Previous studies have shown that human platelets and megakaryocytes carry microRNAs suggesting their role in platelet function and megakaryocyte development, respectively. However, there is limited information on microRNAs' role in zebrafish thrombopoiesis. Zebrafish thrombocytes could be used as a model to study their role in megakaryocyte maturation and platelet function because thrombocytes have both megakaryocyte features and platelet properties. In our laboratory, I identified 15 microRNAs in thrombocytes using single-cell RNA sequencing. Knockdown of three microRNAs, mir-7148, let-7b, and mir-223, by the piggyback method in zebrafish led to an increase in the percentage of thrombocytes. Functional thrombocyte analysis using plate tilt assay showed no modulatory effect of the three microRNAs on thrombocyte aggregation/agglutination. I then verified these findings in zebrafish larvae after the knockdown of the above microRNAs followed by an arterial laser thrombosis assay. I concluded mir-7148, let-7b, and mir-223 are repressors for thrombocyte production. Furthermore, I explored let-7b downstream genes in thrombocytes detected by RNA-seq analysis and chose 14 targets based on their role in cell differentiation (rorca, tgif1, rfx1a, deaf1, zbtb18, mafba, cebpa, spi1a, spi1b, fhl3b, ikzf1, irf5, irf8, and lbx1b) that are transcriptional regulators. The qRT-PCR analysis of expression levels the above genes following let-7b knockdown showed significant changes in the expression of 13 targets. I then studied the effect of the 14 targets on thrombocytes production and identified 5 genes (irf5, tgif1, irf8, cebpa, and rorca) that showed thrombocytosis and one gene ikzf1 that showed thrombocytopenia. Furthermore, I tested whether mir-223 regulates any of the above 13 transcription factors after mir-223 knockdown using qRT-PCR. Six of the 13 genes showed similar gene expression as observed with let-7b knockdown and 7 genes showed opposing results. Thus, our results suggested a possible regulatory network in common with both let-7b and mir-223. I also identified that tgif1, cebpa, ikzf1, irf5, irf8, and ikzf1 play a role in thrombopoiesis. Since the ikzf1 gene showed a opposite expression profiles following let-7b and mir-223 knockdowns (decreased and increased expression, respectively) and knockdown of ikzf1 resulted in thrombocytopenia I confirmed a definitive role for ikzf1 using an ikzf1 mutant obtained from the Zebrafish International Resource Center (ZIRC). The arterial laser thrombosis assay of ikzf1 mutant progeny confirmed our piggyback hybrid knockdown results. Taken together, these studies shed light on understanding the role and the regulatory effects of zebrafish microRNA on thrombopoiesis and identified novel downstream target transcription factors for let-7b and mir-223.

No description available.

<p>The management of thrombosis in the presence of thrombocytopenia is challenging because the inherent risk of bleeding associated with anticoagulant use may increase due to low platelet counts. Guidelines regarding anticoagulant use in this situation are based mainly on expert opinions and anecdotal data. We developed an <em>in-vitro</em> model to study the effect of anticoagulants on plasma clot formation in the presence of low platelet counts. We used thromboelastography (TEG) to measure global viscoelastic properties of clot formation and scanning electron microscopy (SEM) to observe and quantify changes in the fibrin clot structure. Experiments were conducted in plasma with varying platelet concentrations from <10 >– 150 × 10⁹/L. Clotting was activated with tissue factor (TF) and calcium, in the presence of factor XIIa inhibitor, corn trypsin inhibitor. One of the following anticoagulants at therapeutic concentration was added to the mixture: unfractionated heparin (UFH), dalteparin, fondaparinux, rivaroxaban or dabigatran. We found clotting had different sensitivity to TF concentration depending on the anticoagulant present. Effects on TEG parameters varied at a fixed TF concentration with each anticoagulant. UFH had the greatest influence, delaying clotting significantly at low platelet counts. The factor-specific anticoagulants had the least impact on TEG parameters. SEM revealed that UFH had the greatest impact on clot structure. UFH caused significant increase in porosity and fibrin widths and had significantly less fibers when platelets decreased. In conclusion, this study may provide fundamental data to understand clot formation in the presence of anticoagulants at low platelet counts. At low platelets the anticoagulants can jeopardize clot formation, especially UFH. The mechanism of each anticoagulant may contribute to the variation in response to TF initiated clotting. AT-dependent anticoagulants compromised plasma clotting more than the newer factor specific anticoagulants, possibly related to the multiple, non-specific inhibition of coagulation factors.</p> / Master of Science (MSc)

Introduction: Immune thrombocytopenia (ITP) is an autoimmune bleeding disorder characterized by a platelet count less than 100 x 109/L. Platelet-bound autoantibodies are detected in the peripheral blood of only 40-50% of ITP patients. The subset of ITP patients who do not have detectable autoantibodies may truly be seropositive, but autoantibodies may not be detected due to limitations of the assays. Studies have also suggested that autoantibodies might be sequestered in the bone marrow where autoantibodies may impair platelet production. In addition, detecting autoreactive antibody-secreting B cells using the Enzyme-linked Immunospot (ELISPOT) assay was shown to be highly sensitive. In this study, the bone marrow and the peripheral blood compartments of ITP patients were tested for the presence of anti-GPIIbIIIa and anti-GPIbIX IgG autoantibodies and autoreactive B cells. Methods: Bone marrow aspirate and peripheral blood samples were collected from ITP patients (n=12), non-immune thrombocytopenic patient controls (n=3), and healthy controls (n=5). Mononuclear cells were isolated and tested for the presence of anti-GPIIbIIIa and anti-GPIbIX IgG autoreactive B cells before stimulation and after stimulation with R848 and IL-2 using the ELISPOT assay. Anti-GPIIbIIIa and anti-GPIbIX IgG autoantibodies were detected in the bone marrow and the peripheral blood using the direct antigen capture assay. Results: In our study, we detected autoantibodies and autoreactive B cells of known specificity in the bone marrow of a subset of ITP patients. Detecting anti-GPIIbIIIa and anti-GPIbIX autoantibodies in the bone marrow or the peripheral blood had a sensitivity of 42% and testing both compartments increased the sensitivity to 58%, while maintaining 100% specificity. Autoreactive B cells were detected at low frequencies with low specificity in the bone marrow and the peripheral blood of a subset of ITP patients. The majority of the ITP patients without detectable autoantibodies in the peripheral blood did not have autoantibodies in the bone marrow, and autoreactive B cells were not detected in either compartment. Conclusion: Examining both the bone marrow and the peripheral blood compartments for autoantibodies or autoreactive B cells increased the sensitivity. Furthermore, detecting autoantibodies using the antigen capture assay is more sensitive and specific than detecting autoreactive B cells using the ELISPOT assay. / Thesis / Master of Science (MSc)

Heparin is an anticoagulant widely administered to patients undergoing major orthopedic or cardiac surgery. Though heparin is effective at preventing thrombosis, it is paradoxically associated with the development of heparin-induced thrombocytopenia (HIT). HIT is strongly associated with thrombotic complications and is an adverse drug reaction that occurs when heparin binds to the self-protein, platelet factor 4 (PF4) and forms immunogenic multimolecular complexes. As a result, anti-PF4/heparin antibodies are formed, which bind to these complexes, and can cross-linking Fc receptors on platelets and monocytes causing intense platelet activation, thrombocytopenia, and thrombosis. Patients who receive heparin frequently form antibodies against these PF4/heparin complexes; however, most of these antibodies do not cause HIT. Over-diagnosis of HIT is common due to the detection of clinically insignificant non-pathogenic anti-PF4/heparin antibodies. Current enzyme immunoassays (EIAs) cannot distinguish between pathogenic and non-pathogenic anti-PF4/heparin antibodies and will give a false positive result in the presence of the clinically insignificant non-pathogenic anti-PF4/heparin antibodies. Further functional testing is required to identify samples containing the pathogenic anti-PF4/heparin antibodies that will lead to HIT; however, these tests are not readily available in most centres, and delay timely diagnosis. There is little known about the differences between pathogenic and non-pathogenic HIT antibodies. The identification of antigenic determinations of pathogenic HIT antibodies binding to PF4 from this project will have direct implications for patient care. We will be able to accurately and rapidly identify “true” HIT patients from learning more about the pathogenic HIT antibody epitope. / Dissertation / Doctor of Science (PhD) / At least 30% of patients admitted into the hospital will be exposed to the anticoagulant, heparin. 1-3% of these patients develop heparin-induced thrombocytopenia (HIT): an adverse drug reaction. HIT is a major cause of morbidity and mortality in patients receiving heparin if not diagnosed and treated in a timely manner. HIT occurs when patients form antibodies against the platelet protein, platelet factor 4, in complex with heparin leading to an immune response. However, most heparin-exposed patients produce these antibodies but do not have HIT. Current rapid and available diagnostics tools cannot distinguish between antibodies that can or cannot cause the disease. To improve HIT diagnosis, we will identify the molecular differences between the antibodies that cause HIT and those that do not. From this, we can develop a new diagnostic assay that will be able to dictate whether the antibodies found in patients are specific for HIT.

The parasite Rangelia vitalii is the etiologic agent of rangeliosis, a disease that leads to a wide variety of clinical signs, including hemostatic disorders characterized by bleeding. However, the causes of this process have not been established. The aim of this study was to evaluate the production of megakaryocytes, platelet count, clotting time, platelet activity and to determine the activity of enzymes that hydrolyze nucleotides and adenine nucleosides in platelets of dogs experimentally infected with R. vitalii. For this study, 12 dogs were separated in two groups: Group A was composed by five healthy dogs, and group B composed by seven dogs experimentally infected with R. vitalii. After inoculation, the animals were monitored by blood smears. The parasite was found within erythrocytes, neutrophils and monocytes five days post-inoculation (PI). Blood collection to perform platelet count, coagulation tests and measurement of platelet aggregation was performed on days 0, 10 and 20 PI. On days 10 and 20 PI, after this procedure, the dogs were anesthetized for collecting bone marrow samples to evaluate the production of megakaryocytes. To measure the activity of ectonucleotidases and adenosine deaminase, blood samples were collected on days 12 and 21 PI. Blood samples were stored in tubes containing EDTA to quantification of platelets, evaluation of platelet aggregation and measurement of enzymatic activity. The blood was stored in tubes containing citrate for realization of the clotting time. This study revealed a reduction (P<0.01) of platelet numbers in the infected group when compared to control group. Prothrombin time and active partial thromboplastin time showed no significant difference between infected and control animals. There was an increase (P<0.01) in the number of megakaryocytes in infected group when compared with control group. A reduction (P<0.01) of platelet aggregation was observed in dogs infected with R. vitalii. The hydrolysis of ATP, ADP, AMP and deamination of adenosine decreased (P<0.01) on day 12 PI. On day 21 PI, the activity of adenosine deaminase remained reduced (P<0.01), whereas it was observed an increase (P<0.05) in NTPDase levels. From these results, we can conclude that rangeliosis is responsible for severe thrombocytopenia during the acute phase of infection, and this can be due to splenic sequestration and/or immune-mediated thrombocytopenia. Also, alterations in purinergic system contribute to the occurrence of hemostatic disorders observed in this disease. / O parasito Rangelia vitalii é o agente etiológico da rangeliose, uma enfermidade que cursa com uma grande variedade de sinais clínicos, incluindo desordens hemostáticas caracterizadas por sangramentos. Entretanto, as causas das alterações no processo hemostático nessa doença ainda não foram esclarecidas. O objetivo deste estudo foi avaliar os distúrbios hemostáticos através da produção de megacariócitos e contagem de plaquetas, a coagulação sanguínea, a atividade plaquetária e determinar a atividade das enzimas que hidrolisam nucleotídeos e nucleosídeo de adenina em plaquetas de cães infectados experimentalmente com R. vitalii. Para este estudo, 12 cães foram separados em dois grupos: Grupo A foi composto por cinco cães saudáveis, e grupo B com sete cães infectados experimentalmente com R. vitalii. Após inoculação, os animais foram monitorados quanto à parasitemia por esfregaço sanguíneo. O parasito foi encontrado no interior de hemácias, neutrófilos e monócitos cinco dias pós-inoculação (PI). As coletas de sangue para a realização da contagem plaquetária, testes de coagulação e mensuração da agregação plaquetária foram realizadas nos dias 0, 10 e 20 PI. Nos dias 10 e 20 PI, após esse procedimento, os cães foram anestesiados para coleta do aspirado de medula óssea com a finalidade de avaliar a produção de megacariócitos. Para avaliar a atividade das ectonucleotidases e adenosina desaminase, as coletas de sangue foram realizadas nos dias 12 e 21 PI. Este estudo demonstrou redução (P<0,01) no número de plaquetas entre o grupo infectado em relação ao controle. Os tempos de protrombina e de tromboplastina parcial ativado não apresentaram diferença significativa entre animais infectados e controles. Ocorreu aumento (P<0,01) do número de megacariócitos no grupo infectado quando comparado com o controle. Foi observado uma diminuição (P<0,01) na agregação plaquetária em cães infectados por R. vitalii. A hidrólise de ATP, ADP, AMP e desaminação da adenosina foram reduzidas (P<0,01) no dia 12 PI quando comparadas com o grupo controle. No dia 21 PI, a atividade da adenosina desaminase manteve-se reduzida (P<0,01), enquanto que ocorreu aumento (P<0,05) na atividade da ecto-nucleosídeo trifosfato difosfoidrolase (NTPDase). A partir dos resultados, pode-se concluir que a rangeliose é responsável por grave trombocitopenia na fase aguda da infecção, a qual pode estar associada ao sequestro e/ou destruição imunomediada e, que alterações no sistema purinérgico contribuem para a ocorrência das desordens hemostáticas observadas na infecção pelo parasito R. vitalii.

La réponse immune sous héparine (H) est associée à la synthèse d’anticorps (Ac) d’isotype IgG dirigés contre le facteur plaquettaire 4 (FP4) modifié par l’héparine. Ces anticorps se fixent par leur fragment Fc aux récepteurs FcγRIIa des plaquettes et induisent une forte activation plaquettaire. Les héparines de bas poids moléculaire sont constituées d’un mélange hétérogène d’oligosaccharides (OS) dont la structure varie en fonction de leur nombre de sucres et de groupements sulfates. Nous avons montré que seuls les OS ayant dix groupements sulfates ou plus, peuvent modifier le FP4 et permettre la fixation des Ac héparine-dépendants. La chirurgie cardiaque est associée à une forte activation plaquettaire et les patients sont exposés à de fortes concentrations d’héparine qui est neutralisée en fin d’intervention par le sulfate de protamine (SP). Alors que 30 à 50 % d’entre eux développent des Ac anti H/FP4 nous avons montré que 25% développent également des Ac dirigés contre les complexes H/SP et que ces Ac sont capables in vitro d’induire une activation plaquettaire. Le rôle de ces Ac in vivo reste cependant discuté. / The immune response under heparin (H) treatment is associated with IgG antibodies (Abs) synthesis against heparin-modified Platelet Factor 4 (PF4). These Abs bind FcγRIIa receptors via their Fc fragment and promote strong platelet activation. Low Molecular Weight Heparins are complex mixtures of polysaccharide fragments. These oligosaccharides (OS) have a variable structure due to variations in the type of sugar units and the number of sulphate groups. We demonstrated that OS longer than 10 saccharides and with a large number of sulphate groups are likely able to modify PF4 and allow the binding of heparin-dependent Abs. Cardiac surgery is associated with strong platelet activation and high doses of unfractionated heparin are administered to patients during surgery, and then neutralized with protamine sulfate (SP) at the end of the intervention. 30 to 50% of patients develop anti H/PF4 Abs, but we demonstrated that 25% do synthethized anti H/SP Abs able to activate platelets in vitro. The pathogenic role of these Abs to H/SP in vivo is controversial.

Human platelet antigens (HPA) is located on the platelet surface and they are inherited both from the mother and the father. If a mother who is homozygous for HPA-1b carries a child who has inherited HPA-1a from the father, the mother is in danger to form antibodies against HPA-1a on the fetal platelet. This may cause the child to suffer from neonatal alloimmune thrombocytopenia (NAIT) that could lead to death. This can be prevented by platelet transfusion. EVA Biosensor Technology is a new method for detection of HPA-1 that is currently only approved for scientific research. The aim of this study was to evaluate EVAreader R6 and find HPA-1a negative platelet donors that can donate platelets to children born with NAIT. The test material consisted of blood samples from 513 male blood donors with blood group 0. The blood was lysed and tested in EVA-reader R6 from Davos Diagnostics. The result was shown on the screen after 10 min. The results that came out negative or intermediate was analyzed a second time. In total, nine HPA-1a negative donors and 503 HPA-1a positive donors were found. Approximately 2 % of the population is HPA-1a negative, which was reflected in the result. To make sure that the results are correct, a validation with an already existing method has to be made. The conclusion is that the EVA Biosensor Technology could be used for typing of HPA in the future, as long as the results from the validation is correct.

Made available in DSpace on 2016-12-23T13:53:37Z (GMT). No. of bitstreams: 1 Mara Rubia Rocha Pereira Sales.pdf: 980226 bytes, checksum: 42a133fd63917e32301d6df4c9b4fca5 (MD5) Previous issue date: 2012-06-26 / The aim of this study was determined by nested-PCR the presence of Ehrlichia canis in dogs located in the municipality of Alegre-ES and evaluate its correlation with morulae and thrombocytopenia. For this purpose blood samples were collected from 85 dogs, regardless of race, age, sex or health status. With these, slides were obtained for the detection of morulae, thrombocytopenia, and execution of nested-PCR technique. We verified a prevalence of 1.17% to investigated the presence of morulae, 5.6% when using the technique of nested-PCR, and was verified that 17.64% of the CBCs were thrombocytopenia. However, only 40% of positive samples by nested-PCR showed thrombocytopenia. The results presented in this study demonstrate that the introduction of molecular diagnostic techniques such as nested-PCR is an important method to aid in the early diagnosis of diseases / Objetivou-se com este estudo determinar por meio da nested-PCR a prevalência da Ehrlichia canis em cães da região de Alegre-ES, e avaliar sua correlação com a presença de mórulas e trombocitopenia. Para isso, foram colhidas amostras sanguíneas de 85 cães, independente de raça, sexo, idade e estado de saúde. Com estas, foram confeccionadas lâminas para a pesquisa de mórulas, trombocitopenia, e execução da técnica de nested-PCR. Foi verificada uma prevalência de 1,17% ao pesquisar a presença de mórulas, 5,6% ao utilizar a técnica da nested-PCR, e foi verificada que 17,64% dos hemogramas apresentavam trombocitopenia. No entanto, somente 40% das amostras positivas pela nested-PCR, apresentaram trombocitopenia. Os resultados apresentados neste estudo demonstram que a introdução de técnicas de diagnóstico molecular como a nested-PCR é um método importante para o auxílio no diagnóstico precoce de patologias

HPA1a är ett antigen på trombocytytan som kan orsaka alloimmunisering, såsom neonatal alloimmun trombocytopeni och post-transfusion purpura, vilket kan ge svåra blödningssymptom. I fall med antikroppar mot HPA1a måste kompatibla trombocyter finnas tillgängliga. Syftet med studien var att etablera en flödescytometrisk screeningmetod för fenotypning av HPA1a antigen på trombocyter samt att finna HPA1a negativa blodgivare. Före flödescytometrisk analys poolades två till fem blodprover samman till ett prov och vid förekomst av HPA1a negativa trombocyter i poolen analyserades proverna individuellt. Fluorokrommärkta anti-humana antikroppar mot CD42a och CD61 användes för att särskilja HPA1a negativa trombocyter från HPA1a positiva. Totalt fenotypades 177 blodprover varav sju (4%) typades som HPA1a negativa. Av de sju fynden genotypades fyra vid ett externt laboratorium vilket bekräftade att de var HPA1a negativa. Flödescytometrisk screening av HPA1a är snabb, pålitlig och lämplig för storskalig screening. För att fastställa prevalensen av HPA1a negativa individer behöver mer omfattande studier utföras där en större population ingår. Att ha flera HPA1a negativa blodgivare registrerade ger möjligheten att hjälpa patienter i andra regioner. / HPA1a is an antigen on the platelet surface that can cause alloimmunization, such as neonatal alloimmune thrombocytopenia and post transfusion purpura, which can cause severe bleeding symptoms. In case of antibodies against HPA1a, compatible platelets must be available. The purpose of the study was to establish a flow cytometric screening method for phenotyping HPA1a antigen on platelets and to find HPA1a negative donors. Before the flow cytometric analysis, two to five blood samples were pooled into one sample and in the presence of HPA1a negative platelets in the pool, the samples were analyzed individually. Fluorochrome –labeled anti-human antibodies to CD42a and CD61 were used to distinguish HPA1a negative platelets from HPA1a positive. A total of 177 blood samples were phenotyped, of which 7 (4%) were HPA1a negative. Of the seven findings, four samples were genotyped at an external laboratory confirming that they were HPA1a negative. Flow cytometric screening of HPA1a is fast, reliable and suitable for large scale screening. In order to determine the prevalence of HPA1a negative individuals, more extensive studies need to be performed involving a larger population. By having many registered HPA1a negative donors, it can provide opportunities to help patients in other regions.