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Background: the pathogenesis of severe COVID-19 involves the deregulated activation of different compartments of immunothrombosis, which are otherwise important for pathogen eradication and tissue repair. Coagulation activation, angiogenesis and alterations of endothelial barrier (EB) are elements of immunothrombosis that have been shown to be involved in the pathogenesis of COVID-19. Angiopoietins (Ang) 1 and 2 and their receptor Tie2 and VEGF-A are well-known pro-angiogenic mediators that, during inflammation also mediate EB disruption. Recently, it has also been demonstrated that the Ang/Tie2 pathway is involved in coagulation activation. Here we explored whether increased levels of angiogenesis/EB regulators (which have been previously associated with disease severity in COVID-19) are also associated with both EB disruption and coagulation activation in this condition. Methods: the study population consisted of 30 patients with COVID-19 confirmed by RT-PCR and presenting typical CT findings admitted due to hypoxemia. Thirty sex- and age-matched healthy individuals were recruited at the same time, from the same geographic region. Patients were part of a clinical trial (REBEC: U1111-1250-1843) but samples were obtained before any study intervention, within 24 hours from diagnosis confirmation. Circulating levels of angiogenesis/EB regulation mediators and coagulation biomarkers were measured by commercial assays (immunological or functional). Monolayers of endothelial cells from umbilical veins (HUVECs) or lung (HULECs) were used for measurement of EB integrity using an impedance sensor system (ECIS, Electric Cell-substrate Impedance Sensing System). Cells were stimulated with serum from patients or healthy individuals and EB integrity was continuously monitored for 36 hours. Clinical outcomes were obtained from the digital medical records. Results: mean length of hospital stay (LOS) was 12.9 ± 9.8 days. Twelve patients (40%) required intensive care (ICU) and 28/30 patients survived. Mean D-dimer was 3,609 ± 14,440 ng/mL. Circulating levels of Ang1, Ang2, sTie2 and VEGF-A were all significantly increased in patients compared to healthy individuals (Ang1: 463.2 ± 194.6 vs 237.4 ± 104.9 pg/mL, p Figure 1. In (a), endothelial barrier (EB) integrity of HUVEC monolayers upon stimulation by serum from COVID-19 patients and healthy individuals (n=27-30 per group). The lower the normalized resistance, the higher the magnitude of EB disruption. Significant differences (* to ****) are evident from 15 min to 5 hours (Anova corrected for multiple comparisons). In the lower panels, the correlation of EB disruption with clinically relevant outcomes such as length of hospital stay (b) and days of intensive care (c) are shown. Negative correlations (Spearman test) indicate that the magnitude of EB disruption is associated with worse outcomes. Figure 1 Figure 1. Disclosures No relevant conflicts of interest to declare.

Previous evidence suggests that the thromboembolic risk is greater among patients with COVID-19 than among those affected by other types of acute respiratory distress syndrome (ARDS). However, such comparison has been primarily based on historical cohorts. In order to reduce the possible influence of such selection bias, the main goal of this study was to evaluate thromboembolic events in patients with COVID-19 and other ARDS hospitalized in the same time period. For this reason, we have selected patients admitted from March to June, 2020 at the UNICAMP Clinical Hospital who met the ARDS clinical criteria established by the Brazilian Ministry of Health and the Berlin Definition by presenting two or more flu-like symptoms and at least one ARDS-specific manifestation (dyspnea, persistent chest pressure, oxygen saturation lower than 95% at hospital admission, or lip/face cyanosis). Symptom onset or worsening occurred 30 days before hospital admission at the latest, and COVID-19 diagnosis was confirmed or excluded by at least 2 real time polymerase chain reactions or enzyme-linked immunosorbent assays. Descriptive analysis, chi-square and t-tests, as well as binary logistic regression, were used to compare COVID-19 and non-COVID-19 patients. Of the 253 patients hospitalized due to ARDS during this period, 101 COVID-19 and 102 non-COVID-19 patients were included in this study. The remaining patients were excluded due to incomplete medical records (n=16) or absence of COVID-19 testing results (n=34). Table 1 demonstrates the included patients' demographic and clinical baseline features. Both COVID-19 and non-COVID-19 groups showed similar baseline risk of hospital-associated thrombosis (assessed by reduced mobility within the past 3 days or more, previous thromboembolism event, recognized "thrombophilia", and infarction, stroke, trauma or surgery within the past 4 weeks) and oxygen saturation at admission (COVID-19: 92% IQR 90% to 96%; non-COVID-19: 94% IQR 91% to 97%, P=0.44). However, the need for invasive oxygenation support (37.6% vs. 14.7%, P=0.0002) and vasoactive drugs (44.6% vs. 21.6%, P=0.0006) was greater in COVID-19 than in non-COVID-19 patients. Accordingly, those infected by SARS-CoV-2 were more frequently admitted in ICU (55.4% vs. 40.2%, P=0.04) and for a longer period of time (13 days IQR 6 to 22 vs. 3 days IQR 2 to 8.3, P=0.02) than those affected by other types of ARDS. In comparison to the non-COVID-19 group, the COVID-19 group's median total hospital stay was more lasting (15 days IQR 6 to 30.5 vs. 7 days IQR 3 to 16.3, P With respect to coagulation markers (Table 3), activated partial thromboplastin time and C-reactive protein levels were greater in COVID-19 than in non-COVID-19 patients, while the latter presented higher median platelet counts. There was no statistically significant difference between both study groups in regards to prothrombin time, fibrinogen, and D-dimer levels (COVID-19: 1488 ng/mL IQR 726.5 to 3476; non-COVID-19: 1773 ng/mL IQR 807.5 to 4153.8, P=0.57). Although thromboprophylaxis was more commonly administered to COVID-19 (76.2%) than non-COVID-19 patients (41.2%, P By analyzing patients hospitalized in the same time period, we have found that although high, the thromboembolic risk in COVID-19 is similar to that in other types of ARDS, indicating that a hypercoagulable state is inherent to ARDS in general. Additionally, the obtained results show that the use of thromboprophylaxis was significantly higher among COVID-19 patients, and that there was no statistically relevant difference between COVID-19 and non-COVID-19 patients' D-dimer levels, a commonly used coagulation marker. Such findings provide a better understanding of the thromboembolic risk associated with SARS-CoV-2 infection, and suggest that previous evidence of higher thrombosis rates in COVID-19 suffered bias from the use of historical cohorts. Figure 1 Figure 1. Disclosures No relevant conflicts of interest to declare.

Aim: Hydroxychloroquine (HCQ) could form an alternative treatment for thrombotic primary antiphospholipid syndrome (t-PAPS) if HCQ is proven to reduce the risk of thrombosis. We investigated whether HCQ affects immune response and lipid metabolism in t-PAPS. Methods: HCQ at 400mg/day was given to anticoagulated t-PAPS patients for 6 months. After HCQ withdrawal and a wash-out period of 6 months, the same patients were followed for further 6 months. Blood samples taken at baseline and at the end of each 6-months follow-up period were analyzed employing flow cytometry and biochemical assays. Result: Twenty-seven patients were included, mean age was 44 years, 78% were female, 44% had hypertension, 44% dyslipidemia, 7% diabetes, 77% had venous thrombosis, 30% arterial thrombosis and 33% recurrent thrombosis. Triple aPL positivity was detected in 37%. Lymphocyte counts decreased during HCQ use (mean change -318/mm3; 95%CI -438 to -198) and were not changed during non-HCQ use period. CD8+ and CD4+ cells decreased during HCQ treatment and remained unchanged during non-treatment period. In contrast, TREG (FoxP3+) cells increased during HCQ treatment (mean change 0.2U; 95%CI 0 to 0.4) and decreased in the period of non-HCQ treatment (mean change -0.6U; 95%CI -0.9 to -0.1). Serum glucose, total cholesterol and low density lipoprotein cholesterol decreased during HCQ use (7%, 9% and 12% respectively) and did not change substantially during non-HCQ period. Conclusion: HCQ reduces cytotoxic T lymphocytes, increases regulatory T lymphocytes and decreases LDL cholesterol and serum glucose levels in t-PAPS. These HCQ may contribute to reduce the risk of thrombosis. Disclosures No relevant conflicts of interest to declare.

The PLASMIC score was recently developed for rapid diagnosis of thrombotic thrombocytopenic purpura (TTP) and therapeutic decision, as ADAMTS13 is frequently unavailable. The score consists of a scale from 1 to 7 that considers clinical and laboratory factors. PLASMIC score 6 - 7, 4 - 5 and below 4 are associated with high, intermediate and low probability of ADAMTS13 deficiency, respectively. Although the PLASMIC score is validated to predict ADAMTS13 values, its role as a predictor of adverse clinical outcomes in TTP is not established. The primary aim of this study was to evaluate whether the PLASMIC score is associated with neurological complications during TTP episodes. We also evaluated the association of the score with treatment outcomes, such as number of plasma exchange procedures, need for a second line immunosuppression therapy, days in hospital and death. In the present study, we retrospectively applied the PLASMIC score at the time of diagnosis of TTP episodes treated at the UNICAMP Clinical Hospital (University of Campinas - Brazil) between 1995 and 2016. All clinical data were retrieved from medical charts. We grouped the episodes according to the PLASMIC score, calculated at diagnosis, and compared the occurrence of neurological symptoms and other clinical manifestation between the PLASMIC score groups. The association between the PLASMIC score and neurological symptoms was evaluated by regression analysis. A total of 50 episodes of TTP were identified, of these 47 episodes, and 34 patients, were included in the study. Three episodes were excluded due to lack of clinical data. Twenty-seven (79.4%) patients were women, and the mean age was 35.7 years (SD 12.7). At the diagnosis, the mean PLASMIC score was 6, no PLASMIC score below 4 was detected, and the most common clinical features were thrombocytopenia (mean platelet count = 21,029 x 109 / L [SD 18,371 x 109]) and reticulocytosis (mean count = 7.83% [SD 5.29]). Plasma exchange was the main treatment in 98% of the episodes and an immunosuppression therapy was used in 94% of the cases. In 74.5% of the episodes, the patients presented with neurological symptoms at diagnosis or during hospitalization. Clinical features at diagnosis and during hospitalization of all TTP episodes, and of TTP episodes grouped according to the initial PLASMIC score, are presented in Table 1. The incidence of neurological symptoms was higher in PLASMIC score 7 (n= 14 [87, 5%]) and 6 (18 [81, 8%]) when compared with scores 5 (n=1, [16.7%) and 4 (n= 2 (66.7%]). The neurological complications tended to be more severe in PLASMIC scores 6 and 7. Personality change was reported in 2 (9%) TTP episodes in which the PLASMIC score was 6, in 4 (25%) episodes in which the PLASMIC score was 7 and was not reported in PLASMIC scores 4 and 5. Sensitivity loss was reported in 1 (16%) TTP episode in which the PLASMIC score was 5, in 11 (50%) episodes in which the PLASMIC score 6, and in 10 (62%) episodes in which the PLASMIC score was 7 . Seizures were reported in 3 (13.6%) TTP episodes in which the PLASMIC score was 6, in 5 (31.2%) episodes in PLASMIC score 7 and was not reported in PLASMIC scores 4 and 5. Stupor or coma were reported only in PLASMIC scores 6 [n=7 (31%]) and 7 (n= 9 [56%]). The mean number of plasma exchange procedures was 10.67 (SD=4.93) in PLASMIC score 4, 5 (SD=4.94) in PLASMIC score 5, 18.38 (10.61) in PLASMIC score 6 and 7.94 (SD=5.27) in PLASMIC score 7. The length of hospitalization and the days in intensive care unit were similar between groups. Deaths during hospitalization occurred only in cases with PLASMIC score 6 or 7. In the regression analysis, the risk of neurological complications was 9.0-fold increased (95%CI 1.6 - 52.3) in PLASMIC score 6 and was 14-fold increased (95%CI 1.8 - 106.5) in PLASMIC score 7, when compared with PLASMIC score 4 and 5. The risk of severe neurological complications was also higher in PLASMIC score 6 (odds ratio 12.0, 95% CI 1.7 - 83.5) and 7 (odds ratio 18.0, 95% CI 2.0 - 161.0) as compared to PLASMIC score 4 and 5. In conclusion, the frequency and severity of neurological injuries increased with higher PLASMIC scores. These observations suggest that further attention to neurological complications are needed when PLASMIC score is 6 or 7. Awareness of the risk of neurological complications may also improve treatment. Therefore, the PLASMIC score may be an important tool not only to predict ADAMTS13 values but also to provide information on prognosis from a neurological point of view. Disclosures No relevant conflicts of interest to declare.

Glucocorticoids are potent anti-inflammatory drugs used for a variety of chronic diseases and acute conditions that, unfortunately, can also cause severe adverse events. Venous thromboembolism (VTE) has recently been added to the list of clinical complications associated with glucocorticoids. Epidemiological studies reported that the risk for first VTE is increased by 2 to 3-fold with the use of glucocorticoids. However, several confounders may account for the reported association and whether glucocorticoid use increases the risk of recurrent VTE is unknown. To address the issue of confounding, we determined the frequency of a first VTE event associated with the use of oral glucocorticoids employing the self-controlled case-series (SCCS) method. With SCCS every individual is contrasted with him or herself thereby eliminating fixed confounders such as age, sex or chronic comorbidity. In addition, we evaluated the effect of oral glucocorticoids on the risk of recurrent VTE in a cohort design. Patients with VTE from the MEGA study were individually linked to the Dutch Foundation for Pharmaceutical Statistics (SFK). Prescriptions of oral glucocorticoids in the period of the MEGA study were identified. The risk for the first VTE was estimated using SCCS method and conditional Poisson regression. The association between oral glucocorticoids and recurrent VTE was examined using age and sex adjusted Cox regression models. A total of 2547 patients could be linked to the SFK data register, from those 363 received at least one outpatient prescription of oral glucocorticoids. The risk for a first VTE event was 3.5-fold higher in the aggregated period of oral glucocorticoid treatment as compared with baseline periods (incident rate ratio [IRR] 3.5, 95% confidence interval [CI] 2.6-4.8). IRR of a first VTE event was 2.5 (95% CI, 1.1- 5.7) in the week before treatment started, 5.3 (95% CI, 2.9 - 9.5) during the first 7 days with treatment, 3.7 (95% CI, 2.6 - 5.2) until six months with treatment and 1.6 (95% CI, 0.8 - 3.1) after 6 months with oral glucocorticoids, as compared with the baseline period. A dose-dependent relationship between oral glucocorticoid treatment and VTE risk was observed as the IRR increased from 3.4 (95% CI, 2.3 - 5.0) with 30-day cumulative doses below 300mg to 4.9 (95% CI, 1.7 - 14.0) with 30-day cumulative doses above 2000mg, as compared with baseline periods. IRRs for DVT (3.9; 95% CI, 2.9 - 9.5) and PE (3.1; 95% CI, 2.0 - 4.9) were similar and IRR for unprovoked VTE (2.4; 95% CI, 1.3 - 4.7) was lower than the IRR for provoked VTE (4.2; 95% CI, 2.9 - 6.0). The rates of recurrent VTE were elevated in patients with unprovoked VTE and in those who had their first VTE during a period of oral glucocorticoid treatment, either if the first event was otherwise classified as provoked or unprovoked. The adjusted HR for recurrent VTE was 1.6 (95% CI, 1.2 - 2.0) in patients with unprovoked first VTE not using oral glucocorticoids at the time of their first event, 2.1 (95% CI, 1.2 - 3.8) in those who had a provoked first VTE while using oral glucocorticoids and 2.3 (95% CI, 1.2 - 7.0) in those with an unprovoked first VTE while using the drug, as compared with patients with a provoked first event not using oral glucocorticoids at the time of their first event. The risk for recurrent VTE was 2.7-fold increased (95% CI, 1.6 - 4.8) during glucocorticoid treatment periods as compared to baseline periods. We conclude that patients receiving oral glucocorticoids had an approximately three-fold increase in the risk for first and recurrent VTE. The observed risk for VTE was partly associated with the underlying disease (preexposure period) and increased further after oral glucocorticoids were prescribed. These results underscore that treatment strategies to prevent first and recurrent VTE in patients treated with oral glucocorticoids are needed. Disclosures No relevant conflicts of interest to declare.

Introduction: Antiphospholipid (aPL) antibodies are commonly detected in patients with primary immune thrombocytopenic purpura (ITP) and whether the presence of these antibodies affect the prognosis of the disease is not known yet. Nevertheless, knowledge on the role of aPL in primary ITP would contribute to the management of the disease. Aim: Our primary aim was to evaluate if aPL antibodies are associated with either persistence or chronic primary ITP. We also aimed to evaluate the association of aPL antibodies with platelet counts and any bleeding event during the follow-up. Methods: This is a retrospective cohort study that took place in the Hematology Center of the University of Campinas. We collected data on patients with primary ITP diagnosed between January 2008 and January 2016. Lupus anticoagulant (LAC), IgG/IgM anticardiolipin and antibeta2glycoprotein1 (aB2GP1) assays were performed at diagnosis, and repeated within 12 weeks when positive. We compared the baseline characteristics of patients with and without aPL using chi-square test, when variables were categorical, or independent t-test when variables were continuous. We used logistic regression and linear regression to examine the association between aPL and binary and continuous outcomes respectively. Cox regression was used to evaluate the cumulative risk of bleeding. Analysis were adjusted for age and sex. Results: One hundred ninety-six patients were included and the median follow up time was 41 months. Forty-nine patients (21%) were positive for aPL (29 LAC, 20 aB2GP-1, 25 anticardiolipin IgM ou IgG, and 4 triple-positive). Baseline characteristics were similar between groups. Positivity for aPL was not associated with the risk of either persistent (OR=1,5 95%CI= 0.8; 3.0) or chronic ITP (OR=1,2 95%CI= 0.6;2.4). Mean platelet counts at 3 months was 123 x 103 (SD 111 x 103) U/mL in aPL negative patients and 103 x 103 (SD 117 x 103) U/mL in aPL positive patients (mean difference -18 x103 [95%CI= -55 to 18 x103]). Mean platelet counts at 12 months was 127 x 103 U/mL in aPL negative patients and 117 x 103 U/mL in aPL positive patients (mean difference -10 x103 [95%CI= -50 to 29 x103]). aPL positivity was not associated with bleeding events (HR= 1.3 95%CI=0.9; 1.8). Conclusion: Our results demonstrated that the presence of aPL does not influence the clinical course of primary ITP. This suggests that the investigation of aPL may not be useful for the management of primary ITP. Disclosures De Paula: Hematology and Transfusion Medicine Center, University of Campinas: Employment.

Introduction: Primary antiphospholipid syndrome (PAPS) is an autoimmune pro-thrombotic condition that affects different vascular beds, with no detectable underlying diseases. Immunothrombosis is at the basis of thrombosis development in PAPS and neutrophil activation and generation of neutrophil extracellular traps (termed NETosis) have been described as part of the immunological process. NETosis involves the orchestrated participation of several proteins such as peptidyl arginine deaminase (PADI4), neutrophil elastase (ELANE) and myeloperoxidase (MPO). PADI4 mediates histone citrullination, so that inhibition of PADI4 could be considered a potential therapeutic strategy to prevent NETosis. Hydroxychloroquine (HCQ) is an anti-malarial drug prescribed for patients with autoimmune diseases as complementary treatment for prevention of immune activation and thrombosis. Whether HCQ treatment affects the NETosis process is not known. Objective: The aim of this study was to evaluate whether HCQ use is associated with the expression of NETosis regulators proteins PADI4, ELANE, and MPO in patients with PAPS. Methods: This is a cross-over study in which patients with PAPS were selected to receive HCQ at 400mg per day for 6 months and then discontinue the drug. The study had two periods of follow-up: 6 months of HCQ treatment and 6 months of no HCQ treatment. A wash-out period of 6 months between the two study periods was allowed. The mean gene expression and 95% confidence intervals (95% CI) of PADI4, ELANE, and MPO were calculated at baseline and at the end of the study periods. The within periods change in gene expression from baseline to the end of the treatment and the difference in gene expression between periods at the end of the follow-up were determined. Results: The study comprised 20 patients. Mean age was 45 years, 70% were women. In HCQ treatment period, mean relative gene expressions of PADI4, ELANE, and MPO were respectively: 1.0 (SD=0.6), 0.5 (SD=0.7) and 0.6 (SD=0.7) at baseline and 0.8 (SD=0.3), 0.9 (SD=1.5) and 0.6 (SD=0.9) at the end of the period. The mean changes in PADI4, ELANE, and MPO relative gene expressions during HCQ treatment period were: -0.1 (95%CI:-0.4;0.1), 0.4 (95%CI: -0.5;1.4), 0 (95%CI: -0.6;0.5), respectively. In the period when HCQ was not used, mean relative gene expressions of PADI4, ELANE, and MPO were respectively: 1.2 (SD=0.6), 0.6 (SD=1.4) and 0.8 (SD=1.3) at baseline and 0.8 (SD=0.4), 0.4 (SD=0.9) and 0.6 (SD=0.6) at the end of the period. The mean changes in PADI4, ELANE, and MPO relative gene expressions during period of no treatment were: -0.4 (95%CI:-0.6;-0.2), -0.2 (95%CI:-0.5;0.1) and -0.2 (95%CI -0.5;0), respectively. In comparison with no treatment period, treatment with HCQ had no effect on the relative gene expressions of PADI4, ELANE, and MPO. The mean differences in PADI4 ELANE, and MPO relative gene expression between HCQ treatment and no treatment at the end of the periods were 0.1 (95%CI:-0.1; 0.3), 0.5 (95%CI:-0.2; 1.3) and 0.1 (95%CI:-0.3; 0.5), respectively. Conclusion: The results suggest that HCQ treatment has no effect on NETosis process, as measured by the gene expression of NETosis regulators proteins, in patients with PAPS. Disclosures De Paula: Hematology and Transfusion Medicine Center, University of Campinas: Employment.

Neutrophils have a complex migrating process out of the vascular lumen, that consists of chemoattraction and rolling, followed by firm attachment and migration to extravascular tissues. In these sites, neutrophils are capable of promoting phagocytosis, secreting proteases, generating reactive oxygen species (ROS), and probably releasing neutrophil extracellular traps (NETs). Furthermore, neutrophil activation also induces major tissue injury associated with acute and chronic inflammatory disorders, such as the venous thromboembolism (VTE). Recently, animal models and clinical studies in acute VTE have explored the participation of neutrophils in the pathophysiology of VTE. However, VTE has been associated with a chronic inflammatory condition, and it remains unclear whether the activation of neutrophils is persistent after the acute phase of the disease. Furthermore, there are clinical evidences supporting that simvastatin may prevent VTE, since the drug has pleiotropic anti-inflammatory effects. The aim of this study is to evaluate the occurrence of neutrophil activation in patients with VTE compared to health controls and determines the effect of simvastatin in these adhesive properties of the neutrophils. Neutrophils were separated from blood collected over Ficoll-Paque densities. Neutrophils activation was determined by the expression of activated adhesive molecules (LFA-1/CD11a and MAC-1/CD11b) and ROS generation, detected by flow cytometry. Chemotaxis assays (chemoTx, Neuro Probe, Inc) and serum nucleosome, a marker of NETs, were quantified by optical density (OD) (Cell Death Detection ELISAPLUS Kit, Roche). Serum high sensitive CRP (hs-CRP) levels were measured in BN ProSpec System (Siemens) by nephelometry. For CD11a and CD11b integrins expression, cells were evaluated under basal conditions and after TNFα inflammatory stimulus, pretreated, or not, with simvastatin. For the migration assays, neutrophils were treated, or not, with IL-8, a neutrophil chemotactic factor. The results were displayed as mean and standard deviation (±SD). The study group consisted of thirty-seven patients with personal history of VTE, the median time since VTE occurred was 25 months (range 13 - 42 months), the event was spontaneous in 51.35% of the cases and 23 patients presented proximal VTE. Thirty-seven controls, matched with patients according to age, gender and ethnicity, were also included. The mean fluorescence intensity (MFI) of CD11a was higher in VTE patient neutrophils, both in basal conditions (30.84 ± 6.82 vs. 38.72 ± 22.75, P= 0.04) and after TNFα stimulus (34.09 ± 9.64 vs. 45.65 ± 33.06, P= 0.01). Higher MFI of CD11b was observed in patient neutrophils, compared with controls, only after TNFα stimulus (149.10 ± 52.74 vs. 200.0 ± 100.5, P= 0.02), and the stimulus was reverted by pre-treatment with simvastatin (200.8 ± 100.50 vs. 174.60 ± 80.63, P= 0.001). The amount of ROS (MFI) was similar in patients and controls (908.30 ± 423.7 vs. 844.0 ± 312.0, P= 0.83). Neutrophils from VTE patients also presented increased basal chemotaxis (17.55% ± 9.79 vs. 12.64% ± 4.78, P=0.02) and IL-8-stimulated chemotaxis (63.48% ± 29.73 vs. 49.88% ± 19.48%, P=0.06). Serum levels of nucleosomes were similar in patients and controls (1.05 ± 0.81 vs. 0.88 ± 0.62, P= 0.64), however higher levels of circulating nucleosomes were observed in patients with severe post-thrombotic syndrome (PTS), compared to patients with non-severe PTS, without PTS and controls (1.49 ± 0.81 vs. 1.06 ± 0.64 vs. 0.64 ± 0.60 vs. 0.88 ± 0.62, P=0.04). Furthermore, serum levels of hs-CRP were significantly higher in VTE patients when compared with controls (0.59 mg/dl ± 0.58 vs. 0.17 mg/dl ± 0.12, P=0.00). We demonstrated that patients with VTE presented patterns of neutrophil activation long time after the acute thrombotic episode. In particular, the stimuli for neutrophil adhesion and chemotaxis were higher in patients, as detected by the increased activation of adhesive molecules and cell migration. Furthermore, we observed that simvastatin may abrogate the expression of CD11b in inflamed neutrophils. Neutrophil activities associated with ROS generation and the releases of nucleosomes were not increased in these patients. The results may support the hypothesis that increased neutrophils activation is part of the chronic inflammatory condition associated with VTE and may be downregulated by the effects of simvastatin. Disclosures No relevant conflicts of interest to declare.

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.

Introduction: The differential diagnosis of hereditary and acquired thrombocytopenias can be challenging, especially when between immune thrombocytopenia (ITP) and less well characterized hereditary thrombocytopenias (HT) such as MYH9-related disorders. Fundamental differences in the management of these two conditions add clinical relevance to the search for novel parameters that differentiate these conditions. The immature platelet count (IPF) represents the fraction of platelets with higher RNA content, and in analogy to the reticulocyte count for erythropoiesis is a biomarker of thrombopoietic activity. In a recent report (Miyazaki et al, 2015), IPF values that were more than 5-fold higher than those observed in ITP patients were reported in a population of 15 patients with HT. However, whether this increased values represented a real increase in thrombopoietic activity, or reflected a technical limitation of IPF determination in large platelets could not be clarified. Here, we aimed to evaluate the role of IPF determination in the differential diagnosis between HT and several forms of acquired thrombocytopenia in a larger and more diverse population of patients. We also evaluated thrombopoietin (TPO) levels in HT compared to ITP, to further investigate the mechanisms by which extremely large IPF values are observed in HT. Methods: IPF and mean platelet volume (MPV) were prospectively determined using a Sysmex XE5000 hematologic analyzer (as part of the complete blood count) in a cohort of patients with post-chemotherapy thrombocytopenia (n=56), bone marrow failure (myelodysplastic syndromes and aplastic anemia; n=22), ITP (ITP; n=105) and inherited thrombocytopenias (n=27). The latter population consists of a well-defined cohort of individuals with HT thrombocytopenia characterized by clinical, familial, laboratory and molecular data. TPO levels were determined by ELISA (R&D Systems) in 21 HT patients and 22 ITP patients matched for platelet count and age. A group of 178 healthy volunteers were used to determine normal IPF and MPV values. Results: Median platelet counts were similar in post-chemotherapy patients (CTx) (32.0*109/L), bone marrow failure (BMF) (33.5*109/L), ITP (52.0*109/L) and HT (52.0*109/L) (P=0.15). Similar IPF levels were observed in CTx and BMF patients (5.6%; IQR 3.4-8.8% and 6.5%; IQR 3.5-13.7%. Compared to these two groups, higher IPF values were observed in both ITP (12.3%; 7.0-21.0%) and HT patients (29.8%; 17.5-56.4%) (both P values < 0.05). In addition, IPF were significantly higher in HT compared to ITP (Kruskall-Wallis test and Dunn's post test,P=0.001). MPV values were different between HT and CTx/BMF groups, but could not differentiate ITP from HT. TPO levels. The accuracy of IPF to discriminate HT from all other causes of thrombocytopenia estimated by ROC analysis was 0.88 (CI95%0.8-0.96, p Conclusions: IPF represents an informative biomarker for the differential diagnosis of hereditary and acquired thrombocytopenias, and accurately differentiates ITP from the most common HT. As expected, TPO levels in patients with ITP were not higher than in individuals with normal platelets counts. The inverse correlation between TPO and platelet count in these patients confirm a blunted TPO response to thrombocytopenia in these patients. Similarly, patients with HT did not present increased TPO levels compared to healthy individuals. Accordingly, increased IPF levels in these patients cannot be attributed to higher TPO levels. Disclosures No relevant conflicts of interest to declare.

Background: The diagnosis of antiphospholipid syndrome (APS) is based on the persistent positivity of lupus anticoagulant (LA), IgM or IgG anticardiolipin (aCL) or IgG anti-β2 glicoprotein 1 (aβ2GP1) antibodies in patients plasma. Particularly, the role of antibodies directed against the domain 1 of β2GP1 (aβ2GP1-D1) has been described as relevant for the mechanism of immunopathogenesis in APS. However, the role of the aβ2GP1-D1 antibodies in clinical diagnosis and management of APS has not been established. Aim: The aim of this study was to evaluated the association of the presence of aβ2GP1-D1 antibodies with the clinical course of patients with thrombotic APS. Patients and methods: Patientspreviously diagnosed with thrombotic APS were consecutively selected for the study, from December 2013 to July 2014, in the Hemostasis Clinic of the Hematology and Hemotherapy Center of the University of Campinas. Demographic features and clinical conditions were recorded at the inclusion and during the follow-up. The clinical parameters analyzed were APS etiology (primary versus secondary to systemic autoimmune diseases), vascular bed of the thrombosis, history of multiple thrombosis, concomitant obstetrical morbidity, the presence of antinuclear antibodies (ANA) and the profile of the antiphospholipid antibodies. Anti-β2GP1-D1 antibodies were determined in patients plasma by chemiluminescence (BioFlash/AcuStar®, Barcelona, ES). Exact Fisher test and logistic regression were performed for statistical analysis. P < 0.05 were considered statistical significant. Results: Eight-five patients were included in the study, all patients presented venous or arterial thrombosis. The antibodies distribution among patients was: 80% LA positive, 50% aCL positive, 54% aβ2GP1 positive and 26% triple positive. Twenty-one patients (25%) tested positive for aβ2GP1-D1, 94% of them had positive aβ2GP1 antibody, previously detected at diagnosis. The presence of aβ2GP1-D1 was not associated with age or gender. Detected clinical conditions related to APS severity, such as thrombosis recurrence, concomitant obstetrical and vascular morbidity and triple positive antiphospholipid antibodies were evaluated. The positivity for aβ2GPI-DI antibodies was not associated with thrombosis recurrence (OR=1.0, 95%CI=0.37-2.71,P=1.0), concomitant obstetrical and vascular morbidity (OR=1.5, 95%CI=0.33-7.34, P=0.58), or triple positive antibodies (OR=2.79 , 95%CI=0.76 - 8.84, P=0.13). Anti-β2GP1-D1 antibodies were associated with the diagnosis of systemic autoimmune disease, in particular with lupus, (OR= 3.49 , 95%CI=1.25-9.76, P=0.01) and with positive ANA test (OR= 3.3, 95%CI=1.08-10.1, P=0.03). Conclusion: In this study, aβ2GPI-DI antibody was detected mainly in patients who had already tested positive for aβ2GP1 antibody, so it is possible that aβ2GP1-D1 assay may not provide additional sensibility to the diagnosis of APS. However, our results also suggested that the presence of aβ2GP1-D1 antibody might be associated to the diagnosis of secondary APS. The diagnosis of primary APS is based on the exclusion of systemic autoimmune diseases and there are no current laboratory parameters that discriminate between primary and secondary APS. Besides the laboratory criteria for lupus diagnosis, there may be overlapping of the antibodies and hematological features between APS and lupus. Furthermore, after the diagnosis of primary APS, it may take long time of follow-up to detect the underlying autoimmune disease. Therefore, if our findings are confirmed, aβ2GP1-D1 assays may play a role as a laboratory tool for the differential diagnosis between primary and secondary APS. Disclosures No relevant conflicts of interest to declare.

BACKGROUND: Post-thrombotic syndrome (PTS) is a common complication presented by patients with lower limbs deep vein thrombosis (DVT) that is characterized by pain, edema, cramps, itching, paresthesia, hyperpigmentation, and/or wound. PTS results in significant disability, and patients with severe degrees have a quality of life comparable to patients with cancer. From 20-50% of proximal DVT patients develop PTS despite optimal anticoagulant therapy, and between 5 to 10% of them present severe PTS. The diagnosis is based on clinical signs and several clinical classifications have been applied to graduate its severity, whereas the ISTH recommends the use of Villalta scale. PTS physiopathology remains unclear, but the inflammatory response to acute thrombosis, as well as to the fibrosis of venous valves and walls, seem to play an important role. Circulating endothelial cells (CEC) are infrequently detected cells derived from vascular wall or recruited from bone marrow. They have been related to hemostasis, platelet and leukocytes interaction to the vessel wall, and endothelial injury. Increased numbers of CEC are observed in ischemia, vascular trauma and have been associated to angiogenic potential. The aim of this study was to analyze CEC numbers in patients with a history of previous DVT and PTS, comparing them with DVT patients without PTS and healthy controls. We also aimed to compare CEC levels among patients with different degrees of PTS severity. Plasma D-dimer and serum IL8 levels were also determined, respectively by turbidimetric and nephelometric methods. PATIENTS AND METHODS: CEC percentage were determined in PTS patients (N=19), which were subdivided according to their score on Villalta scale; DVT patients with no post-thrombotic syndrome (N=9) and healthy controls (N=19). PTS patients and controls were matched by gender, ethnic origin and age ±5 years, and were also included. Blood (20mL) were collected in EDTA vacuum tubes, and 100μL of whole blood (with leukocyte counting between 5-10x103/μL) were used on flow cytometry analyzes. Different antibodies were included in order to better detect CEC, and two different panels were applied (panel 1: CD45-, CD144+, CD31+, CD133-; panel 2: CD45-, anti-VEGFR2+, CD31+, CD133-). RESULTS: Patients with PTS presented higher percentage of CEC, comparing to the other groups. Mild PTS patients presented higher levels of CEC using both panels in comparison to patients without PTS (P= 0.015, P= 0.019 respectively) as well as to CTR (P= 0.077, P= 0.040 respectively). The moderate+severe PTS patients presented higher levels of CEC using panel 2 both in comparison to patients without PTS (P= 0.008) and CTR (P= 0.013). CD144+ cells were slightly increased in moderate+severe PTS patients comparing to patients without PTS (P= 0.054). Both IL8 (med= 21.85 vs med=13.84 pg/mL; P=0.011) and D-dimer (med= 0.475 vs med= 0.29mg/L; P=0.044) were increased in PTS patients in comparison to patients without PTS, respectively. As CEC could eventually be derived from the residual venous thrombosis (RVT), we also dichotomized patients between those with and without RVT. The levels of CEC were similar between patients with or without RVT. CONCLUSIONS: Our results suggested that PTS may be associated with higher CEC levels. It is believed that CEC has angiogenic properties and that they may be increased on oxidative stress situations. In this context, our findings may suggest that CEC takes part on vascular-mediated PTS pathophysiology. Also the higher levels of D-dimer and IL8 may indicate that PTS patients were experiencing a pro-inflammatory condition. Disclosures No relevant conflicts of interest to declare.

Introduction: The post-thrombotic syndrome (PTS) is a common chronic complication of deep venous thrombosis (DVT) of the lower limbs and may occur even after an appropriate anticoagulant treatment. PTS may develop in the first two years after DVT in about 20-50% of patients; 5–10% of those may present severe manifestations of the disease. PTS is associated to an increased risk for DVT recurrence, morbidity, poor quality of life and significant cost to public health. The diagnosis of PTS is performed by clinical evaluation; particularly, the Villalta scale is recommended by the ISTH for PTS diagnosis and severity evaluation. The PTS-associated medical conditions are poorly understood, and it seems that hypercoagulability and inflammation, after the first episode of DVT, may play an important role in the disease development. Therefore, we performed a comprehensive analysis of clinical parameters, inflammation and ultrasound (US) examination of the affected limb in patients with history of previous DVT and PTS. Objective: The aim of this study was to evaluate possible medical conditions associated to PTS and its severity. Material and Methods: Between February 2013 and October 2013, consecutive patients with history of previous unprovoked DVT of lower limbs (> 6 months from the diagnosis), attended at the Hematology Center of the University of Campinas, Brasil, were included. Patients were submitted to physical evaluation for body mass index (BMI) determination and PTS diagnosis, by Villalta scale. Levels of the IL-8, IL-6 and TNF-α were performed by ELISA, D-dimer by turbidimetry and CRP by nephelometry. US examination was performed by duplex. In patients with residual venous thrombosis (RVT), the thrombus echogenicity was determined by grayscale median (GSM) evaluation, as described for atherosclerotic plaques. The GSM analysis was performed by a computer based US using specific software. Only the region containing the thrombus was analyzed, the image was depicted manually point by point to trace the line surrounding the thrombus, the final GSM values were automatic calculated and translated the thrombus echogenicity. A low GSM value ( 25) suggest subacute thrombosis.. Continuous variables were analyzed by Kruskal-Wallis test and categorical variables were compared using the Fisher´ s exact test. Results: From the 56 patients included, 15 did not present PTS, 23 were classified as mild, 11 as moderate and 7 as severe PTS. Serum levels of CRP was significantly higher in patients with severe PTS when compared to patients with mild+moderate PTS and to those without PTS (respectivelly 0.64mg/dL, 0.31mg/dL and 0.13mg/dL; P=0.02).Serum levels of IL-6 and TNF-α were higher in patients with severe PTS compared to patients with mild/moderate PTS and without PTS (IL-6= 2.81pg/mL vs. 1.48pg/mL vs. 0.80pg/mL respectively, and TNF 1.68pg/mL vs. 1.33pg/mL vs 1.17pg/mL respectively), however the differences did not reach statistical significance. Levels of IL-8 and DD were similar between severe PTS and the other two groups. The presence of RVT was similar between groups, however US-generated GSM was significantly lower in patients with severe PTS compared to patients with mild+moderate PTS and patients without PTS (GSM= 22, 31 and 28.5, respectively; P=0.04). As predicted, the BMI was also higher in patients with severe PTS when compared to mild+moderate PTS and no PTS group (BMI= 34,8 vs. 29,3 vs. 25,9, respectively P=0.007). Conclusion: Our results suggest that severe PTS may be associated with a chronic inflammatory response, characterized by obesity and higher levels of CRP. Besides, we could also observe that severe PTS may be associated with the persistence of a hypoechoic residual thrombus, determined by lower US-generated GSM values. The finding about the persistence of a hypoechoic thrombus in patients with severe PTS, even long time after the acute thrombosis event, is new and may possibly suggest that an adequate thrombus organization process is required to avoid the development of PTS. Disclosures No relevant conflicts of interest to declare.

BACKGROUND: Deep vein thrombosis (DVT) is multi-causal disease associated to a high morbi-mortality due to complications as pulmonary embolism and post-thrombotic syndrome. The identification of factors enrolled on the disease is important to better understand its pathophysiology. As the role of the platelets on DVT is still not completely defined, we previously performed a proteomic study on platelets from 3 DVT patients and compared to results obtained from 1 sibling and 1 neighbor for each patient in order to minimize the genetic and environmental interferences. These high-selected patients presented spontaneous and recurrent episodes of proximal DVT and mentioned a familiar history of thrombotic diseases. METHODS: platelets were washed, lysed, and their proteins were alkylated, reduced, precipitated with acetone and hydrolyzed by trypsin. 100mg of peptides were then separated by hydrophobicity using HPLC, and 8 fractions were obtained and directed to the LTQ-Orbitrap mass spectrometer. The proteins search was performed by Sorcerer-SEQUEST. In this analysis, sorbitol dehydrogenase (SORD) was present only in patients and absent in siblings and neighbors. In order to evaluate the SORD gene expression, qPCR was performed on platelets RNA from the same individuals applying specific primers after the evaluation of a minimal leukocyte contamination with specific primers to CD45. The influence of SORD on platelets adhesion was evaluated with the administration of a direct inhibitor (fidarestat) to 12 mice C57Bl6 by gavage at 10mg/kg and the results were compared to that obtained from 12 animals gavaged in a solution of 5% gum arabic. To the adhesion test, blood was obtained from cava veins in 3.8% sodium citrate and centrifuged at 100G/10’. Platelets were collected, pelleted and washed in Krebs buffer. Platelets at 1.2 x 108/μL were added to 96 wells plates pre-covered with fibrinogen 50μg/mL and their adhesion properties were evaluated with and without stimulation with 50mU of thrombin after incubation at 37°C for 30’. The percentage of adherent cells was calculated by comparing acid phosphatase activity to that of a standard curve with known numbers of platelets (0 – 100%) from the same group at 405nm. On the qPCR, the analyses were performed with fold change, and we considered significant results lower then 0.5x and higher then 1.5x. The Mann-Whitney test was applied to analyze the adhesion results, and P RESULTS: On qPCR, the gene expression of SORD was increased in patients (1x) in comparison to siblings (0.51x), but similar to neighbors (0.77x). The adhesion of platelets unstimulated was similar between both groups of animals (Median fidarestat= 41.6; 2.5-91.6; SD=25.9; median controls = 45; 9.8-91.3; SD=22.6; P=0.69), but after stimulation with thrombin, platelets from treated animals presented increased adhesion (median = 74.4; 42.9-98.8; SD=24) when compared to controls (median = 43.9; 13.6-48.2; SD=12.1; P=0.0148). CONCLUSIONS: SORD was present only in patients with DVT and absent in healthy siblings and neighbors on the proteomic analysis. The increase of SORD gene expression in patients in comparison to all the controls can corroborate the involvement of this protein on DVT physiology. The inhibition of this protein with fidarestat promoted the increase of platelets adhesion to plates covered with fibrinogen only after stimulation with thrombin. Our hypothesis is that SORD may be a protective protein based on the increase of the gene expression, protein levels and adherent properties after thrombogenic stimuli that could be an attempt to decrease the platelet function and minimize the damage. Disclosures No relevant conflicts of interest to declare.

Abstract 4660 Introduction: Thrombotic thrombocytopenic purpura (TTP) is a rare disease characterized by microangiopathic anemia, thrombocytopenia and neurological or kidney damage. TTP occurs due to the deficiency of ADAMTS13, mainly because of autoantibodies against the enzyme. Treatment requires combination of plasmapheresis and steroids. Underline diseases must be ruled out and treated when present. Although well established in the literature, diagnosis and clinical management can be difficult because of the rarity of the disease. Case report: J.C.S., male, 41 years old, previously healthy. In March 2012 he developed malaise, abdominal pain, jaundice, fever, headache and hematuria, being initially treated as having leptospirosis. During the clinical course, he presented renal failure requiring dialysis, transient ischemic attack and thrombocytopenia. Tests showed microangiopathic hemolytic anemia, thrombocytopenia (< 10.000/uL) and increase of lactate dehydrogenase (3 times). The ADAMTS 13 plasma activity was 8% and autoantibody against ADAMTS 13 was positive, confirming the diagnosis of TTP. Plasmapheresis and steroid therapy (prednisone 1mg/kg) were started. Patient achieved clinical remission after seven daily sessions of plasmapheresis and the therapy was discontinued. At this time, the ADAMTS13 activity was 4.4% and the antibody was positive. After extensive etiological investigation, there was no evidence of any associated disease; serological tests, autoimmunity tests and tumor screening were negative. The bone marrow biopsy and CT scan of the thorax were normal and total abdominal ultrasound revealed slight splenomegaly. Ten days after the withdrawn of plasmapheresis therapy, patient had a clinical exacerbation and therapy was intensified with vincristine and a new cycle of plasmapheresis. However, patient did not achieve sustained clinical response to treatment and ADAMTS 13 was 7.8%. A CT scan of the abdomen showed a 15 cm retroperitoneal tumor, whose center was highly vascularized and the periphery was calcified, which was surgically resected. The pathological analysis concluded that it was a primary inflammatory pseudotumor, the hypotheses of Castleman's disease, lymphoma or other neoplasia were all ruled out. After tumor resection, patient had complete TTP remission and ADAMTS 13 normalization (105%), with sustained response during the follow-up (more than 40 days after surgery). Conclusion: In this report, we showed a case of TTP caused by a primary and localized inflammatory pseudotumor. This tumor probably triggered the formation of autoantibodies against ADAMTS 13, leading to the picture of TTP. The evidence that the patient had sustained clinical remission and normalization of ADAMTS 13 only after tumor removal supports this hypothesis. Many cases of TTP triggered by systemic infectious, autoimmune diseases or metastatic neoplasia have been reported before. However, of our knowledge, there are no published cases of TTP secondary to a localized and benign disease so far. This report highlights the importance of a comprehensive etiological investigation in cases of TTP, the role of plasmapheresis as adjuvant therapy and the need for specific treatment of the underlying disease. Disclosures: No relevant conflicts of interest to declare.

Abstract 5251 Deep venous thrombosis (DVT) is a multifactorial disease and in about 30% of patients no risk factor can be identified. Association of inflammation and hemostasis is thought to play a significant role in the pathogenesis of DVT. We hypothesized that distinctive patterns of expression of inflammatory mediators in mononuclear cells from patients with previous DVT could be relevant to the pathogenesis of DVT. cDNA microarray technology (CodeLink Bioarrays) was chosen to study the gene expression profile of inflammatory mediators in DVT patients with clinical characteristics of a high penetrance of any putative hereditary risk, and their healthy siblings. These microarrays were hybridized with synthesized probes from pooled samples collected from patients and control siblings. Patients were divided into two clinical groups: (1) patients with one spontaneous DVT (n=2), and (2) patients with recurrent spontaneous DVT episodes (n=1). Preliminary analysis showed that 2% of approximately 55,000 transcripts contained in the array had significant (higher than 2-fold) differences in expression in spontaneous and recurrent spontaneous DVT relative to their asymptomatic siblings. In group 1, 738 genes were upregulated and 504 genes were downregulated. In group 2, 1229 genes were upregulated and 87 genes were downregulated. Genes with different expression compared to controls were associated to immune and inflammatory response, motility, cell adhesion, cell-cell signaling, defense response, signal transduction and apoptosis. Genes that presented the highest differences compared to controls were interleukin 8 (43-fold increase), chemokine (C-X-C motif) ligand 2 (11-fold increase), major histocompatibility complex, class II, DR beta 5 (8-fold increase), major histocompatibility complex, class II, DQ alpha 1 (29-fold decrease), caspase recruitment domain family, member 15 (7-fold increase), carboxypeptidase A5 (11-fold decrease), cathepsin G (8-fold decrease), and azurocidin 1 (3-fold decrease). A comprehensive list of these genes was generated. Evaluation of the significance of these distinctive gene expression profiles between patients with spontaneous and recurrent DVT compared to their asymptomatic siblings could reveal novel insights into the pathophysiology of the first DVT episode and of DVT recurrence, as well as new biomarkers or therapeutic targets. Disclosures: No relevant conflicts of interest to declare.

Abstract 5246 BACKGROUND: The role of inflammation on the pathophysiology of arterial thrombosis has been extensively studied. However, the relationship between inflammation and deep vein thrombosis (DVT) is not completely understood. Conflicting reports have been published about the levels of proteins involved in the inflammatory response in the context of DVT. Changes in ADAMTS13 levels have been reported in other inflammatory conditions such as sepsis, and this protease could be involved in the interplay between hemostasis and inflammation. OBJECTIVE: To evaluate ADAMTS13 activity in DVT patients and its association with inflammatory markers (IL-6, IL-8, CRP, TNF-α), D-dimer and von Willebrand Factor (VWF) levels. METHODOLOGY: Thirty-eight DVT patients, from 6 months to five years after the diagnosis of DVT, followed at the outpatient unit of thrombotic diseases from University of Campinas and 38 healthy volunteers selected as controls were included in the study. ADAMTS13 activity was determined by the residual binding of VWF to collagen. VWF, IL-6, IL-8 and TNF-alpha levels were determined by ELISA, and D-dimer levels was determined by a turbidimetric method. RESULTS: In this study population DVT was triggered by transient risk factors, particularly the use of oral contraceptives. No patient presented renal, hepatic or malignant disease. Median ADAMTS13 activity was not statistically different between patients (median: 98.2%; range: 70–146) and controls (median: 96.1%; range: 66–117; p=0.35). IL-6 and TNF-alfa levels were also higher in patients (median: 1.0pg/mL and 2.3pg/mL) compared to controls (median: 0.64pg/mL and 1.7pg/mL, p=0,01 and p=0,009). In addition, VWF and D-dimer levels were also significantly higher in patients (all P CONCLUSIONS: This study reinforces the role of inflammation in the pathogenesis of DVT. The correlation of VWF and IL-8 supports the notion that IL-8 stimulates the secretion of VWF. The role of ADAMTS13 in the context of DVT is yet to be determined. Disclosures: No relevant conflicts of interest to declare.

Abstract 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.

Abstract 5256 BACKGROUND: Deep venous thrombosis (DVT) is multi-causal disease associated to a high morbi-mortality due to complications as pulmonary embolism and post-flebitic syndrome. The incidence is about 20 to 30%, and 25% of the patients will present recurrence in 5 years. The identification of new risk factors is important in clinical practice to prevent new thrombotic events. The role of the platelets on DVT is still not well defined. AIM: The objective of this study was to analyze the hole proteins profile of platelets obtained from DVT patients and compare to the same matherial derived from healthy controls. PATIENTS: peripheral blood samples were collected from 3 spontaneous DVT patients and from 1 sibling and 1 neighbor for each patient in order to minimize the genetic and environmental interferences. These patients presented spontaneous and recurrent episodes of lower limbs proximal DVT and all of them mentioned a familiar history of coagulation disorders. METHODS: the platelets were washed, lysed, and the proteins were alkylated, reduced, precipitated with acetone and hydrolyzed by trypsin. 100mg of peptides were then separated by hydrophobicity using HPLC, and 8 fractions were obtained and directed to the LTQ-Orbitrap mass spectrometer. The proteins search was performed by Sorcerer-SEQUEST. RESULTS: We identified 5 proteins that were present on patients and absent in all the controls: Apolipoprotein A1 Binding-Protein, Coatomer (z1 sub-unit), Estradiol 11–17-b Dehydrogenase, Leucotriene A-4 Hydrolase and Sorbitol Dehydrogenase. Western-Blotting was performed with specific antibodies and validated the results. CONCLUSIONS: with this study it was possible to identify proteins up to date non-related to the physiopathology of DVT, that could be involved with metabolic and inflammatory processes. Disclosures: No relevant conflicts of interest to declare.

Abstract 3348 Introduction: Venous thromboembolism (VTE) is a multifactorial disease, and increased levels of coagulation factor VIII (FVIII) has been demonstrated as risk factor for first and recurrent episodes of VTE. Some authors reported that these high levels of FVIII were still persistent after 4 years of the episode, but median follow-up in these studies are relatively short. The aim of the study was investigate if after a long-term follow-up of 4–15 years (median of 10 years), patients with high levels of FVIII after anticoagulant treatment still showed this alteration. Design and Methods: Previously, we selected 174 adult patients with a first episode of acute VTE between January 1990 and September 2004. One hundred seventy four healthy adult individuals selected from blood donors were chosen as controls, from the same geographic area of origin. Of this group of VTE patients, 68 patients with plasma FVIII: C levels above the 90th percentile were selected. FVIII levels (FVIII:C) were measured by a one-stage clotting assay with FVIII-deficient plasma in duplicate in an automated coagulometer. Levels were measured twice, in 2004 and then in 2011. C-reactive protein (CRP) levels were determined in the same samples by a nephelometric method to evaluate the influence of inflammation on FVIII levels. For individuals with CRP values higher than 1mg/dL, an additional blood sample was analyzed. High FVIII levels were only considered for further analysis when in the presence of normal CRP levels. The presence of post-thrombotic syndrome (PTS) was evaluated and classified clinically by the Clinical-Etiologic-Anatomic-Pathophysiologic (CEAP) classification System. Results: 68 patients with VTE and high levels of FVIII (19M:49F) with a median age of 47 years (range 20–70) were included in the study. The control group consisted of 59 subjects (42M:17F) with a median age of 35 years (range 21–56 years). VTE was spontaneous in 26 (38.2%) patients and secondary to an acquired risk factor in 61.8%. In the 1st evaluation, in 2004, patients with VTE had higher plasma levels of FVIII:C (median 235.8 IU/dL vs. 127.0 IU/dL; p Conclusions: Our results show that even after a median of 10 years of VTE, patients still have increased levels of FVIII. Moreover, there seems to be a relationship between severe post-thrombotic syndrome and increased plasma levels of FVIII. Disclosures: No relevant conflicts of interest to declare.

Abstract 1232 Deep venous thrombosis (DVT) is a common and potentially fatal condition affecting 1% to 2% of the population worldwide, with an annual incidence of 1 in 500. Several studies have demonstrated the association between inflammation and DVT. In this study, we performed a comparative analysis of gene expression from mRNA extracted from leukocytes of 20 patients with previous DVT of the lower limbs (14F/6M; mean age: 39.7 years). An array of inflammation-related genes was selected for this study. Patients were divided in subgroups: (1) spontaneous DVT (n=10; 7F/3M). (2) risk factor-associated DVT (n=6; 4F/2M), in whom risk factor included oral contraceptive, pregnancy, protein C or S deficiencies, and FV Leiden heterozigosity. (3) Patients with antiphospholipid syndrome (APS)-associated DVT (n=4; 3F/1M). Nine healthy volunteers (7F/2M) were used as controls. Using bioarray technology, 60 upregulated (UR) and 56 downregulated (DR) genes were found in DVT patients when compared to healthy volunteers. UR genes were related to immune response, inflammation and proteolysis, and DR genes were related to transcription regulation, signal transduction and inflammation. Gene expression of the SLPI, Caspase-4 precursor (CASP4), Cartilage-associated protein precursor (CRATP) and eukaryotic translation elongation factor 1 alpha 2 (EEF1A2) presented the most significant differences in expression and were then analyzed by qPCR for validation of the results. Statistical analysis was performed by the Mann-Whitney test. No significant differences were confirmed in the expression of CASP4, CRTAP and EEF1A2 between patients and controls. However, a three-fold increased in the expression of SLPI was confirmed in DVT patients compared to controls (p=0.0381). Secretory leukocyte protease inhibitor (SLPI) is synthesized and secreted by mononuclear cells to the site of inflammation in response to cytokines and TNF. Increased SLPI expression could be a result of the inflammatory response that follows DVT. SLPI, a protease inhibitor, could function as a protective mechanism that could neutralize any excess protease load and protect host tissue. Further studies to investigate the role of SLPI in the pathogeneis of DVT and its complications are warranted. Disclosures: No relevant conflicts of interest to declare.

Abstract 5064 Introduction Livedoid vasculopathy is a rare skin disease caused by the extensive formation of microthrombi in dermis vessels, which leads to infarction and ulceration of the epidermis. The pathogenesis of livedoid vasculopathy is unknown, and hypercoagulation states have been implicated. Moreover, success using anticoagulation was reported in some patients. We hypothesized that decreased levels of ADAMTS13 activity and Von Willebrand factor (vWF) levels could contribute to the pathogenesis of local thrombus formation in livedoid vasculopathy. Objective Here we evaluated the levels of ADAMTS13 activity and vWF in patients with livedoid vasculopathy. Patients and methods Fourteen patients (93% female) with livedoid vasculopathy, median age of 41.5 years (22 - 48) were included in the study. All patients were evaluated for the presence of classical acquired and hereditary thrombophilia markers. In addition, vWF antigen levels (Elisa) and activity (collagen binding assays), as well as ADAMTS13 activity (evaluated by residual collagen binding) were evaluated. Results nine patients (64%) were carriers of the MTHFR C677T mutation (6 heterozygous and 3 homozygous); 1 patient was a carrier of factor V Leiden mutation, and 1 patient was a carrier of the FII G20210A mutation. None of the patients presented deficiency of antithrombin, protein C or S, and antiphospholipid antibodies were not identified in our study population. ADAMTS13 and VWF activities were normal in all patients. Median vWF antigen was 120.5U/dl (reference value of 40.0 - 232.0%), and vWF activity was 102.3% (reference value of 45.5 - 203.7%). ADAMTS13 activity was 138.9% (reference value of 56.0 - 227.2 %). Two patients had a history of thromboembolic diseases: one patient with 2 strokes and one patient with central retinal artery occlusion. Conclusion We show here that ADAMTS13 activity and vWF levels are not altered in patients with livedoid vasculopathy. The MTHFR C677T mutation is frequent in patients with livedoid vasculopathy but quantification of homocysteine levels is necessary to consider any relationship with this disease. Disclosures No relevant conflicts of interest to declare.

Abstract 3991 Poster Board III-927 Introduction Several risk factors, including increased levels of factor VIII (FVIII) and von Willebrand factor (VWF), have already been identified in patients with deep vein thrombosis (DVT). Recently we published a study showing that in a Brazilian population, plasma levels of FVIII over 180U/dl and VWF over 165U/dl were associated with the occurrence of venous thrombosis (odds ratio = 4.1 and 3.8 respectively) (Mello TB et al, 2009). The level of VWF in plasma and consequently FVIII is the result of genetic and acquired factors. ADAMTS13 (ADisintegrin And Metalloprotease with Thrombospondin type 1 repeats) is an enzyme responsible for cleavage of VWF, and its activity could contribute to VWF and FVIII plasmatic levels in patients with DVT. Objective To evaluate the activity of ADAMTS13 in patients with DVT associated with an increase of VWF and FVIII. Patients and methods: Fifty-six patients with FVIII > 180U/dl or FVW>165U/dl were selected from a cohort of 175 patients with DVT from the study mentioned above. Fifty-four normal subjects were selected as controls. The activity of ADAMTS 13 was performed by binding of residual VWF to collagen; VWF activity was measured by collagen binding, VWF antigen was determined by ELISA and FVIII was measured by a one-stage coagulation assay. Continuous variables were analyzed by Mann-whitney test and categorical variables by the Chi-square test. Results The demographic distribution of patients and controls were similar. Among the 56 patients the median age was 37.5 years, 39 were women, 46 had blood typing “non-O”, 34 had DVT caused by transient risk factor, especially the use of oral contraceptives, 10 patients had a hereditary thrombophilia and 3 were carriers of antiphospholipid antibodies. No patient had renal, hepatic or malignant disease. The median ADAMTS 13 activity was significantly lower in patients (112.9%, 44.4 - 327.6%) when compared to controls (142.9%, 76.7 - 323.6%), P = 0.001. The VWF activity was also higher in patients (109.7%, 26.8 - 422.6%) when compared to controls, (79.1%, 45.5 - 203.8%), P=0.038. The median level of VWF antigen was significantly higher in the group of patients when compared to the control group (178.1U/dl versus 111.9 U/dl respectively, P Disclosures: No relevant conflicts of interest to declare.