Yes / Platelet microparticle (PMP)-induced angiogenesis plays a key role in tumour metastasis and has been proposed to contribute towards cardiovascular disease by enhancing atherosclerotic plaque vulnerability. However, the mechanisms underlying PMP induced angiogenesis are ill defined. Recent reports demonstrate that PMPs deliver micro-RNAs (miRNAs) to recipient cells, controlling gene expression. We therefore evaluated whether miRNA transfer was a key regulator of PMP-induced angiogenesis. Co-culturing PMPs with human umbilical vein endothelial cells (HUVEC) on extracellular matrix gel induced robust capillary like structure formation. PMP treatment altered the release of angiogenesis modulators from HUVEC, including significantly reducing production of anti-angiogenic thrombospondin-1 (THBS-1). Both functional responses were abrogated by treating PMPs with RNase, suggesting the transfer of PMP-derived RNA was a critical event. PMPs were an abundant source of miRNA Let-7a, which was transferred to HUVEC following co-incubation. Using luciferase reporter assays we have shown that Let-7a directly targets the 3’UTR of the THBS-1 mRNA. HUVEC transfection with a Let-7a anti-sense oligonucleotide reduced the ability of PMPs to inhibit THBS-1 release, and significantly decreased PMP induced in vitro angiogenesis. Antibody neutralisation of THBS-1 reversed the anti-angiogenic effect of let-7a inhibition in PMP treated HUVEC, highlighting Let-7a dependent translational repression of THBS-1 drives angiogenesis. Importantly, plasmid overexpression of Let-7a in HUVEC alone induced robust tubule formation on extracellular matrix gel. These data reveal a new role for Let-7a in promoting angiogenesis and show for the first time PMPs induced angiogenic responses occur through miRNA regulation of HUVEC.

Streptococcus sanguinis is a leading cause of infective endocarditis. Bacterial adhesion to platelets is likely important in the pathogenesis of infective endocarditis. Bacterial cell wall-anchored (Cwa) proteins may mediate this adhesion. To begin to test this hypothesis, S. sanguinis adhesion to platelets was examined in vitro. The requirement of 12 Cwa proteins for S. sanguinis-platelet adhesion was individually assessed, measuring adhesion of purified platelets to polystyrene wells coated with S. sanguinis strain SK36 or 12 isogenic Cwa protein mutants. Significantly fewer platelets adhered to wells coated with one mutant strain, VT1614. However, results of a whole-cell enzyme-linked immunosorbent assay (ELISA) showed that 8 mutants, including VT1614, adhered in significantly lower numbers to wells than did SK36. After accounting for unequal bacterial numbers, we determined there was no significant difference in platelet adhesion among the strains. This suggests that none of the Cwa proteins examined were required for S. sanguinis-platelet adhesion.

Os hemoderivados têm sido utilizados com frequência cada vez maior na medicina equina, sendo caracterizados como um produto autólogo, com maior quantidade de fatores de crescimento e que melhora a capacidade de cicatrização de tecidos com pouco aporte sanguíneo, como tendões e articulações, diminuindo o tempo de recuperação do animal. Sabe-se que os fatores de crescimento são derivados das plaquetas, porém a correlação positiva entre o aumento na contagem plaquetária e a maior concentração de fatores de crescimento ainda é motivo de discussão entre os autores. Com o intuito de se obter um produto final com maior contagem plaquetária, é frequente o aumento da velocidade ou número de centrifugações na metodologia empregada, aumentando o risco de agregação plaquetária precoce. O objetivo do presente trabalho é estudar o efeito da dupla centrifugação no preparo de PRP, por meio da comparação entre contagens plaquetárias, concentrações de fator de crescimento TGF-β1, e grau de ativação plaquetária por meio da porcentagem de agregação. Foram utilizados 12 equinos, machos, de 3 a 5 anos, clinicamente sadios. Para tanto foram realizados dois protocolos distintos: um com centrifugação única e o outro com dupla centrifugação. No primeiro, o sangue com anticoagulante foi centrifugado a 141G/12 minutos; enquanto no segundo a primeira centrifugação foi de 300G/5 minutos seguida de 700G/15 minutos, com repouso entre as mesmas e após. Os produtos obtidos após cada centrifugação foram submetidos à contagem plaquetária, teste de agregação e quantificação de TGF--β1 por meio de kit ELISA. Os resultados obtidos demonstraram maior concentração plaquetária quando utilizado protocolo de dupla centrifugação. Agregometria evidenciou maior ativação das plaquetas durante o preparo do PRP quando submetidas a maiores velocidades de centrifugação (força gravitacional) e não ao fato das amostras serem centrifugadas duas vezes. A quantificação do TGF--β1 não mostrou diferença quando realizado em amostras com apenas uma centrifugação, mas demonstrou valores maiores no produto final da segunda centrifugação. A avaliação por meio de coeficiente de determinação e coeficiente de correlação de Pearson evidenciou correlação positiva entre contagem plaquetária e de TGF--β1. O protocolo com dupla centrifugação se mostrou mais eficaz em concentrar plaquetas e TGF--β1, não sendo prejudicado pela ativação precoce dessas plaquetas durante o preparo. / Blood derived products have been used in equine medicine with increasing frequency, being characterized as an autologous product, with greater amount of growth factors and be capable of improvement the healing capacity in tissues with poor blood supply, such as tendons and joints, reducing the time of recovery of the animal. It is known that the growth factors are derived from platelets, but the positive correlation between the increase in platelet count and the higher concentration of growth factors is still a reason for discussion among the authors. In order to obtain a final product with a higher platelet count, it is frequent to increase the speed or number of centrifugations in the methodology employed, increasing the risk of early platelet aggregation. The aim of the present study is to verify the effect of double centrifugation in PRP preparation by comparing platelet counts, TGF-β1 growth factor concentrations, and degree of platelet activation through percentage of aggregation. Twelve horses, male, aged 3 to 5 years-old, clinically healthy were subjected. Two different protocols were performed: one with single centrifugation and the other with double centrifugation. In the first one, the anticoagulated blood was centrifugated at 141G/12 minutes; while in the second one the first centrifugation was 300G/5 minutes followed by 700G/15 minutes, with rest between them and after. The products obtained after each centrifugation were submitted to platelet counting, aggregation test and measurement of TGF-β1 by ELISA kit. The results showed a higher platelet concentration when double centrifugation protocol was used. The aggregometry test evidenced a greater activation of the platelets during the preparation of PRP when submitted to higher centrifugation velocities (times g), and not to double centrifugation. Quantification of TGF-β1 showed no difference when performed on samples with only one centrifugation, but was higher values in the final product of the second centrifugation. The determination coefficient and Pearsons correlation coefficient showed a positive correlation between the platelet count and TGF-β1 concentration. The double centrifugation protocol proved to be more effective at concentrating platelets and consequently higher amounts of TGF-β1, not being impaired by early activation during obtainment.

Platelets are small anucleate cell fragments found in blood whose physiological role is important in maintaining haemostasis. In vivo, platelet surface glycoproteins mediate the mechanistic roles of platelets, and polymorphic changes to these glycoproteins have been observed to have significant effects on the platelet cellular function and such changes may include over-expression, under-expression and antigenicity of the protein. Human platelet antigens (HPA) are a result of polymorphic differences in platelet surface glycoproteins which have been found to be variably expressed in the population. Foetal maternal alloimmune thrombocytopaenia (FMAIT) is a condition that is observed in the unborn foetus and neonates due to HPA incompatibility between the mother and the foetus. HPA incompatibility accounts for a majority of severe thrombocytopaenic cases in neonates, and delayed diagnosis and treatment of such a condition often lead to intracranial haemorrhage. The risk in neonates diagnosed with FMAIT becomes increasingly significant in cases where intra-uterine (during pregnancy) platelet transfusion is the only effective therapeutic option. There are currently no antenatal screening programs for this condition, and laboratory diagnosis of FMAIT relies on the detection of maternal alloantibodies and parental HPA typing. For these reasons a significant amount of research is currently being invested into the isolation of recombinant antibodies with specific reactivity against FMAIT-related platelet antigens. Stable and specific recombinant platelet antibodies have great potential as a diagnostic agent in antenatal screening and broad-scale HPA typing of blood donors for platelet transfusion. Further characterisation of the isolated antibody may lead to a possible therapeutic agent. Studies by previous researchers have shown that the traditional methods (ie. Mouse monoclonal and EBV transformation) of obtaining monoclonal antibodies against FMAIT-related antigens have proven unsuccessful. The continuing progress in the discipline of phage display has produced several novel antibodies against self and non-self antigens. A further advantage in the application of phage display technology for the isolation of novel antibodies is the easy transition from bacterial to mammalian expression for the characterisation of glycosylated antibodies. The main focus of this project was to create and isolate a recombinant human anti-HPA-3a antibody using phage display for its possible application as a therapeutic or diagnostic agent.

Oxidative damage has been suggested to play a key role in the pathogenesis of atherosclerosis and other cardiovascular disease. Increased free radical production induced by oxidative stress can oxidise low density lipoproteins, activates platelets, induces endothelial dysfunction and disturbs glucose transport by consuming endogenous antioxidants. Using a combination, of in vitro and in vivo experimental models, the primary aims of the studies undertaken for this thesis were to examine whether different antioxidants could negate risk factors leading to thrombosis, atherosclerosis and other cardiovascular diseases. The studies utilised the mechanisms involved in platelet activity and glucose uptake by skeletal muscle myotubes. The first study determined if olive leaf extract would attenuate platelet activity in healthy human subjects. Blood samples (n=11) were treated with five different concentrations of extract of Olea europaea L. leaves ranging from 5.4£gg/mL to 54£gg/mL. A significant reduction in platelet activity (pless than0.001) and ATP release from platelets (p=0.02) was observed with 54£gg/mL olive leaf extract. The next crossover study compared the effect of exercise and antioxidant supplementation on platelet function between trained and sedentary individuals. An acute bout of 1 hour exercise (sub maximal cycling at 70% of VO2max) was used to induce oxidative stress in 8 trained and 8 sedentary male subjects, before and after one week supplementation with 236 mg/day of cocoa polyphenols. Baseline platelet count and ATP release increased significantly (pless than0.05) after exercise in all subjects. Baseline platelet numbers in the trained were higher than in the sedentary (235¡Ó37 vs. 208¡Ó34 x109/L, p less than 0.05), whereas platelet activation in trained subjects was lower than sedentary individuals (51¡Ó6 vs. 59¡Ó5%, p less than0.05). Seven days of cocoa polyphenol supplementation did not change platelet activity compared to the placebo group. The third study determined the effect of 5 weeks of either 100mg/day £^-Tocopherol (n=14), 200mg/d £^-Tocopherol (n=13) or placebo (n=12) on platelet function, lipid profile and the inflammatory marker C-reactive protein. Blood £^-tocopherol concentrations increased significantly (pless than0.05) relative to dose. Both doses attenuated platelet activation (pless than0.05). LDL cholesterol, platelet aggregation and mean platelet volume were decreased by 100mg/d £^-tocopherol (all pless than0.05). The final study determined the effect of glucose oxidase induced oxidative stress and £^-tocopherol treatment on glucose transport and insulin signalling in cultured rat L6 muscle cells. One hour treatment with 100mU/mL glucose oxidase significantly decreased glucose uptake both with and without 100nM insulin stimulation (pless than0.05). Pre-treatment with 100ƒÝM and 200ƒÝM £^-tocopherol partially protected cells from the effect of glucose oxidase, whereas 200ƒÝM £^-tocopherol restored both basal and insulin stimulated glucose transport to control levels. Glucose oxidase-induced oxidative stress did not impair basal or insulin stimulated phosphorylation of Akt or AS160, but 200ƒÝM £^-tocopherol improved insulin-stimulated phosphorylation of these proteins. In summary, the results from the studies undertaken for this thesis provide evidence that antioxidant supplementation maintains normal platelet function, exerts a positive effect on blood lipid profile and improves glucose uptake in normal healthy asymptomatic population as well as under conditions of induced oxidative stress. Antioxidants including foods rich in cocoa, olive and gamma tocopherol have the potential to combat oxidative stress induced risk factors leading to cardiovascular diseases.

Atherosclerosis is a major cause of mortality in industrialized nations. Atherosclerosis is characterized by plaque deposition which decreases the lumen diameter into a stenosis. The creation of a restriction increases shear rates pathologic levels exceeding 3,500/s. Following plaque cap rupture, thrombus may form from the accumulation of millions of platelets, occluding the vessel, leading to heart attack and stroke. Studies of high shear thrombosis show that platelet activation, GPIIb/IIIa and vWF are involved. However, some recent studies also suggest that high shear aggregation is not dependent on activation or GPIIb/IIIa. Several antiplatelet pharmaceuticals against activation and GPIIb/IIIa have been proposed, but their efficacy in patients remains mixed. The overall objective of this project is to determine the factors necessary for thrombosis to occlusion in very high shear regions seen in diseased arteries. Our central hypotheses are that platelet activation and the subsequent conformational change in GPIIb/IIIa are necessary for thrombosis, and that higher concentrations of vWF in the plasma will increase thrombosis. To this end, we developed a new high shear hemodynamic model utilizing 30mLs of whole blood and quantified thrombus thickness, volume accumulation and accumulation rates. We demonstrate that thrombosis to occlusion stems from a second phase of Rapid Platelet Accumulation (RPA). Thrombus accumulation is completely prevented by PGE1 inhibition of platelet activation. Similarly, GPIIb/IIIa blockade via abciximab prevented significant thrombus deposition and RPA. We also found that increasing plasma vWF levels in high shear regions increased thrombus thickness and suggestively increased RPA rates. The results clarify the need for activation of mural platelets for long term thrombus accumulation without the activation of circulating platelets.

Platelets are small anucleate cell fragments found in blood whose physiological role is important in maintaining haemostasis. In vivo, platelet surface glycoproteins mediate the mechanistic roles of platelets, and polymorphic changes to these glycoproteins have been observed to have significant effects on the platelet cellular function and such changes may include over-expression, under-expression and antigenicity of the protein. Human platelet antigens (HPA) are a result of polymorphic differences in platelet surface glycoproteins which have been found to be variably expressed in the population. Foetal maternal alloimmune thrombocytopaenia (FMAIT) is a condition that is observed in the unborn foetus and neonates due to HPA incompatibility between the mother and the foetus. HPA incompatibility accounts for a majority of severe thrombocytopaenic cases in neonates, and delayed diagnosis and treatment of such a condition often lead to intracranial haemorrhage. The risk in neonates diagnosed with FMAIT becomes increasingly significant in cases where intra-uterine (during pregnancy) platelet transfusion is the only effective therapeutic option. There are currently no antenatal screening programs for this condition, and laboratory diagnosis of FMAIT relies on the detection of maternal alloantibodies and parental HPA typing. For these reasons a significant amount of research is currently being invested into the isolation of recombinant antibodies with specific reactivity against FMAIT-related platelet antigens. Stable and specific recombinant platelet antibodies have great potential as a diagnostic agent in antenatal screening and broad-scale HPA typing of blood donors for platelet transfusion. Further characterisation of the isolated antibody may lead to a possible therapeutic agent. Studies by previous researchers have shown that the traditional methods (ie. Mouse monoclonal and EBV transformation) of obtaining monoclonal antibodies against FMAIT-related antigens have proven unsuccessful. The continuing progress in the discipline of phage display has produced several novel antibodies against self and non-self antigens. A further advantage in the application of phage display technology for the isolation of novel antibodies is the easy transition from bacterial to mammalian expression for the characterisation of glycosylated antibodies. The main focus of this project was to create and isolate a recombinant human anti-HPA-3a antibody using phage display for its possible application as a therapeutic or diagnostic agent.

Diss. (sammanfattning) Stockholm : Karol inst., 2003. / Härtill 5 uppsatser.

Diss. (sammanfattning) Stockholm : Karol. inst., 2004. / Härtill 4 uppsatser.

Diss. (sammanfattning) Linköping : Univ., 2005. / Härtill 5 uppsatser.