Changes in serum immunoglobulin G glycosylation patterns for antiphospholipid syndrome patients with lectin microarray.

Antiphospholipid syndrome is a rare autoimmune disease characterized by persistent antiphospholipid antibodies. Immunoglobulin G plays a vital role in disease progression, with its structure and function affected by glycosylation. We aimed to investigate the changes in the serum immunoglobulin G glycosylation pattern in antiphospholipid syndrome patients. We applied lectin microarray on samples from 178 antiphospholipid syndrome patients, 135 disease controls (including Takayasu arteritis, rheumatoid arthritis and cardiovascular disease) and 100 healthy controls. Lectin blots were performed for validation of significant differences. Here, we show an increased immunoglobulin G-binding level of soybean agglutinin (p = 0.047, preferring N-acetylgalactosamine) in antiphospholipid syndrome patients compared with healthy and disease controls. Additionally, the immunoglobulin G from antiphospholipid syndrome patients diagnosed with pregnancy events had lower levels of fucosylation (p = 0.001, recognized by Lotus tetragonolobus) and sialylation (p = 0.030, recognized by Sambucus nigra I) than those with simple thrombotic events. These results suggest the unique serum immunoglobulin G glycosylation profile of antiphospholipid syndrome patients, which may inform future studies to design biomarkers for more accurate diagnosis of antiphospholipid syndrome and even for the prediction of clinical symptoms in patients.

Assessment and comparison of the 2023 ACR/EULAR APS criteria with the revised Sapporo criteria.

OBJECTIVE: To analyze antiphospholipid antibody (aPL)-positive patients using the 2023 American College of Rheumatology/The European Alliance of Associations for Rheumatology (ACR/EULAR) antiphospholipid syndrome (APS) classification criteria and compare the revised Sapporo criteria and the 2023 ACR/EULAR criteria and evaluate whether the 2023 ACR/EULAR criteria provide added value over the revised Sapporo criteria. METHODS: In this descriptive study, 94 aPL-positive patients (with or without APS diagnosis) were identified from two hospital-based registries (Gazi and Hacettepe University). Patients were classified into four groups to compare both criteria sets. These four groups are as follows: (1) patients classified with only the revised Sapporo criteria; (2) patients classified with only the 2023 ACR/EULAR APS criteria; (3) patients classified with both two criteria sets; and (4) patients classified with neither two criteria set. RESULTS: Of the 94 patients, 11 were classified with only the revised Sapporo criteria; one with only the 2023 ACR/EULAR APS criteria; 52 with both criteria sets; and 30 with neither set of criteria. For these 94 patients, the operating characteristics of the 2023 ACR/EULAR APS criteria, using the revised Sapporo criteria as the gold standard, the 2023 ACR/EULAR APS entry criteria demonstrated 100% sensitivity, and the 2023 ACR/EULAR APS classification criteria demonstrated 98% specificity and 82.5% sensitivity. CONCLUSION: The study emphasizes the importance of recognizing differences in clinical manifestations, such as early pregnancy loss without severe preeclampsia (PEC) and/or severe placental insufficiency (PI) and calls for a nuanced discussion on anticardiolipin (aCL) and anti-beta 2-glycoprotein-I (anti-ß2GPI) immunoglobulin G (IgG) cutoff values.

Antiphospholipid antibody-related hepatic vasculitis in a juvenile after non-severe COVID-19: a case report and literature review.

Antiphospholipid antibodies (aPL) are both laboratory evidence and causative factors for a broad spectrum of clinical manifestations of antiphospholipid syndrome (APS), with thrombotic and obstetric events being the most prevalent. Despite the aPL-triggered vasculopathy nature of APS, vasculitic-like manifestations rarely exist in APS and mainly appear associated with other concurrent connective tissue diseases like systemic lupus erythematous. Several studies have characterized pulmonary capillaritis related to pathogenic aPL, suggesting vasculitis as a potential associated non-thrombotic manifestation. Here, we describe a 15-year-old girl who develops hepatic infarction in the presence of highly positive aPL, temporally related to prior non-severe COVID-19 infection. aPL-related hepatic vasculitis, which has not been reported before, contributes to liver ischemic necrosis. Immunosuppression therapy brings about favorable outcomes. Our case together with retrieved literature provides supportive evidence for aPL-related vasculitis, extending the spectrum of vascular changes raised by pathogenic aPL. Differentiation between thrombotic and vasculitic forms of vascular lesions is essential for appropriate therapeutic decision to include additional immunosuppression therapy. We also perform a systematic review to characterize the prevalence and clinical features of new-onset APS and APS relapses after COVID-19 for the first time, indicating the pathogenicity of aPL in a subset of COVID-19 patients.

The clinical relevance of different antiphospholipid antibody profiles in pediatric rheumatology patients.

BACKGROUND: The clinical relevance of different antiphospholipid antibody (aPL) profiles, including low level anticardiolipin (aCL) and anti-ß2-glycoprotein-I (aß2GPI) antibodies, is ill-defined in the pediatric population. Our purpose is to describe the demographic, clinical, and laboratory characteristics of aPL positive pediatric patients based on different aPL profiles. FINDINGS: In this single center retrospective cohort study, based on the screening of our pediatric (age ≤ 18) rheumatology electronic medical records (2016-2022), we identified patients who had at least one "positive" aPL (lupus anticoagulant [LA], aCL IgG/M, or aß2GPI IgG/M) result. Patients were grouped into high- (LA positive and/or aCL/aß2GPI IgG/M > 40U [ELISA]) and low-risk (LA negative and aCL/aß2GPI IgG/M 20-39U) aPL profiles; those with persistently positive aPL were descriptively analyzed for demographic and clinical characteristics. Of 57 included patients, 34 (59%) had initial high- and 23 (40%) had initial low-risk profiles. Based on subsequent aPL results available in 42/57 (74%) patients, 25/27 (93%) in the high-, and 7/15 (47%) in the low-risk groups remained still positive. Of these 32 patients with persistently positive aPL, moderate-to-large vessel or microvascular thrombosis occurred in nine (28%) patients with high-risk and in none with low-risk aPL profiles; non-thrombotic aPL-related manifestations were reported in 15 (47%) patients with persistent aPL positivity. CONCLUSION: An initial high-risk aPL profile was persistent in approximately 90% of our cohort, a third of whom had thrombosis, and half had non-thrombotic aPL manifestations. Our results underscore the need for a large-scale effort to better characterize aPL-related manifestations in pediatric patients with persistent high-risk aPL-profiles.

Bibliometric analysis of the global trends in immune-related recurrent pregnancy loss research over the last two decades.

PROBLEM: A comprehensive analysis was conducted to explore the scientific output on immune-related recurrent pregnancy loss (RPL) and its key aspects. Despite the lack of clear explanations for most RPL cases, immune factors were found to play a significant role. METHOD OF STUDY: The study utilized a bibliometric approach, searching the Web of Science Core Collection database for relevant literature published between 2004 and 2023. RESULTS: The collected dataset consisted of 2228 articles and reviews, revealing a consistent increase in publications and citations over the past two decades. The analysis identified the United States and China as the most productive countries in terms of RPL research. Among the institutions, Fudan University in China emerged as the top contributor, followed by Shanghai Jiaotong University. Kwak-kim J was the most prolific author, while Christiansen Ob had the highest number of co-citations. The top 25 co-cited references on diagnosis, treatment, and mechanisms formed the foundation of knowledge in this field. By examining keyword co-occurrence and co-citations, the study found that antiphospholipid syndrome and natural killer cells were the primary areas of focus in immune-related RPL research. Additionally, three emerging hotspots were identified: chronic endometritis, inflammation, and decidual macrophages. These aspects demonstrated increasing interest and research activity within the field of immune-related RPL. CONCLUSIONS: Overall, this comprehensive bibliometric analysis provided valuable insights into the patterns, frontiers, and focal points of global scientific output related to immune-related RPL.

Thrombin Generation Assay in Antiphospholipid Antibodies Positive Subjects as a Personalized Thrombotic Risk Assessment: State of the Art and Perspectives.

PURPOSE OF THE REVIEW: Thrombotic risk assessment in antiphospholipid positive (aPL +) subjects is a major challenge, and the study of in vitro thrombin generation (thrombin generation assays (TGA)) could provide useful information. Activated protein C (APC) sensitivity is involved in thrombotic events in antiphospholipid syndrome patients. We summarized methods used to assess APC sensitivity with TGA and evaluated the prognostic role of APC resistance through literature search. RECENT FINDINGS: APC resistance induced by aPL is a complex pathway. Several cross-sectional studies assessed APC sensitivity to understand thrombotic event mechanisms in aPL + subjects. Only one prospective cohort had investigated the prognostic impact of APC resistance in aPL + subjects, with a positive and significant correlation between APC sensitivity and the risk of thrombosis during the follow up (hazard ratio, 6.07 [95% CI, 1.69-21.87]). APC resistance assessed with TGA could be associated with thrombotic events in aPL + subjects.

Deciphering the clinical significance of longitudinal antiphospholipid antibody titers.

In antiphospholipid syndrome (APS), the risk of clinical manifestations increases with higher titers of antiphospholipid antibodies (aPL). Despite the adoption of aPL titers in the classification approach to aPL-positive subjects, the value of longitudinal monitoring of those titers in the follow-up is still debated, being well studied only in systemic lupus erythematosus (SLE). The literature suggests that the rate of aPL positivity decreases during follow-up in primary APS, estimating that seroconversion occurs in between 8.9 and 59% of patients over time. Negativisation of aPL occurs more frequently in asymptomatic aPL carriers than in patients with full-blown APS as well as in subjects with single aPL positivity or low aPL antibody titers. In patients with SLE, aPL typically behave fluctuating from positive to negative and back again in the course of follow-up. The few studies assessing the longitudinal course of aPL positivity with no associated systemic connective tissue disease reported a progressive decrement of aPL titers over time, in particular of antibodies against ß2 glycoprotein I (antiß2GPI) and cardiolipin (aCL) of IgG isotype. After a thrombotic event, aPL titers tend to decrease, as emerged from cohorts of both primary and secondary APS. Hydroxychloroquine has been identified as the most effective pharmacological agent to reduce aPL titers, with multiple studies demonstrating a parallel reduction in thrombosis rate. This review addresses available evidence on the significance of aPL titer fluctuation from clinical, therapeutic and pathogenic perspectives.

Pediatric antiphospholipid syndrome: clinical features and therapeutic interventions in a single center retrospective case series.

BACKGROUND/PURPOSE: Pediatric antiphospholipid syndrome (APS) is a thromboinflammatory disease characterized by the presence of circulating antiphospholipid antibodies and either thrombotic events or pregnancy morbidity. The objective of this study was to review a large institution's experience to better understand the characteristics of children with APS. METHODS: We conducted a retrospective review of pediatric APS at a tertiary referral center. The electronic medical record system was queried from 2000 through 2019, and 21 cases were included based on meeting the revised Sapporo Classification criteria by age 18 or younger. Comparisons between primary and secondary APS patients were made with two-tailed t-tests. RESULTS: Twenty-one patients were included with a median age at diagnosis of 16 years and median follow-up of 5.8 years. Secondary APS was slightly more common than primary APS (11 vs. 10 cases) and was primarily diagnosed in the context of systemic lupus erythematosus. Two thirds of patients (67%) also had "non-criteria" manifestations of APS including thrombocytopenia, autoimmune hemolytic anemia, and livedo reticularis/racemosa. Almost half of patients (43%) had recurrent thrombosis, typically when patients were subtherapeutic or non-adherent with anticoagulation. Damage Index in Patients with Thrombotic APS (DIAPS) scores indicated a chronic burden of disease in both primary and secondary APS patients. CONCLUSION: This case series of pediatric APS provides important context regarding disease phenotypes displayed by children with APS. High prevalence of non-criteria clinical manifestations highlights the need to consider these characteristics when developing pediatric-specific classification criteria and when considering this relatively rare diagnosis in pediatric practice.

Antiphospholipid antibodies and idiopathic infertility.

OBJECTIVE: The aim of our study was to evaluate obstetric outcome of women affected by idiopathic infertility showing persistently positive antiphospholipid antibodies (aPL). METHODS: : From 2000 consecutive patients undergoing ART, we selected 151 (7.55%) clinical records of patients affected by idiopathic infertility undergoing ICSI and showing positive aPL. RESULTS: Persistently positive aPL were found in 64/151 (42.38%) of the patients: in 34/64 (53.12%) at medium/high titers (group A) and in 30/64 (46.87%) at low titers (group B). Primary or secondary antiphospholipid syndrome (APS) was diagnosed in 25% of the patients, whereas 37.5% women showed clinical and/or laboratory features suggestive of APS, but not fulfilling clinical or laboratory classification criteria. Idiopathic infertility was the sole symptom in 31.25%. In 55% of these infertile patients, a history of recurrent failures of assisted reproductive techniques (ART) was also observed. Eighty-eight percent (88.88%) of women became pregnant and 77.77% gave birth. During pregnancy, an increase of aPL values was observed in 29.41% women of group B. CONCLUSIONS: A careful selection of patients allowed us to confirm that women affected by idiopathic infertility show a high prevalence of aPL, suggesting that these autoantibodies can also affect conception. Considering pregnancy complications and thrombotic risk related to ovarian stimulation, measuring aPL can represent a valid tool to identify among infertile women undergoing ART those at higher risk of pregnancy complications potentially life-threatening for mother and the fetus. In such patients, an accurate diagnosis and an adequate therapy are related to a better ART outcome.

'Non-criteria antiphospholipid antibodies': bridging the gap between seropositive and seronegative antiphospholipid syndrome.

OBJECTIVE: We aimed to analyse the prevalence of non-criteria anti-phospholipid (aPL) antibodies and their role in the diagnosis, treatment and prognosis in a cohort of patients with clinical features consistent with a diagnosis of antiphospholipid syndrome (APS), but persistently negative for criteria aPL - anti-cardiolipin antibodies (aCL), anti-ß2-glycoprotein I antibodies (aß2-GPI) and lupus anticoagulant (LA) - named seronegative APS (SN-APS). METHODS: Sera from SN-APS patients were tested for aCL by TLC-immunostaining, anti-vimentin/cardiolipin (aVim/CL) and anti-phosphatidylserine/prothrombin (anti-PS/PT) by ELISA. Control groups of our study were APS patients and healthy controls. RESULTS: We enrolled 114 consecutive SN-APS patients, 69 (60.5%) resulted positive for at least one non-criteria test in two occasions 12 weeks apart. Among the persistently positive patients to these tests, 97% resulted positive for aCL by TLC-immunostaining, 52.3% for aVim/CL and 17.4% for aPS/PT. SN-APS patients with double positivity (aCL by TLC-immunostaining and aVim/CL) showed a likelihood positive ratio of 8 to present mixed thrombotic and obstetrical features. Among SN-APS patients tested positive, after the therapeutic changes, three cases of recurrent thrombosis were observed [median follow-up 41 months (IQR 39.5)]. Twenty pregnancies were recorded in 17 SN-APS patients after the detection of unconventional aPL and 12 of them (60%) experienced a good outcome under conventional treatment for APS. CONCLUSIONS: This is the largest monocentric study demonstrating that aCL tested by TLC-immunostaining and aVim/CL can detect aPL positivity in SN-APS. It may encourage clinicians to monitor and provide adequate targeted therapy, which improve SN-APS prognosis.

Characteristics of purified anti-ß2GPI IgG N-glycosylation associate with thrombotic, obstetric and catastrophic antiphospholipid syndrome.

OBJECTIVE: Anti-ß-2 glycoprotein I (anti-ß2GPI) antibodies, defined as primary pathogenic antibody in antiphospholipid syndrome (APS). It has been reported that IgG Fc N-glycosylation affects IgG effector, we aim to investigate the association of Fc glycosylation profiles of purified anti-ß2GP1 IgG with clinical features of APS. METHODS: We purify anti-ß2GPI IgG and total IgG from 82 APS patients including nine catastrophic antiphospholipid syndrome (CAPS) patients, as well as total IgG from 103 healthy controls to quantitatively analyse all detectable Fc N-glycanforms of all IgG subclasses with Multiple Reaction Monitoring (MRM) method based on UPLC-ESI-QqQ mass spectrometry. RESULTS: Both purified anti-ß2GPI IgG and APS total IgG showed altered N-glycan profiles when compared with healthy control (HC) IgG. Anti-ß2GPI IgG presented with lower galactosylation, increased bisection and core fucosylation compared with APS total IgG and HC IgG. We found higher galactosylation of aß2GPI IgG2 in thrombotic APS compared with the obstetric APS, and lower galactosylation of aß2GPI IgG2 associated with late pregnancy morbidity. Moreover, low galactosylation of all anti-ß2GPI IgG subclasses, increased bisection and core fucosylation of anti-ß2GPI IgG1/2 were strongly associated with CAPS and triple positivity of antiphospholipid antibodies (aPLs). CONCLUSION: We comprehensively characterize the N-Glycans landscape of both anti-ß2GP1 and total IgG in APS. Altered N-glycan profiles of anti-ß2GPI IgG enables enabled the antibodies with proinflammatory properties. Furthermore, we associated levels of IgG Fc-glycosylation with clinical features antiphospholipid syndrome. These findings could increase our understanding of anti-ß2GPI antibody mediated mechanisms in APS and be used to develop diagnostics and new target treatments.

P2RY8 variants in lupus patients uncover a role for the receptor in immunological tolerance.

B cell self-tolerance is maintained through multiple checkpoints, including restraints on intracellular signaling and cell trafficking. P2RY8 is a receptor with established roles in germinal center (GC) B cell migration inhibition and growth regulation. Somatic P2RY8 variants are common in GC-derived B cell lymphomas. Here, we identify germline novel or rare P2RY8 missense variants in lupus kindreds or the related antiphospholipid syndrome, including a "de novo" variant in a child with severe nephritis. All variants decreased protein expression, F-actin abundance, and GPCR-RhoA signaling, and those with stronger effects increased AKT and ERK activity and cell migration. Remarkably, P2RY8 was reduced in B cell subsets from some SLE patients lacking P2RY8 gene variants. Low P2RY8 correlated with lupus nephritis and increased age-associated B cells and plasma cells. By contrast, P2RY8 overexpression in cells and mice restrained plasma cell development and reinforced negative selection of DNA-reactive developing B cells. These findings uncover a role of P2RY8 in immunological tolerance and lupus pathogenesis.

Effects of anti-beta 2-glycoprotein 1 antibodies and its association with pregnancy-related morbidity in antiphospholipid syndrome.

Antiphospholipid syndrome (APS) is an autoimmune disease characterized by venous, arterial, or small-vessel thrombosis and/or pregnancy-related morbidity, associated with persistent positivity of antiphospholipid antibodies (aPL). Pregnancy-related morbidity in APS patients is characterized by unexplained fetal deaths, premature birth of morphologically normal newborns, and/or consecutive pregnancy losses before the 10th week of gestation. Beta 2-glycoprotein 1 (ß2GP1) is the main antigen recognized by aPL and plays an essential role in the pathogenesis of APS. Antibodies against ß2GP1 (aß2GP1) are involved in damage-generating mechanisms in APS due to their interaction with trophoblasts, decidua, and endothelial cells. aß2GP1 might be used as a prognostic tool for obstetric risk stratification and ß2GP1 could be a target for molecular-targeted treatment to prevent pregnancy morbidity in APS. This review describes these aspects of aß2GP1, including effects on different cellular targets, its association with the severity of obstetric manifestations and the potential of ß2GP1-targeted therapies for APS.

Anti-Phosphatidylserine/Prothrombin Antibodies at Two Points: Correlation With Lupus Anticoagulant and Thrombotic Risk.

Antibodies to phospholipids (aPL) and associated proteins are a hallmark in the diagnosis of anti-phospholipid syndrome (APS). Those included in the classification criteria are the lupus anticoagulant (LA) and the IgG and IgM isotypes of anticardiolipin (aCL) and anti-beta-2 glycoprotein I (ß2GPI) antibodies. Non-classification criteria markers such as autoantibodies that recognize the phosphatidylserine/prothrombin (aPS/PT) complex have been proposed as biomarkers for APS. Studies of aPS/PT antibodies have shown a strong correlation to clinical manifestations and LA. We aimed to study the value and the persistence of aPS/PT IgG and IgM antibodies in a cohort of consecutive patients with clinical suspicion of APS and their utility as thrombotic risk markers. Our study, with 103 patients, demonstrates that persistently positive results for aPS/PT IgG antibodies were significantly associated with APS classification, thrombosis, triple aPL positivity, LA positive result, and the Global APS Score (GAPSS) > than 9 points (p < 0.01, for each condition). On the other hand, no association was seen with pregnancy morbidity (p = 0.56) and SLE (p = 0.07). Persistence of aPS/PT antibodies, defined according to the current laboratory classification criteria, likely improves the diagnosis and clinical assessment of patients with APS.

Antiphospholipid Antibody Assays in 2021: Looking for a Predictive Value in Addition to a Diagnostic One.

Antiphospholipid antibodies (aPL) are mandatory for the diagnosis but are also a risk factor for the antiphospholipid syndrome (APS) clinical manifestations. Lupus anticoagulant (LA), anticardiolipin (aCL), and anti-beta2 glycoprotein I (ß2GPI) assays are the formal laboratory classification/diagnostic criteria. Additional nonclassification assays have been suggested; among them, antiphosphatidylserine-prothrombin (aPS/PT) and antidomain 1 ß2GPI antibodies are the most promising ones although not yet formally accepted. aPL represent the example of a laboratory test that moved from dichotomous to quantitative results consistent with the idea that reporting quantitative data offers more diagnostic/prognostic information for both vascular and obstetric manifestations. Although the general rule is that the higher the aPL titer, the higher the test likelihood ratio, there is growing evidence that this is not the case for persistent low titers and obstetric events. LA displays the highest diagnostic/prognostic power, although some isolated LAs are apparently not associated with APS manifestations. Moreover, isotype characterization is also critical since IgG aPL are more diagnostic/prognostic than IgA or IgM. aPL are directed against two main autoantigens: ß2GPI and PT. However, anti-ß2GPI antibodies are more associated with the APS clinical spectrum. In addition, there is evidence that anti-ß2GPI domain 1 antibodies display a stronger diagnostic/prognostic value. This finding supports the view that antigen and even epitope characterization represents a further step for improving the assay value. The strategy to improve aPL laboratory characterization is a lesson that can be translated to other autoantibody assays in order to improve our diagnostic and prognostic power.

A Novel ELISA Assay for the Detection of Anti-Prothrombin Antibodies in Antiphospholipid Syndrome Patients at High Risk of Thrombosis.

Autoantibodies targeting prothrombin (aPT) can be found in antiphospholipid syndrome (APS) patients. However, their detection has proven difficult to standardize. Here, we developed a new ELISA assay to improve the identification of aPT and compared its performance with currently available anti-phosphatidylserine/prothrombin antibodies (aPS/PT) and autoantibodies targeting prothrombin bound to the plastic plate (aPT-A) assays using a cohort of 27 APS patients at high risk of thrombosis. We generated a novel prothrombin variant, ProTS525A-Biot, carrying an artificial tag at the C-terminus suitable for site-specific biotinylation and added the mutation S525A to improve stability. ProTS525A-Biot was immobilized to neutravidin-coated plates at the desired density and with a defined orientation, i.e., pointing the N-terminal fragment-1 toward the solvent. Antibodies against ProTS525A-Biot (aPT-Bio) were found in 24 out of 27 triple-positive APS patients (88%). When compared to aPS/PT and aPT-A, aPT-Bio showed an excellent linear correlation with aPS/PT (R2 = 0.85) but not with aPT-A (R2 = 0.40). Since aPS/PT but not aPT-A are an emerging biomarker of thrombosis in APS, this method may find utility for detecting pathogenic aPT in APS but also other prothrombotic conditions such as COVID-19.

The Diagnostic Value of New Additional Antiphospholipid Antibodies in Antiphospholipid Syndrome.

BACKGROUND: The antiphospholipid syndrome (APS) is an autoimmune disease that is characterized by thrombosis and/or pregnancy failure and associated with the presence of all or at least one of three standard antibodies (anti-phospholipid (aPL) antibodies, including lupus anticoagulant (LA), anti-cardiolipin (aCL), and anti-ß2-glycoprotein I (anti-ß2GPI)). A growing body of evidence recommends adding additional aPL antibodies, such as anti-phosphatidylserine (aPS), anti-prothrombin (aPT), and anti-annexin A5 (aAA5), to conventional laboratory tests (revised Sapporo criteria), especially in seronegative APS cases. OBJECTIVES: We aimed to compare the diagnostic value, utility, and performance of these three additional antibodies along with the standard aPL antibodies in cases with confirmed and non-criteria APS (seronegative). METHODS: This was a prospective observational study on 59 patients who presented with clinical features of APS at the hematology, medical, rheumatology, and obstetric clinics. LA was detected by standard coagulation tests, while other aPL, IgG, and IgM antibodies (aCL, aß2GPI, aPS, aPT, aAA5) were detected with enzyme-linked immunosorbent assay (ELISA). RESULTS: Anti-PS antibody was more frequent compared to aPT and aAA5 in both confirmed cases (84.6%) and non-criteria (seronegative) (15.4%) APS. As a single test, the aPS antibody was significantly better (P<0.05) than the aPT and aAA5 antibodies in the detection of APS cases. Seven non-criteria patients were confirmed using additional aPL antibodies. Among these patients, four, two, and one patient was confirmed with aPS, aPT, and aAA5 antibodies, respectively. CONCLUSION: Our data support the findings of previously published studies and attribute the clinical significance of additional aPL antibodies, particularly aPS, in identifying non-criteria APS cases. In the future, along with conventional aPL antibodies, these additional antibodies should be included as standard laboratory tests in the revised Sapporo criteria.

Anti-phosphatidyl-serine/prothrombin antibodies (aPS/PT) in isolated lupus anticoagulant (LA): is their presence linked to dual test positivity?

OBJECTIVES: Anti phosphatidylserine/prothrombin antibodies (aPS/PT) are often present in patients with antiphospholipid syndrome (APS) and might be relevant in the pathogenesis of this condition. They are major determinant of lupus anticoagulant (LA) in triple-positive antiphospholipid (aPL) profile. Whether they are present and pathogenic in patients with isolated LA [negative anticardiolipin (aCL) and anti ß2-glycoprotein I (aß2GPI) antibodies] is a matter of debate. METHODS: We measured aPS/PT in a large number of isolated LA with the aim to ascertain whether there is a link between the way isolated LA is assessed and the presence of these antibodies. APS/PT were measured in 86 patients with isolated LA (aCL- and abeta2GPI-). LA was assessed by two test systems, the dilute Russell Viper Venom Time (dRVVT) and the Silica Clotting Time (SCT). RESULTS: Sixty-six (77%) individuals with isolated LA were positive for aPS/PT (IgM 44, IgG and IgM 15, IgG in 7). Diagnosis of LA was made based on positive results in both dRVVT and SCT in 40 patients (Group 1) and based on only one positive test in the remaining 46 patients (Group 2). The rate of positive aPS/PT antibodies was significantly higher in Group 1 (OR=7.2, 95% CI 1.9-27.0, p<0.002). Moreover, the titre of IgM aPS/PT was significantly increased in Group 1 as compared to Group 2 (137 U, IQR 64-179 vs. 43 U, IQR 11-120, p=0.008). CONCLUSIONS: These data indicate an association between LA based on two positive coagulation tests and the presence of aPS/PT antibodies, especially of IgM isotype.

An allosteric redox switch in domain V of ß2-glycoprotein I controls membrane binding and anti-domain I autoantibody recognition.

ß2-glycoprotein I (ß2GPI) is an abundant multidomain plasma protein that plays various roles in the clotting and complement cascades. It is also the main target of antiphospholipid antibodies (aPL) in the acquired coagulopathy known as antiphospholipid syndrome (APS). Previous studies have shown that ß2GPI adopts two interconvertible biochemical conformations, oxidized and reduced, depending on the integrity of the disulfide bonds. However, the precise contribution of the disulfide bonds to ß2GPI structure and function is unknown. Here, we substituted cysteine residues with serine to investigate how the disulfide bonds C32-C60 in domain I (DI) and C288-C326 in domain V (DV) regulate ß2GPI's structure and function. Results of our biophysical and biochemical studies support the hypothesis that the C32-C60 disulfide bond plays a structural role, whereas the disulfide bond C288-C326 is allosteric. We demonstrate that absence of the C288-C326 bond, unlike absence of the C32-C60 bond, diminishes membrane binding without affecting the thermodynamic stability and overall structure of the protein, which remains elongated in solution. We also document that, while absence of the C32-C60 bond directly impairs recognition of ß2GPI by pathogenic anti-DI antibodies, absence of the C288-C326 disulfide bond is sufficient to abolish complex formation in the presence of anionic phospholipids. We conclude that the disulfide bond C288-C326 operates as a molecular switch capable of regulating ß2GPI's physiological functions in a redox-dependent manner. We propose that in APS patients with anti-DI antibodies, selective rupture of the C288-C326 disulfide bond may be a valid strategy to lower the pathogenic potential of aPL.