This study was based on the preparation, characterization, and animal in vivo experiments performed to evaluate nanoparticles of poly(ɛ-caprolactone) (PCL) and chitosan as carriers of enoxaparin. The nanoparticles were characterized and presented satisfactory results in terms of size, polydispersity, and encapsulation efficiency. Anticoagulant activity of the nanoparticles was maintained for 14 hours when the administration was subcutaneous; however no activity was observed after oral administration. There was a significant reduction in thrombus size, in vivo, for both free and encapsulated enoxaparin in comparison with the control group after subcutaneous administration. Oral administration results however were indifferent. In conclusion, the double emulsion method w/o/w was efficient for enoxaparin encapsulation, producing spherical nanoparticles with high encapsulation efficiency. For in vivo studies, the encapsulated enoxaparin showed a sustained anticoagulant activity for a higher period of time compared to free enoxaparin, with an antithrombotic effect when administered subcutaneously.

Platelet-rich Plasma (PRP) has been widely used in different fields of medicine as autologous therapeutic product. The main component that appears to be associated with the therapeutic effect is the presence of growth factors (GF). However, many protocols available induce high methodology variability. In addition, is still unclear what is the best platelet activator and the necessity for the clinical practice. The traditional PRP used (Fresh PRP) can vary to each preparation and it has also been difficult to use in a time manager, especially for emergency care. Freeze-drying processes come out as a PRP standardization possibility, offering a low-risk proliferative microorganisms. This study aims to compare in vitrothe fresh PRP with the lyophilized PRP, in terms of platelet concentration capacity, and the GF potential release. For fresh and lyophilized PRP production, plasma from twenty-two male healthy individuals were obtained, with mean age of 28.6 ± 5.6 years. The blood was collected with ACD tubes (BD Vacutainer), than centrifuged twice: first with a spin at 300 g for 5 minutes and second with 700 g for 17 minutes. At the end of the double spin, the top layer plasma was characterized as platelet poor plasma and the lower layer was considered the PRP. The pellet were homogenized slowly, and adjusted to 1,2x106 platelets/µL before being frozen at -80ºC. For lyophilized PRP a stabilizing buffer were add and samples were frozen for 1 hour at -80ºC. After that, the PRP was lyophilized by Christ Alpha Plus for 20 hours. To compare fresh and lyophilized PRP, the platelets were evaluated for number of concentration, functionality, and the capacity of GF release, such as VEGF, PDGF, EGF and TGFβ. Non-parametric statistics were used in all analysis (Graph Pad 5.0). The PRP was able to recover high concentrations of platelets. The mean of platelet concentration was 1622 x 103 cells/µl, which represents 5.3 folds higher from the basal number (303 x 103 cells/µl). The recovery of platelets after freeze-drying was 54% compared to the initial concentration (1200 x 103 cells/µl). Platelet function was evaluated pre and post fresh PRP preparation and after freeze-drying, with two agonists ADP and epinephrine, to check the capacity of then to induce platelet aggregation. Results were evaluated trough amplitude of aggregation curve. Interestingly, high amplitude was observed only for samples from pre fresh PRP preparation (ADP median 86% from 71% to 100 % and epinephrine median 86 % from 74% to 103%). Despite the high concentration obtained from PRP (5 folds higher the basal number), no amplitude curve for platelet aggregation was observed for samples post fresh PRP preparation (ADP median 19% from 0% to 85% and epinephrine median 2% from 1% to 37%), even for lyophilized PRP (ADP median 1% from 0% to 2% and epinephrine median 1.5 % from 0 % to 3%), figure 1. The GF levels were similar for both products, with any grow factor loss after freeze-drying. The mean and standard deviation for level of GF were: PDGF 49365 pg/ml ± 17410 for fresh PRP and 60207 pg/ml ± 18472 for lyophilized PRP; VEGF 1250 pg/ml ±1171 for fresh PRP and 954,3 pg/ml ± 644,6 for lyophilized PRP; TGFβ 140373 pg/ml ± 91454 for fresh PRP and 111991 pg/ml ± 19827for lyophilized PRP; EGF 771,6 pg/ml ± 320,4 for fresh PRP and 739,1 pg/ml ± 324,1 for lyophilized PRP, figure 2. The results showed that fresh or lyophilized PRP were unable to show normal aggregation function, suggesting that these samples had been already activated by several conditions of preparation, such as the manual manipulation, temperature, pressure that the platelet is subjected inside the needle, among others. This result confirms that platelet activation with thrombin, or calcium chloride before PRP application is not crucial. Anyhow, the GF that is considered an important component for PRP regarding the therapeutic effect, were preserved. In addition, the lyophilized PRP appears as a possible replacement of fresh PRP, adding minor technical variability with a single process production, enabling a large-scale, with shelf life increased. Figure 1 Represents aggregation percentage generated after two antagonists addition. The results demonstrate that post-PRP has no platelet activation. Figure 2. Represents growth factor measurement in Fresh PRP and lyophilized PRP. The only difference between both preparations was PDGF (p=0, 0464). Figure 1. Represents aggregation percentage generated after two antagonists addition. The results demonstrate that post-PRP has no platelet activation. Figure 2. Represents growth factor measurement in Fresh PRP and lyophilized PRP. The only difference between both preparations was PDGF (p=0, 0464). Disclosures No relevant conflicts of interest to declare.

Background: The causes for venous thromboembolism (VTE) remain undetermined in at least 30% of patients with unprovoked VTE. Hypofibrinolysis may be associated to VTE, however the occurrence of hypofibrinolysis in patients with unprovoked, idiopathic, VTE is not well stablished. Aims: To evaluate whether hypofibrinolysis would be associated with unprovoked idiopathic VTE. Methods: Patients with a history of unprovoked VTE without acquired or inherited thrombophilia were included. Global tests of fibrinolysis, such as euglobolin lysis time (ELT) and lysis area on fibrin plate (LAFP), and specific tests of fibrinolysis, such as plasma activity plasminogen, a-2 antiplasmin (a2AP), plasminogen activator inhibitor-1 (PAI-1), and thrombin activatable fibrinolysis inhibitor (TAFI), were performed in patients and healthy controls. We also analyzed the plasma activity of factor (F) XIII. Results: Thirty-one patients and fifty healthy controls were included. ELT results were higher in patients than in controls (median= 295 and IQ= 205-355 minutes vs. median=250 and IQ= 167-295 minutes, respectively, p = 0.006) and LAFP values were lower in patients compared to controls (median=81 and IQ= 56-110 vs. median= 95 and IQ 72-132 respectively, p = 0.0014), suggesting that they were experiencing a hypofibrinolytic state. Plasma activity of plasminogen (median= 131 and IQ= 119-141 vs. median=120 and IQ=111-137, respectively, p = 0.045) and FXIII (median= 103 and IQ 89-127 vs. median= 96 and IQ=80-105, respectively, p = 0.050), were higher in patients than in controls, whereas plasma activity of α-2AP was lower in patients (median= 124 and IQ 114-128 vs. median= 127 and IQ=121-133, p≤0.001). Interestingly, patient's median TAFI activity was lower than in controls (median=16 and IQ 13-18μg/mL vs. median= 19 and IQ= 17-21g/mL, p≤0.001). However, PAI-1 activity did not differ between groups. Conclusions: Hypofibrinolysis may occur in patients with unprovoked idiopathic VTE and may be detected by global tests of fibrinolysis. It is possible that hypofibrinolysis contribute to the pathogenesis of thrombosis in these selected cases. Disclosures No relevant conflicts of interest to declare.

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.

Ao meu marido, Antonio José Bassora Junior, pelo companheirismo, incentivo e amor.vii AGRADECIMENTOS Á Deus por permitir a finalização deste trabalho.À minha orientadora Professora Dra Joyce Maria Annichino-Bizzacchi, exemplo de ética e amor ao trabalho, obrigada pela excelente orientação, por acreditar em meu potencial e pelo seu grande estímulo na realização de todas as etapas do meu doutorado.A Michele Rubin Servais (in memorian), pelo excelente trabalho técnico e intelectual.À coordenação do Curso de Pós-graduação e a secretária do curso de pósgraduação Marcinha em Ciências Médicas área de concentração em Ciências Biomédicas/ Faculdade de Ciências Médicas (FCM)/ Unicamp, pelo apoio em todas as fases de meu doutorado.