ALBERT

Description: 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.

Description: 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.

Description: 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

Description: Introduction: It has been known for more than 50 years that patients with sickle cell disease (SCD) present higher plasma concentrations of heme. More recently, it was shown that heme is capable to activate innate immune response, and to trigger a toll-like receptor-dependent response that involves the expression of several pro-inflammatory genes. Accordingly, the role of heme as critical inflammatory mediator in SCD has been confirmed in different experimental models, suggesting that heme can be a trigger for microvascular occlusion and acute chest syndrome (ACS). The association between innate immune response and coagulation activation dates back to 450 million years in evolution, so that activation of the former is frequently accompanied by activation of the latter. Micro and macrovascular thrombosis are a hallmark of SCD, and the role of heme in the pathogenesis of these events has been recently suggested by demonstrations of heme-induced expression of tissue factor (TF) by endothelial cells and monocytes. However, the functional relevance of heme-induced TF expression on clinically-relevant coagulation markers has not been demonstrated. Methods: herein we evaluated heme-induced TF expression in peripheral blood mononuclear cells (PBMC), and used two different global assays of hemostasis, namely thromboelastometry (TEM) and Thrombin Generation Test (TGT) to evaluate the effect of heme on coagulation activation. Blood from healthy volunteers was drawn from an antecubital vein with minimal stasis in 0.106 sodium citrate tubes (1:10) or heparin. TEM was performed in whole-blood samples (n=10) incubated with 30 µM heme (Sigma-Aldrich) for four hours at 37oC, in a ROTEM equipment (Pentapharm). Coagulation was activated with the addition of CaCl2. Samples from same individuals incubated with vehicle were assayed concomitantly as controls (n=10). TGT was performed in double centrifuged plasma samples, separated from whole blood stimulated with heme or vehicle under the same conditions (n=16). TGT was performed using a Fluoroskan Ascent Flourimeter (Thermolab). Coagulation was activated with TF (5pM) and phospholipids (PPP reagent, Thrombinoscope). Expression of TF was evaluated by qRT-PCR. Heparin-anticoagulated blood was incubated with 30 µM heme (n=6) or vehicle (n=6) for 24 hours. PBMC and neutrophils were then separated by density gradient centrifugation (Ficoll). Non-parametric statistics were used in all analysis. Results: incubation of whole blood with heme 30 µM resulted in a potent induction of TF expression in PBMC compared to vehicle (AU)(0.03±0.06 vs 1.18±0.60; P=0.03). No TF expression could be detected in neutrophils. Heme-induced coagulation activation could be demonstrated by TEM. Heme significantly decreased the coagulation time (sec) (562.1±88.2 to 387±84.3; P=0.002) and the MaxV-t (time to maximum velocity) (651.4±119.2 to 451.1±87.4; P=0.002), which are two indicators of shift towards a hypercoagulable profile. A trend towards a lower clot formation time was also observed (P=0.07). No difference could be observed in the area under the TEM curve. A hypercoagulable profile was also observed in TGT in samples incubated with heme. Statistically significant changes compatible with a shift towards coagulation activation were observed in parameters such as peak thrombin (increased), time to peak thrombin (decreased), velocity index (increased), lagtime (decreased) and StarTail (decreased) (all P

Description: The diagnosis of von Willebrand Disease (VWD) remains a challenge of daily hematology practice. Ristocetin cofactor activity (VWF:RCo) is an important parameter for the diagnosis of VWD and is also essential for its management. However, reproducibility of the available tests for VWF:RCo is still a major issue, as evidenced by coefficient of variations (CV) as high as 30%, 45% and 27% in the ECAT, NEQAS and PALQ external quality assessment program. Classical methods to measure VWF:RCo include light-transmission platelet agregometry (LPA) and visual agglutination with formaldehyde fixed human platelet (VA), and more recently, VWF activity based on automated latex immunoassay (LIA). The glycoprotein (GP) Ibα is the main receptor for von Willebrand factor (VWF) in the platelet membrane. Currently, two automated methods with immobilized GPIbα have been developed to improve the sensitivity and specificity of VWF:RCo. One of them is performed with ristocetin while the other one uses a mutant GPIbα with gain of function and does not require ristocetin. This study aims to compare the two assays using immobilized GPIbα with other four assays for VWF functional determination, in patients with confirmed and under investigation for VWD. We evaluated six different VWF functional assays: VWF:RCo LPA (Chrono-Log); VA (Siemens); VA in house (with ristocetin from Chrono-Log); automated-LIA (Hemosil); in comparison to two assays using immobilized GPIbα with or without ristocetin, the GPIbα-ristocetin (Hemosil), and GPIbα-mutant (Siemens Innovance). Reference ranges for each method were established in 20 healthy adults. Plasma samples collected at the same time from 40 individuals were used in this comparative study, with 25 type 1 VWD, 2 type 3 VWD, and 13 under investigation. Diagnosis of VWD was based on bleeding history (evaluated by MCMDM-1VWD Bleeding Score), historical levels of VWF antigen (VWF:Ag) by ELISA, and VWF:RCo (assayed by LTA or VA) obtained from medical records. Statistical analysis were performed based on linear regression (Spearman correlation), agreement test (Altman Bland), and chi-square test using Prism 6.0. When all 40 patients were evaluated for both methods, GPIbα-ristocetin and GPIbα-mutant, we observed a good coefficient of correlation (r = 0.8954; p

Description: Abstract 3227 Background: Sickle cell anemia (SCA) patients present an elevated rate of thrombotic complications and increased biological markers of hemostatic activation. Hemoglobin SC disease (HbSC) is the second most prevalent hemoglobinopathy after SCA (homozygous HbSS), has specific clinical and pathological characteristics that may differ from SCA, and viscosity appears to be a hallmark of the disease. Studies have shown an increase in thrombotic events in HbSC patients, especially pulmonary embolism, however, not much is known about the coagulation activation in this population. We herein aimed to evaluate the hypercoagulability markers in HbSC disease and their associations with patients' clinical and laboratory characteristics. Patients and Methods: This was a cross-sectional study performed on a cohort of 41 adult HbSC (mean age of 43 years) and 58 adult HbSS patients (mean age of 36 years), all in steady-state, and 25 healthy age-matched controls. We evaluated the expression of tissue factor (TF), the physiological initiator of coagulation, and thrombin-antithrombin complex (TAT), a final marker of coagulation activation. Leukocyte TFmRNA expression was analyzed by real time quantitative PCR and TAT plasma levels were measured by ELISA. Comparisons between the two patient groups and controls were performed using Kruskal-Wallis test followed by Dunn's Multiple Comparisons test. Fisher's exact test and Mann-Whitney's U test were used to compare patients' clinical complications and laboratory characteristics. Spearman's rank test was used for correlations analysis. Results: Relative TF mRNA expression was significantly up-regulated in HbSC patients when compared to controls (2.6 vs. 1.2), however levels of TF were lower in HbSC than HbSS patients (2.6 vs. 3.3) (P

Description: Introduction: Sepsis represents a complex inflammatory response to infection. Gene expression studies based on microarrays have shown that this response can affect more than 80% of cellular functions and pathways, in what has been termed a “genomic storm”. For several years, sepsis was regarded as a pro-inflammatory condition, and this concept resulted in several experimental treatment strategies aimed to block inflammation. However, systematic failure of these therapies and recent evidence demonstrating that anti-inflammatory pathways are also activated during sepsis illustrate the complexity and our incomplete knowledge about the pathogenesis of this condition. In the last decade, microarray-based gene expression studies have been used in attempts to improve our understanding about sepsis. Raw data from most of these studies are now collected in public archives, thus offering a unique opportunity to combine the information from different studies by meta-analysis. It has been shown that by analyzing data from multiple experiments, biases and artifacts between datasets can be cancelled out, potentially allowing true relationships to stand out. Accordingly, an increasing number of bioinformatics protocols and guidelines about meta-analysis of gene expression studies have been published in the last years. In the context of sepsis, several high-quality microarray-based gene expression studies are available. However, no systematic meta-analysis of these studies has been performed. In order to identify genes and pathways robustly associated with the pathogenesis of sepsis, we performed a meta-analysis of gene expression studies in human severe sepsis and septic shock. Material and methods: Microarray data were identified by searching two public databases (Gene Expression Omnibus and Array-Express) using the following search criteria: (“sepsis or “septic shock”) AND (“peripheral blood” or “leukocytes”) AND (“homo sapiens”). Inclusion criteria were: studies in humans with severe sepsis or septic shock; RNA obtained from peripheral blood leukocytes; availability of raw data; and matched healthy controls from the same study. To improve consistency, only studies using similar platforms were compared. We used the R/BioConductor environment to preprocess the datasets using the Robust Multi-array Average algorithm (RMA) implemented in the ‘oligo’ package and to perform meta-analysis through the ‘RankProd’ package implementation. This is a non-parametric statistical method that utilizes ranks of the log-ratio statistics for all genes across different studies to generate a list of differentially expressed (DE) genes between two conditions, and considered superior to alternative methodologies. For this study, we selected genes with fold-change of expression above 2 and false discovery rate below 0.01, calculated based on 10,000 permutations. Gene set analysis was initially performed using WebGestalt and confirmed in similar tools (KEGG, Pathway Commons, WikiPathways). Only pathways identified by more than one tool were considered. Results: Forty-five studies were identified, of which five fulfilled inclusion criteria. Our meta-analysis included data from 259 patients and 132 controls. Out of 22,216 probesets, we observed 352 as candidates for DE, 215 of which were up-regulated and 137 down-regulated. Top 5 up-regulated genes were CD177, MMP8, HP, ARG1 and ANXA3. Top 5 down-regulated genes were FCER1A, YME1L1, TRDV3, LRRN3 and MYBL1. The gene ontology term associated with the set of DE genes in both analysis with higher statistical significance was "immune response” (adjP=2.85e-27), and the most significant pathways identified were “Hematopoietic cell lineage” (adjP=8.69e-13), “TCR signaling pathway” (adjP=3.04e-10) and “immune system” (adjP=1.08e-19). Discussion and conclusion: The combined analysis of data generated by high-throughput experiments is an attractive and validated strategy to improve the sensitivity and specificity of genome-wide expression data. This meta-analysis provides a comprehensive list of genes, pathways and expression signatures associated with severe sepsis and septic shock, confirming several results from individual studies. In addition, our meta-analysis potentially provides new biological insights about sepsis, by listing a comprehensive list of new candidate genes with robust associations with this condition. Disclosures No relevant conflicts of interest to declare.

Description: Abstract 3347 Introduction: Coagulation activation is a consequences of the host response to sepsis. Up-regulation of tissue factor expression and down-regulation of natural anticoagulant and fibrinolytic systems observed in non-clinical and clinical studies of sepsis lead to disseminated intravascular coagulation (DIC), regarded as a contributor to the pathogenesis of sepsis. Nonetheless, clinical strategies targeting coagulation activation have not yet shown benefits for sepsis patients. DIC is a dynamic process, and hypercoagulability is regarded as a hallmark of its early stages. However, assessment of this hypercoagulable state rely on tests that measure specific compartments of hemostasis, such as levels of anticoagulant proteins, fibrinogen and markers of fibrin degradation. Because hemostasis is a complex system, whether these assays actually depict the net result of interactions of all elements in the host is a fair concern. In fact, disconnection between classical assays and global assays of hemostasis is being demonstrated in patients with cirrhosis. Methods: Here we performed laboratory evaluation of hemostasis in 24 patients with hematological malignancies and febrile neutropenia, in the early stages of sepsis. In addition to classical assays used in DIC such as platelet count (PC), fibrinogen (Fib), prothrombin time (PT) and D dimer (DD), thrombin generation (TG) was evaluated using a fluorogenic method (Technoclone, Vienna, Austria) in platelet poor plasma samples. Selection of patients with hematological malignancies and chemotherapy-induced neutropenia allowed us to obtain a baseline sample before the inititiation of chemotherapy, against which all comparisons were made. In addition, this strategy allowed us to obtain the 1st sample within 12h from fever onset, thus standardizing the time of evaluation, a key variable in studies of coagulation in sepsis. Laboratory evaluation was repeated 48h after fever onset. A sepsis severity score (SOFA) was recorder daily. Results: Median age was 42 years-old (16–62). Baseline diagnosis included acute leukemias in 14 and other hematological malignancies in 10. At fever onset, mean neutrophil count was 125/ml (range 20–500). Sepsis was defined according do ACCP/SSCM criteria. SOFA score at fever onset and 48 hours thereafter were 3 (range 0–13) and 4 (range 1–12) respectively. The proportion of patients with overt DIC based on classical ISTH score was overestimated by the presence of severe thrombocytopenia. Using an arbitrary modification to correct for this factor, only 1 patient presented overt DIC. Septic shock developed in 8 patients, in a median of 4 days from fever onset. Classical parameters of coagulation behaved as expected for the early phases of sepsis: PT remained stable, Fib and DD increased progressively. PC analysis was hampered by severe myelotoxicity. Total TG, expressed by the area under the curve of the thrombin generation curve (AUC), peak thrombin and lag time were not changed at fever onset and 48 hours thereafter compared to baseline values, and to values obtained in healthy individuals. An increase in the time to peak thrombin generation and a decrease in the velocity index were the only significant changes observed in our study. A positive correlation was observed between the lag time to TG and the SOFA score, and a negative correlation was observed between the SOFA score and AUC at the time of fever onset (all r〉0.5 with P

Description: Abstract 1068 Sickle cell anemia (SCA) is associated with a hypercoagulable state, through mechanisms not yet clearly defined. SCA patients present an elevated rate of thrombotic complications and increased biological markers of coagulation activation. Currently one of the major pillars of SCA management is hydroxyurea (HU), a drug used primarily as an inductor of fetal hemoglobin (HbF), but with many other pleiotropic effects. Tissue factor (TF) is the major initiator of blood coagulation and has been shown to be up regulated in several inflammatory conditions. In the present study, we evaluated the effect of HU on coagulation activation by studying the expression of TF and final markers of coagulation activation: thrombin-antithrombin complex (TAT) and prothrombin fragment F 1+2 (F 1+2). We also correlated these measurements with HbF levels, lactate dehydrogenase (LDH), markers of endothelial activation (soluble thrombomodulin [sTM]) and inflammation (tumor necrosis factor-alpha [TNF-α] and white blood cell counts, including leukocyte, monocyte and neutrophil counts). We studied a cohort of 48 adult SCA patients (all with genotype SS), median age of 37 years (minimum: 20 – maximum: 50) and 25 healthy age and race matched controls. The patients included were all in steady state and 23 of them were receiving HU (SSHU). We analyzed leukocyte TF mRNA expression by real time quantitative RT-PCR and TF protein plasma levels by ELISA. TAT, F 1+2, sTM and TNF-α were all measured by ELISA. Statistical analyses were performed using Mann-Whitney's U test and Spearman's correlation test. Fisher's exact test was used to compare plasma TF levels, on the basis of detectable levels. Leukocyte TF mRNA expression was up regulated in SCA patients, in comparison to healthy controls (5.29 vs. 1.16; P = 0.0005). HU was effective in inhibiting this expression significantly (5.29 vs. 2.34; P = 0.0083). These results were confirmed by the measurements of protein plasma levels of TF. Only 27.8% (5/18) of SSHU patients had detectable plasma levels of TF, in comparison to 78.5% (11/14) in the group without the drug (P = 0.01). SCA patients also showed higher levels of TAT (11.34 vs. 2.44; P

Description: 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