Bruna de Moraes Mazetto

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.

Increased ADAMTS13 activity in patients with venous thromboembolism

Increased levels of inflammatory markers and clotting factors have been related to the pathogenesis and prognosis of venous thromboembolism. In particular, the imbalance between VWF and ADAMTS13 has been described in patients with arterial thrombosis. In this study, 77 patients with previous VTE and 77 matched controls were selected for the evaluation of the inflammatory markers, FVW, ADAMTS 13 and D-dimer. The presences of post-thrombotic syndrome and residual vein obstruction were also assessed in patients. Serum levels of TNF-α and IL-6 were significantly increased in patients compared to controls (median=2.25 vs 1.59 pg/mL, P ≤ 0.001; 1.16 vs 0.98 pg/ml, P=0.013, respectively). Plasma levels and activity of VWF (median=150.25 vs 95.39 U/dL, P ≤ 0.001; 145.26% vs 92.39%, P ≤ 0.001) and ADAMTS 13 (median=1088.84 vs 950.80 ng/mL, P ≤ 0.001; 96.03 vs 83.64%, P ≤ 0.001) were also higher in patients. We further analysed the subgroups of patients with higher risk for VTE recurrence or VTE sequelae, defined as the presence of high D-dimer levels, RVO or PTS. All inflammatory markers were significantly higher in patients with increased D-dimer. The presence of PTS or RVO was not associated with higher inflammatory or coagulation parameters. The increased levels of inflammatory markers and VWF may suggest that there is a persistence of inflammatory activity in patients even at long periods after the VTE episode. In this context, it may be postulated that increased levels of ADAMTS13 could represent a compensatory mechanism against persistently increased levels of VWF. Moreover, increased inflammatory activity was associated with increased D-dimer levels, thus it is possible that this inflammatory activity may also be related to the risk of VTE recurrence.

Folate, Vitamin B12 and Homocysteine status in the post-folic acid fortification era in different subgroups of the Brazilian population attended to at a public health care center

BackgroundFolate and vitamin B12 are essential nutrients, whose deficiencies are considerable public health problems worldwide, affecting all age groups. Low levels of these vitamins have been associated with high concentrations of homocysteine (Hcy) and can lead to health complications. Several genetic polymorphisms affect the metabolism of these vitamins. The aims of this study were to assess folate, vitamin B12 and homocysteine status in distinct Brazilian individuals after the initiation of folic acid fortification by Brazilian authorities and to investigate the effects of RFC1 A80G, GCPII C1561T and MTHFR C677T polymorphisms on folate, vitamin B12 and Hcy levels in these populations.MethodsA total of 719 individuals including the elderly, children, as well as pregnant and lactating women were recruited from our health care center. Folate, vitamin B12 and Hcy levels were measured by conventional methods. Genotype analyses of RFC1 A80G, GCPII C1561T and MTHFR C677T polymorphisms were performed by PCR-RFLP.ResultsThe overall prevalence of folate and vitamin B12 deficiencies were 0.3% and 4.9%, respectively. Folate deficiency was observed only in the elderly (0.4%) and pregnant women (0.3%), whereas vitamin B12 deficiency was observed mainly in pregnant women (7.9%) and the elderly (4.2%). Plasma Hcy concentrations were significantly higher in the elderly (33.6%). Pregnant women carrying the MTHFR 677TT genotype showed lower serum folate levels (p = 0.042) and higher Hcy levels (p = 0.003). RFC1 A80G and GCPII C1561T polymorphisms did not affect folate and Hcy levels in the study group. After a multivariate analysis, Hcy levels were predicted by variables such as folate, vitamin B12, gender, age and RFC1 A80G polymorphism, according to the groups studied.ConclusionOur results suggest that folate deficiency is practically nonexistent in the post-folic acid fortification era in the subgroups evaluated. However, screening for vitamin B12 deficiency may be particularly relevant in our population, especially in the elderly.

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.

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 = 1.6–8.5, P = 0.002) and monocytosis above 500 u/mm(3) (OR = 2.4, 95% CI = 1.2–5.3, 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.

Increased adhesive properties of neutrophils and inflammatory markers in venous thromboembolism patients with residual vein occlusion and high D-dimer levels

BACKGROUND Venous thromboembolism (VTE) develops via a multicellular process on the endothelial surface. Although widely recognized, the relationship between inflammation and thrombosis, this relationship has been mostly explored in clinical studies by measuring circulating levels of inflammatory cytokines. However, the role of inflammatory cells, such as neutrophils, in the pathogenesis of VTE is not clear in humans. AIMS To evaluate the adhesive properties of neutrophils, erythrocytes and platelets in VTE patients and to correlate findings with inflammatory and hypercoagulability marker levels. METHODS Study group consisted of twenty-nine VTE patients and controls matched according to age, gender and ethnic background. Adhesive properties of neutrophils, erythrocytes and platelets were determined using a static adhesion assay. Neutrophil adhesion molecules expressions were evaluated by flow cytometry. Inflammatory and hypercoagulability marker levels were evaluated by standard methods. Residual vein occlusion (RVO) was evaluated by Doppler ultrasound. RESULTS No significant difference could be observed in platelet and erythrocyte adhesion between VTE patients and controls. Interestingly, VTE patients with high levels of D-dimer and RVO, demonstrated a significant increase in neutrophil adhesion, compared to controls and remaining patients. Inflammatory markers (IL-6, IL-8, TNF-α) were also significantly elevated in this subgroup, compared to other VTE patients. Adhesive properties of neutrophils correlated with IL-6 and D-dimer levels. Neutrophils adhesion molecules (CD11a, CD11b and CD18) were not altered in any of the groups. CONCLUSION These findings not only support the hypothesis of an association between inflammation and hypercoagulability, but more importantly, highlight the role of neutrophils in this process.

The impact of antibody profile in thrombosis associated with primary antiphospholipid syndrome

Triple positivity (TP) for antiphospholipid antibodies(aPL) may identify aPL carriers with poorer prognosis. The clinical impact of TP in primary antiphospholipid syndrome(PAPS) remains unclear and further clinical evidences are needed to validate TP as a marker of severity. The aim of this study was to evaluate the impact of TP on the clinical course of PAPS with thrombosis(t‐PAPS). We performed a retrospective analysis of a cohort of t‐PAPS patients, comparing groups of patients with TP and non‐TP profiles according to their demographic, clinical and laboratory features. We included 105 patients with t‐PAPS, the median follow‐up time of 3.7 years. Twenty‐two patients(21%) had TP; the demographic distribution, the presence of cardiovascular risk factors and the site of thrombosis were similar between TP and non‐TP patients. The frequency of thrombotic events did not differ between TP and non‐TP patients during the study period. Pregnancy morbidities were more frequent in women with t‐PAPS and TP than in those with non‐TP profile (80% vs. 52.8%, P = 0.05). Patients with t‐PAPS and TP presented, at diagnosis, higher dRVVT ratio (median R = 2.44 vs. 1.57, P < 0.0001), higher aCL titer (median = 50UI vs. 35 UI, P < 0.0001), lower C3 levels (median = 1.08 vs. 1.30 mg dL−1, P = 0.001), lower C4 levels (median = 0.22 vs. 0.25 mg dL−1, P = 0.05) and higher frequency of positive ANA test (50% vs. 20%, P = 0.008) than patients with t‐PAPS and non‐TP. Lower‐than‐normal levels of C3 was independently associated with TP (OR = 5.1, P = 0.02). The presence of TP in patients with t‐PAPS was associated with immune derangement, with no effect on the clinical course of the disease.

Inflammatory, immune and lipid transportation proteins are differentially expressed in spontaneous and proximal deep vein thrombosis patients

INTRODUCTION Deep vein thrombosis (DVT) is a multi-causal disease associated with high morbidity and mortality due to complications, and 25% of patients present recurrence within 5 years. The identification of factors involved with DVT can help in the management of patients, prevention of recurrence and in the development of new therapies. The evaluation of plasma components using proteomics potentially provides a window into the individuals state of health. We analyzed the protein profile of plasma samples from 3 DVT patients and compared results to those obtained from 1 sibling and 1 neighbor of each patient. These patients were selected as they presented a personal and family history of spontaneous and recurrent episodes of proximal DVT. MATERIAL AND METHODS Albumin was removed using Affi-Gel Blue Gel, and the proteins were alkylated, reduced, precipitated and hydrolyzed. The peptides were fractionated by SCX chromatography, the 7 fractions obtained were directed to the ESI Q-TOF Premier mass spectrometer. Protein search was performed using the Mascot engine against the IPI human database. RESULTS Proteins that were statistically overexpressed in DVT patients included C4-A plasma protease, C1 inter-alpha-trypsin inhibitor, heavy chain H inhibitor and serum amyloid A. Proteins that were statistically reduced in DVT patients included alpha-2-HS-glycoprotein, isoform 2 of inter-alpha-trypsin inhibitor heavy chain H4 and apolipoprotein A-IV. CONCLUSIONS The evaluation of plasma from patients with spontaneous DVT allows the identification of differently expressed proteins when compared to controls; this expression may be of pathological importance for immune and inflammatory processes in DVT.

Severe postthrombotic syndrome is associated with characteristic sonographic pattern of the residual thrombosis.

Postthrombotic syndrome (PTS) may affect 50% of patients with deep venous thrombosis, 5–10% of them may present severe manifestations. The causes for PTS development and severity have not been well established. This study evaluated whether PTS may be associated with the presence, and echogenicity, of the residual vein thrombosis (RVT). We included patients with a history of deep venous thrombosis in the past 58 months. These patients were further evaluated for PTS diagnosis, clinical comorbidities, plasma levels of D-dimer, serum levels of C-reactive protein and for the presence of RVT. Particularly, RVT was detected by ultrasound examination and the residual thrombi echogenicity was determined by grayscale median (GSM). Fifty-six patients were included, of which 41 presented PTS. Mild PTS was detected in 23 patients, moderate PTS in 11 and severe PTS in seven patients. Patients with severe PTS showed higher body mass index, higher abdominal circumference and higher C-reactive protein levels when compared with the other patients (P = 0.007, P = 0.002, P = 0.02, respectively). The ultrasound-generated GSM was significantly lower in patients with severe PTS compared with patients with mild–moderate PTS or no PTS (median = 24, 35 and 41, respectively; P = 0.04). A GSM value less than 25, which was consistent with a hypoechoic RVT, was the best cut-off value to discriminate patients with severe PTS from those with mild or moderate PTS and those without PTS. RVT is a common finding among patients with PTS and the echogenicity of the RVT may impact the severity of PTS.

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.