Neutrophil activation and circulating neutrophil extracellular traps are increased in venous thromboembolism patients for at least one year after the clinical event.

Neutrophil activation and neutrophil extracellular traps (NETs) have been associated with the pathogenesis of venous thromboembolism (VTE). Considering VTE-associated chronic sequelae, which suggest that some pathological mechanisms remain after the acute episode, we investigated whether neutrophil activation is increased in patients with a prior VTE at least one year before this investigation. Thirty-seven patients with prior VTE and 37 individuals with no history of VTE were included. Neutrophil activity was evaluated by the expression of the adhesive molecule activation-specific epitopes LFA-1 (CD11a) and MAC-1 (CD11b), chemotaxis, reactive oxygen species (ROS) and by MPO-DNA complexes as markers of NETs. The adhesive molecules sICAM-1 and sVCAM-1, involved in the cross talk between neutrophil and endothelial cells, were also evaluated. Patient neutrophils presented increased CD11a expression before and after TNF-α stimulus, whereas increased CD11b expression was observed only after TNF-α stimulus, as compared to controls. Neutrophil chemotaxis on both, basal state and after IL-8 stimulus, on circulating levels of sICAM-1 and sVCAM-1, and on MPO-DNA complexes were also increased in VTE patients. ROS release was similar between patients and controls. This is, to our knowledge, the first study to investigate neutrophil inflammatory activity in VTE patients a long period after an acute event (approximately 2 years). The results showed altered neutrophil activation patterns in these patients. While activated neutrophils can cause endothelial activation and injury, the activated endothelium can induce the release of NETs with consequent endothelial cytotoxicity, creating a vicious cycle of activation between neutrophils and endothelium that can lead to thrombosis. VTE patients (approximately 2 years after the clinical event) present an altered neutrophil activation state evidenced by increased activity of the LFA-1 and Mac-1 adhesive molecules, as well as increased chemotaxis and circulating levels of NETs remnants. Circulating levels of ICAM-1 and VCAM-1, which are endothelial adhesive molecules, are also increased in VTE patients, suggesting not only an exacerbated endothelial activation and dysfunction, but also an interaction of the neutrophil adhesive molecules with their endothelial ligands, favoring the migration process of neutrophil.

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.

Low prevalence of Post-thrombotic syndrome in patients treated with rivaroxaban.

Post-thrombotic syndrome (PTS) is a complication of deep vein thrombosis (DVT). Residual vein thrombus (RVT) on Doppler Ultrasound can be associated with PTS. Limited data are available on the effect of direct oral anticoagulants (DOACs) on the long-term outcome of PTS. This study aimed to compare the prevalence of PTS and RVT, in patients with previous DVT treated with rivaroxaban or enoxaparin/warfarin. A total of 129 patients with previous proximal lower limb DVT and treated with rivaroxaban (n = 71) or enoxaparin/warfarin (n = 58) for at least 3 months were included. The Villalta scale for PTS was performed after treatment. The median duration of the DVT symptoms before anticoagulation was 7 days for both groups. The rate of PTS was 50.7% in the patients treated with rivaroxaban and 69% in the enoxaparin/warfarin group. Enoxaparin/warfarin showed an increased prevalence of PTS (P = .018). An analysis in 3 different models showed that the relative risk of PTS decreased by 76% with rivaroxaban use when compared with enoxaparin/warfarin treatment. In addition, 93 of the 129 patients were evaluated regarding the presence of RVT, of which, 11 (24.4%) and 31 (64.6%) presented with RVT for rivaroxaban and enoxaparin/warfarin, respectively (P < .0001). The RVT analysis excluded the possibility of RVT as a mediator of the association between type of treatment and PTS when comparing rivaroxaban with enoxaparin/warfarin (odds ratio (OR) = 0.14; 95% confidence interval (CI): 0.1-1.0, P = .051) with rivaroxaban compared with enoxaparin/warfarin. Rivaroxaban treatment was associated with a lower risk of PTS when compared to enoxaparin/warfarin; RVT however, was not a mediator in the association between PTS and type of treatment.

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.

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.

BACKGROUND: Folate 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. METHODS: A 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. RESULTS: The 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. CONCLUSION: Our 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.

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.

Circulating progenitor and mature endothelial cells in deep vein thrombosis.

INTRODUCTION: Mature circulating endothelial cells (CEC) and circulating endothelial progenitor cells (EPC) have been described in several conditions associated with endothelial injury. Their role in deep vein thrombosis (DVT) has not been previously evaluated. PATIENTS AND METHODS: In this pilot study we evaluated the time course of CEC and EPC release after vena cava experimental DVT in mice, using the FeCl3 model. We also evaluated their presence in patients with DVT at different phases of the disease (acute and chronic phase). CEC and EPC were evaluated by Flow Cytometry. RESULTS: In mice, both CEC and EPC were increased 24 hours after DVT induction, peaking 48 hours thereafter. After 72 hours, CEC counts decreased sharply, whereas EPC counts decreased less substantially. In DVT patients we observed a significant increase in CEC counts immediately after DVT compared to healthy individuals. Patients with chronic disease also presented a significant elevation of these cell count. In a subgroup of patients for whom serial samples were available, CEC counts decreased significantly after 9-15 months of the acute event. CONCLUSIONS: Our results suggest the participation of these cells in the reparative processes that follows DVT, both at immediate and late time-points. The different kinetics of CEC and EPC release in experimental DVT suggests a heterogeneous role for these cells in the reparative events after DVT.

Severe post-thrombotic syndrome is associated with higher levels of factor VIII.

Increased levels of factor VIII (FVIII) are a prevalent and independent risk factor for deep venous thrombosis (DVT). After a median of 10 years of the first DVT, we evaluated FVIII coagulation levels in 55 patients with DVT of the lower limbs and previous high levels of FVIII and in 74 controls. Subsequently, we analyzed the presence of post-thrombotic syndrome (PTS) in patients and its relationship with FVIII levels. After a median of 10 years of the first DVT, the FVIII levels were still significantly higher in patients when compared to controls (P < .001). Patients with severe PTS showed increased levels of FVIII when compared to patients with moderate or absent PTS (P < .001). We demonstrated a persistent increase in FVIII levels in a subset of patients with DVT, but in a lower magnitude after 10 years of the first DVT episode. Moreover, we observed a significant association between increased FVIII levels and severe PTS.

Polymorphisms and mutations in vWF and ADAMTS13 genes and their correlation with plasma levels of FVIII and vWF in patients with deep venous thrombosis.

BACKGROUND: Increased levels of factor VIII (FVIII) are a prevalent and independent risk factor for deep venous thrombosis (DVT) and are affected by von Willebrand factor (vWF) levels. DESIGN AND METHODS: ADAMTS13 contributes to vWF levels, and we investigated genetic polymorphisms previously described to be associated with decreased levels of these proteins in 435 patients with DVT (126 M and 309 F; median age 37 years, range 18-68 years) and 580 controls (163 M and 417 F; median age 35 years, range 18-68 years). Subsequently, we investigated the relationship between the genotypes and plasma levels of FVIII, vWF, and DVT risk. RESULTS: Patients with DVT showed higher plasma levels of FVIII:C, FVIII:Ag, and vWF:Ag (P < .001) when compared to controls. Patients and controls heterozygous for the 4751A>G polymorphism in the vWF gene presented decreased levels of vWF:Ag, FVIII:Ag, and FVIII:C (P < .001), but this was not a protective factor for DVT. Individuals heterozygous for 1852C>G polymorphism in ADAMTS13 gene, which is associated with reduced levels of ADAMTS13, had significantly elevated levels of vWF:Ag (P = .001), FVIII:Ag (P = .01), and FVIII:C (P = .02). However, this polymorphism was not a risk factor for DVT in our study. Heterozygosis for a new polymorphism identified in ADAMTS13 gene, 1787-26G>A, was significantly associated with elevated levels of FVIII:C (P = .02) when compared to wild type. CONCLUSIONS: Despite the tempting assumption that genetic factors that change ADAMTS13 activity might modulate the risk of DVT by altering vWF and FVIII levels, the polymorphisms analyzed in this study did not correlate with DVT risk among patients investigated.