Association between neutrophil extracellular traps (NETs) and thrombosis in antiphospholipid syndrome.

BACKGROUND: The release of neutrophil extracellular traps (NETs) is the basis of immune-mediated thrombosis. Data on the clinical relevance of NETs in antiphospholipid syndrome-related thrombosis are scarce. OBJECTIVE: The aim of this study was to evaluate whether the NET regulator proteins PADI4, ELANE, and MPO are associated with thrombosis in APS. METHODS: A total of 152 thrombotic APS (t-APS) patients and 123 individuals without thrombosis (controls) were included. The following markers of NETs were evaluated: PADI4, ELANE, and MPO gene expression by qPCR and circulating levels of citrullinated histone H3 (H3cit) and myeloperoxidase-DNA complexes (MPO-DNA) by ELISA. RESULTS: The levels of circulating MPO-DNA and MPO mRNA expression and PADI4 mRNA expression were higher in t-APS patients than in controls. The mean differences were 0.05 OD (95% CI 0.01 to 0.09) in MPO-DNA levels, 1.07 AU (95% CI 0.20 to 1.93) for MPO mRNA and 0.20 AU (95% CI 0.03 to 0.36) in PADI4 mRNA fold-change. These differences were more pronounced in triple-positive patients, who had 56% increased levels of MPO-DNA, 44% increased MPO mRNA expression and 69% increased PADI4 mRNA expression compared to controls. Additionally, circulating MPO-DNA levels and MPO mRNA expression were higher in patients with recurrent thrombosis than in patients with incident thrombosis and controls. In recurrent thrombosis, levels of MPO-DNA were 43.8% higher and MPO mRNA expression was 2-fold higher than in controls. Levels of circulating MPO-DNA and PADI4 mRNA expression did not differ substantially between primary and secondary APS. CONCLUSION: Thrombotic APS was associated with increased NET formation, which was more pronounced among patients with poorer prognosis, such as those with triple antiphospholipid positivity and recurrent thrombosis. Our results provide evidence on the association of NETs and the severity of APS-related thrombosis.

Expression of tissue factor mRNA in thrombosis associated with antiphospholipid syndrome.

Tissue factor (TF) is a procoagulant protein associated with increased risk of thrombotic events in antiphospholipid syndrome (t-APS). The mechanisms by which TF levels are increased in APS have not yet been established. The aim of this study was to investigate whether TF mRNA expression is associated with TF levels and thrombosis in APS. We compared levels of circulating TF and high sensitivity C-reactive protein (hs-CRP) between t-APS and controls (individuals without thrombosis). The association between TF mRNA expression, quantified by real time quantitative polymerase chain reaction, and t-APS was accessed using regression analysis. We included 41 controls and 42 t-APS patients, mean age was 41 years old (SD 14) in both groups. Hs-CRP and TF levels were higher in t-APS patients (mean hs-CRP levels 0.81 mg/dL [SD 1.88] and median TF levels 249.0 pg/mL [IQR 138.77-447.61]) as compared to controls (mean hs-CRP levels 0.24 mg/dL [SD 0.26] and median TF levels 113.0 pg/mL [IQR 81.17-161.53]; P = 0.02 and P < 0.0001, respectively). There was no correlation between TF mRNA expression and TF levels in t-APS (r - 0.209, P = 0.19). TF mRNA expression was not associated with t-APS (adjusted OR 1.16; 95%CI 0.72-1.87). Despite circulating TF levels being higher in patients with t-APS than in controls, TF mRNA expression was similar between groups. The results demonstrate that TF mRNA expression is not associated with levels of circulating TF and hypercoagulability in t-APS.

Increased inflammation and endothelial markers in patients with late severe post-thrombotic syndrome.

INTRODUCTION: Post-thrombotic syndrome (PTS) is a limiting long-term complication present in 20-50% of patients with deep venous thrombosis (DVT) of the lower limbs. A panel of biomarkers with potential relevance to enhance knowledge on the pathophysiology of PTS was investigated. METHODS: This case-control study included 93 patients with DVT in the lower limbs, 31 with severe PTS (cases) and 62 with mild/no PTS (controls), over 24 months after an acute episode. Thirty-one healthy individuals (HI) with no history of DVT were included as a reference to the population. FVIII activity, D-dimer, inflammatory cytokines, endothelial dysfunction markers, matrix metalloproteinases, and their inhibitors, tissue remodeling and growth factor levels were evaluated. The classification of PTS was, by the Villalta scale. RESULTS: Patients with severe PTS showed elevated levels of CRP, sICAM-1, sE-selectin, and decreased MMP-9 and MCP-1 levels when compared to patients with mild/no PTS. Moreover, DVT patients presented higher levels of FVIII and D-dimer when compared to HI. CONCLUSIONS: DVT patients present an inflammatory status, endothelial dysfunction and altered proteolysis MMPs activity, even a long time after the acute thrombotic episode, which is more significant in severe PTS. These results suggest a possible role of these mediators in the maintenance and worsening of PTS severity.

Circulating levels of tissue factor and the risk of thrombosis associated with antiphospholipid syndrome.

The mechanisms behind the severe hypercoagulable state in antiphospholipid syndrome (APS) have not yet been fully elucidated. Knowledge on the etiology of thrombosis in APS is needed to improve treatment. We performed a case control study to evaluate the association of the levels of circulating tissue factor (TF) with thrombotic APS and unprovoked venous thromboembolism (VTE), as compared with controls without a history of thrombosis. Study participants were selected in the same geographic area. Linear regression was used to evaluate possible determinants of TF levels among controls and logistic regression was used to evaluate the association between TF, unprovoked VTE and t-APS. TF levels were grouped into three categories based on: below 50th percentile [reference], between 50-75th percentiles [second category] and 75th percentile [third category]. Two hundred and eighty participants were included in the study; 51 patients with unprovoked VTE, 111 patients with t-APS and 118 control individuals. The levels of TF were not associated with an increased risk of unprovoked VTE, as compared with controls. The adjusted odds ratio for t-APS was 2.62 (95%CI 1.03 to 6.62) with TF levels between 50-75th percentiles and 8.62 (95%CI 3.76 to 19.80) with TF levels above the 75th percentile, as compared with the reference category (below the 50th percentile). In the subgroup analysis, higher levels of TF were associated with both arterial and venous thrombosis in APS and with both primary and secondary APS. Circulating TF is associated with thrombotic complications related to APS, but not with the risk of unprovoked VTE.

Recurrent thrombosis in antiphospholipid syndrome may be associated with cardiovascular risk factors and inflammatory response.

INTRODUCTION: Antiphospholipid syndrome (APS) is a pro-thrombotic autoimmune disease that affects different vascular beds, with potential risk for recurrence. Systemic lupus erythematosus (SLE), specific autoantibodies profile and atherogenic disorders have been described as risk factors for the occurrence of first thrombosis in patients with antiphospholipid antibodies (aPL). However, factors associated with recurrent thrombosis have not yet been completely elucidated in APS. The aim of this study was to evaluate the association of recurrent thrombosis with markers of inflammation, autoimmunity and the presence of atherogenic disorders in APS patients. MATERIALS AND METHODS: We performed a retrospective evaluation of a cohort of APS patients in order to determine if markers of inflammation, autoimmunity and cardiovascular risk were associated with recurrence of thrombosis. RESULTS: One hundred fifteen patients with APS were included, 60% had primary APS. History of recurrent thrombosis was positive in 38.3% of patients, and 40% of them were on oral anticoagulants at the time of recurrence. Independent risk factors associated with recurrent thrombosis were arterial hypertension (OR = 3.7, 95% CI = 1.6­8.5, P = 0.002) and monocytosis above 500 u/mm(3) (OR = 2.4, 95% CI = 1.2­5.3, P = 0.02). These factors were particularly relevant in cases of venous index event. CONCLUSION: The results suggest that arterial hypertension and monocyte counts may be independent factors for thrombosis recurrence in APS. Given the morbidity of recurrent cases, the results may support the evaluation of therapeutic measures to a rigid control of blood pressures and modulation of inflammatory response in APS, as additional prophylaxis against the recurrence of vascular events.

Von Willebrand Factor, ADAMTS13 and D-Dimer Are Correlated with Different Levels of Nephropathy in Type 1 Diabetes Mellitus.

We have investigated whether von Willebrand factor, ADAMTS13 (a disintegrin and metalloproteinase with thrombospondin type 1 motif, member 13), and D-Dimer were associated with different levels of renal function in patients with type 1 diabetes. Patients were classified according to level of renal function through estimated glomerular filtration rate: ≥90 and <130mL/min/1,73m2, n=52 (control group), ≥60 and <90mL/min/1,73m2, n=29 (mild renal dysfunction group), <60mL/min/1,73m2, n=28 (severe renal dysfunction group); and through urinary albumin excretion: normoalbuminuria, microalbuminuria and macroalbuminuria. Von Willebrand factor, ADAMTS13, and D-Dimer plasma levels were determined by enzyme-linked immunosorbent assay. ADAMTS13 activity was determined by fluorescence resonance energy transfer assay. Von Willebrand factor levels were increased in patients with mild (P=0.001) and severe (P<0.001) renal dysfunction as compared to the control group. ADAMTS13 levels were also increased in mild (P=0.029) and severe (P=0.002) renal dysfunction groups in comparison to the control group, while ADAMTS13 activity was increased only in the severe renal dysfunction group as compared to the control group (P=0.006). No significant differences were observed among the groups regarding von Willebrand factor/ADAMTS13 ratio. ADAMTS13 activity/ADAMTS13 levels ratio was reduced in patients with mild (P=0.013) and severe (P=0.015) renal dysfunction as compared to the control group. D-Dimer levels were increased in patients with mild (P=0.006) and severe (P<0.001) renal dysfunction as compared to the control group; it was also higher in patients with severe renal dysfunction as compared to the mild renal dysfunction group (P=0.019). Similar results were found for albuminuria classification. Increased von Willebrand factor, ADAMTS13, and D-Dimer levels and decreased ADAMTS13 activity/ADAMTS13 levels ratio are associated with renal dysfunction in patients with type 1 diabetes, suggesting that endothelial dysfunction and hypercoagulability are associated with nephropathy in type 1 diabetes.

Long-term increased factor VIII levels are associated to interleukin-6 levels but not to post-thrombotic syndrome in patients with deep venous thrombosis.

INTRODUCTION: Increased FVIII levels are a well established risk factor for deep venous thrombosis (DVT), whose etiopathogenesis is not yet well understood. In this study, we aimed to evaluate the possibility that inflammatory markers and post-thrombotic syndrome (PTS) could contribute to FVIII levels in patients with a history of DVT. DESIGN AND METHODS: It is a case-control study that included 68 patients with DVT of the lower limbs 32 months after the acute episode, and 67 healthy adults as controls. We evaluated plasma levels of FVIII, VWF, D-dimer and serum levels of CRP, IL-6, IL-8, TNF-α in patients and controls. The presence of PTS was evaluated by the Villalta scale. RESULTS: Patients with DVT presented higher levels of FVIII, VWF and D-dimer when compared to controls (P ≤ 0.001). Almost 50% of patients presented FVIII levels above 90th percentile. Furthermore, IL-6 (1.19 vs. 0.98 pg/mL, P = 0.01) and TNF-α (2.27 vs. 1.57 pg/mL, P ≤ 0.001) were also higher in patients when compared to controls. In a linear regression multivariate model, VWF and IL-6 levels were independent factors associated with FVIII levels (P ≤ 0.001). FVIII levels were not increased in patients with PTS. Patients with PTS showed higher levels of IL-8 when compared to patients without PTS (23.03 vs. 18.20 pg/mL, P = 0.04). CONCLUSIONS: In conclusion, we demonstrated that DVT is associated with increased levels of inflammatory and coagulation markers, including FVIII, even a long time after the acute episode. Moreover, IL-6 levels were an independent factor associated with FVIII levels. Finally, PTS seems to be related to inflammatory cytokine IL-8, a proinflammatory and proangiogenic chemokine, but not to FVIII levels.