Pharmacological actions of nobiletin in the modulation of platelet function

Background and Purpose The discovery that flavonoids are capable of inhibiting platelet function has led to their investigation as potential antithrombotic agents. However, despite the range of studies on the antiplatelet properties of flavonoids, little is known about the mechanisms by which flavonoids inhibit platelet function. In this study, we aimed to explore the pharmacological effects of a polymethoxy flavonoid, nobiletin in the modulation of platelet function. Experimental Approach The ability of nobiletin to modulate platelet function was explored by using a range of in vitro and in vivo experimental approaches. Aggregation, dense granule secretion and spreading assays were performed using washed platelets. The fibrinogen binding, α-granule secretion and calcium mobilisation assays were performed using platelet-rich plasma and whole blood was used in impedance aggregometry and thrombus formation experiments. The effect of nobiletin in vivo was assessed by measuring tail bleeding time using C57BL/6 mice. Key Results Nobiletin was shown to supress a range of well-established activatory mechanisms, including platelet aggregation, granule secretion, integrin modulation, calcium mobilisation and thrombus formation. Nobiletin was shown to extend bleeding time in mice and reduce the phosphorylation of Akt and PLCγ2 within the collagen receptor (GPVI) - stimulated pathway, in addition to increasing the levels of cGMP and phosphorylation of VASP, a protein whose activity is associated with inhibitory cyclic nucleotide signalling. Conclusions and Implications This study provides insight into the underlying molecular mechanisms through which nobiletin modulates haemostasis and thrombus formation. Therefore nobiletin may represent a potential antithrombotic agent of dietary origins.

Short-term induction of thrombocytopenia delays periodontal healing in rats with periodontal disease: participation of endostatin and vascular endothelial growth factor

Background and Objective: Platelets contain factors, including VEGF and endostatin, that can modulate the healing process. We evaluated the effects of severe thrombocytopenia on periodontal healing in rats and determined the contribution of VEGF and endostatin to the healing process. Material and Methods: Rats were distributed into three test groups and two control groups. Cotton ligatures were placed at the gingival margin level of the lower first molar in the test groups. Sham-operated rats and rats in one of the periodontitis groups were killed 15 days later. Rats in the remaining two periodontitis groups had the ligatures removed in order to study the spontaneous recovery from the periodontal disease 15 days later, and these rats were treated with rabbit antiplatelet serum, in order to induce thrombocytopenia, or normal rabbit serum. An additional group without ligatures received antiplatet serum in the same period. Results: After ligature removal, rats treated with normal rabbit serum showed reduced myeloperoxidase activity, decreased alveolar bone loss and increased numbers of blood vessels. Thrombocytopenia caused a delay in alveolar bone regeneration, a decrease in the number of vessels and a modest decrease in myeloperoxidase activity. In the rats with periodontitis, serum endostatin concentrations were slightly decreased and serum VEGF remained unchanged compared with sham-operated animals. After ligature removal, a significant VEGF increase and endostatin decrease were observed in the rats treated with normal rabbit serum. Thrombocytopenia led to a dramatic fall in both VEGF and endostatin concentrations. Conclusion: Thrombocytopenia leads to a delay of periodontal healing in the situation of experimental periodontitis, which might be mediated in part by a decrease in the serum concentration of VEGF and endostatin derived from the platelets. However, other factors derived from the platelets may also have contributed to a delay of periodontal healing in the rats with thrombocytopenia.

Periodontal Tissue Engineering After Tooth Replantation

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)

Plasma rico em plaquetas no tratamento de lesões condrais articulares induzidas experimentalmente em equinos: avaliação clínica, macroscópica, histológica e histoquímica

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)

Anti-platelet activity of infusion of Tithonia diversifolia's leaves

Tithonia diversifolia, also known as Mexican arnica, has been used in traditional medicine to treat inflammatory refractory with absence of citotoxicity. The possible health risks associated with the consumption of ingestion of the infusion (tea) plant makes it is necessary to identify the potential pharmacological activity or toxicity to prove certain plants that are acclimated in Brazil. Considering the limited number of pharmacological studies regarding the Tithonia diversifolia, the aim of this study was evaluate the effects of this infusion in platelet aggregation. Venous blood was collected with informed consent from healthy volunteers who denied taking any medication in the previous 14 days. Whole blood was transferred into polypropylene tubes containing one-tenth of final volume of acid citrate dextrose (ACD-C; citric acid 3%, trisodium citrate 4%, glucose 2%; 1:9 v/v) and centrifuged at 200g for 15 min. Platelet rich plasma was added of wash buffer solution (NaCl 140mM, KCl 5mM, sodium citrate 12mM, glucose 10mM and saccharose 12mM; pH 6; 5:7 v/v) and centrifuged at 800g for 12 min at 20°C. Platelet pellet was gently resuspended in Krebs-Ringer solution and counts were performed on a Neubauer chamber. Aggregation assay was carried out with 400 μL of platelet suspension (1.2x10 8 platelets/mL) in a cuvette at 37°C with constant stirring. Platelet suspension was incubated for 3 min with aqueous extract infusion (0.6-20μg/mL) prior to addition of thrombin (100 mU/mL). Percentage of platelet aggregation was recorded with an aggregometer (Chrono-log Lumi-Aggregometer model 560-Ca, USA). Our results show an inhibition of thrombin induced platelet aggregation in the presence of 0.6-20 ug/mL Tithonia diversifolia infusion leaves. The Tithonia diversifolia infusion leaves inhibits thrombin induced washed platelet aggregation.

Platelet lysate 3D scaffold supports mesenchymal stem cell chondrogenesis: An improved approach in cartilage tissue engineering

Articular lesions are still a major challenge in orthopedics because of cartilage's poor healing properties. A major improvement in therapeutics was the development of autologous chondrocytes implantation (ACI), a biotechnology-derived technique that delivers healthy autologous chondrocytes after in vitro expansion. To obtain cartilage-like tissue, 3D scaffolds are essential to maintain chondrocyte differentiated status. Currently, bioactive 3D scaffolds are promising as they can deliver growth factors, cytokines, and hormones to the cells, giving them a boost to attach, proliferate, induce protein synthesis, and differentiate. Using mesenchymal stem cells (MSCs) differentiated into chondrocytes, one can avoid cartilage harvesting. Thus, we investigated the potential use of a platelet-lysate-based 3D bioactive scaffold to support chondrogenic differentiation and maintenance of MSCs. The MSCs from adult rabbit bone marrow (n=5) were cultivated and characterized using three antibodies by flow cytometry. MSCs (1×105) were than encapsulated inside 60μl of a rabbit platelet-lysate clot scaffold and maintained in Dulbecco's Modified Eagle Medium Nutrient Mixture F-12 supplemented with chondrogenic inductors. After 21 days, the MSCs-seeded scaffolds were processed for histological analysis and stained with toluidine blue. This scaffold was able to maintain round-shaped cells, typical chondrocyte metachromatic extracellular matrix deposition, and isogenous group formation. Cells accumulated inside lacunae and cytoplasm lipid droplets were other observed typical chondrocyte features. In conclusion, the usage of a platelet-lysate bioactive scaffold, associated with a suitable chondrogenic culture medium, supports MSCs chondrogenesis. As such, it offers an alternative tool for cartilage engineering research and ACI. © 2013 Informa UK Ltd.

Efeito do implante autólogo de plasma rico em plaquetas (PRP) e células tronco mesenquimais na reparação de lesões condrais articulares induzidas experimentalmente em equinos

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)

Osso esponjoso liofilizado de cão utilizado como enxerto puro e associado a plasma rico em plaquetas ou medula óssea em falhas ósseas induzidas em coelhos: estudo experimental

Pós-graduação em Cirurgia Veterinária - FCAV

Uso do plasma rico em plaquetas sob forma de colírio ou tampão no reparo de úlceras de córnea profundas induzidas em coelhos: avaliação clínica e histomorfométrica

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)

Ultra-estrutura e função plaquetária em indivíduos normais e pacientes com aterosclerose obliterante periférica

Pós-graduação em Patologia - FMB

Plaquetas reticuladas na avaliação da trombopoiese medular em cães

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)

Avaliação da influência do plasma rico em plaquetas e do laser de baixa intensidade na cicatrização de defeitos de fenestração periodontal em ratos: estudo histológico e histométrico

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)

Efeito do tratamento com plasma rico em plaquetas em éguas resistentes e susceptíveis à endometrite persistente após inseminação artificial

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)

Cultura de condrócitos para o uso terapêutico: reconstituição de cartilagem

Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES)

Efeito do plasma rico em plaquetas associado ao enxerto autógeno na tíbia de coelhos: estudos histomorfométrico e biomecânico

Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES)