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IntroductionPost-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. MethodsThis 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. ResultsPatients 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. ConclusionsDVT 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.

Although deep vein thrombosis (DVT) recurrence is a common late complication of the disease, there are few predictive markers to risk-stratify patients long-term after the thrombotic event. The accuracy of residual vein thrombosis (RVT) in this context is controversial, possibly due to a lack of a standardized methodology. The objective of the study was to evaluate the accuracy of RVT echogenicity as a predictive marker of late DVT recurrence. To evaluate the accuracy of RVT echogenicity as a predictive marker of late DVT recurrence. This prospective study included patients with history of DVT in the past 33 months. Ultrasound examination was performed to detect the presence of RVT, and its echogenicity was determined by calculating the grayscale median (GSM) of the images. Blood samplings were taken for plasma D-dimer levels. Patients were followed-up for 28 months and the primary end point was DVT recurrence. Deep vein thrombosis recurrence was confirmed or excluded by ultrasound during the follow-up. Fifty-six patients were included, of which 10 presented DVT recurrence during the follow-up. D-dimer levels above 630 ng/mL conferred higher risk for recurrence with a negative predictive value of 94%. The absence of RVT was a protective marker for recurrence with a negative predictive value of 100%. Also, the presence of hypoechoic RVT, determined by GSM values below 24, positively predicted 75% of DVT recurrences. Our results suggest that the persistence of RVT and, particularly, the presence of hypoechoic thrombi (GSM < 24) are predictive markers of the risk of DVT recurrence. Residual vein thrombosis echogenicity, by GSM analysis, could represent a new strategy for the evaluation of recurrence risk in patients with DVT.

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.

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. Graphical abstract 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.

Introduction: Post-thrombotic syndrome (PTS) is a common and chronic complication of deep venous thrombosis (DVT) of the lower limbs, which is present in 20% to 50% of patients, and is associated with a high morbidity, low quality of life and significant cost to the healthcare system. Understanding the mechanisms involved in the pathophysiology of PTS may improve the strategies towards disease prevention and treatment. Thus, the aim of the study was to evaluate blood levels of markers of coagulation, inflammation, endothelial dysfunction and matrix metalloproeinases in patients with different grades of PTS severity. Methods: Between January 2012 and May 2015, 31 out 600 consecutive patients with a history of DVT treated at the Hemostasis and Thrombosis Clinic - University of Campinas - Brazil, presented severe PTS and were selected for this study. The presence of PTS was confirmed and classified by Villalta scale. For each patient with severe PTS included in the study, we also included a patient with mild PTS, a patient without PTS, matched by age, gender and time from last DVT, and a healthy control volunteer matched by age and gender. The coagulation markers included FVIII plasma levels evaluated by one-stage clotting assay, and D-dimer by immunoturbidimetric assay. Multiplex analysis was used to determine parameters related to inflammation (IL-1β, IL-6, IL-8, IL-10 and IL-13, MCP-1, TNF-α, RANTES), endothelial dysfunction (thrombomodulin, endothelin-1, sP-selectin, sE-selectin, sICAM-1, sVCAM-1), angiogenic and cell proliferation (EGF, VEGF-C, VEGF-D, TGF-β, PDGF-AA, PDGF-AB/BB) and matrix metalloproteinases and their inhibitors (MMP-1 MMP-2, MMP-3, MMP-7, MMP-9, MMP-10, MMP-12, MMP-13, TIMP-1, TIMP-2). The inflammatory marker C-reactive protein (CRP) was determined by nephelometry. Results: Thirty-one patients with severe PTS were included: 8 male / 23 female, the median age was 51 years and the median time since the last DVT episode occurred was 51 months before the day of the enrollment for the study. The distribution of the groups was similar according to gender, age, ABO blood group, and the time since the last thrombotic episode occurred. Patients with DVT had higher FVIII and D-dimer levels when compared to controls, but there was no difference according to PTS severity. CRP was the only inflammatory marker increased in patients with DVT when compared to controls (0.33 vs. 0.15 mg/L; p = 0.01), and in patients with severe PTS when compared to mild and no PTS (0.40 vs. 0.31 and 0.26 mg/dL; respectively; p=0.04). Regarding endothelial dysfunction markers, our results showed a significant increase of sICAM-1 and thrombomodulin in DVT patients when compared to controls (53.6 vs. 45.5 ng/mL; p≤0,001; 2.16 vs.1.81 ng/mL, p=0.04, respectively), but there was no difference according to the PTS severity. Patients with severe PTS showed increased sE-selectin when compared to patients without PTS (74.74 vs. 68.71 ng/mL; p=0.02). Patients with severe PTS showed increased MMP-7 (8.8 vs. 7.2 ng/mL; p=0.04) and MMP-10 levels (509.3 vs. 394.6 pg/mL; p = 0.04) when compared to patients without PTS. Furthermore, patients with severe PTS showed decreased MMP-9 levels (101.5 vs. 146.4 ng/mL; p=0.04) when compared to mild PTS. There was no difference in the parameters associated to angiogenesis (VEGF, endotelin-1, EGF) or cell proliferation (TGF-β, PDGF-AA, PDGF-AB/BB) between the groups. Discussion and Conclusions: The results may support the association of coagulometric, inflammatory and endothelial processes in severe PTS, in a well-defined cohort of patients with severe PTS. Previous studies in animal models have shown that matrix metalloproteinases have an important role in the resolution of venous thrombus, and this process leads to changes in vein wall. Our results are consistent with this finding, once we observed increased blood levels of MMPs and markers of endothelial dysfunction/activation in patients with DVT, especially those with severe PTS. We speculate that matrix metalloproteinases can play an important role in the development of severe PTS, culminating in a chronic endothelial dysfunction of the affected limb. Disclosures No relevant conflicts of interest to declare.

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2301 Introduction: Venous Thromboembolism (VTE) is a multifactorial disease that affects 1:1000 individuals worldwide, with a recurrence rate of about 25% in 10 years. Although many risk factors for VTE are well defined, first presentation and recurrence depend, at least in part, on as yet unknown etiologic factors. Studies in animal models show a tight relation between inflammation and hemostasis, as well as the infiltration of neutrophils in the venous wall after the induction of venous thrombosis. Neutrophils also participate in different stages in the formation and resolution of venous thrombosis. Methods: In this study, we investigated the adhesive properties of neutrophils in VTE patients. We hypothesized that increased adhesive properties of these cells, either as an individual baseline characteristic or as an acquired alteration after a previous VTE episode, could be associated with the thrombotic process. The patient population consisted of 22 VTE patients (14F:8M; median age: 46.1 years) that had completed at least 6 months of oral anticoagulation. Twenty-two healthy volunteers matched to VTE patients by age, gender and ethnic background were used as controls. Neutrophil adhesion was measured by a static adhesion assay in triplicate. Peripheral blood was collected with heparin and neutrophils were separated on Histopaque® (Sigma-Aldrich, St. Louis, MO, USA). Isolated neutrophils (2.2×106 cells/ml) were allowed to adhere to fibronectin (FN)-coated 96-well plates (30 min, 37°C, 5%CO2). Non-adherent cells were then removed by washing and adherent cells calculated as the percentage of cells adhered, compared to a standard curve of known cell concentrations and using a colorimetric enzyme assay. Results are expressed as means ± standard error of mean (SEM) and were compared using the Mann-Whitney test. Results: Overall, adhesion of neutrophils from VTE patients (25.40% ±2.35) was not increased when compared to the control group (21.25%±1.20 p=0.2). However when only patients at a higher risk of recurrence (n=13) - here defined as the presence of elevated D-dimer (higher than 0.5mg/L) and residual vein thrombosis - were analyzed, a statistically significant increase in cell adhesion compared to matched controls was observed (26.70%±2.08 and 21.36%±1.26, respectively, p = 0.04). When these patients (higher recurrence risk; n=13) were compared to the remaining VTE patients (standard recurrence risk, n=9), a non significant increase in neutrophil adhesion was observed (26.70%±2.08 vs 23.51%±5.03 respectively, p=0.1). Conclusions: We demonstrate that neutrophil adhesion is increased in patients with VTE with characteristics associated with increased recurrence risk. In addition, we also observed a non-significant difference in neutrophil adhesion in these patients compared to other VTE patients. Our results suggest that the increased adhesive properties of neutrophils in VTE patients could play a role in the exacerbation of inflammation, and in the pathophysiology of VTE. Further studies are warranted to study whether neutrophil adhesiveness could be used as a biomarker of VTE recurrence. Disclosures: No relevant conflicts of interest to declare.