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.

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.

IgA is the predominant isotype of anti-ß2 glycoprotein I in patients with COVID-19.

OBJECTIVE: The aim of this research was to determine the frequency of antiphospholipid antibodies (aPL) in patients with COVID-19. METHODS: The frequency and titers of anticardiolipin antibodies (aCL) and anti-ß2 glycoprotein I antibodies (aß2GPI) were determined in sera of adult patients hospitalized with COVID-19. Immunoglobulin (Ig)G, IgA, IgM aCL, and aß2GPI were measured using enzyme-linked immunosorbent assay. RESULTS: Eighty-three patients were included in the study. The mean age of patients was 62 ± 13.9 years, ranging from 23 to 86 years. Stratification according to severity of infection divided patients in 2 groups: 45 patients with moderate infection and 38 patients with critical or severe infection. Out of the 83 patients suffering from COVID-19, aPL (aCL or aß2GPI) were detected in 24 patients (28.9%). IgG, IgA and IgM aß2GPI were positive in 2.4%, 16.9% and 8.4%, respectively. IgG, IgA and IgM aCL showed positivity in 7.2%, 0%, and 4.8%, respectively. The frequency of aPL was 36.8% in patients with critical/severe infection and 22.2% in patients with moderate infection. In critical/severe patients, the frequency of aß2GPI was significantly higher than aCL (34.2% vs 13.2%, P = .03) and aß2GPI-IgA were significantly more frequent than aß2GPI-IgG (21.1% vs 2.6%, P = .028). CONCLUSION: In this cross-sectional study, aPL and particularly aß2GPI-IgA were common in patients with COVID-19.

Anti-ß2-glycoprotein I and anti-phosphatidylserine/prothrombin antibodies interfere with cleavage of factor V(a) by activated protein C.

BACKGROUND: The acquired thrombotic risk factor known as lupus anticoagulant (LA) interferes with laboratory clotting assays and can be caused by autoantibodies against ß2-glycoprotein I (ß2GPI) and prothrombin. LA is associated with activated protein C (APC) resistance, which might contribute to thrombotic risk in patients with antiphospholipid syndrome. How antibodies against ß2GPI and prothrombin cause APC resistance is currently unclear. OBJECTIVES: To investigate how anti-ß2GPI and antiphosphatidylserine/prothrombin (PS/PT) antibodies induce APC resistance. METHODS: The effects of anti-ß2GPI and anti-PS/PT antibodies on APC resistance were studied in plasma (of patients with antiphospholipid syndrome) and with purified coagulation factors and antibodies. RESULTS: APC resistance was observed in LA-positive patients with anti-ß2GPI or anti-PS/PT antibodies and in normal plasma spiked with monoclonal anti-ß2GPI or anti-PS/PT antibodies with LA activity. Analysis of factor (F)V cleavage patterns after APC incubation indicated that anti-ß2GPI antibodies attenuated APC-mediated FV cleavage at R506 and R306. APC-mediated cleavage at R506 is required for FV cofactor activity during inactivation of FVIIIa. Assays with purified coagulation factors confirmed that anti-ß2GPI antibodies interfered with the cofactor function of FV during FVIIIa inactivation but not with FVa inactivation. Anti-PS/PT antibodies attenuated APC-mediated FVa and FVIIIa inactivation. Analysis of FV(a) cleavage patterns after APC incubation indicated that anti-PS/PT antibodies interfere with APC-mediated cleavage of FV at positions R506 and R306. CONCLUSION: Anti-ß2GPI antibodies with LA activity contribute to a procoagulant state by causing APC resistance via interference with the cofactor function of FV during FVIIIa inactivation. LA-causing anti-PS/PT antibodies interfere with the anticoagulant function of APC by preventing FV(a) cleavage.

'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.

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.

Evaluation of the Diagnostic Value of Non-criteria Antibodies for Antiphospholipid Syndrome Patients in a Chinese Cohort.

Objective: Although specific anti-phospholipid antibodies (aPLs) have been used in the diagnosis of the antiphospholipid syndrome (APS) for years, new biomarkers are required to increase its diagnostic and risk-predictive power. This study aimed to explore the value of several non-criteria aPLs in a Chinese cohort. Methods: A total of 312 subjects, namely, 100 patients diagnosed with primary APS, 51 with APS secondary to SLE, 71 with SLE, and 90 healthy controls, were recruited. Serum anticardiolipin (aCL) IgG/IgM/IgA, anti-ß2-glycoprotein I (aß2GPI) IgG/IgM/IgA, anti-phosphatidylserine/prothrombin antibodies (aPS/PT) IgG/IgM, and anti-annexin A5 antibodies (aAnxV) IgG/IgM were tested using ELISA kits. Results: Of the total number of patients, 30.46% and 6.62% with APS were positive for aCL or aß2GPI IgA, respectively, while 39.07% and 24.50% were positive for aAnxV or aPS/PT for at least one antibody (IgG or IgM). The addition test of aCL IgA and aAnxV IgM assists in identifying seronegative APS patients, and IgG aPS/PT was linked to stroke. Conclusion: Detection of aCL IgA, aß2GPI IgA, aAnxV IgG/M, and aPS/PT IgG/M as a biomarker provides additive value in APS diagnosis and would help in risk prediction for APS patients in medical practice.

A retrospective study on the prevalence of anti-phospholipid antibodies, thrombotic events and cutaneous signs of vasculopathy in 173 hospitalized COVID-19 patients.

BACKGROUND: Hypercoagulability is a risk factor of thromboembolic events in COVID-19. Anti-phospholipid (aPL) antibodies have been hypothesized to be involved. Typical COVID-19 dermatological manifestations of livedo reticularis and digital ischemia may resemble cutaneous manifestations of anti-phospholipid syndrome (APS). OBJECTIVES: To investigate the association between aPL antibodies and thromboembolic events, COVID-19 severity, mortality, and cutaneous manifestations in patients with COVID-19. METHODS: aPL antibodies [anti-beta2-glycoprotein-1 (B2GP1) and anti-cardiolipin (aCL) antibodies] were titered in frozen serum samples from hospitalized COVID-19 patients and the patients' clinical records were retrospectively analyzed. RESULTS: 173 patients were enrolled. aPL antibodies were detected in 34.7% of patients, anti-B2GP1 antibodies in 30.1%, and aCL antibodies in 10.4%. Double positivity was observed in 5.2% of patients. Thromboembolic events occurred in 9.8% of patients, including 11 pulmonary embolisms, 1 case of celiac tripod thrombosis, and six arterial ischemic events affecting the cerebral, celiac, splenic, or femoral-popliteal arteries or the aorta. aPL antibodies were found in 52.9% of patients with vascular events, but thromboembolic events were not correlated to aPL antibodies (adjusted OR = 1.69, p = 0.502). Ten patients (5.8%) had cutaneous signs of vasculopathy: nine livedo reticularis and one acrocyanosis. No significant association was observed between the presence of cutaneous vasculopathy and aPL antibodies (p = 0.692). CONCLUSIONS: Anti-phospholipid antibodies cannot be considered responsible for hypercoagulability and thrombotic events in COVID-19 patients. In COVID-19 patients, livedo reticularis and acrocyanosis do not appear to be cutaneous manifestations of APS.

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.

Cerebrovascular events in patients with isolated anti-phosphatidyl-serine/prothrombin antibodies.

The interest of extra-criteria antiphospholipid antibodies is growing, especially in patients negative for conventional antibodies. In this study we aimed to assess the clinical utility of anti-phosphatidyl-serine/prothrombin antibodies (aPS/PT) testing in patients negative for Beta2-Glycoprotein 1(ß2GPI)-dependent tests, for identifying antiphospholipid syndrome (APS) patients that developed cerebrovascular events (CVE). When screening APS patients attending our center, out of 119 aPS/PT IgG/IgM-positive patients, thus patients negative for aß2GPI and aCL, 42 patients (35%) tested negative for ß2GPI-dependent tests and were tested with thrombin generation assay (TGA). Ten patients (24%), with isolated aPS/PT IgG/IgM, had a history of CVE. Lupus anticoagulant (LA)-positive test was more frequently observed in patients with CVE (8/22 vs. 2/20; p = 0.045). Out of the 10 patients who experienced CVE, 3 patients were aPS/PT IgG positive (all LA positive), and 8 patients were aPS/PT IgM positive (6/8 LA positive). One patient was positive for both aPS/PT IgG and IgM. LA-positive patients had only high titers of aPS/PT IgG/IgM, all of them being ≥ 80 U/ml, while the 2 LA-negative patients were aPS/PT IgM positive with medium titers [40-60 U/ml]. LA-positive patients had significantly altered TGA profile when compared to those who were LA negative, considering all TGA parameters. LA-positive patients had significantly higher tLag (8.4 ± 3.3 min vs. 6.6 ± 1.8 min; p = 0.046), higher tPeak (14 ± 4.3 min vs. 11 ± 2.7 min; p = 0.015) and lower Peak (207 ± 152 nM vs. 356.3 ± 104.7 nM; p < 0.001) and lower AUC (2109.7 ± 1006.9 nM vs. 2772.5 ± 776.8 nM; p = 0.033). The use of aPS/PT might be of help in identifying patients with CVE and APS, as also confirmed by TGA testing.

Comparison of Different Test Systems for the Detection of Antiphospholipid Antibodies in a Chinese Cohort.

Background: Diagnosis of antiphospholipid syndrome (APS) is based on the positivity of laboratory criteria antiphospholipid antibodies (aPLs). Test results for aPLs could be contradictory among different detection methods as well as commercial manufacturers. This study aimed to assess and compare the diagnostic and analytic performances of four commercial assays prevalently used in China. Methods: A total of 313 patients including 100 patients diagnosed with primary APS, 52 with APS secondary to SLE, 71 with SLE, and 90 health controls were recruited. Serum IgG, IgM, and IgA for aCL, and aß2GPI antibodies were detected with two ELISA and two CLIA systems, and test system with the best diagnostic value was explored of its correlation with key clinical features. Results: CLIA by YHLO Biotech Co. was considered as the system with the best predictive power, where 58.55 and 57.89% of APS patients were positive for aCL or aß2GPI for at least one antibody (IgG or IgM or IgA). Overall, CLIA showed better performance characteristics than traditional ELISA test systems. Conclusion: CLIA was considered as a better platform for aPL detection in APS diagnosis. A combination of other detection platforms could assist in differential diagnosis as well as in identifying high-risk patients.

The Role of Antiphospholipid Antibodies in COVID-19.

PURPOSE OF THE REVIEW: Elevated levels of anti-phospholipid (aPL) antibodies are the most important criterion in the diagnosis of anti-phospholipid syndrome (APS) and are usually responsible for promoting the risk of thrombotic complications. Now, in the course of the global coronavirus disease 2019 (COVID-19) pandemic, measurable aPL antibodies have also been detected in a noticeable number of patients showing a variety ranging from studies with only isolated positive tests to cohorts with very high positivity. Thus, the question arises as to whether these two different clinical pictures may be linked. RECENT FINDINGS: The ambivalent results showed a frequent occurrence of the investigated aPL antibodies in COVID-19 patients to an individually varying degree. While some question a substantial correlation according to their results, a number of studies raise questions about the significance of a correlation of aPL antibodies in COVID-19 patients. Within the scope of this review, these have now been described and compared with each other. Ultimately, it is necessary to conduct further studies that specifically test aPL antibodies in a larger context in order to make subsequent important statements about the role of APS in COVID-19 and to further strengthen the significance of the described comparisons.

Therapeutic plasma exchange for anticoagulant-refractory antiphospholipid syndrome with severe ischemic and necrotic skin lesions: A case series.

Antiphospholipid syndrome (APS) is a systemic autoimmune disease characterized by clinical findings including thrombosis and/or obstetric complication and laboratory findings, e.g. ≥1 positive antiphospholipid antibodies (aPL) (lupus anticoagulant, anticardiolipin IgG/IgM and/or anti-ß2-glycoprotein IgG/IgM). A rare APS clinical entity is severe necrosis which is difficult to treat and often does not respond to anticoagulant therapy. Three consecutive patients with primary or secondary APS who presented with necrotic skin lesions secondary to APS were treated with therapeutic plasma exchange (TPE), glucocorticoids and low-molecular-weight heparin. All patients had a rapid-onset, either full or significant recovery of their APS-related necrotic lesions. Upon treatment, one patients showed resolution of lupus anticoagulant. Two patients had a decrease of at least 88 % in aPL titers after the initial treatment, and were kept on TPE maintenance every 5-6 weeks. None of the patients experienced significant side effects of the TPE. This is the first case series showing the clinical benefits of TPE in patients with ischemic and necrotic skin lesions due to severe anticoagulant-refractory vascular APS.

Clinical relevance of nitrated beta 2-glycoprotein I in antiphospholipid syndrome: Implications for thrombosis risk.

Β2-Glycoprotein I (ß2GPI) is an important anti-thrombotic protein and is the major auto-antigen in the antiphospholipid syndrome (APS). The clinical relevance of nitrosative stress in post translational modification of ß2GPI was examined.The effects of nitrated (n)ß2GPI on its anti-thrombotic properties and its plasma levels in primary and secondary APS were determined with appropriate clinical control groups. ß2-glycoprotein I was nitrated at tyrosines 218, 275 and 309. ß2-glycoprotein I binds to lipid peroxidation modified products through Domains IV and V. Nitrated ß2GPI loses this binding (p < 0.05) and had diminished activity in inhibiting platelet adhesion to vWF under high shear flow (p < 0.01). Levels of nß2GPI were increased in patients with primary APS compared to patients with either secondary APS (p < 0.05), autoimmune disease without APS (p < 0.05) or non-autoimmune patients with arterial thrombosis (p < 0.01) and healthy individuals (p < 0.05).In conclusion tyrosine nitration of plasma ß2GPI is demonstrated and has important implications with regards to the pathophysiology of platelet mediated thrombosis in APS. Elevated plasma levels of nß2GPI in primary APS may be a risk factor for thrombosis warranting further investigation.

Anti-vimentin/cardiolipin IgA in the anti-phospholipid syndrome: A new tool for 'seronegative' diagnosis.

Anti-phospholipid syndrome (APS) is a systemic autoimmune disorder defined by the simultaneous presence of vascular clinical events, pregnancy morbidity and anti-phospholipid antibodies (aPL). In clinical practice, it is possible to find patients with APS who are persistently negative for the routine aPL tests (seronegative APS; SN-APS). Recently, the identification of aPL immunoglobulin (Ig)A and/or anti-ß2-glycoprotein-I (ß2-GPI) IgA was shown to represent a further test in SN-APS patients. In this study we analyzed the presence of anti-vimentin/cardiolipin (aVim/CL) IgA in a large cohort of patients with SN-APS, evaluating their possible association with clinical manifestations of the syndrome. This study includes 60 consecutive SN-APS patients, 30 patients with APS and 40 healthy donors. aVim/CL IgA were detected by enzyme-linked immunosorbent assay (ELISA). Results show that 12 of 30 APS patients (40%) and 16 of 60 SN-APS patients (26.7%) resulted positive for aVim/CL IgA. Interestingly, SN-APS patients who tested positive for aVim/CL IgA showed a higher prevalence of arterial thrombosis (p = 0.017, likelihood positive ratio = 5.7). This study demonstrates for the first time, to our knowledge, the presence of aVim/CL IgA in sera of patients with APS. In particular, they revealed a potential usefulness in identification of a significant proportion of SN-APS patients. Moreover, as patients tested positive for aVim/CL IgA reported a high likelihood ratio to have the clinical features of APS, this test may be considered a suitable approach in the clinical evaluation of SN-APS.

Specific domain V reduction of beta-2-glycoprotein I induces protein flexibility and alters pathogenic antibody binding.

Beta-2-glycoprotein I (ß2GPI) is a blood protein and the major antigen in the autoimmune disorder antiphospholipid syndrome (APS). ß2GPI exists mainly in closed or open conformations and comprises of 11 disulfides distributed across five domains. The terminal Cys288/Cys326 disulfide bond at domain V has been associated with different cysteine redox states. The role of this disulfide bond in conformational dynamics of this protein has not been investigated so far. Here, we report on the enzymatic driven reduction by thioredoxin-1 (recycled by Tris(2-carboxyethyl)phosphine; TCEP) of ß2GPI. Specific reduction was demonstrated by Western blot and mass spectrometry analyses confirming majority targeting to the fifth domain of ß2GPI. Atomic force microscopy images suggested that reduced ß2GPI shows a slightly higher proportion of open conformation and is more flexible compared to the untreated protein as confirmed by modelling studies. We have determined a strong increase in the binding of pathogenic APS autoantibodies to reduced ß2GPI as demonstrated by ELISA. Our study is relevant for understanding the effect of ß2GPI reduction on the protein structure and its implications for antibody binding in APS patients.