BackgroundDengue cases have been classified according to disease severity into dengue fever (DF) and dengue hemorrhagic fever (DHF). Although DF is considered a non-severe manifestation of dengue, it has been recently demonstrated that DF represents a heterogeneous group of patients with varied clinical complications and grades of severity. Particularly, bleeding complications, commonly associated to DHF, can be detected in half of the patients with DF. Although a frequent complication, the causes of bleedings in DF have not been fully addressed. Thus, the aim of this study was to perform a comprehensive evaluation of possible pathophysiological mechanisms that could contribute to the bleeding tendency observed in patients with DF.MethodsThis is a case–control study that enrolled adults with DF without bleeding and adults with DF and bleeding complications during the defervescence period. Healthy controls were also included. Peripheral blood counts, inflammatory, fibrinolysis and endothelial cell activation markers, and thrombin generation were evaluated in patients and controls.ResultsWe included 33 adults with DF without complications, 26 adults with DF and bleeding and 67 healthy controls. Bleeding episodes were mild in 15 (57.6%) and moderate in 11 (42.4%) patients, 8 (30.7%) patients had bleedings in multiple sites. Patients with DF and bleedings had lower platelet counts than DF without bleeding (median = 19,500 vs. 203,500/mm3, P < 0,0001). Levels of TNF-α, thrombomodulin and VWF were significantly increased in the two dengue groups than in healthy controls, but similar between patients with and without bleedings. Plasma levels of tPA and D-dimer were significantly increased in patients with bleedings (median tPA levels were 4.5, 5.2, 11.7 ng/ml, P < 0.0001 and median D-dimer levels were 515.5, 1028 and 1927 ng/ml, P < 0.0001). The thrombin generation test showed that patients with bleeding complications had reduced thrombin formation (total thrombin generated were 3753.4 in controls, 3367.5 in non-bleeding and 2274.5nM in bleeding patients, P < 0.002).ConclusionsDF can manifest with spontaneous bleedings, which are associated with specific coagulation and fibrinolysis profiles that are not significantly present in DF without this complication. Particularly, thrombocytopenia, excessive fibrinolysis and reduced thrombin formation may contribute to the bleeding manifestations in DF.

The evaluation of patients with a bleeding tendency represents a challenge as the routinely available tests for evaluating bleeding disorders are limited, complicating the laboratory determination of the clinically observed bleeding tendency. As a result, some bleeding disorders remain undiagnosed. The aim of the study was to evaluate whether global coagulation tests would contribute to the laboratory analysis of patients with undiagnosed bleeding disorders. Patients were evaluated for coagulation and fibrinolysis activities by thrombin generation test and euglobulin lysis time. In addition, plasma activity of factor XIII, plasminogen, α-2 antiplasmin, plasminogen activator inhibitor-1, and thrombin-activatable fibrinolysis inhibitor was also obtained. Forty-five patients were included. Eight per cent presented a mild bleeding disorder and 20% a moderate bleeding disorder. The thrombin generation test results were similar between patients and controls. Euglobulin lysis time results, however, were lower in patients than in controls, both before (median 175 vs. 250 min, respectively; P = 0.003) and after (median 145 vs. 115 min, respectively; P ≤ 0.001) arm constriction, suggesting that they were experiencing hyperfibrinolysis. Interestingly, patients' median thrombin-activatable fibrinolysis inhibitor activity was higher than in controls (21.2 vs. 19.46 μg/ml; P = 0.016). However, plasminogen, α-2 antiplasmin, plasminogen activator inhibitor-1, and factor XIII activities did not differ between the groups. Global coagulation and fibrinolysis tests proved to be limited in detecting the hemostatic disorders in some patients with a relevant bleeding tendency and may not be adequate to address their bleeding risk. Bleeding scores are currently the available medical approach for the evaluation of these patients.

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Bleeding complications in dengue may occur irrespective of the presence of plasma leakage. We compared plasma levels of modulators of the endothelial barrier among three dengue groups: bleedings without plasma leakage, dengue hemorrhagic fever, and non-complicated dengue. The aim was to evaluate whether the presence of subtle alterations in microvascular permeability could be detected in bleeding patients. Plasma levels of VEGF-A and its soluble receptors were not associated with the occurrence of bleeding in patients without plasma leakage. These results provide additional rationale for considering bleeding as a complication independent of endothelial barrier breakdown, as proposed by the 2009 WHO classification.

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 <0.0001), F 1+2 (301.5 vs. 145.2; P = 0.0003), sTM (3.11 vs. 2.58; P = 0.0008) and TNF-α (2.49 vs. 0; P < 0.0001) when compared to controls. HU therapy was able to effectively reduce all of these markers (TAT: 11.34 vs. 6.53, P = 0.019; F1+2: 301.5 vs. 216.7, P = 0.05; sTM: 3.11 vs. 2.52, P = 0.0075; TNF-α: 2.49 vs. 0.27, P = 0.0003). Levels of TF mRNA showed a strong negative correlation with HbF levels (r=−0.47) and hemoglobin (r=−0.54), and a positive correlation with sTM (r=0.6), TNF-α (r= 0.52), leukocyte (r=0.41), neutrophil (r=0.46) and monocyte (r=0.54) counts (P values ≤0.01). TAT and F 1+2 presented a positive association with LDH and TAT also presented a significant negative association with HbF levels (P values <0.05). sTM showed a significant negative correlation with HbF and a positive correlation with LDH and inflammation markers (P values <0.05). Additionally, TNF-α presented a negative association with HbF and a positive association not only with TF and sTM, but also with LDH and white blood cell counts (P values < 0.001). Our results clearly demonstrated that HU therapy reduces the hypercoagulability encountered in SCA. We showed that HU was capable of inhibiting TF expression and decreasing both TAT and F1+2 levels, final markers of thrombin generation. In addition, HU reduced the endothelial marker sTM, as well as the pro-inflammatory marker TNF-α. Correlation analyses indicated that TF inhibition was proportional to the increase in HbF levels and a reduction in LDH, a relevant marker of hemolysis and disease severity in SCA. In parallel, TF down-regulation was associated with a reduction in the endothelial marker sTM, the inflammatory markers TNF-α and white blood cell counts. Hemolysis, endothelial activation and inflammation are pathways closely connected to each other and to the activation of coagulation. Thus, as HU is a drug capable of modulating all of these pathways, most likely all of these mechanisms are involved in the inhibitory effect of HU on activation of coagulation. Disclosures: No relevant conflicts of interest to declare.

Lower D-dimer and longer time to peak thrombin generation in new users of DMPA suggest a positive profile against hypercoagulability.

Hemoglobin SC (HbSC) disease is the second most prevalent hemoglobinopathy after sickle cell anemia (SCA – homozygous HbSS). Despite its high prevalence, most of the knowledge about the pathophysiology of HbSC is inferred from studies focused primarily on SCA. In general, HbSC is considered a milder form of SCA. Chronic inflammatory activity is clearly seen in SCA, but not much is known about the inflammation present in HbSC. In the present study we aimed to evaluate inflammatory markers in HbSC patients and their associations with clinical and laboratory characteristics of the disease. This was a cross-sectional study performed on a cohort of 56 HbSC patients (mean age of 41 years), 39 SCA patients (mean age of 34 years), and 24 healthy age-matched controls. All of the patients were in steady state, with no history of painful crisis, hospitalization or transfusions during the preceding 3 months. None of the patients were in use of hydroxyurea. We evaluated the inflammatory markers, tumor necrosis factor-alpha (TNF-α) and interleukin 8 (IL8). Levels of inflammatory markers were correlated with hemolysis markers, blood counts, coagulation markers (tissue factor expression [TF], thrombin-antithrombin complex [TAT], d-dimer [DD]) and endothelial activation marker (soluble thrombomodulin [sTM]). TNF-α, IL8, TAT, DD and sTM were all measured by ELISA. Leukocyte TF mRNA expression was analyzed by real time quantitative PCR. Statistical analyses were performed by Mann-Whitney’s U test and Spearman’s rank correlation test. HbSC patients presented higher TNF-α levels than those of controls (2.72 vs. 0 pg/mL; P <0.0001), which were similar to the levels seen in SCA patients (2.72 vs. 2.74 pg/mL; P = 1.0). IL8 levels were similar between HbSC patients and controls (2.54 vs. 2.29 pg/mL; P = 0.16) and also similar between HbSC and SCA patients (2.54 vs. 2.82 pg/mL; P = 0.45). In the analyses of the HbSC cohort, IL8 levels presented significant positive correlations with: leukocyte (r=0.4; P=0.02), monocyte (r=0.5; P=0.001), and platelet counts (r=0.6; P=0.0002); and hemolysis markers: indirect bilirubin (r=0.4; P=0.04) and lactate dehydrogenase levels (r=0.4, P=0.04). TNF-α levels presented no significant correlations with laboratory markers. We also evaluated associations between IL8 and TNF-α levels and clinical complications; stroke, pulmonary arterial hypertension, acute thoracic syndrome, retinopathy, osteonecrosis, leg ulcers and autosplenectomy. HbSC patients with osteonecrosis had significantly higher IL8 levels (3.8 vs. 2.4; P=0.01) when compared with patients without this complication. IL8 levels were also higher in patients with autosplenectomy (3.8 vs. 2.0; P=0.0003). Our results indicate that HbSC disease patients present elevation of inflammation markers, similar to alterations seen in SCA. In our cohort, patients with higher peripheral blood counts and a more intense hemolytic activity, such as patients with autoesplenectomy, have higher levels of inflammatory markers. Our data suggest that inflammation may be a factor contributing to the pathophysiology of a very prevalent chronic complication of HbSC disease, osteonecrosis. Studies focusing on the pathophysiology of HbSC disease are lacking, and in view of the high prevalence of this disease we believe it should be the focus of future studies. Disclosures: No relevant conflicts of interest to declare.

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<0.0001). Confirming the biological relevance of TF expression, TAT plasma levels were also higher in HbSC patients in comparison to controls (4.2 vs. 2.4), and lower in HbSC than HbSS patients (4.2 vs. 7.3); (P<0.0001). In the analyses of the HbSC cohort, TAT levels presented significant positive correlations with inflammation markers: leukocyte (r=0.5; P=0.001), monocyte (r=0.4; P=0.01), and platelet counts (r=0.5; P=0.002); and hemolysis markers: reticulocyte counts (r=0.4; P=0.01) and lactate dehydrogenase levels (r=0.6, P=0.0001). We also evaluated associations between TAT levels and clinical complications: stroke, pulmonary arterial hypertension, acute thoracic syndrome, retinopathy, osteonecrosis, leg ulcers, autosplenectomy and microalbuminuria. HbSC patients with retinopathy had significantly higher TAT levels (4.7 vs. 3.9; P=0.03) when compared with patients without this complication. TAT levels were also higher in patients with autosplenectomy (4.8 vs. 3.8; P=0.004), and in patients with osteonecrosis, although this had borderline statistical significance (4.6 vs. 3.9; P=0.06). TF expression significantly correlated with monocyte counts (r=0.6; P=0.01). Conclusions: Our results indicate that HbSC disease patients present elevated coagulation activation markers when compared to controls, although not as intense as seen in SCA. Thrombotic complications in HbSC patients have been mainly linked to the hyperviscosity present in this disease. Our data suggest that inflammation and hemolysis are also important factors contributing to hemostatic activation, which may participate in the pathophysiology of very prevalent chronic complications of HbSC disease: retinopathy and osteonecrosis. Interestingly, in our cohort, patients with autosplenectomy had higher levels of pro-coagulant markers, possibly due to a higher intravascular hemolytic rate and elevated peripheral blood counts. Although HbSC disease is considered a milder form of SCA, autopsy studies have shown that mortality by pulmonary embolism is more frequent in HbSC disease than in SCA, being the second cause of mortality in these patients (Manci et al, 2003). Studies addressing the pathophysiology of coagulation activation in HbSC disease are lacking. Low numbers of cases of HbSC disease are usually included in studies focusing mainly in SCA, and the results are very variable, probably due to the small numbers of patients. In view of the high prevalence and morbimortalilty of thrombotic complications in this population, we believe that future studies should focus on a better understanding of hypercoagulability in HbSC disease. Disclosures: No relevant conflicts of interest to declare.