Made available in DSpace on 2014-06-11T19:27:57Z (GMT). No. of bitstreams: 0 Previous issue date: 2001Bitstream added on 2014-06-13T18:32:16Z : No. of bitstreams: 1 franco_emg_me_botfm.pdf: 1141908 bytes, checksum: 5c0af3cff17baf1fc15b5658534eb386 (MD5) / O objetivo do presente estudo foi analisar plaquetas de pacientes do sexo masculino, portadores de aterosclerose obliterante periférica (GAOP) no estágio de claudicação intermitente, comparando-as com grupo controle de indivíduos normais (GC2) do mesmo sexo e faixa etária. As plaquetas foram estudadas do ponto de vista funcional, através da agregação plaquetária espontânea, e morfológico, através da ultra-estrutura e da morfometria plaquetária. Também foram analisadas plaquetas de indivíduos normais, de faixa etária inferior a 50 anos (GC1), para verificar eventual influência do fator idade na função plaquetária. Para a análise morfológica e morfométrica, sedimentos plaquetários foram processados para microscopia eletrônica de transmissão (MET) e varredura (MEV), segundo técnicas convencionais. Para a prova funcional, o plasma rico em plaquetas foi agitado em agregômetro de plaquetas, sem a adição de agonistas. Todos os indivíduos foram avaliados clínica e laboratorialmente. Com relação à função plaquetária, não houve diferença significativa na agregação plaquetária espontânea, entre os grupos analisados (p>0,05). A morfometria das plaquetas também não acusou diferença na forma das plaquetas, entre os grupos estudados (p>0,05). À MEV, as plaquetas dos indivíduos do grupo GC1 apresentaram freqüência de alterações na forma significativamente maior (42,70±4,54), quando comparadas às dos outros grupos, GC2 (32,31±6,22) e GAOP (37,76±8,91), p>0,05. A dosagem de fibrinogênio do grupo GAOP (262,90±47,86) revelou-se significativamente maior que do grupo GC1 (209,80±47,02), p>0,05; a dosagem de HDL colesterol do grupo GAOP (30,00±5,33) foi significativamente menor que do grupo GC2 (42,70±12,58), p>0,05. Nossos resultados mostram que as plaquetas circulantes dos pacientes portadores de AOP não... / This study aims to analyze the platelets of patients with peripheral obliterative atherosclerosis (POAG), at the intermittent claudication stage and comparing this platelets with the control group of healthy age and sex-matched subjects (CG2). The platelets were studied in relation to: their function, by the spontaneous platelet aggregation, and their morphology, by the platelet ultrastructure and morphometry. We also analyzed the platelets of younger healthy subjects aged less than 50 years (CG1) to find out a probable influence of age factor on platelet function. Morphological and morphometric analyses were performed using platelet pellets processed by transmission electron microscopy (TEM) and scanning electron microscopy (SEM), according to conventional procedures. The functional test was performed using the platelet-rich plasma, mixed in platelet aggregometer without the addition of stimulating agents. All the healthy subjects and patients submitted to clinical and laboratory evaluations. Platelet function did not show significant difference in the studied groups (p>0.05). Platelet morphometry did not show difference in the analysed groups (p.>0.05). The CG1 platelets showed major alteractions in shape (42.70±4.54) than the other groups: CG2 (32.31±6.22) and POAG (37.76±8.91), p>0.05. The POAG fibrinogen determination (262.90±47.86) was significantly higher than CG1 group (209.80±47.02), p>0.05. The POAG HDL determination (30.00±5.33) was significantly higher than CG2 (42.70±12.58), p>0.05. Our results reveal: the POAG circulating platelets do not show functional or morphological alteractions when compared to the age-matched healthy subjects; and the freqüency of circulating platelet with alteractions in shape using SEM preparations were greater in CG1. This suggests an influence of the age factor on function platelet.

Made available in DSpace on 2014-06-11T19:23:01Z (GMT). No. of bitstreams: 0 Previous issue date: 2010-02-08Bitstream added on 2014-06-13T20:29:38Z : No. of bitstreams: 1 padovani_jl_me_botfm.pdf: 451979 bytes, checksum: 3efa92ac18ae071ea9b4fd67de660268 (MD5) / Ministério da Saúde / Fundação para o Desenvolvimento Médico e Hospitalar (Famesp) / Secretaria do Estado da Saúde de São Paulo / A hepatite C é uma doença crônica resultante da infecção por um vírus, o VHC, que infecta hepatócitos pela interação com um receptor de superfície celular, o CD81. No entanto, algumas proteínas de adesão já foram associadas com a entrada do vírus em hepatócitos e, têm sido relacionadas com a entrada de outros vírus em suas células alvo. O RNA do VHC já foi encontrado em plaquetas, as quais não expressam CD81, embora esta proteína esteja presente em megacariócitos. As plaquetas expressam determinantes antigênicos polimórficos em sua superfície denominados HPA. Alguns HPA residem em integrinas. Já foi demonstrada uma associação entre HPA-5b e a infecção pelo VHC. O objetivo deste estudo foi determinar a presença de RNA do VHC em plaquetas coletadas de sangue periférico de 50 doadores de sangue com RT-PCR negativa para o vírus. Estas plaquetas foram infectadas com VHC in vitro e incubadas a 37ºC por 8-48 horas. RNA extraído foi utilizado para detectar a presença do vírus por RT-PCR e, avaliar as diferenças quantitativas na carga viral de acordo com o perfil HPA por qRT-PCR. Todas as plaquetas foram positivas para o RNA do VHC após a incubação com o vírus, o que demonstra que o vírus interage com as plaquetas in vitro. Foi observado um desvio no equilíbrio de Hardy-Weinberg no sistema HPA-1. A freqüência do alelo HPA-1b foi significativamente inferior (p <0,05) em plaquetas com carga viral acima de 18,4 UI/mL, sugerindo que o alelo b pode estar relacionado com menores níveis de carga viral / Hepatitis C is a chronic disease caused by a virus, the HCV, which usually infects hepatic cells by interaction with a cell surface receptor, a tetraspanin (CD81). However, adhesion proteins already were associated with the virus entry in hepatocytes and have been related with the entry of the others virus in their host cell, too. The HCV RNA already found in platelet but it doesn’t express CD81, although this protein is present in megakaryocytes. Platelets express polymorphic antigenic determinants on their surface, named HPA. Some HPA resides in transmembrane glycoprotein of integrin family. Preview study demonstrated an association between HPA -5b and HCV infection. The goal of this study was to determine the presence of HCV RNA in platelets collected from peripheral blood from 50 blood donors with RT-PCR negative for the virus. These platelets were infected in vitro with HCV and incubated at 37oC for 8-48 hours. RNA was then extracted and used for RTPCR and qRT-PCR to detect the presence of the virus and evaluate quantitative differences in viral load according with the HPA profile. All platelets were positive for HCV RNA after incubation with the virus, demonstrating that the virus interacts with the platelets in vitro. There was deviation from Hardy–Weinberg equilibrium in the HPA-1 system. The allele frequency of HPA-1b was found to be significantly lower (p<0.05) in platelet with viral load upper 18.4 UI/mL, suggesting that allele b could be related with lower viral load levels

Orientador: Elisa Aparecida Gregório / Resumo: O objetivo do presente estudo foi analisar plaquetas de pacientes do sexo masculino, portadores de aterosclerose obliterante periférica (GAOP) no estágio de claudicação intermitente, comparando-as com grupo controle de indivíduos normais (GC2) do mesmo sexo e faixa etária. As plaquetas foram estudadas do ponto de vista funcional, através da agregação plaquetária espontânea, e morfológico, através da ultra-estrutura e da morfometria plaquetária. Também foram analisadas plaquetas de indivíduos normais, de faixa etária inferior a 50 anos (GC1), para verificar eventual influência do fator idade na função plaquetária. Para a análise morfológica e morfométrica, sedimentos plaquetários foram processados para microscopia eletrônica de transmissão (MET) e varredura (MEV), segundo técnicas convencionais. Para a prova funcional, o plasma rico em plaquetas foi agitado em agregômetro de plaquetas, sem a adição de agonistas. Todos os indivíduos foram avaliados clínica e laboratorialmente. Com relação à função plaquetária, não houve diferença significativa na agregação plaquetária espontânea, entre os grupos analisados (p>0,05). A morfometria das plaquetas também não acusou diferença na forma das plaquetas, entre os grupos estudados (p>0,05). À MEV, as plaquetas dos indivíduos do grupo GC1 apresentaram freqüência de alterações na forma significativamente maior (42,70±4,54), quando comparadas às dos outros grupos, GC2 (32,31±6,22) e GAOP (37,76±8,91), p>0,05. A dosagem de fibrinogênio do grupo GAOP (262,90±47,86) revelou-se significativamente maior que do grupo GC1 (209,80±47,02), p>0,05; a dosagem de HDL colesterol do grupo GAOP (30,00±5,33) foi significativamente menor que do grupo GC2 (42,70±12,58), p>0,05. Nossos resultados mostram que as plaquetas circulantes dos pacientes portadores de AOP não... (Resumo completo, clicar acesso eletrônico abaixo) / Abstract: This study aims to analyze the platelets of patients with peripheral obliterative atherosclerosis (POAG), at the intermittent claudication stage and comparing this platelets with the control group of healthy age and sex-matched subjects (CG2). The platelets were studied in relation to: their function, by the spontaneous platelet aggregation, and their morphology, by the platelet ultrastructure and morphometry. We also analyzed the platelets of younger healthy subjects aged less than 50 years (CG1) to find out a probable influence of age factor on platelet function. Morphological and morphometric analyses were performed using platelet pellets processed by transmission electron microscopy (TEM) and scanning electron microscopy (SEM), according to conventional procedures. The functional test was performed using the platelet-rich plasma, mixed in platelet aggregometer without the addition of stimulating agents. All the healthy subjects and patients submitted to clinical and laboratory evaluations. Platelet function did not show significant difference in the studied groups (p>0.05). Platelet morphometry did not show difference in the analysed groups (p.>0.05). The CG1 platelets showed major alteractions in shape (42.70±4.54) than the other groups: CG2 (32.31±6.22) and POAG (37.76±8.91), p>0.05. The POAG fibrinogen determination (262.90±47.86) was significantly higher than CG1 group (209.80±47.02), p>0.05. The POAG HDL determination (30.00±5.33) was significantly higher than CG2 (42.70±12.58), p>0.05. Our results reveal: the POAG circulating platelets do not show functional or morphological alteractions when compared to the age-matched healthy subjects; and the freqüency of circulating platelet with alteractions in shape using SEM preparations were greater in CG1. This suggests an influence of the age factor on function platelet. / Mestre

Resumo: A hepatite C é uma doença crônica resultante da infecção por um vírus, o VHC, que infecta hepatócitos pela interação com um receptor de superfície celular, o CD81. No entanto, algumas proteínas de adesão já foram associadas com a entrada do vírus em hepatócitos e, têm sido relacionadas com a entrada de outros vírus em suas células alvo. O RNA do VHC já foi encontrado em plaquetas, as quais não expressam CD81, embora esta proteína esteja presente em megacariócitos. As plaquetas expressam determinantes antigênicos polimórficos em sua superfície denominados HPA. Alguns HPA residem em integrinas. Já foi demonstrada uma associação entre HPA-5b e a infecção pelo VHC. O objetivo deste estudo foi determinar a presença de RNA do VHC em plaquetas coletadas de sangue periférico de 50 doadores de sangue com RT-PCR negativa para o vírus. Estas plaquetas foram infectadas com VHC in vitro e incubadas a 37ºC por 8-48 horas. RNA extraído foi utilizado para detectar a presença do vírus por RT-PCR e, avaliar as diferenças quantitativas na carga viral de acordo com o perfil HPA por qRT-PCR. Todas as plaquetas foram positivas para o RNA do VHC após a incubação com o vírus, o que demonstra que o vírus interage com as plaquetas in vitro. Foi observado um desvio no equilíbrio de Hardy-Weinberg no sistema HPA-1. A freqüência do alelo HPA-1b foi significativamente inferior (p <0,05) em plaquetas com carga viral acima de 18,4 UI/mL, sugerindo que o alelo b pode estar relacionado com menores níveis de carga viral / Abstract: Hepatitis C is a chronic disease caused by a virus, the HCV, which usually infects hepatic cells by interaction with a cell surface receptor, a tetraspanin (CD81). However, adhesion proteins already were associated with the virus entry in hepatocytes and have been related with the entry of the others virus in their host cell, too. The HCV RNA already found in platelet but it doesn't express CD81, although this protein is present in megakaryocytes. Platelets express polymorphic antigenic determinants on their surface, named HPA. Some HPA resides in transmembrane glycoprotein of integrin family. Preview study demonstrated an association between HPA -5b and HCV infection. The goal of this study was to determine the presence of HCV RNA in platelets collected from peripheral blood from 50 blood donors with RT-PCR negative for the virus. These platelets were infected in vitro with HCV and incubated at 37oC for 8-48 hours. RNA was then extracted and used for RTPCR and qRT-PCR to detect the presence of the virus and evaluate quantitative differences in viral load according with the HPA profile. All platelets were positive for HCV RNA after incubation with the virus, demonstrating that the virus interacts with the platelets in vitro. There was deviation from Hardy-Weinberg equilibrium in the HPA-1 system. The allele frequency of HPA-1b was found to be significantly lower (p<0.05) in platelet with viral load upper 18.4 UI/mL, suggesting that allele b could be related with lower viral load levels / Orientador: Rejane Maria Tommasini Grotto / Coorientador: Maria Inês de Moura Campos Pardini / Banca: Paula Rahal / Banca: Isabel Maria Vicente Guedes de Carvalho / Mestre

Bacterial infection is a leading cause of morbidity and mortality arising from platelet transfusions (1, 2). Storage of platelet products at room temperature (20 to 24ºC) provides ideal conditions for bacterial proliferation (1, 3-6). Furthermore, platelets are stored in plasma containing bioavailable iron that bacteria require to survive (7). Thus we hypothesize that the inclusion of iron chelators will bind and remove iron, thereby inhibiting bacterial growth in both culture medium and platelet concentrates. Additionally, we hypothesize that residual red blood cells (RBCs) in platelet units may contribute bioavailable iron that promotes bacterial growth. To test these hypotheses, we first assessed growth of Staphylococcus epidermidis in culture medium after treatment with the iron chelators deferoxamine (DFO) or phytic acid. DFO significantly inhibited bacterial growth in a dose dependent manner (p < 0.009). Conversely, phytate only inhibited bacterial growth at concentrations ≥ 100 mM (p < 0.001); at ≤ 5 mM, phytate supplied S. epidermidis with additional nutrients and significantly promoted growth (p < 0.001). Subsequently, we monitored the change in RBCs over time. Hemolysis, methemoglobin, and iron levels all significantly increased over the 7-day storage period (p < 0.001) releasing bioavailable iron. Indeed, we found that S. epidermidis growth in iron-poor medium drastically increased with the addition of RBCs, thus supporting our second hypothesis. Surprisingly, the inclusion of DFO in minimal medium did not demonstrate a bacteriostatic effect in the presence of RBCs. The inhibitory effect of DFO was likely overcome by iron released from the elevated methemoglobin levels arising from the direct interaction of DFO with hemoglobin. Previous studies demonstrate that methemoglobin releases iron more quickly than normal hemoglobin (8). Lastly, we evaluated the effect of DFO on microbial growth in platelet concentrates using the BacT/ALERT system. The presence of DFO significantly inhibited S. epidermidis growth in buffy coat platelets in a dose dependent manner (p < 0.001). With these findings, the inclusion of iron chelators is a promising approach to preventing transfusion-transmitted bacterial infection and providing patients with a safer platelet product. / Medicine, Faculty of / Pathology and Laboratory Medicine, Department of / Graduate

Bacterial contamination of platelet concentrates (PCs) poses the major transfusion-associated infectious risk. Coagulase negative staphylococci (CoNS), the predominant platelet contaminants, are recognized as one of the leading causes of hospital-acquired infections due to their ability to form biofilms (surface-attached aggregates). In this study, 29 CoNS strains were characterized for their growth and biofilm formation abilities in media and PCs. Twenty-five strains were aerobic including Staphylococcus epidermidis, S. capitis, and S. chromogenes, while four were identified as the anaerobe S. saccharolyticus. Biofilm-associated icaA and icaD genes were amplified from eight strains. Interestingly, only six of those strains were biofilm-positive. Sequencing of S. capitis icaD revealed no mutations that could explain differences in biofilm phenotypes. Growth of CoNS in PCs varied significantly between strains. This study provides preliminary evidence that slow-growing biofilm-positive S. epidermidis are more likely to be missed during platelet culture, highlighting the need for improved screening methods.

Immune thrombocytopenia (ITP) is a heterogeneous immune-mediated blood disorder with multiple pathologies that cause thrombocytopenia. The primary source of this thrombocytopenia is platelet destruction by antiplatelet autoantibodies. Although several treatment options are available for ITP, they are often transient, and responses can be difficult to predict. Different studies show ITP plasma and autoantibodies can also inhibit platelet production, but the mechanism and its impact in causing thrombocytopenia remains unknown. By identifying the different mechanisms causing ITP thrombocytopenia, it may be possible to identify more effective and patient specific treatment options, as well as identify patients who could be at an increased risk of bleeding. To study the antibody mediated inhibition of platelet production, I developed a peripheral blood based megakaryopoiesis assay that used the patient’s own hematopoietic stem and progenitor cells (HSPC) as a starting cell source to grow megakaryocytes. I demonstrate this assay can use a small amount of peripheral blood to grow mature megakaryocytes that are capable of thrombopoiesis. Using this assay, I investigated the effect patient plasma had on platelet production. As such, this study is the first autologous investigation of the effect ITP plasma has on platelet production. I found no inhibition of megakaryopoiesis, but did find an effect on thrombopoiesis, indicating that the plasma is affecting the end stages of platelet production. Secondary observations also show that some ITP HSPC have an enhanced megakaryopoiesis potential, generating more mature megakaryocytes than what was observed with healthy donors. While screening monoclonal antiplatelet antibodies, I discovered an anti-GPIb antibody that inhibited megakaryocyte maturation and found this affect was also present with the Fab antibody fragment. From my research I have developed several tools that can be used to investigate impaired platelet production in ITP and further our understanding of this pathology. / Thesis / Doctor of Philosophy (PhD) / Immune thrombocytopenia (ITP) is an immune mediated blood disorder where autoantibodies target and destroy platelets, cells that are crucial for preventing blood loss. Evidence from different studies show that ITP autoantibodies are also affecting the cells producing platelets (megakaryocytes), but the mechanism of this effect remains unknown. To study antibody mediated inhibition of megakaryopoiesis, I developed a peripheral blood based megakaryopoiesis assay that used the patient’s own cells as a starting source to grow megakaryocytes. With this assay, I investigated the effect patient plasma had on platelet production. I found no inhibition of megakaryocyte growth but did find an effect on their ability to produce platelets. I also found a model antibody that affected the maturity of the megakaryocytes during their development. These tools can now be used to further investigate impaired platelet production in ITP patients and determine the impact this inhibition has on ITP pathology.

No description available.

Platelets permeabilized by high voltage electric discharges have provided a valuable model system in which to analyse the roles of Ca²⁺ ions and guanine nucleotides in the regulation of secretion by exocytosis. In the present study, the effects of fluoride or fluoroaluminate and of vanadate or pervanadate on secretion of platelet dense granule constituents, and the roles of activation of phospholipase D (PLD), phospholipase C (PLC) and protein kinase C (PKC) in secretion, have been investigated. Electropermeabilized human platelets containing [¹⁴C]5-HT in their dense granules were suspended in a glutamate medium containing ATP and incubated for 10 min at 25°C with, variously, Ca²⁺ buffers, KF/AlCl₃, vanadate/H₂O₂, guanine nucleotides and phorbol 12-myristate 13-acetate (PMA). KF/AlCl₃, which activates heterotrimeric G proteins but not low-M, GTP-binding proteins, caused a Ca²⁺ -dependent [¹⁴C]5-HT secretion; maximal effects were obtained with 10 mM KF plus 10 μM AlCl₃ at a pCa of 6, when 53% of [¹⁴C]5-HT was released. Secretion induced by KF/AlCl₃ in the presence of Ca²⁺ correlated with the stimulation of [3H]diacylglycerol accumulation in permeabilized platelets containing [³H]arachidonate-labelled phospholipids. KF/AlCl₃ also stimulated the phosphorylation of pleckstrin (P47) in permeabilized platelets incubated with [γ-³²P]ATP, indicating the activation of PKC. In the absence of Ca²⁺ (pCa > 9), KF/AlCl₃ caused none of the above effects. These actions of KF/AlCl₃ were attnbutable to the activation of PLC, since KF/AlCl₃ also stimulated the formation of [³H]inositol phosphates in [³H]inositol-labelled permeabilized platelets in the presence of Ca²⁺. PLD activity, measured as the formation of[³H]phosphatidylethanol (PEt) from [³H]arachidonate-labelled phospholipids in the presence of ethanol, could not be detected after stimulation of platelets by KF/AlCl₃ in the absence or presence of Ca²⁺. However, KF/AlCl₃ inhibited the [³H]PEt formation (PLD activity) induced by GTPγS. In the absence of Ca²⁺ (pCa >9), the inhibitory effects of KF/AlCl₃ on [¹⁴C]5-HT secretion induced by GTPγS alone or GTPγS plus PMA correlated well with their inhibitory effects on [³H]PEt formation. At pCa 6, KF/AlCl₃ had only a small inhibitory effect on GTPγS-induced secretion and inhibited GTPγS-induced PLD activity more strongly than GTPγS-induced PLC activity. These results suggest that PLD is important for Ca²⁺ -independent secretion, and that, although both PLD and PLC may play roles in Ca²⁺ -dependent secretion, PLC is likely to be the more important. In the presence of Ca²⁺, either vanadate or H₂O₂ caused concentration-dependent stimulations of [¹⁴C]5-HT secretion, [³H]DAG formation and [³H]PEt formation. At pCa 6, low concentrations of vanadate and H₂O₂, which would be expected to form pervanadate, acted synergistically to stimulate [¹⁴C]5-HT secretion, which correlated with [³H]DAG formation. However, vanadate with H₂O₂ had a biphasic effect on PLD activity that did not correlate with secretion. In addition, at pCa 6, GTPγS-induced PLD activity was abolished by vanadate with H₂O₂, whereas GTPγS-induced secretion and PLC activity were only partially inhibited. These results support the idea that both PLC and PLD are involved in the regulation of secretion but have different contributions to Ca²⁺ dependent and Ca²⁺ -independent secretion. The results are consistent with activation of platelet PLC by a heterotrimeric G protein, but suggest that different mechanisms, possibly involving a low-M, GTP-binding protein, may be involved in the regulation of PLD activity. / Thesis / Master of Science (MSc)

The use of an appropriate anticoagulation regiment for the treatment of venous thromboembolism (VTE) in patients with concomitant thrombocytopenia is based on anecdotal evidence and the opinions of managing physicians. The current guidelines suggest that therapeutic levels of anticoagulants may be safely administered to patients who have a minimum platelet count of 50 x 109/L. However, it has recently been suggested that the minimal platelet threshold for safe anticoagulation treatment can be provided at a reduced platelet count of 30 x 109/L. Thus, in order evaluate these platelet threshold we used a thromboelastography (TEG) model to evaluate the clotting parameters of whole blood at predefined platelet counts in the presence of unfractionated heparin (UFH) and low-molecular weight heparin (LMWH). Due to the importance of red blood cells on hemostasis a whole blood TEG model was designed in order to mimic in vivo hemostasis. Clotting was initiated using different concentrations of tissue factor for each anticoagulant at therapeutic and prophylactic levels of UFH and LMWH at predefined platelet counts. In the presence of therapeutic concentrations of either UFH or LMWH, there were no significant differences in TEG parameters of whole blood clots between platelet counts of 30 x 109/L and 50 x 109/L when clotting was driven by the extrinsic pathway. At prophylactic levels of LMWH clot formation was less compromised. Furthermore, no significant difference was noted between platelet-depleted blood (PDB; <10 x 109/L) and 30 x 109/L with respect to r-time. This suggests LMWH at prophylactic levels has no significant bearing on clot formation at a lower platelet threshold versus therapeutic levels of LMWH. Overall, it shows that clot formation is similar for UFH and LMWH when platelet counts are reduced from 50 x 109/L to 30 x 109/L. This work provides insight on the potential for anticoagulation at a reduced platelet threshold in thrombocytopenic conditions. / Thesis / Master of Science (MSc)