Tesis sobre el tema "Platelet resistance"

Platelets play a pivotal role in haemostasis and at sites of vascular injury arrest bleeding. Unregulated haemostasis leads to thrombosis and coronary artery disease (CAD). The 'gold-standard' preventative measure to combat CAD is administration of antiplatelet compounds that target TxA2/ ADP amplification pathways. Some CAD patients are 'resistant' to anti platelet therapy and these patients often have hyperactive platelets. In combination with physiological stimuli, primers potentiate platelet functional responses. Primers are known to function via a Pl3K-dependent mechanism and may induce antiplatelet resistance. The aims of this PhD project were: (i) establish the role of primers in anti platelet resistance and (ii) characterise the PI3K isoform involved in primer-mediated platelet potentiation. First, I studied the role of primers in anti platelet resistance by treating platelets with ARC66096 (P2Y12 inhibitor) and/or aspirin in the presence/absence of primers (IGF-l, TPO and adrenaline). This study demonstrated that primers could drive antiplatelet resistance. Furthermore Pl3K-mediated mechanisms were responsible for IGF-l/TPO-mediated resistance, while Pl3K-dependent and independent mechanisms were responsible for adrenaline-mediated resistance.

Ischemic heart disease (IHD) is the most common cause of death around the world. The underlying cause of IHD is myocardial ischemia as a result of progressive narrowing of coronary arteries due to atherosclerosis with potential thrombotic complications mediated by platelets. In addition to the role in hemostasis, platelets are increasingly recognized as an important mediator in this atherothrombotic disease. Basic management of IHD lies on medical therapy and coronary revascularization procedures. Percutaneous coronary intervention (PCI) is a commonly used revascularization procedure in the treatment of IHD especially for relief and reduction of symptoms. On the other hand, antiplatelet therapy is often administrated to patients undergoing PCI in an attempt to prevent major adverse cardiac events (MACE) following the procedures. However not all patients respond to the same degree of the antiplatelet therapy and some still develop MACE or stent thrombosis in the presence of the treatment with antiplatelet drugs. Recently a flow cytometric-based assay has been developed to monitor the effect of the antiplatelet drug, particularly the P2Y12 receptor antagonist, in patients treated with this kind of drug. This assay measures the activity of platelets as platelet reactivity index (PRI) based on the phosphorylation state of an intracellular platelet protein called vasodilator stimulated phosphoprotein (VASP). The measured value of PRI is inversely related to the response of patient to the antiplatelet drug. In this study, the response of patients to the P2Y12 receptor antagonist Clopidogrel was investigated following PCI. The PRI of patients was found to be significantly lower than normal subjects without taking this drug, indicating the therapeutic effect of this drug on the patients. However nearly one-third of patients (17 out of 59) studied were found to be non-responsive to clopidogrel treatment based on a cut-off established in this study for classifying patients into responders or non-responders. Furthermore, significant difference between the two types of stents used in PCI procedure, namely bare metal stent (BMS) and drug eluting stent (DES), was observed in the study. Patients receiving DES had nearly three times higher percentage of being non-responsive to clopidogrel than the BMS counterpart (45% vs. 16%, p<0.028). This study provides evidence that DES may be implicated in the non-responsiveness or drug resistance of clopidogrel in patient undergoing PCI.
published_or_final_version
Pathology
Master
Master of Medical Sciences

Bacterial contamination of platelet concentrates (PCs) represents the highest post-transfusion infectious risk. The skin flora bacterium Staphylococcus epidermidis has been reported to be the predominant aerobic contaminant of PCs. The Ramirez' group has shown that S. epidermidis can form surface-attached bacterial aggregates known as biofilms, and can outcompete other coagulase-negative staphylococci, such as Staphylococcus capitis, in PCs. The ability of S. epidermidis to form biofilms has been linked to increased pathogenicity and missed detection during PC screening with an automated culture system (BacT/ALERT). This thesis aimed at investigating the proliferative advantage and resistance mechanisms displayed by S. epidermidis in the PC milieu. Furthermore, in an effort to enhance PC safety for transfusion patients, I studied the anti-biofilm properties of essential oils and antimicrobial peptides (AMPs). My studies aimed at improving PC safety by focussing on both the point of introduction of bacterial contaminants (blood collection), and the stage at which bacterial contaminants can form biofilms and proliferate (PC storage). S. epidermidis can be found in the skin of blood donors as biofilms, which are resistant to the blood donor skin disinfectant currently used by Canadian Blood Services, chlorhexidine-gluconate and isopropyl alcohol (CHG-IPA). Here, several plant-extracted essential oils were evaluated for their ability to enhance the anti-biofilm activity of CHG-IPA. Data revealed that the Lavandula multifida oil and its main component (linalool) greatly enhanced the activity of CHG-IPA against S. epidermidis biofilms. Furthermore, the ability of a combination of three synthetic AMPs to inhibit S. epidermidis biofilm formation during PC storage was assessed These results showed that the combination of AMPs could inhibit biofilm formation but was ineffective against pre-formed S. epidermidis biofilms. The accumulation associated protein (Aap) encoded by the aap gene, found in most S. epidermidis strains and absent in S. capitis, plays a role in biofilm formation. When S. epidermidis aap is transformed into S. capitis, this bacterium displayed increased biofilm formation and proliferated to higher concentrations compared to untransformed S. capitis and to a S. epidermidis aap deletion mutant. Based on these results, aap appears to play a role in providing S. epidermidis a proliferative advantage in PCs by enhancing biofilm formation. Lastly, the GraRS system and SepA were studied for their role in S. epidermidis resistance to platelet-derived AMPs using the synthetic AMP PD4 as a model molecule. Results indicate that the GraS mechanism is involved in resistance towards PD4. The work presented in my thesis provides further insights into why S. epidermidis has a proliferative advantage in the PC storage environment and allows for the proposal of alternative methods to enhance PC safety for transfusion patients.

Coronary blood flow (CBF) is principally controlled by changes in coronary vascular resistance (CVR). Low CVR helps to maintain myocardial perfusion in the presence of epicardial stenosis, therefore factors that impair the reduction of CVR will have a direct effect on CBF leading to either a narrower “effective” perfusion pressure range or reduced ability to compensate for increased demand on myocardial contraction. There are several mechanisms which may be important in the control CVR in humans including both endothelial dependent production of NO and reduction of the simple anion inorganic nitrite (a metabolite of NO) back to NO (via several putative mechanisms). The synthesis of NO by both nitric oxide synthase (NOS) and the reduction of nitrite in the coronary circulation has been the subject of many animal and human clinical studies. Reduced systemic endothelial dependent production of NO has an association with worse cardiovascular outcomes in humans, the number of potential mechanisms are large and perhaps central is the effect on CVR. The reduction of nitrite to NO is seen, in some ways, to be the perfect compensatory mechanism, particularly when endothelial function is impaired. It is easy to hypothesise that this stoichiometric balance of NO production might be responsible for the perfect regulation of CVR and thus CBF. Methods This thesis investigates the influence and effect of both endothelial production of NO and the reduction of nitrite to NO on CVR in man. First, an observational study assessing the impact of these mechanisms in patients undergoing percutaneous coronary intervention (PCI), in the treatment of both non-ST-elevation myocardial infarction and stable angina. Specifically, the metabolites of NO were measured from aortic root to coronary sinus together with the associated CVR both before and after PCI. Second, using a systemic infusion of sodium nitrite (NaNO2) in NSTEMI patients to assess the effect of nitrite reduction on CVR during PCI. The systemic nitrite concentration was increased 8-fold in the same experimental conditions as the observational study. Third, beyond CVR control the influence of NO and nitrite was also assessed in terms of platelet reactivity and systemic inflammatory cytokines in the NSTEMI cohort both with and without NaNO2 infusion. Results NSTEMI patients have a net increase in NO metabolites across the coronary circulation unlike healthy controls (historical data) and stable angina patients. This net increase is lost following PCI and is associated with a significant rise in CVR. Stable angina patients appear to compensate with increase collateral circulation and not NO synthesis. An 8-fold increase in nitrite concentration has no effect on CVR or platelet reactivity in NSTEMI patients. Conclusions In NSTEMI patients a net aorta to coronary sinus NO synthesis appears to be important to maintain a low CVR and thus CBF when haemodynamically significant epicardial disease is present. After the epicardial disease is treated this net increase in NOx (Nitric Oxide metabolites), is lost and is associated with an acute increase in CVR. Stable angina patients have no net increase in NOx across the coronary circulation and after revascularisation have no change in CVR, this may reflect an alternate mechanism of compensation and microvascular perfusion maintained by collateral circulation as evident by the increase CFI. Despite the perfect environment for the reduction of nitrite to NO we saw no influence of an 8-fold increase in serum nitrite concentration on CVR in patients with NSTEMI either before or after PCI, suggesting that nitrite reduction to NO plays no role in CBF regulation in NSTEMI patients. Nitrite reduction depends on conditions that are found predominantly in the capillary bed or venules, thus any mechanism would need to rely on a feedback mechanism to signal back to the arterioles (where much of resistance change is created). Despite hypotheses by others that this may occur by the close arrangement of venules to arterioles, this appears not to be the case in NSTEMI patients. Other clinically relevant and important mechanisms also appear to be unaffected by this increase in serum nitrite, residual platelet function and cytokine concentrations at 24 hours.

Um dos desafios recorrentes na prática clínica da Oncologia é o processo de repopulação, no qual células tumorais resistentes à terapia são capazes de proliferar e reconstituir o tumor. No entanto, os mecanismos envolvidos neste fenômeno ainda foram pouco explorados, sendo necessário melhor compreendê-los para evitar a falha terapêutica. Melanomas são bons modelos para estudar repopulação devido às baixas taxas de sobrevida livre de progressão e às altas taxas de resistência às terapias associadas a este tipo de tumor sólido. Sabe-se que a exposição de células tumorais a condições estressoras do microambiente, como hipóxia e hipóxia/reoxigenação, bem como ao próprio tratamento antitumoral, são pressões seletivas frequentemente encontradas em tumores sólidos que favorecem a resistência às terapias e a repopulação tumoral. Estudos prévios indicam que a sinalização mediada pelo Fator de Ativação de Plaquetas (PAF), um lipídio bioativo relacionado à diversas funções fisiológicas, e de seu receptor, PAFR, está associada com a resistência de células de melanoma aos tratamentos citotóxicos e com crescimento tumoral. Portanto, este trabalho teve como objetivo investigar o envolvimento da sinalização de PAFR frente às condições estressoras descritas acima no fenômeno de repopulação de melanomas. Os resultados de espectrometria de massa indicaram que hipóxia aumentou a geração de PAF nas linhagens de melanoma humano SKmel05 e A375, mas não na A375M, embora este aumento não tenha sido observado após a reoxigenação. Além disso, mostraram que SKmel37 exibiu os maiores níveis basais de PAF, aumentando substancialmente sua geração em diferentes tempos de exposição à hipóxia e hipóxia/reoxigenação. Investigamos também a geração de outros ligantes de PAFR, porém nenhum deles foi encontrado nas amostras. Os resultados de detecção de PAFR por Western Blot e/ou citometria de fluxo revelaram que os níveis proteicos não foram modulados por estas condições em nenhuma das linhagens e que, em termos basais, SKmel37 e SKmel05 apresentaram os maiores níveis. Estas linhagens foram, portanto, submetidas a ensaios de proliferação, os quais evidenciaram que frente ao tratamento com o antagonista de PAFR, WEB2086, em condições de hipóxia e hipóxia/reoxigenação, apenas as células SKmel37 tiveram sua proliferação reduzida e morfologia associada à morte. Ensaios de incorporação de iodeto de propídio indicaram que o tratamento destas células expostas à hipóxia/reoxigenação com WEB2086 levou a acúmulo em SG2M, morte celular e sensibilização à cisplatina. Além disso, imunofluorescência de cortes congelados de tumores induzidos com SKmel37 revelou que houve acúmulo de PAFR em áreas hipóxicas e em seu entorno. Em relação ao modelo de exposição à tratamentos antitumorais, por sua vez, curvas de concentração e tempo com Vemurafenib mostraram que SKmel37 e A375 foram resistentes à droga, ao passo que SKmel05 e UACC62 foram sensíveis. Além disso, WEB2086 potencializou o efeito de morte induzido por Vemurafenib nas linhagens sensíveis, mas não afetou as resistentes. Considerando os aspectos clínicos de resposta inicial com posterior desencadeamento de repopulação, seguimos com as linhagens sensíveis e verificamos por citometria que esta droga aumentou ROS em ambas as linhagens, mas só aumentou PAFR extracelular na SKmel05. O tratamento combinado potencializou a geração de ROS e levou a ativação de caspase3/7 apenas na SKmel05. Esta linhagem foi então submetida a ensaios clonogênicos cujos resultados mostraram que o tratamento com Vemurafenib reduziu o número de clones e que WEB2086 não potencializou este efeito. Assim, o conjunto de resultados apresentados evidencia que a sinalização de PAFR participa dos desfechos de sobrevivência frente à hipóxia/reoxigenação e/ou tratamentos antitumorais, podendo, de alguma forma, contribuir com a repopulação de melanomas
One of the recurrent challenges in the clinical practice of Oncology is the process of repopulation, in which therapy-resistant tumor cells can proliferate and reconstitute the tumor. However, the mechanisms involved in this phenomenon were still little explored. The understanding of these events is, therefore, needed to avoid therapeutic failure. Melanomas are good models for studying repopulation due to the low rates of progression-free survival and the high rates of resistance to therapies associated to this type of solid tumor. It is known that the exposure of tumor cells to microenvironmental stress conditions, such as hypoxia and hypoxia/reoxygenation, as well as the exposure to antitumor treatment itself, are selective pressures frequently found in solid tumors that favor therapy resistance and tumor repopulation. Previous studies have indicated that the signaling mediated by the Platelet Activation Factor (PAF), a bioactive lipid related to various physiological functions, and its receptor, PAFR, is associated with resistance of melanoma cells to cytotoxic treatments and with tumor growth. Therefore, the aim of this study was to investigate the involvement of PAFR signaling upon the adverse conditions described above in the phenomenon of melanoma repopulation. Mass spectrometry results indicated that hypoxia increased the generation of PAF in human melanoma cell lines SKmel05 and A375, but not in A375M, although this increase was not observed after reoxygenation. In addition, they showed that SKmel37 exhibited the highest PAF basal levels, whose generation increased substantially after different times of hypoxia and hypoxia/reoxygenation exposure. We also investigated the generation of other PAFR ligands, but none were found in the samples. The results of PAFR detection by Western Blot and/or flow cytometry revealed that protein levels were not modulated by these conditions in any of the cell lines and that, at baseline, SKmel37 and SKmel05 showed the highest levels. These lines were therefore submitted to proliferation assays, which showed that the treatment with the PAFR antagonist, WEB2086, under conditions of hypoxia and hypoxia/reoxygenation, led to proliferation reduction and death-associated morphology in SKmel37 cells only. Propidium iodide incorporation studies indicated that the treatment of these cells exposed to hypoxia/reoxygenation with WEB2086 led to accumulation in SG2M, cell death and cisplatin sensitization. In addition, immunofluorescence of frozen sections of SKmel37-induced tumors revealed that PAFR was found accumulated in hypoxic areas and its surroundings. Regarding the model of exposure to antitumor treatments, concentration and time curves with Vemurafenib showed that SKmel37 and A375 were resistant to the drug, whereas SKmel05 and UACC62 were sensitive. In addition, WEB2086 potentiated the effect of Vemurafenib-induced death on sensitive cell lines but did not affect the resistant ones. Considering the clinical aspects of initial response with subsequent repopulation triggering, we continued using the sensitive cell lines and we verified by cytometry that this drug increased ROS in both cell lines but only increased extracellular PAFR in SKmel05. The combined treatment potentiated the generation of ROS and led to the activation of caspase3/7 in SKmel05 only. This cell line was then submitted to clonogenic assays whose results showed that treatment with Vemurafenib reduced the number of clones and that WEB2086 did not potentiate this effect. Thus, the set of results presented highlights that PAFR signaling participates in the survival outcomes upon hypoxia/reoxygenation and/or antitumor treatments, and may, in some way, contribute to the repopulation of mel

Orientador: Mario Jose Abdalla Saad
Dissertação (mestrado) - Universidade Estadual de Campinas, Faculdade de Ciências Médicas
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Resumo: A obesidade é hoje considerada um problema de saúde pública. Essa condição é caracterizada pelo aumento do peso corporal, mais especificamente do tecido adiposo branco. A adipogênese (diferenciação do pré-adipócito em adipócito) é um fenômeno complexo e não muito bem caracterizado. Recentes estudos mostraram que os préadipócitos estão localizadas nas paredes dos vasos que irrigam o tecido adiposo. Estas células estão presentes exclusivamente neste tecido e expressam alguns marcadores, dentre eles o PDGFRß. O PDGFRß é um receptor tirosina quinase cujo papel na migração, proliferação e diferenciação de diversos tipos celulares tem sido extensivamente estudado. O AG1296 (6,7- dimetoxi-2-fenil-quinoxalina) é um potente inibidor do receptor PDGF, pertencente à classe da quinoxalinas. Deste modo, levando-se em consideração o papel do receptor PDGF no crescimento e proliferação celulares e o fato de que as células PDGFR_ positivas provenientes do tecido adiposo possuem alto potencial adipogênico, neste estudo investigamos o efeito do AG1296 na adipogênese de camundongos submetidos à dieta hiperlipídica e à dieta padrão. Nós também investigamos se essa inibição afetaria a sensibilidade à insulina desses grupos estudados. Para tanto, camundongos Swiss machos com seis semanas de vida foram divididos em quatro grupos: o grupo Controle que recebeu dieta padrão, o grupo C+AG1296 que recebeu dieta padrão e tratamento com AG1296, o grupo DH que recebeu dieta hiperlipídica somente e o grupo DH+AG1296 que recebeu dieta hiperlipídica e tratamento com AG1296. Peso corpóreo e ingestão alimentar foram medidos diariamente durante o tratamento (7 ou 15 dias). Através de Western blot, foram quantificadas as principais proteínas pró-adipogênicas (SREBP-1c, C/EBP? e PPAR?) e a fosforilação das principais proteínas da via da insulina (IR, IRS1 e AKT). Nossos resultados indicaram que nos animais controle, após 15 dias de tratamento com AG1296, houve uma redução nas três frações de tecido adiposo, associada a uma redução em algumas das proteínas adipogênicas, além de uma melhora na sinalização insulínica em fígado e músculo e uma redução na glicemia de jejum. Além disso, nos animais submetidos à dieta hiperlipídica, após 7 dias de tratamento com AG1296, foi possível observar uma redução nas proteínas adipogênicas e uma redução na fração epididimal do tecido adiposo. Houve também uma melhora na sinalização insulínica e na tolerância à glicose. Com isso, podemos sugerir que a inibição do PDGFRß pode ter um papel importante na adipogênese e na sinalização insulínica e pode ser um alvo potencial para prevenção da obesidade e resistência à insulina
Abstract: Obesity can be defined as a disease in which body fat is excessively accumulated. Adipogenesis is a complex and not completely known phenomenon. Recent studies showed that adipocyte progenitor cells are exclusively found in adipose tissue and express some markers like PDGFRß (Platelet-derived growth factor ß). AG1296 (6,7-dimethoxy-2-phenyl-quinoxaline) is a potent and selective inhibitor of PDGF receptor kinase. In this context, the main objective of this work was to investigate if the inhibition of PDGF receptor through AG1296 would be able to affect white adipose tissue generation in high-fat-diet-fed and standard-chow-fed mice. We also investigated if this inhibition would have an effect on the insulin sensitivity in these studied groups. For this purpose, six-week-old male Swiss mice were divided into four groups and assigned to receive the following diet and/or treatment: the control group (C) received standard rodent diet, the second group (C + AG1296) received standard rodent diet plus AG1296 (50 mg/Kg/day by gavage), the third group (HFD) received high fat diet (55% calories from fat, 29% calories from carbohydrate and 16% from protein) and the fourth group (HFD+AG1296) received high fat diet plus AG1296. Body weight and food intake were measured during the treatment (7 and 15 days). After that, tissues (epididymal, retroperitoneal and mesenteric adipose tissue, liver and muscle) were extracted and processed. Through Western blot analysis, we were able to quantify the main proteins related to adipogenesis (SREBP-1c, C/EBP? e PPAR?) and the phosphorylation of the main proteins from insulin pathway (IR, IRS1 and Akt). Our results indicated that on control mice, after 15 days of treatment with AG1296, there was a reduction on adipose fat pad, associated with reduction in some adipogenic proteins, an increase in insulin signaling in liver and muscle and a reduction in fasting plasma glucose. Futhermore, on mice fed a high fat diet, after 7 days of treatment with AG1296, it was possible to observe a reduction on adipogenesis proteins and a reduction in epididymal fat pad. Also, there was an improvement in insulin signaling pathway and in glucose tolerance. In conclusion, our results suggest that PDGFRß inhibition might have an important role in adipogenesis and in insulin signaling and could be a potential target for preventing obesity and insulin resistance
Mestrado
Biologia Estrutural, Celular, Molecular e do Desenvolvimento
Mestre em Ciências

INTRODUÇÃO: Racional: A alta atividade plaquetária residual (AAPR) em uso do AAS é considerada um fator de mau prognóstico em portadores de síndrome isquêmica miocárdica instável (SIMI). Adicionalmente, as taxas de prevalência de AAPR verificadas em diferentes estudos realizados na fase aguda das SIMI são consideradas elevadas em relação às verificadas em portadores de doença arterial coronariana estável. Todavia, não está bem demostrado se essa elevada prevalência de AAPR diagnosticada na fase aguda das SIMI representa um fenômeno transitório, desaparecendo na fase tardia, ou se é um estado permanente, independente da fase aguda. MÉTODOS: O objetivo primário do presente estudo foi o de comparar, em pacientes com SIMI sem supradesnível do segmento ST, a resposta antiplaquetária ao AAS nas fases aguda e tardia na mesma população. Foram incluídos 70 pacientes com SIMI sem supradesnível de ST (77% com angina instável e 22% com IAM sem supra de ST), com idade média de 64,97 anos, sendo 54% do sexo feminino, todos em uso de AAS na dose de 100 a 200mg por pelo menos sete dias anteriores à inclusão. Os pacientes foram submetidos a cinco testes de agregação plaquetária na fase aguda, e os mesmos testes foram repetidos na fase tardia, três meses depois: VerifyNowAspirin®, agregometria de sangue total (AST) com ácido aracdônico (AA) e colágeno, tromboxane B2 sérico, PFA-100. RESULTADOS: De acordo com os testes COX-1 específicos (VFN e AST com AA), a AAPR em uso do AAS foi mais prevalente na fase aguda das SIMI do que na fase tardia (VFN: 32,1% versus 16%, p=0,049; e AST com AA: 31,4% versus 12,8%, p=0,015). Os testes não específicos (AST com colágeno, PFA) e o teste bioquímico não conseguiram demonstrar diferenças entre as fases. A correlação entre os cinco testes realizados foi considerada fraca ou moderada. CONCLUSÃO: A alta prevalência de AAPR, apesar do uso da AAS durante as SIMI, reflete mais provavelmente um estado de hiper-reatividade plaquetária transitória, que se reverte na fase crônica e estável da DAC, de acordo com os testes COX-1 específicos. A correlação entre os testes plaquetários foi apenas moderada nos dois cenários
BACKGROUND: The high residual platelet activity (HRPA) in response to acetylsalicilic acid (ASA) is considered a poor prognostic factor in patients with acute coronary syndromes (ACS). Additionally, the HRPA prevalence rates reported by different studies in ACS patients are considered high compared to those reported in patients with stable coronary artery disease. However, it is not well demonstrated whether this high HRPA prevalence diagnosed during the acute phase represents a transient phenomenon, disappearing in the late phase, or if it is a permanent state, independent of the acute phase. The aim of this study was to compare platelet aggregation in response to ASA during the ACS acute phase with the platelet aggregation in chronic stable phase. METHODS: Inclusion of patients with non ST ACS who were on aspirin at a dose of 100mg to 200mg per day for at least seven days prior to inclusion. We conducted five tests of platelet aggregation in the first 48 hours and repeated them three months later: VerifyNow Aspirin® (VFN), Whole Blood aggregometry (WBA) with arachidonic acid (AA) and collagen, thromboxane B2, PFA-100®. We analyzed 70 patients (77% with unstable angina and 22% with non ST AMI), mean age 64.97 years, 54% female. According to the COX-1 specific tests, the HRPA was more frequent in the acute phase than in the chronic phase (VerifyNowAspirin®: 31.4% versus 12.8%, p=0.015; and WBA with AA: 32.1% versus 16%, p=0.049; respectively). The non specific tests (AST with collagen and PFA) and the biochemical test sTXB2 failed to show differences between the phases. The correlation between the five tests was considered weak or moderate. CONCLUSION: The high prevalence of RPA despite the use of aspirin during the acute phase of the SCA most likely reflects a state of transient platelet hyperreactivity, which is reversed in the chronic phase. The correlation between platelet tests was only moderate in both scenarios

Orientador: Iguatemy Lourenço Brunetti
Banca: Luís Carlos Spolidorio
Banca: Marília Afonso Rabelo Buzalaf
Banca: Maria Teresa Pepato
Banca: Maria Lúcia Rubo de Rezende
Resumo: O tratamento cirúrgico da fissura congênita do processo alveolar superior compreende o enxerto ósseo, um procedimento bem aceito e de grande importância na restauração da forma e da função perdidas. Associado ao enxerto ósseo tem-se utilizado um produto atóxico, não imunoreativo e de fácil obtenção, denominado plasma rico em plaquetas (PRP). Neste estudo foi analisado o comportamento dos marcadores fosfatase alcalina, fosfatase alcalina isoforma óssea, osteocalcina e fosfatase ácida tartarato resistente em 50 pacientes, com idade entre 10 e 20 anos e que foram submetidos à cirurgia de enxerto ósseo autógeno alveolar pelo serviço de Cirurgia Buco-maxilofacial do Hospital de Reabilitação de Anomalias Craniofaciais da Universidade de São Paulo. O objetivo foi acompanhar de forma sistêmica e em curto período a formação ou reabsorção óssea após a realização do enxerto ósseo alveolar, bem como avaliar a eficácia do uso do plasma rico em plaquetas no processo de formação óssea. O estudo concluiu que as propriedades restauradoras do PRP não puderam ser demonstradas por nenhum dos marcadores bioquímicos do metabolismo ósseo nos primeiros 70 dias do ato cirúrgico; a análise temporal dos marcadores de formação óssea testados demonstrou uma tendência de queda com 35 dias e retorno próximo aos níveis basais com 70 dias do ato cirúrgico nos dois grupos estudados; não houve uma correlação significativa dos marcadores com o número de plaquetas e nem com a área da fissura e o resultado do exame ao raio X foi considerado inconclusivo para a presença ou não de trabeculado ósseo organizado em fase inicial de formação.
Abstract: The surgical treatment of the congenital cleft of the upper alveolar process understands the bone graft, a well accepted procedure of great importance in the restoration of the lost form and function. Together with the bone graft it is being used a non-toxic, non imunoreactive and easily obtained product, denominated platelet-rich plasma (PRP). In this study it was analysed the behavior of the alkaline phosphatase, bone alkaline phosphatase, osteocalcin and tartrate-resistant acid phosphatase markers in 50 patients, with age between 10 and 20 years and that were undergone to alveolar autogenous bone graft performed by the Bucomaxillofacial Service of the "Hospital for Rehabilitation of Craniofacial Anomalies, University of São Paulo". The aim was follow in a sistemic and early way the bone formation or reabsorption after the accomplishment of the alveolar bone graft, as well as to evaluate the effectiveness of the use of the platelet-rich plasma in the process of bone formation. The study concluded that the restorative properties of the PRP could not be demonstrated by of the biochemistry markers of the bone metabolism in the first 70 days of the surgery; the temporal analisys of the bone formation markers tested demonstrated a fall tendency in 35 days with return near to basal levels in 70 days in the two studied groups; there was not a significant correlation between markers and the number of platelets and neither with the area of the cleft and the result of the x-ray examination was not considered conclusive for the presence or not of organized bone trabeculae in the initial phase of formation.
Doutor

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Universidade Estadual Paulista (UNESP)
O tratamento cirúrgico da fissura congênita do processo alveolar superior compreende o enxerto ósseo, um procedimento bem aceito e de grande importância na restauração da forma e da função perdidas. Associado ao enxerto ósseo tem-se utilizado um produto atóxico, não imunoreativo e de fácil obtenção, denominado plasma rico em plaquetas (PRP). Neste estudo foi analisado o comportamento dos marcadores fosfatase alcalina, fosfatase alcalina isoforma óssea, osteocalcina e fosfatase ácida tartarato resistente em 50 pacientes, com idade entre 10 e 20 anos e que foram submetidos à cirurgia de enxerto ósseo autógeno alveolar pelo serviço de Cirurgia Buco-maxilofacial do Hospital de Reabilitação de Anomalias Craniofaciais da Universidade de São Paulo. O objetivo foi acompanhar de forma sistêmica e em curto período a formação ou reabsorção óssea após a realização do enxerto ósseo alveolar, bem como avaliar a eficácia do uso do plasma rico em plaquetas no processo de formação óssea. O estudo concluiu que as propriedades restauradoras do PRP não puderam ser demonstradas por nenhum dos marcadores bioquímicos do metabolismo ósseo nos primeiros 70 dias do ato cirúrgico; a análise temporal dos marcadores de formação óssea testados demonstrou uma tendência de queda com 35 dias e retorno próximo aos níveis basais com 70 dias do ato cirúrgico nos dois grupos estudados; não houve uma correlação significativa dos marcadores com o número de plaquetas e nem com a área da fissura e o resultado do exame ao raio X foi considerado inconclusivo para a presença ou não de trabeculado ósseo organizado em fase inicial de formação.
The surgical treatment of the congenital cleft of the upper alveolar process understands the bone graft, a well accepted procedure of great importance in the restoration of the lost form and function. Together with the bone graft it is being used a non-toxic, non imunoreactive and easily obtained product, denominated platelet-rich plasma (PRP). In this study it was analysed the behavior of the alkaline phosphatase, bone alkaline phosphatase, osteocalcin and tartrate-resistant acid phosphatase markers in 50 patients, with age between 10 and 20 years and that were undergone to alveolar autogenous bone graft performed by the Bucomaxillofacial Service of the Hospital for Rehabilitation of Craniofacial Anomalies, University of São Paulo. The aim was follow in a sistemic and early way the bone formation or reabsorption after the accomplishment of the alveolar bone graft, as well as to evaluate the effectiveness of the use of the platelet-rich plasma in the process of bone formation. The study concluded that the restorative properties of the PRP could not be demonstrated by of the biochemistry markers of the bone metabolism in the first 70 days of the surgery; the temporal analisys of the bone formation markers tested demonstrated a fall tendency in 35 days with return near to basal levels in 70 days in the two studied groups; there was not a significant correlation between markers and the number of platelets and neither with the area of the cleft and the result of the x-ray examination was not considered conclusive for the presence or not of organized bone trabeculae in the initial phase of formation.

Objetivos: Este estudo teve por objectivo fazer uma comparação directa da resistência à tração entre membranas produzidas com o protocolo L-PRF (Leucocyte-Platelet Rich Fibrin) e A-PRF (Advanced-Platelet Rich Fibrin). Materiais e Métodos: Após a colheita de sangue de uma pessoa saudável e sem histórico de toma de anticoagulantes, procedeu-se à produção de membranas segundo os protocolos de L-PRF e A-PRF, previamente descritos na literatura. De seguida, as membranas (13 para cada protocolo) foram submetidas a um teste de tração, para os quais foram obtidos valores de tração máxima e de tração média. A análise estatística dos dados foi feita com recurso a teste t não pareado. Resultados: Relativamente à tração média o protocolo A-PRF obteve 0.0288 N.mm-2 e o L-PRF 0.0192 N.mm-2 (p<0.05 utilizando teste-t não pareado; n=13). Para a tração máxima registou-se para o protocolo A-PRF o valor de 0.0752 N.mm-2 e L-PRF 0.0425 N.mm-2 (p<0.05 utilizando teste t não pareado; n=13). Conclusão: Com este estudo foi possível concluir que o protocolo A-PRF permite obter membranas com valores de tração máxima e tração média mais elevados do que os obtidos para o protocolo L-PRF, apontando assim para uma maior resistência quando duas forças opostas são aplicadas sobre a membrana. Este facto, associado à otimização das suas propriedades celulares e biológicas, fazem do protocolo A-PRF uma escolha melhor em detrimento do L-PRF.
Purpose: This study aimed at comparing the resistance traction between membranes produced with the protocol L-PRF (Leucocyte-Platelet Rich Fibrin) versus the protocol A-PRF (Advanced-Platelet Rich Fibrin). Materials and Methods: After blood collection of a healthy individual with no history of anticoagulant usage, we produced fibrin membranes according to the protocols L-PRF and A-PRF, previously described in the literature. Afterwards the membranes (13 for each condition) were submitted to a traction test, assessing the maximal traction and the average traction obtained for each membrane. The data was analyzed using unpaired t-test. Results: Regarding average traction, the A-PRF protocol obtained a value of 0.0288 N.mm-2 and L-PRF 0.0192 N.mm-2 (p<0.05 using unpaired t-test; n=13). For maximal traction A-PRF obtained 0.0752 N.mm-2 and L-PRF 0.0425 N.mm-2 (p<0.05 using unpaired t-test; n=13). Conclusion: With this study, we conclude that the A-PRF protocol generates membranes with higher maximal traction average traction scores, which indicates an increased resistance when two opposing forces are applied to it. This fact, associated with the optimization of the cellular and biological properties, make A-PRF a better protocol for the preparation of fibrin membranes.

Objetivos: O presente estudo teve como objetivo comparar as propriedades mecânicas de resistência à tração e estrutura entre as membranas produzidas por A-PRF (Advanced- Platelet Rich Fibrin) e A-PRF+ (Advanced-Platelet Rich Fibrin+). Materiais e métodos: Recorrendo à colheita de sangue de um dador saudável sem história de uso de anticoagulantes ou imunossupressores, realizou-se a preparação das membranas seguindo as indicações protocolares definidas na literatura para A-PRF e A-PRF+. De um N=16/grupo, 13 membranas de A-PRF e 12 de A-PRF+ foram submetidas ao teste de tração, para a obtenção de valores referentes à tração máxima e tração média. Os dados obtidos foram analisados estatisticamente com o teste t não pareado. Após avaliação desta variável, as membranas foram observadas em MEV (Microscopia Eletrónica de Varredura). Resultados: Foram obtidos em relação à tração máxima, 0.0020 N.mm-2 para o A-PRF e 0.0022 N.mm-2 para A-PRF+. Relativamente à tração média, A-PRF obteve 0.0012 N.mm-2 , enquanto o A-PRF+ obteve 0.0015 N.mm-2 (p˂0,01). Nas observações de superfície com MEV, A-PRF+ mostrou ser o concentrado plaquetário mais poroso, com maior abundância de fibras e preservação celular. Conclusão: Este estudo permitiu concluir que o protocolo A-PRF+ foi capaz de produzir membranas com valores de tração máxima superiores aos obtidos pelo A-PRF, sendo os resultados indicativo de que o protocolo apresentou membranas com maior resistência e capacidade elástica ao serem tracionadas por duas forças opostas. A este fenómeno acrescenta-se a arquitetura demonstrada na matriz de A-PRF+ e as propriedades biológicas otimizadas descritas na literatura.
Purpose: This study aimed to formulate a comparison of the mechanical properties of tensile strength and structural organization between membranes produced by A-PRF (Advanced Platelet-Rich Fibrin) and A-PRF+ (Advanced Platelet-Rich Fibrin+). Materials and Methods: Blood was collected from a healthy donor with no history of anticoagulant or immunosuppressant use, the membranes were prepared following the protocol indications defined in the literature for A-PRF and A-PRF+. From an N=16 for each protocol, 13 membranes of A-PRF and 12 of APRF+ were submitted to the traction test, evaluating maximum and average traction. Data was statistically analyzed using the unpaired t test. Membranes were then carefully observed in SEM (Scanning Electron Microscopy). Results: For maximum traction were obtained 0.0020 for A-PRF and 0.0022 for A-PRF+. Regarding the average traction, A-PRF scored 0.0012 while A-PRF+ obtained 0.0015 (p=0.01 unpaired t-test). Surface morphology observations with SEM, A-PRF+ showed to be the most porous platelet concentrate, with greatest fiber abundance and cell preservation. Conclusion: This study allowed to conclude that A-PRF+ protocol was able to produce membranes with higher maximum traction results than those found for A-PRF, indicating that the protocol with low centrifugation time, presented membranes with better viscoelastic strength when they are stretched by two opposed forces. To this phenomenon is added the architecture demonstrated in the A-PRF+ matrix and the optimized biological properties described in literature. A-PRF+, by the view of the developed findings in this work, a better option compared to A-PRF. Keywords: “Platelet-Rich Fibrin”, “Viscoelastic”, “Tensile Strength”, “Rupture”, “Porosity”, “Low-Speed Centrifugation Concept”.

The identification of Nitric oxide (NO) as an endothelium-derived relaxing factor stimulated research into the physiology of this most important biological messenger, which maintains a healthy vascular endothelium and an anti-thrombotic intravascular environment. Healthy endothelial cells constantly produce NO to create ‘basal’ vasorelaxation via the classical L-arginine/sGC/cGMP activation cascade. Under physiological conditions this NO pathway is the fundamental to maintenance of normal cardiovascular health, and conversely it is the substrate for development of many cardiovascular disease states, when the balance in this system becomes impaired. Endothelial dysfunction, with the closely associated phenomenon of “NO resistance”, can affect any NO-sensitive tissues including blood vessels and platelets, and is now believed to trigger atherogenesis and thrombogenesis. Treatment of cardiovascular diseases associated with this phenomenon utilizing NO donors often has proved to be ineffective. Furthermore, treatment with organic nitrates is subject to development of nitrate tolerance, limiting efficacy of this class of agents. Several agents can ameliorate NO resistance over days or weeks, but there remains a problem in circumventing NO resistance in cardiac emergencies. In this thesis we demonstrate for the first time in humans partial circumvention of NO resistance with nitroxyl, a structural analogue of NO. Additionally, another NO sibling nitrite (NO₂⁻) has been attracting substantial interest in the last decade. Evidence has been accumulating that effects of nitrite are increased during hypoxia: - nitrite becomes a potent vasodilator and anti-aggregant when compared to normoxic environment. This is especially important in the situation of chronic tissue hypoxia or in acute vascular emergencies. Key findings from the experiments in this thesis are: 1. Nitrite is a potent vasodilator compared to GTN: in general nitrite vasodilator effects are significantly potentiated in hypoxia in human saphenous veins. However, in human internal mammary arteries, nitrite-induced vasodilation is not potentiated under hypoxia. Prolonged exposure of human saphenous vein to nitrite does not cause tolerance or cross-tolerance to GTN. Nitrite effects in saphenous veins are substantially inhibited by ODQ, suggesting that they are largely mediated by soluble guanylate cyclase. Haemoglobin, myoglobin and red blood cells significantly increase hypoxic potentiation of nitrite vasodilator effects in human saphenous veins. Hypoxic potentiation of nitrite is diminished when saphenous vein intrinsic myoglobin is blocked by ferricyanide. 2. In platelets, the anti-aggregatory effects of nitrite are markedly and selectively potentiated under hypoxia. However, nitrite is subject to “NO resistance”. Antiaggregatory actions of nitrite are more potent in venous relative to arterial blood and correlate with (greater) deoxyhaemoglobin levels. Deoxyhaemoglobin is the primary nitrite reductase in blood. We have also presented evidence that continuous generation of NO from endogenous nitrite is important in homeostasis of platelet aggregability. 3. Nitroxyl is a more potent anti-aggregant than SNP. Anti-aggregatory effects of nitroxyl are partially sGC mediated. Nitroxyl partially circumvents the phenomenon of “NO resistance” in platelets. Nitroxyl is also a potent dilator of human saphenous veins. Its effects are not NO-mediated but partially sGCmediated.
Thesis (Ph.D.) -- University of Adelaide, School of Medicine, 2015

碩士
國立臺灣大學
生理學研究所
101
Introduction: Aspirin is integral in the primary and secondary prevention of coronary artery disease and acute coronary syndrome. However, the effect of aspirin on antiplatelet is not very well in diabetes called aspirin resistance (AR). In this study, we investigate whether AR in diabetes is associated with platelet surface receptors, monocyte surface receptors and microparticle. Materials and methods: A total of 70 patients with cardiovascular disease were enrolled. Twenty-four patients had diabetes and six of them had AR. Forty-six patients were diabetes and eight of them were AR. Blood samples were obtained twice for this study, first at baseline (pre-ASA), and second was after at least two weeks following the post-ASA. All subjects received one 100 mg aspirin. (Bokey EM /cap 100 mg or Tapal/tab 100 mg ). We use the PFA-100 to evaluate platelet aggregation and flow cytometry to evaluate CD62p (P-selectin), PAC-1, CD31, CD42a, CD14 and CD62E. Results: The PAC-1 expression was enhanced in diabetes with AR in post-aspirin than in pre-aspirin (0.12±0.05, 0.03±0.04, p<0.05). The CD62E counts reduced in post-aspirin compared to pre-aspirin (171.30±79.00, 237.73±123.82, p<0.05). Conclusion: AR in diabetes is associated with high PAC-1 expression. Although the effect of aspirin on antiplatelet is not very well in diabetes, it still is a vasoprotective agent for CVD.

碩士
中原大學
化學工程研究所
96
The effective control of a material surface that affect biological response is of fundamental importance for the development of biomaterials, especially when living systems encounter synthetic surfaces. In this work, we demonstrate biofouling-resistance polymeric membrane grafted with surface-immobilized poly(ethylene glycol) methacrylate for human plasma protein and blood platelet repulsions. In the first part of this disseration, the work describes the surface modification and characterization of poly(vinylidene fluoride) (PVDF) microfiltration (MF) membranes grafted with poly(ethylene glycol) methacrylate (PEGMA) via surface-activated ozone treatment and thermally induced graft copolymerization. The chemical composition and microstructure of the surface-modified PVDF membranes were characterized by Fourier transform infrared spectroscopy (FT-IR), contact angle, and atomic force microscopy (AFM) measurements. Blood compatibility of the modified membranes was evaluated by the biofouling property of the platelet adhesion observed by scanning electron microscopy (SEM) and the plasma protein adsorption determined by an enzyme-linked immunosorbent assay (ELISA). In general, the grafting density of the copolymerized PEGMA and the hydrophilicity on the surface of PVDF MF membranes increase with increasing macromonomer concentration of PEGMA in the reaction solution. The grafting distribution of PEGMA on the resulting membranes was found to form a uniform polymer hydrogel-like layer controlled by sufficient high content of PEGMA in the reaction solution, while their surface roughness was kept lower than that of the virgin membrane. For the platelet adhesion test, a remarkable suppression of the platelets adhered to the PVDF MF membranes grafted with PEGMA polymer was observed. In the water flux experiments, the PEGMA-grafted hydrophilic PVDF MF membranes exhibited good anti-fouling properties to substantially reduce the irreversible membrane fouling caused by platelet adhering and plasma protein adsorption as compared with the virgin hydrophobic PVDF MF membranes. In the second part of this disseration, we prepare the polymeric materials with high blood compatibility, which can provide the ability to prevent the thrombin activation of catheters in the future. This work describes the surface modification and characterization of segmented polyurethane (SPU) films grafted with poly(ethylene glycol) methacrylate (PEGMA) respectively via surface-activated ozone treatment and thermally induced graft copolymerization. The chemical composition and microstructure of the surface-modified SPU films were characterized by Fourier transform infrared spectroscopy (FT-IR), contact angle, and atomic force microscopy (AFM) measurements. Blood compatibility of the modified membranes was evaluated by the biofouling property of the platelet adhesion observed by scanning electron microscopy (SEM) and the plasma protein adsorption determined by an enzyme-linked immunosorbent assay (ELISA). This study not only determines the grafting quality with PEGMA, but also provides a fundamental understanding of various grafting density governing the effects on hydrophilicity, surface morphology and plasma proteins adoption of film surfaces.

碩士
中原大學
化學工程研究所
104
The target of this study focuses on the molecular structure effects of hydroxyl and amide groups on the resistance of protein adsorption and blood-cells adhesion. Three types of hydrogel monomer structures were prepared with different hydroxyl and amide functional groups, the first type of monomer structure contains only one hydrophilic hydroxyl group: (1) 2-Hydroxyethyl methacrylate (HEMA) and (2) 2-Hydroxyethyl acrylate (HEA); the second type of monomer structure contains only one hydrophilic amide group: (3) Acrylamide (AAm) and (4) Methacrylamide (MAA); the third type of monomer structure contains both hydroxyl and amide group: (5) N-(2-Hydroxypropyl) methacrylamide (HPMA) and (6) N-(2-Hyroxyethyl)acrylamide (HEAA). After the preparation of three types of monomers into hydrogel systems, the resistance of protein adsorption, platelet adhesion, and leukocyte attachment was evaluated to illustrate the effects of molecular structures combined from different hydrophilic functional groups. In this study, differential scanning calorimetry (DSC) was used to determine the differences of bound water structures in micro scale from three types of hydrogel systems. It was found that HPMA and HEAA hydrogels have best hydration capability and higher amounts of non-freezable bound water. From the analysis of protein adsorption on the prepared hydrogels by enzyme-linked immunosorbent assay (ELISA), the results showed that HPMA and HEAA hydrogels perform excellent resistance of plasma protein adsorption. From the observation of human blood cells on HPMA hydrogels, the results indicated that extremely low amounts of fibrinogen adsorption resulting the resistance of platelet activation and leukocyte attachment.

MMed, Haematology, Faculty of Health Sciences, University of the Witwatersrand, 2009
Objective: Stroke is the second most common cause of death in most countries.1 In South Africa, which has a population undergoing demographic and epidemiological transition, stroke is the third most common cause of death.2 Platelet response to therapeutic doses is not uniform, although aspirin remains an essential part of treatment. Some patients exhibit aspirin resistance and develop secondary thrombotic events. It was decided to determine the prevalence of aspirin resistance and/or platelet hypersensitivity, as determined by platelet aggregometry, in sixty Caucasian patients who have suffered one or more strokes and/or Transient Ischaemic Attacks (TIAs) as compared with sixty control subjects. Methods: Aspirin resistance was determined by platelet aggregation (>20%) to one or more of the four agonists, namely arachidonic acid (1.5mM), adrenaline (0.05mg/ml), collagen (0.2mg/ml) or ADP (0.1x10-5 M). Results: Two patients demonstrated ‘complete aspirin resistance’ (non-responder to aspirin) with resistance to arachidonic acid (high concentration) noted. Three patients demonstrated ‘partial aspirin resistance’ (semi-responder to aspirin). One control subject showed ‘complete aspirin resistance’. There is a 1.67% chance of a control subject being resistant to aspirin in a general South African Caucasian population. A history of prior stroke or transient ischaemic attack was associated with a statistically significant increase in risk of aspirin resistance with an odds ratio of 5.36. Conclusion: These results essentially concur with those of the studied literature in showing an 8.3% prevalence (statistically significant) of aspirin resistance (complete and partial) in South vi African Caucasian patients with previous atherothrombotic cerebrovascular events i.e.CVAs and/or TIAs. The current study shows an increased prevalence of aspirin resistance in people who have had prior strokes / TIAs and raises the question whether people who have had these events are somehow predisposed to vascular events or indeed recurrent vascular events. ‘Aspirin resistant’ patients or ‘poor responders’ to aspirin must be considered at heightened risk of atherothrombotic events and laboratory monitoring of antiplatelet therapy may become clinically useful.

Introduction : L’effet biologique variable de l’aspirine a été attribué à un état de résistance pharmacologique. L’incidence de cette « résistance » varie selon la population ou la technologie étudiée. Méthodes : Nous avons déterminé la performance de 5 techniques évaluant l’effet de l’aspirine chez des sujets sains, non fumeurs et ne prenant aucune médication pouvant interférer avec la fonction plaquettaire. Des spécimens de sang et d’urine ont été obtenus avant et après 8-10 jours de prise de 80 mg d’aspirine. Résultats: Chez 45 sujets de 19-59 ans, la sensibilité (SE), la spécificité (SP), et la valeur optimale de coupure (CO) pour détecter l’effet de l’aspirine sont : agrégométrie par transmission optique induite avec 1,6 mM d’acide arachidonique (ATO-AA) - SE 100%, SP 95,9%, CO 20%; ATO-ADP 10 μM - SE 84,4%, SP 77,7%, CO 70%; VerifyNow® Aspirin - SE 100%, SP 95,6%, CO 550 ARU; agrégation en tube - SE 82,2%, SP 86,7%, CO 55%; TEG® - SE 82,9%, SP 75,8%, CO 90%; et le dosage de 11-dehydrothromboxane B2 urinaire - SE 62,2%, SP 82,2%, CO 60 pg/ml. Conclusions: La résistance à l’aspirine chez les sujets sains définie par ATO-AA et VerifyNow® Aspirin est rare. Puisque les autres techniques étudiées discriminent de façon sous optimale l’effet de l’aspirine, leur utilité dans la définition de la résistance pharmacologique à l’aspirine semble marginale. Ces résultats suggèrent qu’une proportion de la variabilité de l’incidence rapportée de “résistance à l’aspirine” est artefactuelle et reliée aux limitations technologiques de certaines analyses.
Background: Variable biological effect of aspirin is suggested to be related to pharmacological resistance. The incidence of this so-called “resistant” state varies with the study population and the assay used. Methods: We determined performance features of five assays used to assess aspirin effects in non smoking healthy volunteers not taking any drug known to interfere with platelet function. Blood and urine samples were obtained immediately before and after 8-10 days of aspirin 80 mg intake. Results: Forty-five participants 19-59 years old were enrolled. The sensitivity (SE), specificity (SP), and optimal cut-off (CO) value to detect the effect of aspirin were: light transmission aggregometry (LTA) with 1.6 mM arachidonic acid - SE 100%, SP 95.9%, CO 20%; LTA with ADP 10 μM - SE 84.4%, SP 77.7%, CO 70%; VerifyNow® Aspirin - SE 100%, SP 95.6%, CO 550 ARU; platelet count drop - SE 82.2%, SP 86.7%, CO 55%; TEG® - SE 82.9%, SP 75.8%, CO 90%; and urinary 11-dehydrothromboxane B2 levels - SE 62.2%, SP 82.2%, CO 60 pg/ml. Conclusions: Aspirin resistance in normal individuals as defined by arachidonic acid-induced LTA and the VerifyNow® assay is rare. Because the other assays discriminate suboptimally aspirin effect, they should not be used to define pharmacological “aspirin resistance”. These results suggest that a proportion of the variability in the reported incidence of aspirin resistance is artefactual and related to technical limitations of some assays.

We sought to identify possible clinical and laboratory predictors of bleeding and ischaemic risk in a real-world population with concomitant atrial fibrillation and PCI, discharged from our cardiology ward with double antithrombotic therapy (DAT) or triple antithrombotic therapy (TAT). Nell'ambito della tesi sono stati ricercati predittori clinici o laboratoristi di eventi ischemici o emorragici in una popolazione di pazienti con storia di fibrillazione atriale, sottoposti ad angioplastica, e dimessi in duplice o triplice terapia antitrombotica.