Antiphospholipid Syndrome Patients Research Articles

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.

Antiphospholipid syndrome, thrombosis, and vaccination in the COVID-19 pandemic.

Thrombosis is one of the many signs of antiphospholipid syndrome (APS) and COVID-19 infection. Although the mechanisms contributing to thrombosis in APS and COVID-19 are relatively similar, this remains an open subject. Even now (when the COVID-19 pandemic has subsided), there is no conclusive solution to APS and COVID-19 co-occurrence. The presence of newly generated antiphospholipid antibodies (aPLs) in COVID-19 infection may or may not be connected to the diagnosis of APS. The prevalence of aPLs is substantial in severe COVID-19 but not related to thrombosis or a worse outcome. Adequate monitoring of antibody positivity over time is recommended for APL diagnosis. On the other hand, thrombosis and thrombocytopenia can rarely occur with vaccination with mRNA vaccines. Some studies have shown that COVID-19 immunization is well tolerated among APS patients who are triple-positive for aPL, which may comfort patients and referring physicians and lessen hesitation in unvaccinated APS/aPL-positive patients. In this narrative review, we will give an overview of the interaction between aPL-APS-COVID-19-thrombosis and related diagnostic insights learned during the pandemic.

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

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. 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.

7-year follow-up atherosclerotic plaque progression in patients with antiphospholipid syndrome versus diabetes mellitus and healthy controls.

Patients with antiphospholipid syndrome (APS) carry a substantial burden of cardiovascular disease and subclinical atherosclerosis. We aimed to assess a 7-year follow-up atherosclerotic plaque progression in APS patients vs diabetes mellitus (DM) and healthy controls (HC). Eighty-six patients with thrombotic APS, 86 with DM and 86 HC (all age- and sex-matched) who underwent a baseline ultrasound of carotid and femoral arteries were invited for a 7-year follow-up ultrasonography examination. We compared atherosclerosis progression among the three groups and examined determinants of plaque progression in APS patients. Sixty-four APS patients (75% females, 43.8% with primary APS), 58 patients with DM and 66 HC were included in the 7-year ultrasound re-evaluation. New plaque was detected in 51.6%, 36.2% and 25.8% of APS, DM and HC subjects, respectively. After adjusting for traditional cardiovascular risk factors (CVRFs) and baseline plaque presence, APS patients showed a 3-fold (OR = 3.07, p= 0.007) higher risk for atherosclerosis progression vs HC and 2-fold (OR = 2.25, p= 0.047) higher risk than DM patients. In multivariate analysis in the APS group, plaque progression was independently associated with systemic lupus erythematosus (SLE) co-existence (OR = 7.78, p= 0.005) and number of CVRFs (OR = 3.02, p= 0.002), after adjusting for disease-related parameters and CVRF-related medications. Sustained low-density lipoprotein target attainment reduced plaque progression risk (OR = 0.34, p= 0.021). Half of APS patients develop new atherosclerotic plaques over a 7-year follow-up, having a three-times higher risk vs HC. Concomitant SLE and number of traditional CVRFs are associated with plaque progression, supporting the need for thorough CVRF assessment and control.

Taipan snake venom time has high sensitivity for lupus anticoagulants in non-anticoagulated, triple positive antiphospholipid syndrome patients.

Dilute Russell's viper venom time (dRVVT) and activated partial thromboplastin time (APTT) are the mainstay assays in lupus anticoagulant (LA) detection yet they have limitations, particularly in relation to interferences and specificity. The recently validated Taipan snake venom time (TSVT) screening with ecarin time (ET) confirmatory assays overcome many of those limitations due to the innate specificity engendered from direct prothrombin activation, and insensitivity to the effects of vitamin K antagonists (VKA). The present study aimed to further evidence diagnostic utility of TSVT/ET by performing them in samples from 116 nonanticoagulated patients with established triple-positive antiphospholipid syndrome (APS). Samples were identified in three expert centres who performed dRVVT, APTT and solid phase antiphospholipid antibody assays with reagents from a variety of manufacturers. All samples additionally received TSVT/ET analysis using standardised reagents. Ninety seven of 116 (83.6%) were dRVVT- and APTT-positive, 85/97 (87.6%) of which were TSVT/ET-positive, 9/116 (7.8%) were dRVVT-positive only, 6 of which were TSVT/ET-positive, and 10/116 (8.6%) were APTT-positive only, 5 of which were TSVT/ET-positive. 96/116 TSVT/ET-positivity returned a high sensitivity for LA of 82.8%. Low coefficients of determination revealed weak relationships between LA potency and anticardiolipin and anti-β2-glycoprotein I antibody titres for all three LA assays. TSVT/ET has high sensitivity for the clinically significant LA found in triple positive APS patients. TSVT/ET can establish multiple LA assay positivity in nonanticoagulated patients negative for one of dRVVT or APTT, and is the only assay pairing insensitive to VKAs, the recommended anticoagulation for APS.

Renal function is associated with endothelial dysfunction and increase in NT-proBNP in systemic lupus erythematosus and antiphospholipid syndrome patient: Pilot study.

Renal function is associated with endothelial dysfunction and increase in NT-proBNP in systemic lupus erythematosus and antiphospholipid syndrome patient: Pilot study.

Antiphospholipid antibodies as a potential factor of tumor progression

Introduction. Current generally accepted clinical and laboratory criteria for antiphospholipid syndrome (APS) have been clearly determined, which include vascular thrombosis and pregnancy complications in patients with circulating antiphospholipid antibodies (aPLs). However, in the last several years, aPLs have become a common finding in patients with malignancies. Accumulating data provide strong evidence for such association and suggests that thrombosis in cancer patients may be related to aPLs activity. According to global publications, aPLs circulation in cancer patients varies from 15 to 74 %, which may be due to differences in clinical characteristics of cancer patients examined as well as distinct interpretations on aPLs diagnostic tests.Aim: to determine aPLs profile in patients with malignant neoplasms of the female reproductive system, identify an association between aPLs and thrombosis as well as degree of disease progression and outcome.Materials and Methods. A single-center observational study was conducted with 130 women, among which 70 subjects had adenocarcinoma of the uterine body, cervix and ovaries. 60 age-matched apparently healthy women lacking thrombotic complications were included into control group. All study participants were examined for circulating lupus anticoagulantas well as anti-cardiolipin antibodies (aCLs), anti-β2-glycoprotein 1 antibodies (anti-β2-GР1), annexin V antibodies, and anti-phosphatidylserine-prothrombin complex antibodies (anti-PS-PT) IgG and IgM by using enzyme-linked immunosorbent assay.Results. Moderate or low aPLs titers were found in 34.2 % of patients with uterine, cervical and ovarian cancer. Ten (14.2 %) of 70 women in main study group had thrombosis so that aPLs were detected only in 5 of 10 women with thrombosis. No significant differences between patients with thrombosis and without thrombotic complications in gynecological cancer were observed. In addition, assessed parameters had no impact on relapse-free survival in cancer patients. However, a significant relation was found between circulating aCLs (IgG, IgM) and anti-PS-PT (IgG, IgM) as well as degree of oncological process. In addition, a significant association was found between aCLs isotype IgG (p = 0.017) and disease relapse.Conclusion. Although thrombosis along with acute thrombosis is a hallmark of APS patients, they demonstrate other non-thrombotic manifestations, one of which is the impact on tumor growth invasion and progression.

Stroke and Risk Factors in Antiphospholipid Syndrome.

Stroke is considered one of the most common and life-threatening manifestations of antiphospholipid syndrome (APS), which leads to high mortality and permanent disability. This study investigated the prevalence and the potential risk factors of stroke in APS. We enrolled 361 APS patients retrospectively from 2009 to 2022 at Peking University People's Hospital. Stroke was found in 25.8% (93/361) of the participants. The multivariate logistic regression showed that hypertension, diabetes, livedo reticularis, and other central nervous system involvements were significant related factors for stroke. The use of hydroxychloroquine appeared to relate to a lower incidence of stroke. During a median follow-up of 3.0 years, 11.8% (11/93) of the individuals with a previous stroke developed stroke recurrence, and thrombocytopenia seemed to be a predictor of stroke recurrence.

Thrombosis recurrence and major bleeding in non-anticoagulated thrombotic antiphospholipid syndrome patients: Prospective study from antiphospholipid syndrome alliance for clinical trials and international networking (APS ACTION) clinical database and repository (“Registry”)

BackgroundLong-term anticoagulant therapy is generally recommended for thrombotic antiphospholipid syndrome (TAPS) patients, however it may be withdrawn or not introduced in routine practice. ObjectivesTo prospectively evaluate the risk of thrombosis recurrence and major bleeding in non-anticoagulated TAPS patients, compared to anticoagulated TAPS, and secondly, to identify different features between those two groups. Patients/MethodsUsing an international registry, we identified non-anticoagulated TAPS patients at baseline, and matched them with anticoagulated TAPS patients based on gender, age, type of previous thrombosis, and associated autoimmune disease. Thrombosis recurrence and major bleeding were prospectively analyzed using Kaplan-Meier method and compared using a marginal Cox's regression model. ResultsAs of June 2022, 94 (14 %) of the 662 TAPS patients were not anticoagulated; and 93 of them were matched with 181 anticoagulated TAPS patients (median follow-up 5 years [interquartile range 3 to 8]). The 5-year thrombosis recurrence and major bleeding rates were 12 % versus 10 %, and 6 % versus 7 %, respectively (hazard ratio [HR] 1.38, 95 % confidence interval [CI] 0.53 to 3.56, p = 0.50 and HR 0.53; 95 % CI 0.15 to 1.86; p = 0.32, respectively). Non-anticoagulated patients were more likely to receive antiplatelet therapy (p < 0.001), and less likely to have more than one previous thrombosis (p < 0.001) and lupus anticoagulant positivity (p = 0.01). ConclusionFourteen percent of the TAPS patients were not anticoagulated at recruitment. Their recurrent thrombosis risk did not differ compared to matched anticoagulated TAPS patients, supporting the pressing need for risk-stratified secondary thrombosis prevention trials in APS investigating strategies other than anticoagulation.

Quantification of Antiphospholipid Antibodies: The Importance of Using an Appropriate Methodology for Each Clinical Profile.

The presence of antiphospholipid antibodies (aPLs) is associated with antiphospholipid syndrome (APS), characterized by thrombosis and obstetric morbidity. aPLs included in APS classification criteria are lupus anticoagulant, anti-cardiolipin and anti-beta-2-glycoprotein-I of IgG or IgM isotypes. Enzyme-linked immunosorbent assay is the most used diagnostic technique to determine aPLs. Recently, new automated technologies mainly based in antigen-coated beads have been developed. The aim is to compare a fluorescence enzyme immunoassay (M1) and an antigen-coated bead assay (M2) in obstetric and thrombotic APS patients. All samples from the first 1020 patients received in the Immune Service Laboratory (Hospital 12 de Octubre) during the recruitment period, without exclusions, were analysed for aPLs. The weighted kappa for both methods in all the patients was 0.39 (0.30-0.47). Agreement increased to 0.56 (0.38-0.73) in patients with autoimmune disease. Sensitivity and specificity obtained for M1 were 17.1% and 89.3%, respectively, and 12.7% and 91.4% for M2. The sensibility and specificity of IgG isotypes were higher than the IgM ones. Regarding obstetric patients, M1 obtained significant diagnostic performance and had more sensitivity 23.75 (14.95-34.58) compared to M2 12.50 (6.16-21.79). In conclusion, clinical suspicion-based method selection for aPLs should be considered. To identify obstetric APS patients, solid phase methods remain more preferable.

Regulatory B Cells Evaluation in Systemic Lupus Erythematosus Patients with Subclinical Atherosclerosis and Secondary Antiphospholipid Syndrome.

The current knowledge of human studies that address B cells in Systemic Lupus Erythematosus (SLE) patients with subclinical atherosclerosis remains insufficient. We aimed to evaluate the contribution of Breg cells in SLE and secondary antiphospholipid syndrome (APS) patients taking into consideration its relation to subclinical atherosclerosis and the disease activity. Thirty SLE patients and 23 controls were included. Systemic Lupus Erythematosus Disease Activity Index-2000 was estimated. Evaluation of Breg cells percentage using flow cytometry was done. All participants underwent carotid doppler ultrasound examination for measurements of the intima-media thickness of the common carotid artery (cIMT). The coronary artery calcium scoring was calculated using the Agatston method. The mean± SD of age was 32.60±8.34 years, while of the age of onset was 28.27±7.60 years. Twenty-three patients (76.7%) had subclinical atherosclerosis. There was a highly significant difference in Breg cells between SLE and APS patients with subclinical atherosclerosis and controls (P= 0.001, 0.005). SLE and APS patients had significantly higher mean cIMT than control (P=0.01, 0.050). Breg cells had 70% sensitivity and 87% specificity for diagnosing of SLE (P=0.01). Multivariate regression analysis indicated that low Breg cells were predictive for the disease activity (OR=1.76, 95% CI=1.21- 2.85; P= 0.01). SLE patients had a high frequency of subclinical atherosclerosis, those and patients with secondary APS had a high risk of plaque formation. We found a contribution of Breg cells in SLE patients with subclinical atherosclerosis. Breg cells are considered a good predictor of diagnosis of SLE.

Prevalence, Domain Specificity, and Residue Specificity of IgG, IgM, and IgA Anti-β2GPI Antibodies in Tetra-Positive Antiphospholipid Syndrome Patients: Identification of Two Non-Overlapping Epitopes in Domain I of β2GPI

Background. Risk stratification of antiphospholipid syndrome (APS) patients involves several laboratory tests, including lupus anticoagulant (LAC), anti-cardiolipin antibodies (anti-CL), anti-β2GPI antibodies (anti-β2GPI), and, more recently, anti-phosphatidylserine/prothrombin antibodies (anti-PS/PT). Patients with positive results for all four tests, known as tetra-positive APS patients, are considered at high risk for thrombotic recurrence. β2GPI is a protein composed of five domains: DI, DII, DIII, DIV, and DV, arranged in a flexible elongated structure. Previous studies have demonstrated that DI is frequently targeted by IgG in thrombotic APS patients. However, the prevalence, domain specificity, and residue specificity of the three different isotypes of anti-β2GPI antibodies in tetra-positive APS patients remain unknown. Objective. To establish the prevalence, domain specificity, and residue specificity of IgG, IgM, and IgA anti-β2GPI antibodies in tetra-positive APS patients. Methods. Plasma samples from 32 clinically confirmed tetra-positive APS patients and 8 healthy controls were analyzed for levels of IgG, IgM, and IgA using an enzyme-linked immunosorbent assay (ELISA) with immobilized β2GPI as the antigen. Domain and residue mapping were investigated using five deleted domain variants and eight single-point mutations. Mutants lacking specific disulfide bonds were used to assess whether the epitopes targeted by the antibodies are linear or conformational. Results. Among the 32 patients tested, 31 (96%) had IgG, 18 (56%) had IgM, and 18 (56%) had IgA anti-β2GPI antibodies. Ten patients (31%) had all three isotypes, 7 (22%) had IgG and IgM, and 8 (25%) had IgG and IgA. Six patients (19%) had only IgG, 1 had only IgM, and none had only IgA. The removal of DI from the β2GPI molecule led to an almost complete loss of binding (~80%) for the majority of IgG antibodies. There was a lesser but still significant reduction in binding when DII and DV were removed. However, no loss of binding was observed with variants lacking DIII and DIV. Notably, IgA antibodies behaved similarly to IgG but differently from IgM, as their reactivity towards DI and DV was significantly attenuated. To investigate the contribution of specific residues to binding, charged residues K19, E23, E26, E27, R39, R43, K44, and K59 in DI were individually replaced with alanine (A). The most important residue for IgG and IgA antibodies was R43, followed by R39 and K44. In contrast, most residues contributed to optimal binding for IgM antibodies, suggesting a less specific epitope definition. Interestingly, the mutations R39A and K44A often led to loss of binding in conjunction with the mutation R43A, but rarely simultaneously, suggesting the existence of two distinct epitopes for anti-DI antibodies-one located north and one south of R43. Finally, IgG, IgM, and IgA antibodies did not react against the mutant C32S/C60S but showed good reactivity against the mutant C288S/C326S, indicating that epitopes in DI are conformational and require the integrity of the disulfide bond 32-60. Conclusions. In tetra-positive APS patients, IgG anti-β2GPI antibodies are the most prevalent isotype, followed by IgA and IgM. The IgG and IgA isotypes exhibit a pronounced preference for DI, while the IgM isotype demonstrates lower specificity, which aligns with IgM's role as initial responders in infections. Notably, our research revealed that anti-DI antibodies are diverse, as they recognize epitopes within DI that are closely related but physically distinct. Ongoing studies aim to further characterize these antibody subtypes and understand their significance in APS. This knowledge may inform improved diagnostic and therapeutic approaches for APS patients.

Complement Activation Is Increased in Antiphospholipid Syndrome-Related Ischemic Stroke with Higher Risk Features

Introduction: Antiphospholipid syndrome (APS) patients with arterial thrombosis, mostly ischemic stroke, have concerningly high rates of recurrent thrombosis despite antithrombotic treatment, with over 20% thrombosis recurrence within two years. Complement activation is increased in thrombotic APS patients, although its role has not been fully investigated in APS-related stroke. This study aimed to evaluate the degree of complement activation in APS-related ischemic stroke, transient ischemic attack (TIA) or other ischemic brain injury patients and whether complement activation correlated with APS severity and platelet hyperreactivity as a potential mechanism for increased thrombotic risk. Method: Patients with confirmed APS by Sydney Criteria and imaging-proven ischemic stroke, TIA or other ischemic brain injury were recruited, subcategorized as high-risk APS (HR-APS, n=30) and lower-risk APS (LR-APS, n=41). HR-APS included triple antiphospholipid antibody (aPL) positive, anticoagulant refractory or both arterial and venous thromboembolism patients. LR-APS included patients with a single thrombotic event who were single or double aPL positive. Ischemic stroke patients without aPL (n=40) and healthy donors (n=40) were utilized as controls. Patients with active cancer, inflammation, infection or use of hydroxychloroquine, immunosuppression and heparins were excluded. Complement activation markers were measured as Bb fragment (Bb), C3a-desArg (C3a), C5a-des-Arg (C5a), and SC5b-9 using ELISA (MicroVue kits, Quidel Corp, Pathway Diagnostics Ltd). Complement-mediated cell killing was measured by the modified Ham (mHam) test, which determines the degree of cell lysis after adding patient serum to a paroxysmal nocturnal hemoglobinuria cell line (PIGA-null TF-1 cells) lacking cell surface complement regulators. A positive result was considered 20% or greater cell killing. Flow cytometry was used to evaluate whether complement activation correlated with platelet hyperreactivity. For this, plasma from mHam positive patients was added to healthy donor whole blood and procoagulant platelet response was measured as the absolute change in proportion of platelet events that were positive for annexin V and P-selectin following stimulation with thrombin 2U/mL. Results: There was no significant difference in median age between the patient cohorts, ranging from 49 to 56 years, although the healthy controls were younger, with median age 36 (range 25-73, p&amp;lt;0.001). Circulating complement activation markers were all significantly increased in the APS cohorts compared to healthy controls (p&amp;lt;0.01). The most prominent increase was observed in C3a levels: HR-APS median (interquartile range - IQR) 215 ng/mL (137.1-297.4), LR-APS 290.8 ng/mL (120.3-450.3) and healthy controls 62 ng/mL (27.9-121.1)(p=0.004 and p&amp;lt;0.0001 compared to healthy controls, respectively); and Bb fragment levels: HR-APS 1.2 µg/mL (IQR 1.0-1.4), LR-APS 1.3 µg/mL (IQR 1.0-1.5) and healthy controls 0.8 µg/mL (IQR 0.7-0.9)(both p&amp;lt;0.0001 compared to healthy controls). However, only C3a, SC5b-9 and Bb fragment levels in LR-APS remained significantly elevated when compared to stroke controls (p=0.0009, p=0.01 and p=0.03, respectively). There were no differences between HR-APS and LR-APS, nor between stroke controls and healthy controls. Complement-mediated cell killing was also significantly higher in APS patients overall, median (IQR) proportion of non-viable cells 18% (IQR 12-33), compared to stroke controls: 10% (IQR 8-15, p&amp;lt;0.0001) and healthy controls: 12.5% (IQR 6.3-17, p=0.0009). Notably, HR-APS had significantly higher proportion of positive mHam tests (60%), compared to LR-APS: 36.6%, stroke controls: 12.5%, and healthy controls: 17.5% (p&amp;lt;0.0001, Figure 1A). Importantly, HR-APS patients with positive mHam results (n=5) induced a substantially higher increase in procoagulant platelets, median 17.2% (IQR 11.2-28.6), whereas healthy controls (n=4) only resulted in median change of -0.7% (IQR -7.0-1.4, p=0.02) (Figure 1B). Conclusion: Preliminary data suggest that APS-related ischemic stroke, TIA or ischemic brain injury are associated with increased, functionally important, complement activation, more prominent in higher risk disease. Complement-mediated platelet hyperreactivity may have a contributory role that remains to be established.

Domain Specificity of Anti-Prothrombin Antibodies That Express Lupus Anticoagulant Activity

Background: The antiphospholipid syndrome (APS) is an autoimmune disorder characterized by the persistent presence of antiphospholipid antibodies (aPL). aPL are a heterogeneous group of autoantibodies directed against negatively charged phospholipids or phospholipid-binding proteins. Some of these autoantibodies exert lupus anticoagulant (LAC) activity, a laboratory phenomenon characterized by the prolongation of phospholipid-dependent coagulation tests. Recently, our group identified that LAC-positive anti-prothrombin antibodies activate platelets via the platelet receptor, FcγRIIA. However, a lot is still unclear regarding the mechanism by which these antibodies cause LAC and why some, but not all, antibodies in this heterogeneous group express LAC activity. Furthermore, experimental data on the binding site of these pathogenic anti-prothrombin antibodies are lacking. Aims: In this study we aimed to investigate the binding domain of LAC-positive monoclonal anti-prothrombin antibodies and APS patient derived anti-prothrombin antibodies. Method: Recombinant fragments of prothrombin were designed and produced in Chinese hamster ovary cells. Prothrombin fragment 1, fragment 2 and fragment 1.2 were coated on ELISA plates to study epitope specificity. Two LAC-positive monoclonal anti-prothrombin antibodies (3B1 and 28F4), two LAC-negative anti-prothrombin monoclonal antibodies (11H2 and 9C6), isolated anti-prothrombin antibodies derived from APS patients and plasma from APS patients were studied. A modified dilute Russell's viper venom time (DRVVT) test was used to evaluate whether the recombinant prothrombin constructs could neutralize the effect of LAC-positive monoclonal and patient derived anti-prothrombin antibodies. Furthermore, a fragment 1.2 nanobody was produced by immunizing Llama's followed by phage display. The ability of this nanobody to compete with LAC-positive monoclonal and patient derived anti-prothrombin antibodies for the prothrombin binding site was tested using a modified DRVVT test. Results: LAC-positive monoclonal anti-prothrombin antibodies recognized prothrombin fragment 2. Interestingly, LAC-negative anti-prothrombin antibodies bound exclusively to prothrombin fragment 1. In addition, LAC-ratios of patient samples correlated significantly with anti-fragment 2 antibody levels ( p&amp;lt;0.05, ρ=0.33), but not with anti-fragment 1 antibody levels. Furthermore, incubation with the prothrombin F1.2 nanobody almost completely reversed the prolonged DRVVT clotting time in plasma containing monoclonal and patient derived LAC-positive anti-prothrombin antibodies, but not in plasma containing LAC-positive anti-β2GP1 antibodies. This neutralising effect was also observed when the monoclonal anti-prothrombin antibody was incubated with prothrombin fragment 2. In contrast, the LAC-activity of anti-prothrombin antibodies was not inhibited when incubated with prothrombin fragment 1. Conclusion: Our data show that LAC-positive anti-prothrombin antibodies react with fragment 2 of prothrombin. Furthermore, a novel nanobody directed against prothrombin fragment 1.2 and prothrombin fragment 2 were able to neutralize a prothrombin-dependent LAC.

Discovery of Unexpected Phospholipid-Binding Proteins in Antiphospholipid Syndrome Patients

INTRODUCTION: The antiphospholipid syndrome (APS), initially described nearly 4 decades ago, remains enigmatic with unclear pathophysiology. In addition to the lack of a mechanistic diagnostic test, APS exhibits considerable phenotypic heterogeneity, including the occurrence of “non-consensus” clinical manifestations (i.e. other than vascular thrombosis and pregnancy complications). Despite the evidence that phospholipid-binding factors are highly likely to be central to APS pathogenesis, the full complement of phospholipid-binding proteins has yet to be systemically assayed. Proteomic profiling by label-free quantitation (LFQ) may identify patterns of protein binding to phospholipid that are central to APS pathogenesis and thereby elucidate a basis for the heterogeneous clinical presentations of APS. METHODS: C18 silica beads were coated with phospholipid by incubation with a suspension of 30% phosphatidylserine/70% phosphatidylcholine. Plasmas from 4 groups: 1) APS patients (n=11), 2) APL positive patients who lacked clinical manifestations (“APL”) (n=11), 3) non-APL patients with thrombosis (“nonAPL”)(n=11), and 4) normal healthy controls (“normal”) (n=6) were incubated with the phospholipid-coated beads, and eluates were processed by gel electrophoresis followed by tryptic digestion. The resulting peptides were analyzed by LC-MS/MS on a Thermo Orbitrap Fusion Lumos. Protein identification, false discovery rate (FDR) estimation, and LFQ were performed using Proteome Discoverer 2.2 (Thermo). LFQ values were normalized by overall peptide abundance, and relative quantitation was obtained. Groupwise comparisons were performed using PD 2.2 in order to identify differentially enriched proteins. RESULTS: A total of 1,210 proteins were identified, with 875 proteins identified at high confidence (q&amp;lt;0.01). Based on gel electrophoresis, all samples had comparable amounts of total protein, and normalization of LFQ data on the basis of total proteins was therefore performed. Differentially expressed proteins between groups were identified using both fold-change and -log p value cutoffs (Figure 1a). Primary analysis focused on identifying proteins that were consistently preferentially bound from the APS plasmas compared to the normal controls. Novel findings included the following proteins related to coagulation, immune response, and to lipid binding that bound preferentially from APS plasmas compared to normal plasmas (APS:control ratios shown in parentheses): coagulation factor VII (10.4), heparin cofactor 2 (9.6), CD14 (6.1), apolipoprotein L1 (6.0), and apolipoprotein L2 (5.5). Additional proteins that preferentially bound from APS plasmas included OAF (13.7), BRSK1 (13.2), MINPP1 (12.5), fetuin B (10.5), SHBG (8.9), ADAMTS-like protein 4 (8.5), SERPIN A4 (7.6), alpha-2-HS glycoprotein (6.7) and inter-alpha-trypsin inhibitor heavy chains (H3, H4 and H1; 8.5, 6.6, 6.5 respectively). Consistent with prior knowledge of APS, and validating this LFQ approach immunoglobulins and complement proteins were also among the preferentially bound proteins and included: Ig heavy chain 1-3 (15.6), complement C9 (13.3), complement C4-B, (7.1), complement component C8 beta chain (5.6), Ig kappa variable 1D-8 (5.6), and Ig lambda-like polypeptide 1 (3.9). Interestingly, a number of identified proteins showed intermediate abundance (between APS and control) in the APL positive patients without clinical manifestations (“APL”) and APL negative patients with thrombosis (“nonAPL”) (see figure 1B-D for examples), suggesting a potential dose response effect. CONCLUSIONS: 1) These results are a first demonstration of concept for the ability of LFQ proteomics to address the complexity of APS through analysis of phospholipid-bound proteins. 2) A number of phospholipid-binding proteins, many of which have not been previously implicated in APS pathogenesis, are preferentially present in APS patient plasma 3) Some plasmas from APL and non-APL patients also show increased binding of some of these proteins compared to the normal controls, suggesting that LFQ may identify markers for subgroups of patients within these 2 categories.

Pomab a Novel Monoclonal Type-I Antiphospholipid Antibody, Reduces Thrombin Generation By Selectively Binding to the Open Form of Prothrombin

Background. Anti-prothrombin (anti-PT) antibodies are a type of antiphospholipid antibodies frequently found in antiphospholipid syndrome (APS) patients. Previously, we identified two types of anti-PT antibodies, one binding to epitopes in kringle-1 that are hidden in the closed form but available in the open form (Type-I), and another one in which the epitopes are located in fragment-1 but accessible in both forms (Type-II). Our understanding of their functional characteristics remains limited. Goal. To identify Type-I monoclonal antibodies for mechanistic studies. Methods. Type-I anti-prothrombin antibodies were generated by immunizing mice with fragment-1 and producing monoclonal antibodies using hybridoma technology. Binding mechanisms and epitope recognition for one representative Type-I antibody, POmAb, were elucidated using surface plasmon resonance (SPR), single-molecule FRET (smFRET), and cryo-electron microscopy (cryo-EM). Functional evaluations were performed in human plasma and with purified components. In vivo hemostatic activity was assessed in a laser injury model of the cremasteric arteriole followed by intravital microscopy. Results. Type-I antibodies recognizing kringle-1 were obtained from immunization experiments. One of them, POmAb (Prothrombin Open monoclonal Antibody), was selected for in-depth mechanistic and structural studies. SPR studies showed that POmAb binds to the open form of prothrombin with low nanomolar affinity, and binding was impaired by the mutations S91A and Y93A in kringle-1. smFRET provided evidence of conformational selection, where POmAb preferentially selects the open conformation of prothrombin, thereby shifting the equilibrium towards the open form. Cryo-EM studies of the POmAb-prothrombin complex revealed an extended binding interface in kringle-1 which overlaps with the region occupied by the serine protease domain in the closed form, explaining why POmAb binds to the open form. In human plasma, POmAb prolonged the activated partial thromboplastin time and Russel's viper venom time, though the effect was modest. Further investigations using purified proteins and antibody fragments confirmed that POmAb reduces thrombin generation, although it does not completely abolish it, as the inhibition of thrombin generation occurs through a non-competitive mechanism with prothrombinase. In vivo studies corroborated in vitro findings, further proving reduced fibrin accumulation at the site of injury. Conclusions. We discovered POmAb, a Type-I antiphospholipid antibody. We demonstrated that it selectively binds to the open form of prothrombin, resulting in reduced thrombin generation. POmAb represents a novel tool for studying the role of Type-I antibodies in APS and advances the provocative new idea of conformational trapping of prothrombin as a novel class of anticoagulants.

Cardiovascular risk in rare autoimmune diseases, lessons from a primary antiphospholipid syndrome patients

Abstract Background Antiphospholipid syndrome is a thrombo-inflammatory disease driven by circulating autoantibodies (aPL) that recognize cell surface phospholipids and phospholipid-binding proteins. In the absence of an underlying connective tissue disorder, the persistent presence of these antibodies associated with recurrent fetal losses and arterial or venous thrombosis is defined as a primary antiphospholipid syndrome (PAPS) with a prevalence of 1/2000 people, predominantly female. Purpose Estimation of cardiovascular risk presence based on markers of subclinical cardiovascular impairment. Methods 100 PAPS patients, with an average age of 47.70±13.14y, 93.7% females, were included in the study. Anticardiolipin antibodies (aCL IgG/IgM), anti-ß2 glycoprotein-I (anti-ß2GPI IgG/IgM), and lupus anticoagulant (LAC) were determined. Abnormal cut-off values used for left ventricular diastolic dysfunction (LVDD) were septal E ́&amp;lt;7 cm/sec, lateral E ́ &amp;lt;10 cm/sec, average E/E ́ ratio &amp;gt;14, LA volume index (LAVI) &amp;gt;34 mL/m2, and peak tricuspid regurgitation velocity &amp;gt;2.8 m/sec. LVDD was present if more than half parameters were with abnormal values. Endothelial dysfunction was assessed through ultrasound measurement of brachial artery flow-mediated dilation (FMD) and atherosclerosis progression by ultrasound assessment of intima-media thickness (cIMT) on carotid arteries (common carotid artery, CCA, internal carotid artery, ICA). The results were compared to 90 healthy, age and sex-matched controls. Results LVDD was significantly more prevalent in PAPS patients compared to healthy controls (24.8% vs. 2.2%, p=0.001). FMD was significantly lower in PAPS patients (10.76±5.15% vs. 14.20±6.76%, p=0.0001) and cIMT values were significantly higher (left ICA 0.80±0.29mm vs 0.56±0.20mm, p=0.001). All markers of subclinical atherosclerosis were significantly related to age and standard atherosclerotic risk factors present. Regarding aPL profile, anti-ß2GPI IgG positive PAPS patients had higher levels of cIMT ICA left (p=0.049) and LAC positive higher prevalence of LVDD (p=0.047) CONCLUSIONS: PAPS patients impose significantly higher prevalence of subclinical cardiovascular involvement compare to healthy population. Timely recognition, along with vigorous control of standard atherosclerotic risk factors and a proper therapeutic (antithrombotic and/or anticoagulant) regime, is of great importance.

Cytokine Profiling in Antiphospholipid Syndrome Demonstrates Persistent Immune Dysregulation and a Procoagulant Phenotype

Background: The antiphospholipid syndrome (APS) is an autoimmune thromboinflammatory disease characterized by thrombosis and the persistent detection of antiphospholipid antibodies (aPL). The rate of recurrent thrombosis in APS is between 3 and 24% despite standard of care anticoagulation with a vitamin K antagonist, suggesting a persistent prothrombotic milieu in APS which may be driven by altered cytokine signaling. Objective: To test the hypothesis that cytokine signaling is dysregulated in APS and contributes to thrombotic risk, we profiled cytokine levels in patients with thrombotic APS and healthy individuals. Methods: We measured a panel of 46 serum cytokines using the Meso Scale Discovery electrochemiluminescent platform, a validated measurement system that provides linear readouts of analytes over several logs of concentration, in 21 APS patients and 12 healthy individuals. All APS patients met the Revised Sapporo Criteria for thrombotic APS. Measurements were performed in triplicate and the log2 transformed averages were analyzed, setting significance at a FDP-adjusted p value of &amp;lt;0.05, while controlling for age and sex. Cytokine values were correlated with clinical history and antibody isotype profile. Results: Our cohort was 67% female and 85% identified as white. All APS patients had thrombotic disease: 57% with history of VTE, 19% with ATE, and 24% with both VTE and ATE. 14% had catastrophic APS. 67% were triple-positive for anti-cardiolipin (aCL), anti-β2 glycoprotein 1 (aβ2GP1), and lupus anticoagulant (LAC). 10% had isolated IgM isotypes of aCL or aβ2GP1. We found significantly elevated levels of TNF-α, IL-6, IL-7, IL-8, IL-10, IL-16, IL-12/IL-23p40, soluble VCAM-1, soluble ICAM-1, PIGF, SAA and CRP and significantly decreased levels of IL-17genB, IL-22 and IL-31 in APS compared with healthy controls (Figure 1). Cytokine levels were measured a median of 5 months (1-240) from the last thrombotic event, demonstrating persistence of the inflammatory response. To determine if cytokine levels were predictive of thrombotic risk, we correlated the number and pattern of positive aPL isotypes and LAC with cytokine levels. We found that TNF-α, IL-8, IL-10, sVCAM-1, sICAM-1, VEGF, IL-7, IL-12/IL-23p40, IL-4, IL-6, IL-16, PIGF, IL-23 and IL-17A each correlated with aPL positivity, with higher levels in triple-positive APS, and the highest levels observed in patients positive for all five measured antibody isotypes (aCL IgG, IgM, IgA; aβ2GP1 IgG, IgM) plus the functional LAC (Figure 2). IL-31 level inversely correlated to the number of positive aPL tests. A sub-analysis of cytokines in patients with isolated IgM aCL or aβ2GP1 antibodies with or without LAC showed significantly increased levels of TNF-α compared to healthy controls, and did not differ from APS patients with other patterns of aPL positivity. This suggests that a similar proinflammatory or procoagulant background is present in patients with isolated antiphospholipid IgM antibodies as those with IgG. Discussion: The sustained differences in cytokine levels measured in APS patients suggests a picture of ongoing immune dysregulation and compensation capable of driving vascular cell activation, and tipping the hemostasis balance into thrombosis. We find upregulation of proinflammatory cytokines, such as TNF-α and IL-6, capable of activating endothelial cells and triggering amplification loops leading to chemokine and acute phase reactant release, cell adhesion molecule expression, recruitment of leukocytes, and accumulation of complement and coagulation proteins, with the net effect of driving inflammation and thrombosis. Decreased levels of IL-17, IL-22 and IL-31 are indicative of dysregulation of the Th17 CD4+ T cell response, which has been implicated in autoimmune pathogenesis. The correlation of cytokine levels with triple or greater aPL positivity suggests a model for advancing risk stratification and has utility as a biomarker of thrombosis. Our study, although limited by size, represents one of the most extensive profiling of cytokines in thrombotic APS.

Do Patients with Antiphospholipid Syndrome Have More Significant Venous Thromboembolic Clot Burden?: A Single-Center Retrospective Analysis

Background: Antiphospholipid syndrome (APS) is a prothrombotic condition defined by arterial or venous thrombotic events and/or obstetrical morbidity in the presence of persistently positive antiphospholipid antibodies (aPL). Venous thromboembolic disease (VTE), comprised mostly of deep vein thrombosis (DVT) and pulmonary embolism (PE), is the initial presenting clinical manifestation in approximately 35% of APS diagnoses. VTE is a heterogenous disease and can present with varying degrees of clot burden. Lower extremity DVT can present with thrombus in the iliofemoral, femoropopliteal, and/or distal calf veins; pulmonary embolism can present with thrombus in the main pulmonary arteries, lobar branches, segmental, and/or subsegmental branches. In both DVT and PE, more proximal clot burden portends higher morbidity and mortality. While VTE is a commonly encountered clinical presentation, the decision regarding whom to test for APS is often challenging. Current guidelines recommend that clinicians consider testing for APS in patients with unexplained venous or arterial thrombotic events, especially young patients, or when a patient has one or more specific adverse outcomes related to pregnancy. To date, no data exist to suggest that patients with APS are more likely to present with more extensive venous thromboembolic clot burden as compared to patients without APS. Purpose: To assess clot burden in patients with a co-diagnosis of VTE and APS in comparison to patients with VTE without APS to determine if clot burden should be considered when deciding whom to test for APS. Methods: This retrospective cohort study included adult patients with a history of VTE who had also been tested for APS within a multihospital academic healthcare system from December 1, 2019 through January 31, 2022. All patients in the study had a history of VTE and were considered to be positive for APS if they had persistently positive aPL and/or lupus anticoagulant in accordance with the revised Sapporo APS Classification Criteria. Patients were excluded if they had confounding provoking risk factors for thrombosis. Patients were stratified as APS and non-APS. Significant clot burden was defined as PE involving the main and/or lobar pulmonary arteries or DVT involving the iliofemoral veins. Assessment of clot burden was performed by review of radiology reports of the sentinel clotting event.Patients were further stratified based on age, BMI, insurance status, severity of APS (triple-positive APS versus non-triple-positive APS), and type of APS (primary versus secondary). Exploratory analysis of the patient groups was performed. Summary statistics are presented as percentages for categorical data and median with interquartile range for quantitative data. Chi square test was used to compare percentages and t-test was used to compare means. Univariate and multivariate regression models were used to analyze the predictors of significant clot burden. Results: We identified 748 patients with a history of VTE who had also been tested for APS. The study population was comprised of 53.1% females with a median age of 59 (IQR 46-69). A majority of the patients were Caucasian (85%). APS was detected in 75 (10%) patients. Significant clot burden was present in 29 (38.7%) APS patients and 269 (40.0%) non-APS patients (OR 0.95, 95% CI 0.58-1.56; P = 0.85). On univariate analysis, age (OR 1.01, 95% CI 1.00-1.02; P = 0.02) and being uninsured (OR 9.2, 95% CI 1.10-76.10; P = 0.04) were associated with more significant clot burden. No predictors for significant clot burden were found on multivariable analysis. Triple positive APS (OR 0.83, 95% CI 0.16-4.21; P = 0.82) and primary APS (OR 0.72, 95% CI 0.15-3.45; P = 0.68) were not associated with more significant clot burden. Conclusions: In this retrospective cohort of 748 patients with a history of VTE, the presence of antiphospholipid syndrome was not associated with more significant VTE clot burden as compared to non-APS patients. Based on these data, VTE clot burden may not be a reliable predictor for the presence of APS. Study limitations include selection bias, retrospective design, and lack of precise volumetric measurement of blood clots.

Evaluation of the Efficacy and Safety during Treatment with TPO-Ras in 40 SLE-ITP and APS-ITP Patients

Objectives: Thrombopoietin receptor agonists (TPO-RAs) have emerged over the last decade as a therapeutic option in primary immune thrombocytopenia (ITP). TPO-RAs have demonstrated their efficacy in more than 70% of cases and are taking an increasingly important place in second line therapy. However, to date, no prospective cohort has been able to question the efficacy and safety of TPO-RAs in systemic lupus erythematosus (SLE-ITP) and antiphospholipid syndrome (APS-ITP) patients, particularly regarding venous and arterial thrombosis risks in this population. Methods: This multicenter retrospective cohort study was conducted from 2012 to 2023 in seven hospitals belonging to the French national network for adult ITP. We assessed the efficacy and the tolerance of TPO-RAs on a large retrospective cohort with a long follow-up of patients with ITP associated with 1) SLE or 2) primary or secondary APS or 3) with antinuclear antibodies (ANA) and anti-dsDNA or anti-Sm antibodies or 4) with ANA and at least one antiphospholipid antibody (APA) without thrombotic events (TEs). We studied the efficacy according the international criteria of response (Rodeghiero et al, Blood 2009) and the incidence of TEs, the risk factors for the occurrence of thrombosis, the management of TPO-RAs and their combination with antiplatelet agents or anticoagulants. Results: We included 40 ITP patients treated with TPO-RAs; 28 SLE-ITP, four APS-ITP, four with both SLE and APS, and four with significant ANA and APA positivity without definite SLE or APS. Five patients were triple positive for APA. The median duration of follow up was 46 months (1-132). The overall response rate was 84.2% for patients treated with romiplostim and 71% for those treated with eltrombopag after failure of standard treatments such as corticosteroids, intravenous immunoglobulins, rituximab and immunosuppressive agents. Eight TEs occurred in seven (17.5%) patients at a median of 119 days (IQR=171.5) after eltrombopag (n=4) or romiplostim (n=4) initiation with a median exposure duration of 37 days (13-119) for romiplostim and 255 days (30-807) for eltrombopag. Two venous TEs were observed including one cerebral vein thrombosis and one pulmonary embolism. We observed six arterial thrombotic events, such as myocardial infarctions and ischemic strokes including one fatal catastrophic APS. Four of these patients had a history of APS, and most had thrombotic risk factors and positive APA. TPO-RAs were continued after TEs in two patients for a total duration of 20 and 53 months in association with an antiplatelet agent for one patient and a combination of an antiplatelet agent and an anticoagulant for the second one without a new TE thereafter. TPO-RAs were transiently stopped and then reintroduced due to ITP flare in two patients. A new episode of myocardial infarction occurred in one of them despite association with antiplatelet agents requiring TPO-RAs definitive arrest. Conclusion: We confirmed the effectiveness of TPO-RAs in over 70% of SLE-ITP and APS-ITP patients who failed to respond to corticosteroids, intravenous immunoglobulins, rituximab and/or standard immunosuppressive agents. Nevertheless, these findings highlight the need to carefully consider the potential benefits and thrombotic risks before initiating TPO-RAs, especially in elderly patients with thrombotic risk factors and positive APA or a confirmed APS. Further studies are needed to evaluate more precisely the TEs risk factors of these agents in this specific population, in order to better select SLE and APS-ITP patients who could benefit from them.