Disentangling the riddle of systemic lupus erythematosus with antiphospholipid syndrome: blood transcriptome analysis reveals a less-pronounced IFN-signature and distinct molecular profiles in venous versus arterial events.

INTRODUCTION: Systemic lupus erythematosus with antiphospholipid syndrome (SLE-APS) represents a challenging SLE endotype whose molecular basis remains unknown. METHODS: We analysed whole-blood RNA-sequencing data from 299 patients with SLE (108 SLE-antiphospholipid antibodies (aPL)-positive, including 67 SLE-APS; 191 SLE-aPL-negative) and 72 matched healthy controls (HC). Pathway enrichment analysis, unsupervised weighted gene coexpression network analysis and machine learning were applied to distinguish disease endotypes. RESULTS: Patients with SLE-APS demonstrated upregulated type I and II interferon (IFN) pathways compared with HC. Using a 100-gene random forests model, we achieved a cross-validated accuracy of 75.6% in distinguishing these two states. Additionally, the comparison between SLE-APS and SLE-aPL-negative revealed 227 differentially expressed genes, indicating downregulation of IFN-α and IFN-γ signatures, coupled with dysregulation of the complement cascade, B-cell activation and neutrophil degranulation. Unsupervised analysis of SLE transcriptome identified 21 gene modules, with SLE-APS strongly linked to upregulation of the 'neutrophilic/myeloid' module. Within SLE-APS, venous thromboses positively correlated with 'neutrophilic/myeloid' and 'B cell' modules, while arterial thromboses were associated with dysregulation of 'DNA damage response (DDR)' and 'metabolism' modules. Anticardiolipin and anti-ß2GPI positivity-irrespective of APS status-were associated with the 'neutrophilic/myeloid' and 'protein-binding' module, respectively. CONCLUSIONS: There is a hierarchical upregulation and-likely-dependence on IFN in SLE with the highest IFN signature observed in SLE-aPL-negative patients. Venous thrombotic events are associated with neutrophils and B cells while arterial events with DDR and impaired metabolism. This may account for their differential requirements for anticoagulation and provide rationale for the potential use of mTOR inhibitors such as sirolimus and the direct fIIa inhibitor dabigatran in SLE-APS.

Single-cell transcriptome analyses reveal disturbed decidual homoeostasis in obstetric antiphospholipid syndrome.

OBJECTIVES: Obstetric antiphospholipid syndrome (OAPS) is an autoimmune disease characterised by the presence of antiphospholipid antibodies in circulation and pathological pregnancy. However, the pathogenesis of OAPS remains unknown. We aimed to reveal cellular compositions and molecular features of decidual cells involved in the development of OAPS using single-cell RNA sequencing (scRNA-seq). METHODS: We performed unbiased scRNA-seq analysis on the first-trimester decidua from five OAPS patients and five healthy controls (HCs), followed by validations with flow cytometry, immunohistochemical staining and immunofluorescence in a larger cohort. Serum chemokines and cytokines were measured by using ELISA. RESULTS: A higher ratio of macrophages but a lower ratio of decidual natural killer (dNK) cells was found in decidua from OAPS compared with HCs. Vascular endothelial cells shrinked in OAPS decidua while having upregulated chemokine expression and conspicuous responses to IFN-γ and TNF-α. Macrophages in OAPS had stronger phagocytosis function, complement activation signals and relied more on glycolysis. dNK cells were more activated in OAPS and had enhanced cytotoxicity and IFN-γ production. Downregulation of granules in OAPS dNK cells could be associated with suppressed glycolysis. Moreover, stromal cells had a prosenescent state with weakened immune surveillance for senescent cells in OAPS. In addition, the cellular interactions between decidual immune cells and those of immune cells with non-immune cells under disease state were altered, especially through chemokines, IFN-γ and TNF-α. CONCLUSION: This study provided a comprehensive decidual cell landscape and identified aberrant decidual microenvironment in OAPS, providing some potential therapeutic targets for this disease.

History of pre-eclampsia does not appear to be a risk factor for vascular phenotype in women with systemic sclerosis.

BACKGROUND: Vascular phenotype is associated with a poor prognosis in systemic sclerosis (SSc). The identification of its risk factors could facilitate its early detection. OBJECTIVES: To explore risk factors for a vascular phenotype of SSc, among them a history of pre-eclampsia. METHODS: This observational multicentre case-control study enrolled adult women fulfilling European Alliance of Associations for Rheumatology 2013 diagnosis criteria for SSc and having a pregnancy history≥6 months before SSc diagnosis in 14 French hospital-based recruiting centres from July 2020 to July 2022. Cases had specific vascular complications of SSc defined as history of digital ischaemic ulcers, pulmonary arterial hypertension, specific cardiac involvement or renal crisis. Women with SSc were included during their annual follow-up visit and filled in a self-administered questionnaire about pregnancy. A case report form was completed by their physician, reporting data on medical history, physical examination, clinical investigations and current medication. The main outcome was the presence/absence of a personal history of pre-eclampsia before SSc diagnosis, according to the validated pre-eclampsia questionnaire. RESULTS: 378 women were included: 129 cases with a vascular phenotype and 249 matched controls. A history of pre-eclampsia was reported in 5 (3.9%) cases and 12 (4.8%) controls and was not associated with a vascular phenotype (OR=0.96, 95% CI 0.28 to 3.34, p=0.9). Besides, Rodnan skin score and disease duration≥5 years were risk factors for vascular phenotype. CONCLUSIONS: In women with SSc and a pregnancy history≥6 months before SSc, a history of pre-eclampsia is not associated with a vascular phenotype.

Therapy with direct oral anticoagulants for secondary prevention of thromboembolic events in the antiphospholipid syndrome: a systematic review and meta-analysis of randomised trials.

OBJECTIVE: Antiphospholipid syndrome (APS) is a systemic autoimmune disorder characterised by venous thrombosis (VT) or arterial thrombosis (AT) and/or pregnancy morbidity and the presence of antiphospholipid antibodies. Direct oral anticoagulants (DOACs) hold several advantages to vitamin K antagonists (VKAs) for prevention of thrombosis and we wish to evaluate DOACs compared with VKAs in secondary prevention of thromboembolic events in patients with APS. METHODS: We conducted searches of the published literature using relevant data sources (MEDLINE, Embase and Cochrane CENTRAL), and of trial registers for unpublished data and ongoing trials. We included randomised trials examining individuals >18 years with APS classified according to the criteria valid when the trial was carried out. Randomised controlled trials had to examine any DOAC agent compared with any comparable drug. We tabulated all occurrences of events from all eligible randomised trials. Due to few events, ORs and 95% CIs were calculated using the Peto method. RESULTS: 5 randomised trials comprising 624 patients met the predefined eligibility criteria. The primary outcome measure was new thrombotic events, a composite endpoint of any VT or AT, during the VKA-controlled phase of treatment. According to the I2 inconsistency index, there was evidence of statistical heterogeneity across the studies (I2=60%). Across trials, 29 and 10 thrombotic events were observed in 305 and 319 patients with APS treated with DOAC and VKA, respectively, corresponding to a combined Peto OR of 3.01 (95% CI 1.56 to 5.78, p=0.001). There was a significantly increased risk of AT while treated with DOACs compared with VKA (OR 5.5 (2.5, 12.1) p<0.0001), but no difference in the risk of VT (p=0.87). We found no significant difference in risk of bleeding. CONCLUSIONS: DOACs were associated with a significant increase in the risk of a new thrombotic event, especially AT, favouring standard prophylaxis with warfarin. PROSPERO REGISTRATION NUMBER: CRD42019126720.

2023 ACR/EULAR antiphospholipid syndrome classification criteria.

OBJECTIVE: To develop new antiphospholipid syndrome (APS) classification criteria with high specificity for use in observational studies and trials, jointly supported by the American College of Rheumatology (ACR) and EULAR. METHODS: This international multidisciplinary initiative included four phases: (1) Phase I, criteria generation by surveys and literature review; (2) Phase II, criteria reduction by modified Delphi and nominal group technique exercises; (3) Phase III, criteria definition, further reduction with the guidance of real-world patient scenarios, and weighting via consensus-based multicriteria decision analysis, and threshold identification; and (4) Phase IV, validation using independent adjudicators' consensus as the gold standard. RESULTS: The 2023 ACR/EULAR APS classification criteria include an entry criterion of at least one positive antiphospholipid antibody (aPL) test within 3 years of identification of an aPL-associated clinical criterion, followed by additive weighted criteria (score range 1-7 points each) clustered into six clinical domains (macrovascular venous thromboembolism, macrovascular arterial thrombosis, microvascular, obstetric, cardiac valve, and hematologic) and two laboratory domains (lupus anticoagulant functional coagulation assays, and solid-phase enzyme-linked immunosorbent assays for IgG/IgM anticardiolipin and/or IgG/IgM anti-ß2-glycoprotein I antibodies). Patients accumulating at least three points each from the clinical and laboratory domains are classified as having APS. In the validation cohort, the new APS criteria vs the 2006 revised Sapporo classification criteria had a specificity of 99% vs 86%, and a sensitivity of 84% vs 99%. CONCLUSION: These new ACR/EULAR APS classification criteria were developed using rigorous methodology with multidisciplinary international input. Hierarchically clustered, weighted, and risk-stratified criteria reflect the current thinking about APS, providing high specificity and a strong foundation for future APS research.

Thrombin generation assay and lupus anticoagulant synergically distinguish populations of patients with antiphospholipid antibodies.

AIM: To apply thrombin generation assay (TGA) in a large cohort of antiphospholipid antibodies (aPL)-positive patients. MATERIAL AND METHODS: 108 patients were tested with TGA and lupus anticoagulant (LA) testing and divided according to their aPL profile. Briefly, 21 patients were positive for anti-phosphatidylserine (aPS)/prothrombin (PT) IgG/IgM (group1), 29 for anti-ß2-glycoprotein-I (aß2GPI) and anti-cardiolipin (aCL) IgG/IgM (group2), 31 for aPS/PT, aß2GPI and aCL IgG/IgM (group3), 27 for aPS/PT and/or aß2GPI+aCL IgM at low-titres (group4). 31 healthy donors (HDs) and 24 controls treated with vitamin K antagonists (VKA) were included. RESULTS: The most deranged TGA and LA profiles were observed in tetra-positive patients (group3) that differed significantly to the other groups, thus those with isolated, double or triple aPL-positivity. Moreover, when comparing the TGA profile of all antiphospholipid syndrome (APS) patients, aPL-carriers, HDs and VKA controls, we observed that the aPL+ patients (especially APS) showed a distinctive profile that allowed to distinguish them from the other groups with significantly higher tLag and tPeak, and lower Peak and area under the curve.When focusing on APS clinical manifestations, patients with a high-risk profile (group3) showed significant differences from those presenting low-titres aPL (group 4) regarding the number of venous events (p=0.04), recurrence of any thrombotic event (p=0.01), of arterial events (5 vs 0, p=0.02), the occurrence of TIA (p=0.04), DVT (p=0.02) and, when analysing extracriteria manifestations, of peripheral artery disease (p=0.04). CONCLUSIONS: TGA seems a valuable approach to stratify aPL+ patients according to their risk profile. The differences among different populations of autoantibodies specificities could be considered a translational validation of the increased thrombotic risk of patients with triple or tetra aPL-positivity.

Antibodies to FXa and thrombin in patients with SLE differentially regulate C3 and C5 cleavage.

OBJECTIVES: The significance of antibodies directed against activated factor X (FXa) and thrombin (Thr) in patients with SLE and/or antiphospholipid syndrome (APS) is unknown. FXa and Thr are coregulated by antithrombin (AT) and activate complement. Therefore, we studied the ability of anti activated factor X (aFXa) and/or anti-(a)Thr IgG from patients with SLE±APS to modulate complement activation. METHODS: Patients with SLE±APS were selected on the basis of known aThr and/or aFXa IgG positivity, and the effects of affinity-purified aFXa/aThr IgG on FXa and Thr-mediated C3 and C5 activation were measured ±AT. Structural analyses of FXa and Thr and AT-FXa and AT-Thr complexes were analysed in conjunction with the in vitro ability of AT to regulate aFXa-FXa and aThr-Thr-mediated C3/C5 activation. RESULTS: Using affinity-purified IgG from n=14 patients, we found that aThr IgG increased Thr-mediated activation of C3 and C5, while aFXa IgG did not increase C3 or C5 activation. Structural analysis identified potential epitopes and predicted a higher likelihood of steric hindrance of AT on FXa by aFXa IgG compared with the AT-Thr-aThr IgG complex that was confirmed by in vitro studies. Longitudinal analysis of 58 patients with SLE (±APS) did not find a significant association between positivity for aFXa or aTHr IgG and C3 levels or disease activity, although there was a trend for patients positive for aFXa IgG alone or both aFXa and aThr IgG to have lower levels of C3 compared with aThr IgG alone during clinical visits. CONCLUSIONS: We propose a novel method of complement regulation in patients with SLE±APS whereby aFXa and aThr IgG may have differential effects on complement activation.

Right Atrial Thrombus Mimicking a Myxoma: Synergism of Hormonal Contraceptives and Antiphospholipid Antibodies.

Cardiac thrombus, the most common intracardiac mass, is typically seen in the left side of the heart in the presence of atrial fibrillation, mitral stenosis, or impaired global wall motion. Right atrial thrombus, which is rarer, is usually associated with central venous catheter placement or pulmonary embolism. We present the case of a 24-year-old woman with a history of mitral valve prolapse who presented with fatigue and palpitations. Echocardiograms and cardiac magnetic resonance images revealed a right atrial mass compatible with a myxoma. However, after surgical excision of this and a second mass discovered intraoperatively, pathologic evaluation confirmed organized thrombus rather than myxoma. The patient's only risk factor was her use of oral contraceptive pills. Test results for hypercoagulable disorders revealed the presence of antiphosphatidylserine, an uncommon antiphospholipid antibody. The patient stopped taking the contraceptive. This case suggests the need to examine further the role of antiphosphatidylserine antibodies in the diagnosis of antiphospholipid syndrome.

Pregnancy outcomes in antiphospholipid antibody positive patients: prospective results from the AntiPhospholipid Syndrome Alliance for Clinical Trials and InternatiOnal Networking (APS ACTION) Clinical Database and Repository ('Registry').

OBJECTIVES: To describe the outcomes of pregnancies in antiphospholipid antibody (aPL)-positive patients since the inception of the AntiPhospholipid Syndrome Alliance for Clinical Trials and InternatiOnal Networking Registry. METHODS: We identified persistently aPL-positive patients recorded as 'pregnant' during prospective follow-up, and defined 'aPL-related outcome' as a composite of: (1) Preterm live delivery (PTLD) at or before 37th week due to pre-eclampsia (PEC), eclampsia, small-for-gestational age (SGA) and/or placental insufficiency (PI); or (2) Otherwise unexplained fetal death after the 10th week of gestation. The primary objective was to describe the characteristics of patients with and without aPL-related composite outcomes based on their first observed pregnancies following registry recruitment. RESULTS: Of the 55 first pregnancies observed after registry recruitment among nulliparous and multiparous participants, 15 (27%) resulted in early pregnancy loss <10 weeks gestation. Of the remaining 40 pregnancies: (1) 26 (65%) resulted in term live delivery (TLD), 4 (10%) in PTLD between 34.0 weeks and 36.6 weeks, 5 (12.5%) in PTLD before 34th week, and 5 (12.5%) in fetal death (two associated with genetic anomalies); and (2) The aPL-related composite outcome occurred in 9 (23%). One of 26 (4%) pregnancies with TLD, 3/4 (75%) with PTLD between 34.0 weeks and 36.6 weeks, and 3/5 (60%) with PTLD before 34th week were complicated with PEC, SGA and/or PI. Fifty of 55 (91%) pregnancies were in lupus anticoagulant positive subjects, as well as all pregnancies with aPL-related composite outcome. CONCLUSION: In our multicentre, international, aPL-positive cohort, of 55 first pregnancies observed prospectively, 15 (27%) were complicated by early pregnancy loss. Of the remaining 40 pregnancies, composite pregnancy morbidity was observed in 9 (23%) pregnancies.

Risk factors of disease flares in a Chinese lupus cohort with low-grade disease activity.

OBJECTIVE: Recurrent disease flare is one of the key problems in lupus patients. A Chinese Flare-Prevention Lupus Initiative Cohort (FLIC) was established. Risk factors of disease flare were evaluated accordingly. METHODS: Patients with low-grade disease activity (the Safety of Estrogens in Lupus Erythematosus National Assessment-SLE Disease Activity Index (SELENA-SLEDAI) =≤6, daily prednisone ≤20 mg, no British Isles Lupus Assessment Group A or no more than one B organ domain score) from January 2014 to August 2020 were included in the FLIC. Disease flares were defined by the modified SELENA--SLEDAI Flare Index. Low disease activity status (LDAS) and remission were also assessed. The cumulative flare rate was estimated by an event per 100 person-years analysis. Cox proportional hazards models were performed to identify risk factors of subsequent disease flares after adjusting clinical confounders. Survival was assessed with the Kaplan-Meier method. RESULTS: 448 eligible patients with low-grade disease activity were included in FLIC. During a mean follow-up of 30.4 months, 170 patients flared. The cumulative lupus flare rate was 22.2 events per 100 patient-years. Compared with patients without flare, those with lupus flares were taking more prednisone, had higher disease activity index and with less patients attained LDAS/remission at baseline. They also had higher rates of antiphospholipid antibodies (aPLs) and antiribosomal P antibody. Cox regression analysis confirmed that attainment of either LDAS or remission at baseline were independent protective factors against subsequent disease flare (LDAS but not in remission: HR 0.58, 95% CI 0.38~0.88; remission: HR 0.46, 95% CI 0.30~0.69), while aPL was a risk factor of lupus flares (HR 1.95, 95% CI 1.36~2.78). Kaplan-Meier curves indicated that attaining LDAS or remission and absence of aPL at baseline had the least flare risk. CONCLUSIONS: In our real-world cohort study, not attaining LDAS or remission at baseline and aPL positivity was associated with higher risk of disease flares in patients with low-grade SLE.

Current status and role of antiphospholipid antibody testing in cryptogenic stroke.

BACKGROUND AND PURPOSE: Although it is not recognized as essential to test for antiphospholipid antibody (aPL) in stroke of unknown cause, aPL-related stroke may account for a considerable number of cryptogenic strokes. We aimed to assess the current status and diagnostic value of aPL testing in cryptogenic stroke patients. METHODS: Consecutive patients admitted with acute ischemic stroke were examined to confirm the factors associated with performing aPL testing and with positive aPL test results in real-world practice. Cryptogenic stroke patients were separately examined in the same manner. The antibody profiles of cryptogenic stroke patients with aPL positivity were compared by age. RESULTS: Among 2947 patients, 606 (20.6%) were tested for aPLs and 129 (21.3%) were positive. Physicians tended to perform aPL testing in patients aged <50 years and in cryptogenic stroke patients. Cryptogenic stroke was a strong predictor of positive aPL results (adjusted odds ratio 3.70, 95% confidence interval 2.38-5.76). However, aPL positivity did not differ by age in stroke patients. Among 283 cryptogenic stroke patients, 136 (48.1%) were tested for aPLs and 56 (41.2%) were positive. aPL tests were performed predominantly in patients aged <50 years rather than in older patients, even among cryptogenic stroke patients. The two age groups had similar positivity rates of >40% (<50 years: 43.2%; ≥50 years: 40.4%; p = 0.92) and their antibody profiles were similar. CONCLUSIONS: A significant number of patients with cryptogenic stroke had positive aPL results regardless of age. aPL testing may offer additional diagnostic opportunities in cryptogenic stroke patients, and thus may reduce the incidence of cryptogenic stroke.

Correlations of anti-cardiolipin antibody and anti-β2 glycoprotein I antibody with ischemic stroke in patients with systemic autoimmune diseases / 中国基层医药

Objective:To correlate anti-cardiolipin antibody (aCL) and anti-β2 glycoprotein I antibody (aβ2GPI) with ischemic stroke (IS) in patients with systemic autoimmune diseases (SADs).Methods:A total of 104 patients with SADs who received treatment in the Affiliated Hospital of North Sichuan Medical College during January to December 2019 were included in this study. They were divided into two groups whether they had IS (IS group, n = 42) or not (non-IS group, n = 62). aPL positive rate was qualitatively compared between the IS and non-IS groups. aCL and aβ2GPI expression levels were quantitatively compared between the IS and non-IS groups. Logistic regression analysis was performed to evaluate the risk factors for IS in patients with SADs. Results:aPL positive rate in the IS group was significantly higher than that in the non-IS group [61.9% (26/42) vs. 40.3% (25 /62), χ2 = 4.66, P = 0.031]. The aCL-IgM and aβ2GPI-IgM levels in the IS group were (22.82 ± 27.27) RU/mL and (18.70 ± 23.95) RU/mL, respectively, which were significantly higher than those in the non-IS group [(13.34 ± 8.43) RU/mL, (7.61± 5.80) RU/mL, t = -2.18, -2.76, P = 0.034, 0.009]. Logistic regression analysis showed that aPL is an independent risk factor for IS ( P = 0.037). Conclusion:aCL and aβ2GPI are closely related to the occurrence of IS and are the independent risk factors for IS in patients with SADs.