Anna Vikerfors

Cigarette smoking, antiphospholipid antibodies and vascular events in Systemic Lupus Erythematosus

Objective Smoking can induce autoantibodies in persons who are genetically predisposed to rheumatoid arthritis. We investigated the association between smoking and antiphospholipid antibodies (aPL) in systemic lupus erythematosus (SLE), a question not previously addressed. Further, we explored the relationship between smoking, aPL and vascular events (arterial and venous, VE). Methods In this cross-sectional study, clinical evaluation and questionnaire data were collected from 367 prevalent SLE patients. At the same time, we measured aPL (anticardiolipin (aCL), anti-β2 glycoprotein-1 (aβ2GP1) antibodies IgG/IgM/IgA, and lupus anticoagulant (LA)), and a large set of other SLE-associated autoantibodies for comparison. Association analyses using logistic regression models with smoking, (ever, former and current with never as reference) and antibody status as outcome variable were performed. As a secondary outcome, we investigated the associations between aPL, smoking and VE. Results In multivariable-adjusted models ever, and in particular former, cigarette smoking was associated with the most pathogenic aPL; LA, aCL IgG and aβ2GP1 IgG. Other SLE-associated autoantibodies were not associated with smoking. The combination of smoking and aPL was strongly associated with VE. We noted a positive interaction between smoking-LA and smoking-‘triple aPL’ positivity for previous VE. Conclusions We investigated a large set of commonly occurring autoantibodies in SLE, but only aPL were positively associated with a history of smoking. This association was especially apparent in former smokers. Among ever regular smokers who were aPL positive, we observed a strikingly high frequency of former VE. The underlying mechanisms and temporality between smoking, aPL and VE need further investigations.

Clinical manifestations and anti-phospholipid antibodies in 712 patients with systemic lupus erythematosus: evaluation of two diagnostic assays

OBJECTIVES To evaluate the agreement and performance of two tests for aPLs with regard to association with manifestations of the APS in patients with SLE. METHODS We investigated 712 SLE patients and 280 population controls. Cardiolipin and β(2) glycoprotein-I antibodies were measured with routine ELISA and a new automated method. Three positivity cut-offs (99%, 90% of controls and recommended cut-off by manufacturers) were used. Associations with previous thrombotic events, thrombocytopenia and, in a subgroup of patients, obstetric morbidity (n = 296) were evaluated. Results were compared with the LA test, performed in 380 patients. RESULTS Inter-test agreement was moderate (demonstrated by κ-values 0.16-0.71). Performance of the two tests was similar: at the 99th percentile cut-off, sensitivity for any thrombotic event ranged from 3.7% to 24.8%, while specificity was 84.7-97.7%. Regardless of assay, IgG isotypes were associated with venous thrombosis and ischaemic cerebrovascular disease, whereas aPLs of IgM isotype were weakly associated with ischaemic heart disease. Associations were greatly affected by aPL level. LA performed better than the specific aPL tests. LA was associated with any thrombotic event, odds ratio 5.4 (95% CI 3.1, 9.4), while the specific aPL tests ranged from non-significant to an odds ratio of 1.9 (95% CI 1.03, 3.4) using criteria cut-off. LA was also convincingly associated with other APS manifestations. CONCLUSION In relation to thrombotic manifestations, there was moderate agreement but no clear advantages when comparing a routine aPL ELISA with an automated method. APL isotype and titre as well as LA positivity are important for risk assessment in SLE patients.

Studies of microparticles in patients with the antiphospholipid syndrome (APS)

Objectives: To study circulating platelet, monocyte and endothelial microparticles (PMPs, MMPs and EMPs) in patients with antiphospholipid syndrome (APS) in comparison with healthy controls. Material and method: Fifty-two patients with APS and 52 healthy controls were investigated. MPs were measured on a flow cytometer (Beckman Gallios) and defined as particles sized < 1.0 µm, negative to phalloidin, positive to lactadherin and positive to either CD42a (PMPs), CD144 (EMPs) or CD14 (MMPs). Exposure of CD142 (TF) was measured on CD144 positive MPs. Results: Total number of MPs (i.e. lactadherin positive particles) was higher in APS patients versus controls (p < 0.001). An increased number of EMPs (p < 0.001), increased TF-positive EMPs (p < 0.001) and increased MMPs (p < 0.001) were also observed. PMP numbers did not differ between the groups. None of the MP types differed in numbers between obstetric and thrombotic APS patients. Conclusion: We observed a high number of EMPs expressing TF in APS patients. The numbers of MMPs and total EMPs were also higher as compared with healthy controls but in contrast to previous reports, the number of PMPs did not differ between groups.

Microparticles in the blood of patients with systemic lupus erythematosus (SLE): phenotypic characterization and clinical associations

Systemic lupus erythematosus (SLE) is a prototypic autoimmune disease characterized by circulating autoantibodies and the formation of immune complexes. In these responses, the selecting self-antigens likely derive from the remains of dead and dying cells, as well as from disturbances in clearance. During cell death/activation, microparticles (MPs) can be released to the circulation. Previous MP studies in SLE have been limited in size and differ regarding numbers and phenotypes. Therefore, to characterize MPs more completely, we investigated 280 SLE patients and 280 individually matched controls. MPs were measured with flow cytometry and phenotyped according to phosphatidylserine expression (PS+/PS−), cellular origin and inflammatory markers. MPs, regardless of phenotype, are 2–10 times more abundant in SLE blood compared to controls. PS− MPs predominated in SLE, but not in controls (66% vs. 42%). Selectively in SLE, PS− MPs were more numerous in females and smokers. MP numbers decreased with declining renal function, but no clear association with disease activity was observed. The striking abundance of MPs, especially PS− MPs, suggests a generalized disturbance in SLE. MPs may be regarded as “liquid biopsies” to assess the production and clearance of dead, dying and activated cells, i.e. pivotal events for SLE pathogenesis.

Systems biology of SLE : biochemical characterisation of subgroups within SLE for improved diagnosis and treatment

Systemic lupus erythematosus (SLE) is a systemic autoimmune disease with symptoms ranging from skin related problems to more severe cardiovascular effects. The heterogenous representation of the disease might be the reason for the lack of efficient treatment. The authors hypothesise that subgroups of SLE can be characterised by different biochemical pathways and that specific biomarkers along these pathways can be identified. In this study, the authors have utilised the Karolinska lupus cohort that consists of 320 SLE patients and 320 age-matched controls. Two main subgroups were defined: One group was defined as having SSA and SSB antibodies and a negative lupus anticoagulant test (LAC), that is, a ‘Sjögren-like’ group. The other group was defined as being negative for SSA and SSB antibodies but positive in the LAC test. According to previous studies these patients are at increased risk for cardiovascular events as compared to the ‘Sjörgren-like’ group. A pilot study was designed and EDTA-plasma from selected patients in these two groups and controls were analysed using a proteomic and metabolomic approach. Pathway analysis was then performed on the obtained data. The pilot study showed that it was possible to differentiate the two subgroups of SLE based on the proteomic profile. From the proteins found to be significantly different between the groups, several proteins known to be involved in SLE were detected, for example, Apolipoprotein A1 and complement factor 3. In addition, proteins that to our knowledge have not been reported earlier to correlate with SLE, for example, Apolipoprotein M, were detected and are subject for further investigations. Apolipoprotein E was one of the proteins that was found to be significantly different between the two subgroups of SLE and will be investigated in the entire cohort. Preliminary data from metabolomics demonstrate that it is possible to separate patients from controls and the authors found for example that tryptophan levels were lower in SLE patients. Pathway analyses of proteomics and metabolomics data strongly predict that the changes in SLE patients compared to controls are associated with inflammation and immunity related pathways. This project will provide new knowledge about SLE taking several complex systems into account simultaneously. Using selected biomarkers it will be possible to identify more homogenous patient populations for clinical trials and thereby increase the efficacy. The systems biology approach is likely to identify pathways that may lead to better understanding of the disease, identification of novel drug targets and biomarkers supporting improved diagnosis of SLE.

Thrombin activatable fibrinolysis inhibitor (TAFI) — A possible link between coagulation and complement activation in the antiphospholipid syndrome (APS)

BACKGROUND Thrombosis and complement activation are pathogenic features of antiphospholipid syndrome (APS). Their molecular link is Plasma carboxypeptidase-B, also known as thrombin activatable fibrinolysis inhibitor (TAFIa), which plays a dual role: anti-fibrinolytic, by cleaving carboxyl-terminal lysine residues from partially degraded fibrin, and anti-inflammatory, by downregulating complement anaphylatoxins C3a and C5a. AIM To investigate the levels of TAFI (proenzyme) and TAFIa (active enzyme) in relation to complement activation, fibrin clot permeability and fibrinolytic function in clinical and immunological subsets of 52 APS patients and 15 controls. RESULTS TAFI (p<0.001), TAFIa (p<0.05) and complement factor C5a (p<0.001) were increased, while fibrin permeability (p<0.01) was decreased and clot lysis time (CLT) was prolonged (p<0.05) in APS patients compared to controls. Furthermore, TAFIa was increased (p<0.01) in samples from APS patients affected by arterial thrombosis compared to other APS-phenotypes. Positive associations were found between TAFI and age, fibrinogen and C5a, and between TAFIa and age, fibrinogen and thrombomodulin. CONCLUSION TAFI and TAFIa levels were increased in patients with APS as a potential response to complement activation. Interestingly, TAFI activation was associated with arterial thrombotic APS manifestations. Thus, TAFIa may be considered a novel biomarker for arterial thrombosis in APS.

SAT0004 Increased Levels of Thrombin Activatable Fibrinolysis Inhibitor - TAFI Correlate with Complement Activation in Patients with the Antiphospholipid Syndrome

Background The Antiphospholipid syndrome (APS) is diagnosed when arterial/venous/small vessel thrombosis or obstetric morbidity occur together with positive laboratory tests for antiphospholipid antibodies (aPL, including anticardiolipin (aCL) and/or anti-β2glycoprotein-I (a β2GPI) and/or the functional lupus anticoagulant (LA) test). The pathophysiology behind aPL associated thrombosis is complex. Thrombin activatable fibrinolysis inhibitor (TAFI) represents a link between coagulation and fibrinolysis. Active form of the enzyme removes carboxyl-terminal lysine residues from partially degraded fibrin, thus maintaining fibrin clots and having a prothrombotic function. TAFI has been considered a potential acute phase protein while it may mediate anti-inflammatory effects by regulating complement anaphylatoxins C3a and C5a which are also of importance for the pathogenesis of APS. Objectives To investigate TAFI levels in relation to the fibrin clot tightness and fibrinolytic function as well as complement activation in patients with APS. Methods 50 patients with APS and 15 controls were included. The levels of pro-TAFI (proenzyme) and TAFIa/TAFIai (complex of active and inactive form which corresponds to the concentration of the active form that cannot be measured because of instability) were analyzed with a chromogenic assay for TAFI and a specific ELISA for TAFIa/ai (both from Diagnostica Stago, Asnieres, France). Fibrin permeability was assessed by flow measurement and clot lysis time (CLT) was analysed by a turbidimetric lysis assay. C5a was analysed by a specific ELISA (Becton Dickinson Bioscience, NJ, USA). Results The levels of proenzyme - Pro-TAFI (%) as well as the levels of active enzyme TAFIa/TAFIai (ng/mL) were significantly increased in patients with APS compared to controls (114±2.7 vs 88.7±16.5; p<0.001 and 18.9±1.6 vs 12.2±0,7; p<0.001, respectively). Fibrin permeability expressed as permeability coefficient Ks (cm2x10-9) was lower in samples from APS-patients compared to controls (6.4±0.6 vs 9.8±0.8; p<0.001) indicating a tighter fibrin structure. CLT (seconds) was longer in APS-samples compared to controls (374±34.3 vs 278±42.2; p<0.001, respectively). TAFI levels did not correlate with CLT, neither with Ks in the investigated samples. However, moderately significant correlation was found between pro-TAFI and C5a (r=0.37; p<0.01). Conclusions Increased levels of TAFI-proenzyme were found in patients with APS as a potential response to increased complement activation. In spite of increased TAFI activation it does not seem that active form of TAFI significantly contributes to fibrinolysis impairment in APS. However further studies are required to elucidate TAFIs role in the pathophysiology of APS. Disclosure of Interest None declared DOI 10.1136/annrheumdis-2014-eular.3908

THU0183 Cigarette smoking and antiphospholipid antibodies in systemic lupus erythematosus

Background Patients with systemic lupus erythematosus (SLE) have an increased risk for cardiovascular disease (CVD). In previous prospective studies both smoking and antiphospholipid antibodies (aPL) had a significant impact on CVD risk in SLE. Smoking can in other settings induce autoantibodies. Objectives To investigate the potential association between smoking and aPL in SLE patients. Methods 367 prevalent SLE patients from a single center were included. Clinical evaluation and data on smoking habits were recorded at inclusion. aPL (anticardiolipin antibodies (aCL) IgG/IgM, anti-b2 glycoprotein-1 (ab2GP1)) and the lupus anticoagulant (LAC) were measured using standard methods. Logistic regressions evaluated the association between smoking (ever, former, current) at inclusion and antibody status. Never smokers were used as reference. Results In multivariable models adjusted for age, sex and age at disease onset, ever smoking was associated with LAC, aCL IgG and ab2GP1 IgG, and this association was primarily driven by former smokers (p<0.05 for all). Conclusions Smoking is known to accelerate atherosclerosis, additionally we demonstrate that a history of smoking is associated with pro-thrombotic aPL among SLE patients. This association was particularly evident among former smokers. Further studies are needed to investigate mechanisms behind these observed associations. Disclosure of Interest None Declared

SAT0383 Microparticles in 290 SLE Patients and Matched Controls and Their Relationship to Vascular Disease

Background Microparticles (MPs) are circulating cellular blebs released by activated and apoptotic cells. MPs have been associated with autoimmune diseases (1) and with vascular events in the general population (2). Objectives We investigated the number and phenotype of MPs in patients with systemic lupus erythematosus (SLE) and population based controls. We hypothesised that the MP profile could identify SLE subgroups with previous vascular events and/or antiphospholipid syndrome (APS). Methods 290 consecutive SLE patients and 290 controls, individually matched to the patients were included. Previous vascular events, clinical characteristics and presence of antiphospholipid antibodies (aPL) were tabulated. MPs positive for phosphatidylserine (PS) were identified with lactadherin and measured by flow cytometry. They were phenotyped according to cellular origin (platelet, endothelial or leukocyte) and protein expression (CD40 ligand=CD40L, tissue factor=TF, vascular cell adhesion molecule 1=VCAM-1 or high-mobility group protein B1=HMGB1). C4d expression was measured on endothelial and platelet MPs, regardless of PS expression. Results There was a highly significant difference between SLE patients and controls for all MPs, regardless of origin, p<1x 10-40 for all. Moreover, MPs exposing inflammation and/or activation markers CD40L, TF, VCAM-1, HMGB1 or C4d were also significantly increased in the SLE patients, p<1 x 10-15 for all. Associations with borderline p-values (p=0.044 and p=0.047) between endothelial MPs and previous vascular events were noted. Conclusions Patients with SLE had 2-10 times elevated levels of MPs of various cellular origins in blood. Associations with borderline p-values between MPs of endothelial origin and previous vascular events were found but the subgroup with APS could not be identified by MP profile. Though, analytical challenges remain; the remarkably high levels of MPs in SLE patients may provide new insights into the pathogenesis of SLE, and MPs merit further evaluation as candidate diagnostic biomarkers for SLE. References Pisetsky DS, Ullal AJ, Gauley J, Ning TC. Microparticles as mediators and biomarkers of rheumatic disease. Rheumatology (Oxford). 2012;51(10):1737-46. Lacroix R, Dubois C, Leroyer AS, Sabatier F, Dignat-George F. Revisited role of microparticles in arterial and venous thrombosis. J Thromb Haemost. 2013;11 Suppl 1:24-35. Acknowledgements The work in this abstract will be part of Dr Vikerfors thesis, defended 24 Feb and distributed both electronically and in paper format 30 Jan. Disclosure of Interest None declared

THU0167 Evaluation of two assays for antiphospholipid antibodies in 712 SLE patients; clinical associations depend on isotypes and cut-off levels

Background There are several problems with the current methods for detection of antiphospholipid antibodies (aPL) and there is an on-going discussion about the interpretation of different aPL-tests in everyday practice. (1) Objectives I. To evaluate agreement and performance of two tests for aPL, including a new automated assay. II. To study the importance of isotypes and titers with respect to thrombotic events in patients with Systemic Lupus Erythematosus (SLE). Methods We investigated 712 SLE-patients and 280 controls. Antibodies against cardiolipin and b2 glycoprotein-I were analysed by a routine ELISA method (Orgentec, Mainz, Germany) and a new, automated method (Elia Cardiolipin IgG/IgM, Elia –β2 –Glycoprotein I IgG/IgM performed on Phadia 250, Phadia AB, now Thermo Fisher Scientific, Germany). We used three cut-offs for positivity and calculated specificity, sensitivity and odds ratio (OR) for objectively verified previous thrombotic events. Results were compared with outcomes for Lupus anticoagulant (LAC) performed in 380 of the patients. Results Agreement between and performance of both aPL-methods were modest with no strong advantage in performance for either test. Using a cut-off corresponding to established criteria (2) sensitivity for the individual aPL-tests for any previous thrombosis ranged from 3.7 to 24.8% while specificity ranged from 84.7% to 97.7%. Regardless of assay, antibodies of IgG-isotype were associated with venous thrombosis and ischemic cerebrovascular disease while IgM-antibodies were associated with ischemic heart disease. Associations were highly affected by the positivity cut-off-level. ORs for LAC were generally higher than for the specific aPL-tests. For any previous thrombosis, OR (95% Confidence Interval) for LAC was 5.4 (3.1-9.4) while OR for the individual aPL-tests using a cut-off corresponding to established criteria ranged from non-significant to 1.9 (1.03-3.4) with the highest OR for routine ELISA cardiolipin IgG. Conclusions The most interesting finding in this study is that aPL of IgG and IgM isotypes were associated with different types of thrombotic events. Only moderate agreement between, but no clear advantage in relation to thrombotic manifestations, was observed when comparing a routine ELISA for aPL with a new automated method. The LAC test performed better than both aPL-assays. References Galli M. Clinical utility of laboratory tests used to identify antiphospholipid antibodies and to diagnose the antiphospholipid syndrome. Semin Thromb Hemost. 2008 Jun;34(4):329-34. Miyakis S, Lockshin MD, Atsumi T, Branch DW, Brey RL, Cervera R, et al. International consensus statement on an update of the classification criteria for definite antiphospholipid syndrome (APS). J Thromb Haemost. 2006 Feb;4(2):295-306. Disclosure of Interest None Declared