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.

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.

Noncriteria antiphospholipid antibodies in antiphospholipid syndrome.

Antiphospholipid syndrome (APS) is an autoimmune disease characterized by thrombotic manifestations and/or obstetric complications in patients with persistently positive antiphospholipid antibodies (aPL). aPL are a heterogeneous group of autoantibodies, but only lupus anticoagulant, anticardiolipin (aCL), and antibeta2-glycoprotein I antibodies (aß2GPI) IgG or IgM are included as laboratory classification criteria. Seronegative APS patients are usually defined as patients with the clinical symptoms of APS but who test negative for aPL. The negativity to classic aPL criteria does not exclude the presence of other aPL. Several noncriteria aPL have been identified. Some noncriteria aPL are well studied, such as IgA aCL and aß2GPI, the antiphosphatidylserine-prothrombin (aPS/PT) antibodies, and the antibodies against the domain I of beta2-glycoprotein I (aDI), both latter groups receiving more attention for their role in thrombotic events and pregnancy complications. Other noncriteria aPL that have been studied are antibodies against annexin V, prothrombin, phosphatidylethanolamine, phosphatidic acid, phosphatidylserine, phosphatidylinositol, vimentin-cardiolipin complex, anti-protein S/protein C. Measurement of some of these noncriteria aPL (aPS/PT, aDI) is useful in the laboratory work-out of APS in specific situations. We have to differentiate between patients who are positive for noncriteria aPL only, and patients who have both criteria and noncriteria aPL to enable us to study their role in the diagnosis or risk stratification of APS. The research on noncriteria aPL is continually developing as the clinical relevance of these antibodies is not yet fully clarified.

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.

T cell involvement in antiphospholipid syndrome.

Antiphospholipid syndrome (APS) is a systemic autoimmune disorder characterized by arterial and venous thrombosis, and obstetric complications in the presence of antiphospholipid antibodies (aPL), including lupus anticoagulant, anticardiolipin and anti-ß2-glycoprotein I antibodies. APS manifests as single, often as recurrent events, and rarely as a catastrophic condition. Most studies of APS pathogenesis to date have focused on the prothrombotic role of aPL, while innate immune responses such as monocyte, complement and neutrophil activation have been also recognized as part of the thrombo-inflammatory cascade in APS. While the presence of autoreactive T cells against ß2-glycoprotein I has been long known, less data are available on their pathogenetic role in APS. In this review, we summarize current knowledge on the involvement of T cells in APS pathophysiology, alterations of T cell subsets in peripheral blood, and clinical associations. We also highlight potential therapeutic opportunities by targeting T helper-B cell interactions in these patients.

Cryo-EM structure and functional basis of prothrombin recognition by a type I antiprothrombin antiphospholipid antibody.

ABSTRACT: Antiprothrombin antibodies are found in antiphospholipid patients, but how they interact with prothrombin remains elusive. Prothrombin adopts closed and open forms. We recently discovered type I and type II antibodies and proposed that type I recognizes the open form. In this study, we report the discovery and structural and functional characterization in human plasma of a type I antibody, POmAb (prothrombin open monoclonal antibody). Using surface plasmon resonance and single-molecule spectroscopy, we show that POmAb interacts with kringle-1 of prothrombin, shifting the equilibrium toward the open form. Using single-particle cryogenic electron microscopy (cryo-EM), we establish that the epitope targeted by POmAb is in kringle-1, comprising an extended binding interface centered at residues R90-Y93. The 3.2-Å cryo-EM structure of the complex reveals that the epitope overlaps with the position occupied by the protease domain of prothrombin in the closed state, explaining the exclusive binding of POmAb to the open form. In human plasma, POmAb prolongs phospholipid-initiated and diluted Russell's viper venom clotting time, which could be partly rescued by excess phospholipids, indicating POmAb is an anticoagulant but exerts a weak lupus anticoagulant effect. These studies reveal the structural basis of prothrombin recognition by a type I antiphospholipid antibody and uncover an exciting new strategy to achieve anticoagulation in human plasma.

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.

Endothelial Cell-Activating Antibodies in COVID-19.

OBJECTIVE: While endothelial dysfunction has been implicated in the widespread thromboinflammatory complications of COVID-19, the upstream mediators of endotheliopathy remain, for the most part, unknown. This study was undertaken to identify circulating factors contributing to endothelial cell activation and dysfunction in COVID-19. METHODS: Human endothelial cells were cultured in the presence of serum or plasma from 244 patients hospitalized with COVID-19 and plasma from 100 patients with non-COVID-19-related sepsis. Cell adhesion molecules (E-selectin, vascular cell adhesion molecule 1, and intercellular adhesion molecule 1 [ICAM-1]) were quantified using in-cell enzyme-linked immunosorbent assay. RESULTS: Serum and plasma from COVID-19 patients increased surface expression of cell adhesion molecules. Furthermore, levels of soluble ICAM-1 and E-selectin were elevated in patient serum and correlated with disease severity. The presence of circulating antiphospholipid antibodies was a strong marker of the ability of COVID-19 serum to activate endothelium. Depletion of total IgG from antiphospholipid antibody-positive serum markedly reduced the up-regulation of cell adhesion molecules. Conversely, supplementation of control serum with patient IgG was sufficient to trigger endothelial activation. CONCLUSION: These data are the first to indicate that some COVID-19 patients have potentially diverse antibodies that drive endotheliopathy, providing important context regarding thromboinflammatory effects of autoantibodies in severe COVID-19.

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.

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.

Thrombotic microangiopathy in primary antiphospholipid syndrome is linked to stroke and less deep venous thrombosis.

OBJECTIVE: To compare clinical and laboratory data obtained from patients with primary Antiphospholipid Syndrome (pAPS) with and without Thrombotic Microangiopathy (TMA). PATIENTS AND METHODS: A cross-sectional study with 66 (83.3% female) pAPS patients was performed. Demographic, clinical, drug use, antiphospholipid antibodies data were evaluated. Patients were subdivided into one of two groups: pAPS with TMA and pAPS without TMA and were compared. RESULTS: In this sample, 5/66 (7.6%) of patients had TMA. Primary APS with TMA group exhibited a higher frequency of arterial events (100% vs. 54.1%, p=0.02), stroke (100% vs. 32.8%, p=0.001) and a lower frequency of deep venous thrombosis (0 vs. 68.9%, p=0.0009) compared to the patients without TMA. Analysis of therapy used in these patients showed a higher frequency of current (40% vs. 6.6%, p=0.0006) and previous glucocorticoid use (80% vs. 36%, p=0.0007) and statin use (50% vs. 22.9%, p=0.037) in the first group. The two groups exhibited no differences in the frequency of positive autoantibodies, except for higher IgG anticardiolipin titers (86 ± 52 vs. 34.5 ± 39 GPL, p=0.003). CONCLUSIONS: Patients with pAPS and TMA have distinct clinical and laboratory spectra from those without TMA, that is characterized by an increased frequency of arterial events, stroke, and higher titers of IgG anticardiolipin; they have deep venous thrombosis less frequently.

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.

Systematic Review of Antiphospholipid Antibodies in COVID-19 Patients: Culprits or Bystanders?

PURPOSE OF REVIEW: COVID-19 patients have a procoagulant state with a high prevalence of thrombotic events. The hypothesis of an involvement of antiphospholipid antibodies (aPL) has been suggested by several reports. Here, we reviewed 48 studies investigating aPL in COVID-19 patients. RECENT FINDINGS: Prevalence of Lupus Anticoagulant (LA) ranged from 35% to 92% in ICU patients. Anti-cardiolipin (aCL) IgG and IgM were found in up to 52% and up to 40% of patients respectively. Anti-ß2-glycoprotein I (aß2-GPI) IgG and IgM were found in up to 39% and up to 34% of patients respectively. Between 1% and 12% of patients had a triple positive aPL profile. There was a high prevalence of aß2-GPI and aCL IgA isotype. Two cohort studies found few persistent LA but more persistent solid phase assay aPL over time. aPL determination and their potential role is a real challenge for the treatment of this disease.

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.