not reduced in premenopausal women with isolated aPL positivity. Moreover, when granulating the aPL profile in terms of risk assessment, using the GAPSS, no impact on fertility was observed.

The effect of hydroxychloroquine on thrombosis prevention and antiphospholipid antibody levels in primary antiphospholipid syndrome: A pilot open label randomized prospective study.

Sporadic studies suggest hydroxychloroquine (HCQ) may be effective for thrombosis prevention in patients with primary antiphospholipid syndrome (PAPS) and may lead to antiphospholipid antibody (aPL) titer reduction but data from randomized studies are lacking. We conducted a pilot open-label randomized prospective study aiming to evaluate the safety and efficacy of HCQ for thrombosis prevention in 50 patients with PAPS allocated 1:1 to HCQ plus standard care (systemic anticoagulation and/or antiplatelet therapy) vs. standard care alone, as well as the effect of HCQ on aPL titers of 50 PAPS patients and 15 asymptomatic aPL carriers. HCQ use plus standard care was associated with lower incidence rate of thrombosis than standard care alone (0.001 vs. 0.007, log-rank p=0.048) over an average 2.6-year follow-up, and a multivariate hazard ratio of 0.09 (95% CI=0.01-1.26, p=0.074) after adjusting for the effect of age, sex, traditional cardiovascular risk factors, triple aPL positivity, history of recurrent thrombotic events at baseline, and poor anticoagulation quality (INR levels within therapeutic range for ≤80% of follow-up). No significant difference in safety outcomes was observed between the two groups. Long-term HCQ use was associated with a decrease in aPL titers except for IgM anticardiolipin antibodies, which tended to decrease overtime regardless of treatment allocation. HCQ may represent an effective adjuvant treatment for thrombosis prevention in patients with PAPS, which may be mediated via a reduction in aPL titers. Larger randomized trials are needed in order to corroborate this finding and investigate the thromboprotective role of HCQ in asymptomatic aPL carriers.

Identifying phenotypes of patients with antiphospholipid antibodies: results from a cluster analysis in a large cohort of patients.

To identify the aggregation of patients with aPL into different subgroups sharing common features in terms of clinical and laboratory phenotypes. We applied a hierarchical cluster analysis from the multiple correspondence analysis to determine subgroups of patients according to clinical and laboratory characteristics in a cohort of subjects with confirmed aPL positivity who presented to our outpatient clinics from 2006 to 2018. A total of 486 patients [403 women; age 41.7 years (26)] were included, resulting in five clusters. Cluster 1 (n= 150) presented with thrombotic events (65.3% with venous thrombosis), with triple aPL positivity found in 34.7% of them (the highest rate among the different clusters). All the patients from cluster 2 (n = 91) had a confirmed diagnosis of SLE and the highest rate of anti-dsDNA positivity (91.7%). Cluster 3 included 79 women with pregnancy morbidity. Triple positivity was present in 3.8%, significantly lower when compared with Cluster 1 (34.7% versus 3.8%, P<0.01). Cluster 4 included 67 patients, 28 (41.8%) of whom with APS. Thrombotic events were observed in 23.9% patients. Cluster 4 had the highest rate of cytopenia, with thrombocytopenia as high 41.8% with no anti-dsDNA antibodies. Cluster 5 included 94 asymptomatic aPL carriers. While clusters 1, 2, 3 and 5 corresponded to well-known entities, cluster 4 might represent a bridging condition between pure primary APS and defined SLE, with lower thrombotic risk when compared with primary APS but higher general features such as ANA and cytopenia (mainly thrombocytopenia).

Preporuke Evropske lige protiv reumatizma (EULAR) za profilaksu i lečenje antifosfolipidnog sindroma kod odraslih

Antiphospholipid syndrome (APS) is a rare disease characterised by venous and/or arterial thrombosis, pregnancy complications and the presence of specific autoantibodies called antiphospholipid (aPL) antibodies. The European League Against Rheumatism, EULAR, has published a set of recommendations for the management of antiphospholipid syndrome in adults, based on the systematic literature reviews (SLRs) results and expert opinions. Low dose aspirin (LDA) is recommended for asymptomatic aPL carriers, patients with systemic lupus erythematosus without prior thrombotic or obstetric APS, and nonpregnant women with a history of obstetric APS only, all with high-risk aPL profiles. Patients with APS and the first unprovoked venous thrombosis should receive long-term treatment with vitamin K antagonists (VKA) with a target international normalised ratio (INR) of 2-3. In patients with APS with the first arterial thrombosis, treatment with VKA with INR 2-3 or INR 3-4 is recommended, considering the individual's bleeding/ thrombosis risk. For patients with recurrent arterial or venous thrombosis despite adequate treatment, addition of LDA, increase of INR target to 3-4 or switch to low molecular weight heparin may be considered. In women with prior obstetric APS, combination treatment with LDA and prophylactic dosage heparin during pregnancy is recommended. In patients with recurrent pregnancy complications, increase of heparin to therapeutic dose, addition of hydroxychloroquine or addition of low-dose prednisolone in the first trimester may be considered. These recommendations aim to guide treatment in adults with APS.

Anti-beta-2 glycoprotein I epitope specificity: from experimental models to diagnostic tools.

Beta-2 glycoprotein I (β2GPI) is the main antigenic target for anti-phospholipid antibodies (aPL), the serological markers of anti-phospholipid syndrome (APS). Conformational changes of the molecule seem to be essential for exposing the cryptic epitope for aPL binding and to trigger pathogenic pathways. There is increasing evidence that a conformational epitope located in the Domain I (DI) of the molecule is the main epitope targeted by human autoantibodies. The pathogenic role of the DI epitope has been recently supported by invivo models and by immuno-histopathological findings in APS patients. Antibodies targeting β2GPI-DI are more frequently detected in patients with full-blown APS compared to asymptomatic aPL carriers or patients with infectious diseases who have antibodies directed against the whole molecule. Anti-DI antibodies are positively correlated with medium to high titres of aPL, with the presence of lupus anticoagulant and thrombotic and pregnancy manifestations, enabling identification of patients at higher risk of clinical events. However, some APS patients develop antibodies reacting against β2GPI epitopes other than DI, suggesting that other anti-β2GPI antibody subsets may be clinically relevant. Although preliminary results suggest that anti-DI antibodies can be detected by different assays in a comparable manner, further prospective studies are needed to support their use in the clinical setting and their predictive value.

Clinical characterization of antiphospholipid syndrome by detection of IgG antibodies against β2 -glycoprotein i domain 1 and domain 4/5: ratio of anti-domain 1 to anti-domain 4/5 as a useful new biomarker for antiphospholipid syndrome.

It has been suggested that only antibodies against domain 1 (D1) of β2 -glycoprotein I (β2 GPI) are pathogenic and diagnostic. The role of antibodies against other β2 GPI domains is still debated. This study was undertaken to evaluate the clinical relevance of domain specificity profiling of anti-β2 GPI IgG antibodies in antiphospholipid syndrome (APS) patients and in control groups of patients with systemic autoimmune rheumatic diseases and in asymptomatic antiphospholipid antibody (aPL) carriers. We evaluated 159 subjects with persistently positive, medium or high-titer anti-β2 GPI IgG, including 56 patients with thrombotic (obstetric or nonobstetric) primary APS, 31 women with obstetric primary APS, 42 aPL-positive patients with systemic autoimmune rheumatic diseases, and 30 asymptomatic aPL carriers. One hundred healthy donors were included. Anti-β2 GPI D1 and D4/5 IgG were tested on research enzyme-linked immunosorbent assays containing recombinant β2 GPI domains. As compared to other groups, aPL carriers displayed higher frequency/titer of anti-D4/5 IgG. Unlike anti-D4/5, anti-D1 IgG antibodies were more frequent and at higher titer in triple than in single or double aPL-positive subjects. An anti-D1 to anti-D4/5 ratio of ≥1.5 was predictive of systemic autoimmunity (odds ratio 3.25 [95% confidence interval 1.45-7.49], P = 0.005). Neither anti-D1 nor anti-D4/5 antibodies were associated with APS clinical criteria. Anti-D1 IgG is the preferential specificity not only in vascular and obstetric primary APS, but also in patients with systemic autoimmune rheumatic disease with no clinical features of APS. Conversely, aPL carriers do not have a polarized profile toward D1. Combined testing for anti-β2 GPI IgG with different domain specificity allows a more accurate aPL profiling, with polarization toward anti-D1 IgG as a possible fingerprint of systemic autoimmunity.

Primary prophylaxis to prevent obstetric complications in asymptomatic women with antiphospholipid antibodies: a systematic review.

Obstetric complications are common in patients with antiphospholipid syndrome. However, the impact of antiphosholipid antibodies (aPL) in the pregnancy outcomes of asymptomatic aPL carriers is uncertain. The aim of this systematic review is to assess whether primary prophylaxis is beneficial to prevent obstetric complications during pregnancy in asymptomatic women positive for aPL who have no history of recurrent pregnancy loss or intrauterine fetal death. Studies evaluating the effect of prophylactic treatment versus no treatment in asymptomatic pregnant aPL carriers were identified in an electronic database search. Design, population and outcome homogeneity of studies was assessed and meta-analysis was performed. The pooled Mantel-Haenszel relative risk of specific pregnancy outcomes was obtained using random effects models. Heterogeneity was measured with the I(2) statistic. All analyses were conducted using Review Manager 5.3. Data from five studies involving 154 pregnancies were included and three studies were meta-analysed. The risk ratio and 95% confidence interval (CI) of live birth rates, preterm birth, low birth weight and overall pregnancy complications in treated and untreated pregnancies were 1.14 (0.18-7.31); 1.71 (0.32-8.98); 0.98 (0.07-13.54) and 2.15 (0.63-7.33),respectively. Results from the meta-analysis revealed that prophylactic treatment with aspirin is not superior to placebo to prevent pregnancy complications in asymptomatic aPL carriers. This systematic review did not find evidence of the superiority of prophylactic treatment with aspirin compared to placebo or usual care to prevent unfavourable obstetric outcomes in otherwise healthy women with aPL during the first pregnancy.

Persistent antiphospholipid antibody (aPL) in asymptomatic carriers as a risk factor for future thrombotic events: a nationwide prospective study.

The long-term prognosis of individuals fulfilling the laboratory criteria, but not clinical criteria, of antiphospholipid syndrome (APS) has not been widely investigated. The primary aim of this study was to evaluate the incidence of first thrombotic event (deep venous thrombosis (DVT), pulmonary embolism (PE), myocardial infarction (MI), stroke or transient ischaemic attack (TIA) in a nationwide antiphospholipid antibody (aPL) carrier cohort. We conducted a prospective nationwide cohort study. The aPL profile of participants was recorded from the laboratory database. Information was collected about thrombotic and pregnancy complications, subsequent medical history, other risk factors for thrombosis, use of prophylactic antithrombotic medication and general health. Participants included adult asymptomatic aPL carriers recognized in Finland during 1971-2009. The main outcome measure was incidence of first thrombotic event. A total of 119 (89% female) aPL carriers were followed for mean (SD) of 9.1 (7.5) years (range 3-41 years). Sixty-one per cent of the study participants had autoimmune disease, most often systemic lupus erythematosus (SLE). Thirty-six of 119 (30%) were either double or triple positive, 56% single lupus anticoagulant (LA) positive, and 8% and 5% single anticardiolipin antibodies (aCL) and anti-β2glycoprotein I antibodies (aβ2GPI) positive, respectively. Nine (7.6%) study patients experienced a first thrombotic event (five DVT, one PE, two MI, one TIA) mean (SD) 7.2 (8.3) years (range 1-26 years) after aPL detection (annual incidence rate 0.8%). All individuals who developed thrombotic complications had autoimmune disease. Annual rate of first thrombotic event in carriers of single positivity (0.65%) was equal to the known risk of thrombosis in the healthy Caucasian population, whereas the rate was two times higher in carriers of double or triple positivity (1.27%). Sixteen of 79 (20%) women experienced pregnancy complications. Double or triple positivity for aPL is a risk factor for future thrombotic events, especially in individuals with an underlying autoimmune disease, whereas single positivity does not seem to carry an elevated risk of thrombosis.

New Tests to Detect Antiphospholipid Antibodies: Anti-Domain I Beta-2-Glycoprotein-I Antibodies

Beta-2 glycoprotein I (β2GPI) is the main antigenic target for antiphospholipid antibodies (aPL), the serological markers of antiphospholipid syndrome (APS). Domain I (DI) of β2GPI has lately been identified as the main epitope targeted by antibodies reacting against β2GPI. DI is a cryptic epitope, becoming available for autoantibody binding when β2GPI opens from a circular to a fish-hook configuration. Antibodies targeting β2GPI-DI are more frequently detected in patients with a full-blown syndrome than in asymptomatic aPL carriers or in patients with infectious diseases that have reactivity toward the whole molecule. Interestingly, anti-DI antibodies are strongly positively correlated with thrombotic and pregnancy manifestations, enabling identification of patients at higher risk of clinical events. However, available tests to detect anti-DI antibodies still lack standardization. Moreover, some APS patients develop antibodies reacting against β2GPI epitopes other than DI, suggesting that other anti-β2GPI antibody subsets may be clinically relevant. Available evidence on anti-DI antibodies in APS is herein critically reviewed.

A5.5 Antibodies against Domain I of β2 Glycoprotein I in Antiphospholipid Antibody Syndrome

<h3>Background and Objectives</h3> antibodies anti-phospholipids (aPL) react to proteins bound to PL, mainly β2glycoprotein I (β2GPI). Antibodies against β2GPI (aβ2GPI) exert a pathogenic role and represent a risk-factor for clinical manifestations of anti-phospholipid syndrome (APS). However, some aβ2GPI-positive subjects never develop APS-related clinical manifestations. This observation may be explained by the heterogeneity of aβ2GPI population, with autoantibody subgroups targeting different β2GPI epitopes. In particular, antibodies anti-domain I (aDI) but not domains IV and V (aDIV/V) of β2GPI have been associated with thrombotic events. Therefore, the aim of this study was to assess the prevalence of aDI and aDIV/V IgG in a cohort of aPL-positive patients. <h3>Material and Methods</h3> 58 patients with a diagnosis of primary APS (PAPS) according to the 2006 Sydney criteria have been included in this study. 38 PAPS patients (65.5%) presented with venous and/or arterious thrombothic events while 20 subjects (34.5%) had obstetric manifestations only. 15 aPL asymptomatic carriers were also recruited. All samples had been tested for LA and for aCL and aβ2GPI with home-made assays according to international guidelines. In the thrombotic PAPS group, 35/38 subjects (92.1%) were aβ2GPI IgG positive; aβ2GPI IgG positivity rate was 85% in the obstetric PAPS group (17/20 women); 80% of the asymptomatic aPL carriers displayed aβ2GPI IgG. IgG specificities against whole β2GPI, DI and DIV/V have been evaluated with a novel solid-phase chemiluminiscent assay (BioFlash and ELISA, INOVA Diagnostics). <h3>Results</h3> Out of the 73 aPL positive patients: 21% were positive for aβ2GPI, aDI and aDIV/V; 41% were positive for aβ2GPI and aDI but negative for aDIV/V; 4% were positive for aβ2GPI and aDIV/V but negative for aDI; 21% were aβ2GPI positive only; 4% were positive for aDIV/V; 9% were negative for antibodies against the whole molecule and the studied domains. The prevalence of aDI was 74% among patients with thrombotic pAPS and 60% among women with obstetric manifestations. 40% of aPL asymptomatic carriers were positive for aDI. We observed a strong correlation between aβ2GPI and aDI (p &lt;&lt; 0.01, r = 0.836) but not aDIV/V (p = 0.07, r = 0.216). <h3>Conclusions</h3> Most of the aβ2GPI positive sera displayed reactivity against DI, while aDIV/DV were detected in a low rate of patients. Our data suggest that DI is the immunodominant β2GPI epitope and that aDI are the main antibody population in APS patients. Future studies are warranted to better define the diagnostic and prognostic role of anti-DI in APS.