ALBERT

Description: Background: Systemic Lupus Erythematosus (SLE) is an autoimmune disorder of unknown origin with a high mortality pattern due to the development of a premature cardiovascular disease. The presence in these patients of a dysfunctional endothelium together with a hypercoagulable milieu may contribute to an increased incidence of thrombotic events. Increased thrombin generation, elevated levels of circulating microparticles and plasmatic levels of PAI-1 may contribute to the prothrombotic phenotype of the disease but data are scarce. Objectives: 1-To characterize the prothrombotic state in SLE by rotational thromboelastometry (ROTEM) and thrombin generation linked to tissue factor bearing microparticles. 2- To evaluate endothelial damage in patients with SLE and its relationship with the prothrombotic state of the disease. 3- To evaluate the influence of PAI-1 in the prothrombotic state of SLE. Material and methods: 39 patients with SLE and 25 sex and age matched healthy subjects were included. Whole blood was drawn in standard BD sodium citrate tubes (3.2%). ROTEM was performed in naTEM condition. Clotting time (CT, time from start of measurement until initiation of clotting [in seconds], alpha angle, which reflects the rate of fibrin polymerisation (tangent to the curve at 2-mm amplitude [in degrees]), maximum clot firmness, which reflects the maximum tensile strength of the thrombus (MCF, [in mm]), the time that clot takes to increase from 2mm above baseline to 20mm above baseline (CFT) and A5, amplitude at 5 min, were recorded. To evaluate the presence of tissue factor bearing microparticles, thrombin generation was determined by Calibrated Automated Thrombogram (CAT) in presence of 4 mM phospholipid (MP-Reagent, Diagnostica Stago, Spain). The endogenous thrombin potential (ETP, the total amount of thrombin generated over time); the lag time (the time to the beginning of the explosive burst of thrombin generation); the peak height of the curve (the maximum thrombin concentration produced); and the time to the peak were evaluated. Antigenic levels of E-selectin and PAI-1 were determined by ELISA (R&D Systems, MN, USA and Affymetrix eBioscience, Vienna, Austria) respectively. Results: ROTEM parameters showed a hypercoagulable profile in LES patients. CT and CFT were shorter (p

Description: Introduction: Systemic lupus erythematosus (SLE) is a chronic autoimmune disease of unknown origin characterized by a hypercoagulable state and a high mortality rate. Mechanisms that cause the accelerated deterioration of cardiovascular health in SLE are unknown. Objectives: to characterize the prothrombotic state in SLE patients by global coagulation assays and the contribution of platelets, endothelial damage, microparticles and neutrophil extracellular traps (NETs) in their prothombotic profile. Material and methods: 72 patients and 90 healthy controls were recruited. Patients were classified according to clinical characteristics in: 32 with lupus (SLE group), 29 with SLE and antiphospholipid antibodies (aFL, SLE+aFL group) and 12 who met the criteria for SLE and antiphospholipid syndrome (APS, SLE+APS group). Experimental protocol was approved by La Paz University Hospital Ethics Committee. Venous blood collected in BD sodium citrate tubes (3.2%) was centrifuged at 150 g for 20 min at 23ºC to obtain platelet-rich plasma (PRP). PPP was obtained by centrifugation at 1500 g for 15 min at 23ºC. To obtain neutrophils, whole blood was centrifuged to 1600 rpm 25 min using a Ficoll gradient and red cells were lysed. Rotational thromboelastometry (ROTEM®) was performed in naTEM condition. Clotting time (CT, time from start of measurement until initiation of clotting [in seconds]); alpha angle (tangent to the curve at 2-mm amplitude [in degrees]), Ax (clot firmness at time x, [in mm]) and maximum clot firmness (MCF, [in mm]) were recorded. Procoagulant activity associated to microparticle's content of tissue factor was determined in PPP by Calibrated Automated Thrombogram (CAT) using MP-reagent (4 mM phospholipids, Diagnostica Stago, Spain). We evaluated the endogenous thrombin potential (ETP, the total amount of thrombin generated over time); the lag time (the time to the beginning of the explosive burst of thrombin generation); the peak height of the curve (the maximum thrombin concentration produced) and the time to the peak. Thrombin generation associated to NETs was also measured by CAT. Neutrophils from healthy controls or from LES patients were stimulated with 100 nM PMA in RPMI medium during 45 min at 37º and then cocultivated with PRP adjusted to 105 platelets/µL. NETs formation was verified by fluorescent microscopy performed with DAPI and an anti-myeloperoxidase antibody. Plasma levels of LDL-ox, E-Selectin and PAI-1 were determined by Elisa (R&D Systems, MN, USA and Affymetrix eBioscience, Vienna, Austria, respectively). Platelet activation was analysed by flow cytometry (FCM, FACScan, BD Biosciences). Fibrinogen receptor activation was evaluated through PAC1-FITC binding and release of granule's content was assessed with monoclonal antibodies (mAbs) anti-CD63 and anti P-selectin in quiescent and 100 µM TRAP and 10 µM ADP stimulated platelets. Data were analysed with Graphpad prism and p ≤0.05 was stablished as statistical significance. Results: PAI-1 plasma level was increased in all patient's groups, whereas LDL-ox and E-selectin showed no differences with control cohort (Fig.1). ROTEM demonstrated a procoagulant profile in SLE and SLE+aPL but not in SLE+APS group (Fig. 2). PAI-1 levels correlated with several ROTEM parameters (Table 1). SLE patients and SLE+aFL showed a basal platelet activation. Moreover, SLE group exposed more P-selectin and CD63 than controls (Fig.3). Regarding thrombin generation associated to tissue-factor content of microparticles, no differences were observed between SLE patients and healthy controls. On the other hand, SLE patients had an increased peak of thrombin generation related to NETs formation (control group: 170.3± 58.0, SLE patients: 230.6±39.3, p=0.019). Conclusions: ROTEM® detected a hypercoagulable state in SLE and SLE+aPL patients. The hypercoagulable state might be linked to increased PAI-1 plasma levels and basal platelet activation in SLE and SLE+aPL groups. Moreover, neutrophils from SLE patients seemed to present a basal activation that induced a NETs-related procoagulant state in these patients. SLE+APS patients did not show a hypercoagulable state perhaps because of the presence of lupus anticoagulant and/or to therapeutic treatment of these patients. This work was supported by grants from the FIS-FONDOS FEDER (PI15/01457, NB). NVB holds a Miguel Servet tenure track grant from FIS-FONDOS FEDER (CP14/00024). Disclosures Fernandez-Bello: Novartis, Pfizer, ROCHE, Stago: Speakers Bureau. Robles:ABBVIE, SANDOZ FARMACEUTICA: Speakers Bureau. Álvarez Roman:Sobi: Consultancy, Speakers Bureau; CSL Behring: Consultancy, Speakers Bureau; Roche: Consultancy, Speakers Bureau; Pfizer: Consultancy, Speakers Bureau; Bayer: Consultancy, Speakers Bureau; Novartis: Consultancy, Speakers Bureau; Amgen: Consultancy, Speakers Bureau; Takeda: Research Funding; NovoNordisk: Consultancy, Speakers Bureau. Canales:Celgene: Honoraria; Gilead: Honoraria; Novartis: Honoraria; Janssen: Honoraria, Speakers Bureau; Sandoz: Honoraria; iQone: Honoraria; Takeda: Speakers Bureau; SOBI: Research Funding; Karyopharm: Honoraria; F. Hoffmann-La Roche Ltd: Honoraria, Speakers Bureau. Jimenez-Yuste:Bayer, CSL Behring, Grifols, Novo Nordisk, Octapharma, Pfizer, Roche, Sobi, Shire: Consultancy, Honoraria, Other: reimbursement for attending symposia/congresses , Research Funding, Speakers Bureau. Butta:Novartis: Consultancy; Roche, Pfizer: Speakers Bureau.

Description: Abstract 3313 Objectives: Behçet's disease (BD) is a chronic inflammatory disease of unknown etiology associated with an increased risk of venous and arterial thrombosis. To date, no studies for haemostasis have been conducted in this population by rotational thromboelastometry and thrombin generation test. These methods appear to be more sensitive and specific than routine coagulation tests in detecting defects of the coagulation system and could provide new elements for better understanding the mechanisms involved in the hypercoagulable state observed in BD patients. On this basis, this work aims to study haemostasis in patients with BD by rotational thromboelastometry (ROTEM ®) and thrombin generation test (CAT). Methods: Twenty-six patients with BD were included. Ages were between 25–86 years (mean ± SD: 48.60 ± 15.66 years) and 73% were female. Eight patients had active disease at the time of enrollment and 6 had a history of thrombosis. Control group included 20 healthy individuals aged between 28–55 years (42.85 ± 9.02 years) and 65% were female. Rotational thromboelastometry was performed in whole blood with ROTEM® coagulation analyzer (Pentapharm, Munich, Germany) at INTEM condition (activation of coagulation mainly throughout intrinsic pathway). Samples were allowed to rest 1 hour at room temperature and heated for 4 minutes at 37 ° C immediately before testing. Thrombin generation was measured in platelet-free plasma by the method of Hemker (Calibrated Automated Thrombography, CAT). Activation was performed with a final concentration of 4 mM of phospholipids and 1 pM of tissue factor which favored the activation of coagulation mainly throughout intrinsic pathway. Normal distribution for continuous variables was assessed with the Shapiro-Wilk testComparisons of quantitative variables were made with non-paired Student's t-test or Mann-Whitney's U test as appropriate. Results: Values of thromboelastometry parameters were increased in samples from BD patients. The higher clot consistency at 5 and 10 minutes (p=0.0452 and p=0.0179 respectively), and the pronounced maximal clot firmness (p=0.0064) suggest that platelet function may be altered in our patient cohort. Moreover, values higher than control ones for a angle (p=0.0266), maximum clot formation velocity (p=0.0454) and area under the curve at 5 and 10 minutes (p=0.0206 and p=0.0220 respectively) point to an increased thrombin generation in BD patients. In order to verify this hypothesis, thrombin generation was evaluated in samples from these patients. CAT experiments showed that BD patients had an increased endogenous thrombin potential (p = 0.0251) and reached higher maximum levels of thrombin (peak height, p = 0.0119) than controls. To determine if haemostatic profile in BD patients evaluated by rotational thromboelastometry and CAT correlates with disease activity, Spearman's rank correlation coefficient was calculated. There was a significant but mild correlation between disease activity and area under the curve at 5 minutes (r=0.3818, p=0.0494) and the maximal clot firmness (r=0.3978, p= 0.0399) (thromboelastometry parameters) and the peak height (r=0.4615, p=0.0251) (CAT parameter). Conclusions: Rotational thromboelastometry at INTEM condition and thrombin generation test confirmed that there is a hypercoagulable state in Behcet's disease. Procoagulant/anticoagulant protein equilibrium and platelet function may be altered in this pathology as indicated by several parameter measured by both tests. Disclosures: No relevant conflicts of interest to declare.