Anticoagulants are used as the first line therapy for management and prevention of thrombotic disorders. Thrombin and factor Xa have been the prime targets for regulation of the coagulation cascade. In this work, a small library of 17 benzofuran derivatives were synthesized and screened against thrombin and factor Xa. The derivatives that displayed inhibitory potential were docked on the exosite-II of factor Xa using a docking protocol that was developed in our research group. These compounds were based on the β-5 structural unit found in the oligomer -'CDSO3‘, which was prepared in our lab and was found to inhibit both thrombin and factor Xa by an exosite-II mediated allosteric disruption of the catalytic triad.The results revealed that these β-5 like derivatives are inhibitory against thrombin and factor Xa, although their potency is weak. Thrombin and factor Xa appear to recognize different structural features suggesting a significant selectivity in recognition. Furthermore, a slight preference for the benzofuran scaffold was observed with factor Xa. Probing the mechanism of inhibition using Michaelis-Menten kinetics reveal that these compounds display uncompetitive inhibition of these proteases and the mechanism of inhibition is allosteric. Docking of these compounds on factor Xa were done using GOLD (Genetic algorithm for ligand docking) and the results, explain the observed inhibition profile. The computed docked poses also give an idea of the residues on the exosite-II of factor Xa critical for inhibition. The molecules studied here are radically different in terms of structure and mechanism of inhibition from any other ligand described in literature. This represents an opportunity to discover novel molecules with a possibly different pharmacological and toxicological profile.

Glycosaminoglycans (GAGs) are complex biopolymers that play important roles in inflammation, coagulation, angiogenesis, cell adhesion and viral invasion by interacting with several different proteins.1,2 Structurally, GAGs are built up of several different sulfated disaccharide units.3 Specific GAG sequences that uniquely recognize their cognate proteins exist. Such specificity typically arises from the binding of unique sulfation patterns on the linear GAG chain to highly electropositive protein domains. Thus, these highly charged, sulfated biopolymers potentially represent a new class of therapeutics. Yet, the major stumbling block to the development to these agents is their extremely complicated and tedious chemical synthesis. We hypothesized that replacing the saccharide skeleton with an equivalent non-saccharide and readily synthesized organic skeleton would usher in an era of new, GAG-based therapeutics. This challenge has been addressed on two fronts, computational design and chemical synthesis, by focusing on the heparin pentasaccharide-antithrombin system that represents an exhaustively studied model GAG-protein system. With respect to chemical synthesis, a microwave-based synthetic procedure that can rapidly introduce multiple sulfate groups on a poly-hydroxyl substrate within minutes was developed.4 Using this method, the synthesis of a previously designed activator (IAS5), which otherwise proved to be problematic, was successfully completed. Biochemical screening of IAS5 and its analogs revealed that these molecules could activate antithrombin up to 30-fold in comparison to the 300-fold activation by the heparin pentasaccharide. In an effort to develop more potent antithrombin activators, a new method to predict high affinity GAG sequences for a given GAG-binding protein based on combinatorial virtual-library screening was developed.5 This combinatorial virtual-library screening method was applied to a library of 24,576 non-saccharide, sulfated molecules that were created using the structure of IAS5 as a template. Thirty seven‘hits’ that had common structural features were identified from this study. Interestingly, all these ‘hits’ bind to antithrombin similarly and orient the 4 negative charges identical to the corresponding groups in the heparin pentasaccharide. The synthesis of selected targets is currently in progress and several synthetic steps have already been optimized.

Le traitement AVK concerne plus d’1% de la population française. Il représente un problème de santé publique majeur en raison de son importante iatrogénie. Les causes peuvent être notamment reliées à la complexité du parcours de soins des patients, à leurs erreurs, méconnaissances et incompréhensions. L’éducation thérapeutique (ETP) permettrait de contribuer à rendre ce patient autogestionnaire de ses propres risques. Cette recherche qualitative vise à proposer un modèle d’ETP en en identifiant les compétences d’auto-soins et d’adaptation à la maladie et en en précisant le programme, les méthodes pédagogiques et d’évaluation. Le protocole de recherche comporte une revue de la littérature sur les connaissances des patients, des entretiens de type semi-directifs de patients traités par AVK et de soignants-éducateurs, et enfin des entretiens avec un groupe d’experts. Trente-six entretiens associés à une revue extensive de la littérature ont permis d’élaborer un référentiel de huit compétences. Vingt-et-un objectifs pédagogiques découlent de ces compétences. Les principales difficultés des patients concernaient la mise en lien des concepts constitutifs du paradigme du traitement par AVK. Les huit compétences du référentiel correspondent à la gestion intelligible et sans danger d’un traitement anticoagulant. Les différents types de soignants-éducateurs envisagés pour ce modèle d’ETP se retrouvent dans le parcours de soins habituel des patients. Notre modèle pédagogique se veut applicable à différents contextes de soins. Les propositions tentent de répondre à la problématique de l’intelligibilité du traitement AVK et de rendre accessible aux patients l’éducation thérapeutique. / The VKA treatment concerns more than 1% of the french population. It represents a major public health problem beacause of its consirable drug related iatrogny....

Os anticoagulantes orais são fármacos que agem aumentando o tempo de coagulação sanguínea, úteis na ocorrência de certas doenças que levam a formação de trombos intravasculares. Portanto, controles constantes dos níveis sanguíneos são necessários para um tratamento seguro. Algumas estratégias, como a intervenção educativa, vem demonstrando bons resultados. No presente estudo, o objetivo principal foi avaliar a qualidade de vida relacionada à saúde, adesão ao tratamento e autoeficácia de pacientes que iniciaram o uso de anticoagulantes orais segundo a participação em um programa educativo (Grupo Intervenção) ou o recebimento do cuidado de rotina (Grupo Controle). Como objetivos secundários, comparamos o estado de saúde percebido e a presença de sintomas de ansiedade e depressão entre os grupos. Trata-se de um estudo experimental com designação aleatória em dois grupos (Intervenção ou Controle). Há aprovação do Comitê de Ética em Pesquisa e registro na base ClinicalTrials.gov. Realizado no Hospital Estadual de Ribeirão Preto, foram incluídos pacientes que iniciaram o uso de anticoagulantes orais para tratamento clínico pela primeira vez, maiores de 18 anos e com avaliação cognitiva adequada. A estratificação e a aleatorização dos sujeitos foram obtidas por The Outpatient Bleeding Risk Index e por blocos, respectivamente. Envelopes coloridos foram usados na designação dos grupos. O programa educativo foi norteado pela Teoria de Bandura e composto por informações verbais e por escrito do tratamento dados durante a internação e contatos telefônicos para o reforço dessas informações na primeira e quarta semanas após a alta hospitalar do participante. Na coleta dos dados foram aplicados os instrumentos: caracterização sociodemográfica e clínica, Duke Anticoagulation Satisfaction Scale, Medidas de Adesão ao Tratamento, Hospital Anxiety and Depression Scale, Escala Visual Analógica. As análises foram feitas pelo IBM Statistical Package for Social Science (SPSS) version 21.0. Foram realizadas análises descritivas de frequência simples, de posição e de dispersão. Para a comparação das variáveis entre os grupos foi usado o teste T de Student não pareado. Para a comparação das frequências dos sintomas de ansiedade e depressão foi usada o teste Qui-quadrado ou o T de Student. Já a comparação das variáveis no início e no final do estudo em cada grupo foi obtida pelo teste T de Student pareado ou seu similar não paramétrico (teste de Wilcoxon). O Qui-quadrado foi aplicado para a comparação da ansiedade e depressão, antes e depois, em cada grupo. O nível de significância adotado foi de 5%. A comparação dos grupos mostrou características semelhantes, maioria de mulheres, casadas, idade próxima a 60 anos, Fibrilação Atrial ou Trombose Venosa Profunda como indicação para o início do anticoagulante oral. O Grupo Intervenção apresentou melhor qualidade de vida relacionada à saúde, maior adesão, maior autoeficácia diante do novo tratamento, o que possivelmente refletiu em menor ansiedade e depressão em relação ao Grupo Controle. Concluímos que a participação em um programa educacional proporcionou melhores desfechos em relação àqueles que receberam o cuidado de rotina. / Oral anticoagulants are drugs that act by increasing blood clotting time and are useful in the presence of certain diseases that lead to the formation of intravascular thrombi. Thus, constant controls of blood levels are necessary for a safe treatment. Some strategies such as educational intervention are showing good results. The main objective of the present study was to assess the quality of life related to health, adhesion to treatment and self-efficacy of patients who had started the use of oral anticoagulants according to the participation in an educational program (Intervention Group) or receiving routine care (Control Group). Secondary objectives were to compare the health status perceived and the presence of anxiety and depression symptoms between groups. This was an experimental study of random design conducted on two groups (Intervention and Control). The Research Ethics Committee approved the study, which was registered in the ClinicalTrials.gov database, and all subjects gave written informed consnet to participate. The study was conducted at the State Hospital of Ribeirão Preto and included patients who had started the use of oral anticoagulants for clinical treatment for the first time, who were older than 18 years and who had adequate cognitive function. The subjects were stratified and randomized using the Outpatient Bleeding Risk Index and by block, respectively. Colored envelopes were used for group allotment. The educational program was based on Bandura\'s theory and consisted of verbal and written information about the treatments applied during hospitalization and of telephone contacts for the reinfordcement of this information during the first and fourth weeks after hospital discharge of the patient. The following instruments were applied during data collection: sociodemographic and clinical characterization, Duke Anticoagulation Satisfaction Scale, Measurements of Adhesion to Treatment, Hospital Anxiety and Depression Scale, and Visual Analogue Scale. The analyses were carried out using the IBM Statistical Package for the Social Sciences (SPSS) version 21.0. Descriptive analyses of simple frequency, position and dispersal were performed. The unpaired Student t-test was used to compare the variables between groups, and the Chi-square or Student t-test was used to compare the frequency of anxiety and depression symptoms, while the paired Student t-test or the non-parametric Wilcoxon test was used to compare the variables at the beginning and at the end of the study in each group. The Chi-square test was applied to compare anxiety and depression before and after the study in each group. The levels of significance was set at 5%. Group comparison showed similar characteristics, a majority of women who were married and aged close to 60 years, and atrial fibrillation or deep venous thrombosis as the indication of the use of an oral anticoagulant. The Intervention Group showed better quality of life related to health, greater adhesion, and more self-efficacy when receiving the new treatment, a fact that possibly reflected lower anxiety and depression compared to the Control Group. We conclude that the participation in an educational program provided better outcomes compared to patients who received routine care.

Objetivo: Avaliar o uso de antitrombóticos em pacientes com fibrilação atrial (FA) em hospital cardiológico no Brasil (InCor).Métodos e resultados: Um estudo observacional transversal analisou os prontuários de todos os pacientes atendidos no InCor em cada um de 5 dias separados no ano de 2002 (Fase 1), sendo prospectivamente reanalisados após 1 ano (Fase 2). A prevalência da FA nos 3764 prontuários analisados foi de 8%. Antiplaquetários foram prescritos para 21,26% e 19,93%, anticoagulantes para 46,51% e 57,81%, e 32,23% e 22,26% não usavam nenhum antitrombótico nas Fases 1 e 2, respectivamente. Somente 15,60% e 23,25% apresentavam níveis de RNI terapêuticos.Conclusão: A anticoagulação é subutilizada nos pacientes com FA apesar do fato de serem tratados por cardiologistas em um hospital universitário / Objective: To assess antithrombotic therapy among atrial fibrillation (AF) patients in a Brazilian University Heart Hospital (InCor).Methods and results: A cross sectional study analyzed the charts of all patients treated at InCor in 5 separate days of 2002 (Phase 1), and prospectively reviewed them after one year (Phase 2). The prevalence of AF in the 3,764 assessed charts was of 8.0%. Antiplatelets were prescribed to 21.26% and 19.93%, anticoagulants to 46.51% and 57.81%, and 32.23% and 22.26% were not receiving any antithrombotic in Phases 1 and 2, respectively. Only 15.60% and 23.25% were within INR therapeutic range.Conclusion: Anticoagulation is underused in AF patients besides the fact of being treated by cardiologists in a University Hospital

A varfarina é um anticoagulante oral, antivitamina K, amplamente utilizado na prevenção de trombos intravasculares de diferentes etiologias. Exige um controle rigoroso e complexo em decorrência da sua interação com outros fármacos e alimentos, o que pode interferir na Qualidade de Vida Relacionada à Saúde (QVRS) dos usuários. Intervenções educativas têm sido utilizadas para diminuir o impacto dessa terapia na QVRS. O objetivo principal deste estudo foi comparar o efeito na QVRS de duas intervenções educativas em pacientes que iniciaram o uso de varfarina pela primeira vez durante a internação, aos três e seis meses após a alta. Como objetivos secundários, comparamos a presença de sintomas de ansiedade e depressão e a adequação do valor do International Normalized Ratio (INR) na faixa terapêutica indicada, entre os grupos. Este foi um estudo experimental com designação aleatória nos dois grupos. Os pacientes do grupo intervenção (GI) receberam o programa educativo com seguimento por telefone após a alta e os do grupo controle (GC) receberam o programa educativo sem o acompanhamento telefônico. O estudo foi aprovado por Comitê de Ética em Pesquisa e foi registrado na base ClinicalTrials.gov. A pesquisa foi conduzida no Hospital Estadual de Ribeirão Preto e no Hospital Estadual de Américo Brasiliense. Foram incluídos pacientes que iniciaram o uso da varfarina, pela primeira vez, maiores de 18 anos e com telefone para contato. O programa educativo foi norteado pela teoria de Bandura e composto por informações verbais e escritas sobre o tratamento, as quais foram apresentadas durante a internação. Os participantes do GI receberam cinco contatos telefônicos para o reforço dessas informações, após a alta hospitalar. Ambos os grupos tiveram dois encontros presenciais, aos três e aos seis meses, para avaliação das variáveis desfechos: QVRS (avaliada pela versão brasileira da Duke Anticoagulation Satisfaction Scale - DASS) e sintomas de ansiedade e de depressão (avaliados pelas subescalas da Hospital Anxiety and Depression Scale - HADS). Para comparar o DASS e as escalas do HADS, realizamos teste t de Student para amostras independentes. Para compararmos os grupos ao longo do tempo, em relação à QVRS, foi realizada Análise da Variância (ANOVA) para medidas repetidas, tendo como fatores o tempo (três e seis meses), o grupo (intervenção ou controle) e uma interação de tempo por grupo. O nível de significância adotado foi de 0,05. Os grupos eram similares quanto às caracterizações sociodemográfica e clínica. A maioria dos participantes eram casados, do sexo feminino e com média de idade de 55 anos (D.P=15). As indicações mais frequentes para o início da varfarina foram trombose venosa profunda e tromboembolismo pulmonar. Não encontramos diferenças estatisticamente significantes entre as médias de QVRS, ansiedade e depressão dos dois grupos, aos três e aos seis meses após a alta / Warfarin is an oral anticoagulant, antivitamin K, widely used in the prevention of intravascular thrombi of different etiologies. It requires rigorous and complex control as a result of its interaction with other drugs and foods, which may interfere with users\' Quality of Life Related to Health (HRQoL). Educational interventions have been used to lessen the impact of this therapy. The main objective was to compare the HRQoL, at three and six months after discharge, of two groups of patients who started using warfarin for the first time during hospitalization. Secondary objectives, were to compare the presence of anxiety and depression symptoms and the adequacy of the International Normalized Ratio (INR) value in the indicated therapeutic range, between the groups. It is an experimental study with random designation in two groups. The patients in the intervention group (IG) received the educational program with telephone follow-up after discharge and those in the control group (CG) received the educational program without telephone follow-up. The study was approved by the Research Ethics Committee and was enrolled in the ClinicalTrials.gov database. The research was conducted at the State Hospital of Ribeirão Preto and the Hospital Estadual de Américo Brasiliense and included patients who started using warfarin for the first time, over 18 years and with a telephone to contact. The educational program was guided by the Bandura Theory and was composed of verbal and written information about the treatment and approached during hospitalization. GI participants received five telephone contacts to reinforce this information after discharge. Both groups had two face-to-face meetings at three and six months for the evaluation of the outcome variables: HRQOL (assessed by the Brazilian version of the Duke Anticoagulation Satisfaction Scale - DASS) and symptoms of anxiety and depression (assessed by the subscales of Hospital Anxiety and Depression Scale - HADS). To compare the DASS and the HADS scales, we performed Student\'s t-test for independent samples. In order to compare the groups over time, in relation to HRQoL, we performed Variance Analysis (ANOVA) for repeated measures, taking as factors the time (three and six months), the group (intervention or control) and a time interaction per group. The level of significance was set at 0.05. The groups were similar in sociodemographic and clinical characterization. The majority of the participants were married, female and with an average age of 55 years (D.P = 15). The most frequent indications for the initiation of warfarin were deep venous thrombosis and pulmonary thromboembolism. We did not find statistically significant differences between the means of HRQoL, anxiety and depression of the two groups at three and six months after discharge

A manutenção do RNI (Relação Normalizada Internacional), um exame específico feito em usuários de anticoagulantes orais, determinam os resultados individuais de quem se mantém na faixa terapêutica ou não. De acordo com as diretrizes Europeias (2015) e Americanas (2014), a faixa terapêutica recomendada para Fibrilação Atrial e demais eventos tromboembólicos é de 2 a 3, e para portadores de prótese valvar mecânicas, de 2,5 a 3,5. As complicações referentes ao exceder a faixa são classificadas em sangramentos, caso venha interferir e diminuir a faixa a possibilidade de ocorrer eventos trombóticos. As inferências relacionadas às complicações devidas à alteração do RNI estão associadas ao uso inadequado do medicamento, interações medicamentosas e alimentares, problemas de saúde, procedimentos invasivos e cirúrgicos, dentre outros fatores como estresse, atividade física, emagrecimento e aumento de peso. A intervenção baseada na educação em saúde por meio de um instrumento que estratifique e aponte com precisão os fatores de não permanência na faixa facilitará a adesão ao tratamento e, consequentemente, diminuirá os riscos de vida desta população. O presente objetivo principal foi avaliar e identificar os fatores que interferem na adesão de usuários de ACO (Anticoagulação Oral) e propor um escore de adesão aos usuários de Anticoagulante Oral para a manutenção na faixa terapêutica. Trata-se de um estudo analítico, observacional, transversal e abordagem quantitativa, realizado em um Centro de Anticoagulação Oral de um Hospital Público especializado em Cardiologia vinculado a Secretaria do Estado de São Paulo. Participaram deste estudo 607 usuários de ACO que atenderam aos critérios de inclusão: idade acima de 18 anos, alfabetizados e cadastrados no Sistema de ACO e que iniciaram o uso após duas semanas. Para o modelo de desenvolvimento, optou-se pelo desfecho o RNI alterado e as variáveis preditoras foram determinadas em manipulação do medicamento, interações medicamentosas e da dieta, atividade física, procedimentos e cirurgias, uso adequado do medicamento, problemas de saúde, outros fatores. A coleta de dados foi feita pelo pesquisador e foram realizadas, na parte I, questões relacionadas à caracterização e, na parte II, questões de perfil clínico. Antes, aplicou-se o TCLE após o consentimento e aprovação do usuário. Os dados do perfil sociodemográficos foram apresentados em absolutos e porcentagens, foram consideradas as variáveis que apresentaram nível de significância menor que 5% na análise exploratória ou que foram consideradas de relevância clínica, foram submetidas a um modelo de regressão logística múltipla. Participaram 607 usuários de ACO; o perfil sociodemográfico da população foi de 52% gênero feminino, 57% casados, faixa etária maior que 60 anos 62%, ensino fundamental incompleto 42%, provenientes de São Paulo 93%. As variáveis com nível de significância menor que 5% na análise multivariada: escolaridade, renda familiar, uso inadequado, interação medicamentosa, procedimentos invasivos, interações alimentar, atividade física, condições clínicas, outros fatores e complicações próprias do uso do ACO. Foram identificadas variáveis prognósticas: renda familiar, uso inadequado, procedimento invasivo, interação medicamentosa, hábitos alimentares, condições clínicas, outros fatores (estresse, emagrecimento, perda de peso). O C Statistic para o Escore de Adesão Simonetti & Mancussi foi de 0,94. Em usuários de Anticoagulante oral, o escore de adesão Simonetti & Mancussi mostrou-se de aplicabilidade fácil e exequível, com alto valor preditivo mediante os fatores intervenientes da adesão e permitiu o direcionamento para tomadas de decisões objetivas e direcionadas para o problema, facilitando a melhoria da adesão e manutenção na faixa ideal. / Maintaining the INR (International Normalized Ratio), a specific blood test performed in users of oral anticoagulants, determines the individual outcomes of those within or not the therapeutic range. According to the European (2015) and American (2014) guidelines, the therapeutic range recommended for atrial fibrillation and other thromboembolic events is 2 to 3. For those with mechanical valve prosthesis, the therapeutic range is 2.5 to 3.5. Bleeding can occur as a complication when the therapeutic range is exceeded whereas thrombotic events can occur when the therapeutic range is not reached. The inferences related to the complications due to changes in the INR are associated with inappropriate use of medication, drug and food interactions, health problems, invasive and surgical procedures, and other factors such as stress, physical activity, weight loss and weight gain. Interventions based on health education through instruments that stratify and accurately identify the factors keeping people out of the therapeutic INR range will facilitate adherence to treatment and consequently reduce the risks to life in this population. This main objective was to assess and identify the factors interfering in adherence to users of OAC (oral anticoagulants) and to propose an adherence score for users of OAC to maintain the therapeutic range. It is an observational, analytical, cross-sectional and quantitative study performed in a center of oral anticoagulation of a public hospital specialized in Cardiology associated with the Secretaria of the State of São Paulo. The study included 607 users of OAC that met the inclusion criteria: 18 years old or older, literate and registered in the OAC system and started using OAC two weeks later. For the development model the chosen outcome was altered INR and the predictor variables were medication handling, drug and food interactions, physical activity, surgeries and procedures, adequate use of medication, health issues, other factors. Data were collected by the researcher. Part I of the questionnaire contained questions related to characterization and Part II questioned the clinical profile. Before data collection, the users signed the informed consent form. Data on the sociodemographic profile were presented in absolute frequencies and percentages. Variables with a significance level less than 5% in the exploratory analysis or those considered clinically relevant were submitted to a multiple logistic regression model. Six hundred seven OAC users participated in the study. The sociodemographic profile of the population was: 52% female, 57% married, 62% over 60 years old, 42% with incomplete primary education, 93%from São Paulo. The variables with a significance level less than 5% in the multivariate analysis were: education, family income, inadequate use, drug interactions, invasive procedures, food interactions, physical activity, clinical conditions, other factors and OAC-associated complications themselves. The identified prognostic variables were: family income, inadequate use, invasive procedure, drug interactions, dietary habits, clinical conditions, other factors (stress, getting thinner, weight loss). The C statistic for the Adherence score Simonetti & Mancussi was 0.94. In users of OAC, the adherence score Simonetti & Mancussi proved to be of easy and practical applicability, with high predictive value given the factors intervening on adherence and allowed for objective and problem-focused decision making, thereby facilitating improvement of adherence and maintenance of the optimal range.

多發性骨髓瘤(Multiple myeloma) 為骨髓內漿細胞異常增生的惡性腫瘤，到目前為止仍然難以治癒。其發生發展是一個複雜的多步驟事件，涉及腫瘤細胞中遺傳和表觀遺傳的改變，以及骨髓微環境的支持。現已確定骨髓瘤細胞和骨髓微環境之間的相互作用對於骨髓瘤的病理發生，以及骨髓瘤細胞的生長，遷移和抗藥性起著關鍵作用。血小根因子四(Platelet factor 4, or PF4) 是一種抗血管生成的趨化因子。它不僅在體外抑制血管內皮細胞增殖和遷移，而且在體內抑制腫瘤的生長。此前，我們發現PF4 基因在多發性骨髓瘤中等位缺失以及DNA 高度甲基化，因而導致其在骨髓瘤病人及細胞系中的表達缺失或降低。在本研究中，我們利用體內和體外實驗鑒定了PF4 對骨髓瘤細胞以及血管生成的作用，並闡明了其作用機制。 / 首先，我們在體外鑒定了PF4 在骨髓瘤細胞中的功能。我們發現PF4 抑制骨髓瘤細胞系以及從病人骨髓中分離出來的骨髓瘤細胞的生長，以及促進其凋亡。其促凋亡活性與caspase-3 和PARP 的激活有關。我們也檢測了PF4 在骨髓瘤中對血管生成的作用。我們首先分離了病人骨髓中的內皮細胞。結果顯示PF4抑制骨髓瘤內皮細胞的生長和管狀物的形成。這些結果證明PF4 在骨髓瘤中可能是一個抑癌因子。 / 接下來我們進一步檢測了PF4 在體內的抑癌功能。在第一種模型中，骨髓瘤細胞被皮下移植到重症聯合兔疫缺陷型(NOD-SCID) 小鼠中。尾靜脈注射200ngPF4 明顯的抑制了腫瘤的生長，並延長了小鼠的成活率。第二種小鼠模型稱為兔鼠融合模型(SCID-rab model) 。在這一模型中，大白兔的腿骨先被皮下移植到(NOD-SCID) 小鼠中，再將骨髓瘤細胞注射入已植入的大白兔腿骨的骨腔中。兩周後，小鼠被尾靜脈注射入20 或200ng PF4 。結果顯示200ng PF4 顯著抑制了腫瘤的生長。通過兔疫組化分析大白兔腿骨切片，我們進一步證明了PF4 在腫瘤細胞中的增瘟，凋亡以及血管生成的作用。我們的發現因此證實了PF4 是骨髓瘤中的一個抑制因子。 / 為了鑒定PF4 在骨髓瘤中的作用機制，我們用Protein/DNA 微陣列(Protein/DNA array) 分析了PF4 參與的信號通路。結果顯示PF4 調節了若干個轉錄因子，其中包括STAT3 。凝膠遷移(EMSA) 和螢光素酪報告基因(luciferase reporter assay )檢測進一步證實PF4 抑制了STAT3 的DNA 結合能力以及轉錄活性。因此PF4 可能通過抑制STAT3 信號通路而抑制骨髓瘤的生長。我們進一步發現PF4 能抑制組成性的以及自介素6 (IL-6) 誘導的STAT3的激活。我們發現PF4 下調了STAT3 下游的靶基因，包括Mc1-1， Survivin 以及血管內皮細胞生長因子(VEGF)。而過表達組成性激活的STAT3 能逆轉PF4 所誘導的細胞凋亡。在兔鼠敵合模型中，通過兔疫組化分析大白兔腿骨切片，我們發現PF4 能抑制STAT3 的入核。SOCS3 是STAT3 其中的一個抑制因子，我們發現PF4 能誘導SOCS3 的表達。而干擾掉SOCS3 能使PF4 喪失其抑制STAT3 激活的能力。這些結果表明PF4 可能通過誘導SOCS3 的表達，從而抑制STAT3 信號通路，引起骨髓瘤的生長抑制以及抗血管生成。 / 總而言之，本研究表明PF4 是骨髓髓中一個重要調節因子。在體外和體內，PF4 通過抑制STAT3 信號通路，從而抑制腫瘤細胞的生長，促進凋亡以及抑制血管生成。本文為PF4 的臨床研究，作為一種新的治療骨髓瘤藥物，提高骨髓瘤病人的治療效果提供基礎。 / Multiple myeloma (MM) is an incurable hematological malignancy characterized by accumulation of clonal plasma cells in bone marrow (BM). The development and progression of MM is a complex multistep tumorigenic event involving both genetic and epigenetic changes in the tumor cell as well as the support by the BM microenvironment. It has been well established that the physical interaction of MM cells with the BM milieu are crucial for MM pathogenesis, MM cell growth, survival, migration and drug resistance. Platelet factor 4 (PF4), a potent antiangiogenic chemokine, not only inhibits endothelial cell proliferation and migration in vitro but also solid tumor growth in vivo. Our group previously demonstrated loss of PF4 expression in patient MM samples and MM cell lines due to concurrent allelic loss and DNA hypermethylation. In this study, we characterized the effects of PF4 on MM cells and angiogenesis in the BM milieu both in vitro and in vivo and elucidated the mechanism of PF4 effects on MM. / To characterize the effects of PF4 on MM cells in vitro, assays on cell growth, cell cycle arrest and apoptosis were performed and we found that PF4 inhibited growth and induced apoptosis in both MM cell lines and MM cells from patients. The proapoptotic activity of PF4 is associated with activation of caspase-3 and poly (ADP) ribose polymerase (PARP). We also investigated the effects of PF4 on angiogenesis in MM using endothelial cells isolated from patient's BM aspirates (MMECs). Our results showed that PF4 suppressed MMECs growth and tube formation on matrigel in a dose-dependent manner. / Given the ability of PF4 to suppress MM cell growth and angiogenesis in vitro, we evaluated its tumor suppressive function in vivo. In human subcutaneously matrigel xenograft mouse model, tail vein injection of 200ng PF4 significantly reduced MM tumor growth and prolonged survival. We next used the SCID-rab mouse model which recapitulates the human BM milieu in vivo. In this model, MM cells were directly injected into the rabbit bone which was subcutaneously implanted into the NOD-SCID mice. Two weeks after injection, SCID mice were treated with various dose of PF4 (20 or 200ng per injection, three times per week) or PBS by tail vein injection. ELISA assay for hIg (lambda) showed that tumor growth in 200ng PF4-treated mice was markedly reduced by 58% compared with the control group, which was further confirmed by immunohistochemistry analysis of CD 138 staining on rabbit bone section. Consistent with the in vitro results, induction of apoptosis in MM cells and inhibition of angiogenesis by PF4 could also be demonstrated in vivo, as evidenced by the findings on ki67, Cleaved caspase-3, CD31 and VEGF staining on rabbit bone sections from treated versus control mice. Our findings thus confirmed that PF4 is a novel tumor suppressor in MM. / However, the molecular mechanism of how PF4 inhibits MM tumorigenesis is still unclear. To identify the signal pathway PF4 involved in MM, Protein/DNA array was performed. We found that PF4 regulated several transcription factors including STAT3 in U266 cells. EMSA and luciferase reporter assay further confirmed that PF4 suppressed STAT3 DNA binding and transcriptional activity. So it is possible that PF4 mediates its tumor suppressive function, through suppressing STAT3 pathway in MM cells. We further found that pre-treatment of PF4 blocked both constitutive and interleukin-6-induced STAT3 activation in a time-dependent manner in human MM cells. PF4 could also down-regulate the STAT3-regulated gene products including Mcl-I, Survivin and vascular endothelial growth factor (VEGF). Moreover, enforced expression of constitutively active STAT3 rescued cells from PF4-induced apoptosis. In SCID-rab mouse model, we also found that PF4 inhibited STAT3 nuclear translocation by immunostaining of rabbit bone sections. When examined further, we found that PF4 induced the expression of one of the STAT3 inhibitor SOCS3, and gene silencing of SOCS3 by small interfering RNA abolished the ability of PF4 to inhibit STAT3 activation, suggesting a critical role of SOCS3 in the action of PF4. Our findings therefore suggest that by inducing SOCS3 expression, PF4 abrogates STAT3 activity, thus induces tumor growth inhibition and anti-angiogenesis. / Together, these novel studies have shown that PF4 is an important regulator of MM tumorigenesis. By abrogating STAT3 signaling it targets cell growth, induces apoptosis, suppresses angiogenesis both in vitro and in vivo in MM. These scientific observations provide the framework for clinical studies of this chemokine, as a novel drug for treatment of MM to improve patient outcome in MM. / Detailed summary in vernacular field only. / Detailed summary in vernacular field only. / Detailed summary in vernacular field only. / Detailed summary in vernacular field only. / Detailed summary in vernacular field only. / Liang, Pei. / "November 2011." / Thesis (Ph.D.)--Chinese University of Hong Kong, 2012. / Includes bibliographical references (leaves 139-161). / Electronic reproduction. Hong Kong : Chinese University of Hong Kong, [2012] System requirements: Adobe Acrobat Reader. Available via World Wide Web. / Abstract also in Chinese. / Abstract in English --- p.I / Abstract in Chinese --- p.IV / List of Publications --- p.VI / Acknowledgements --- p.VII / List of abbreviations --- p.IX / List of Tables --- p.XII / List of Figures --- p.xm / Table of Contents --- p.XV / Chapter Chapter1 --- Introduction and Literature Review --- p.1 / Chapter 1.1 --- Multiple myeloma-General description --- p.1 / Chapter 1.1.1 --- Epidemiology of MM --- p.1 / Chapter 1.1.2 --- Stages of MM --- p.1 / Chapter 1.2 --- The bone marrow (BM) microenvironment in MM --- p.3 / Chapter 1.3 --- Signal pathways in MM cells --- p.5 / Chapter 1.3.1 --- JAK/STAT3 in cancers and MM --- p.5 / Chapter 1.3.1.1 --- IL-6 and its receptor --- p.7 / Chapter 1.3.1.2 --- Activation of downstream signals-The "on" signals --- p.9 / Chapter 1.3.1.3 --- Inactivation of downstream signaling --- p.11 / Chapter 1.3.1.3.1 --- Phosphatases --- p.12 / Chapter 1.3.1.3.2 --- SOCS family --- p.13 / Chapter 1.3.1.3.3 --- The PIAS family --- p.14 / Chapter 1.3.2. --- NF-κB pathway --- p.15 / Chapter 1.3.3 --- RAS-MAPK pathway --- p.17 / Chapter 1.3.4 --- Phosphatidyl inositol-3 kinase (PI3K)/AKT --- p.18 / Chapter 1.4 --- Angiogenesis in MM --- p.18 / Chapter 1.4.1 --- The process of angiogenesis --- p.18 / Chapter 1.4.2 --- Angiogenesis in caner --- p.20 / Chapter 1.4.3 --- Angiogenesis in MM --- p.22 / Chapter 1.5 --- Animal models in MM --- p.24 / Chapter 1.6 --- Treatment of MM --- p.27 / Chapter 1.6.1 --- Chemotherapy --- p.27 / Chapter 1.6.2 --- Autologous stem cell transplantation --- p.28 / Chapter 1.6.3 --- Biologically based therapies --- p.28 / Chapter 1.7 --- Platelet factor 4 (PF4) --- p.30 / Chapter 1.8 --- Structure of PF 4 --- p.30 / Chapter 1.9 --- Role of PF4 in physiological process --- p.32 / Chapter 1.9.1 --- Inhibition of megakaryocytopoiesis --- p.32 / Chapter 1.9.2 --- PF4 and coagulation --- p.33 / Chapter 1.10 --- Role of PF4 in pathological process --- p.34 / Chapter 1.10.1 --- PF4 and cancer --- p.34 / Chapter 1.10.2 --- PF4 is an angiogenic inhibitor --- p.35 / Chapter 1.11 --- Clinical applications of PF4 --- p.37 / Chapter 1.12 --- Summary and project aims --- p.37 / Chapter Chapter 2 --- Materials and Methods --- p.40 / Chapter 2.1 --- Reagents and antibodies --- p.40 / Chapter 2.2 --- MM Cell lines --- p.40 / Chapter 2.3 --- CD138⁺ primary MM cells --- p.41 / Chapter 2.4 --- CD31⁺ MM endothelial cells (MMECs) --- p.42 / Chapter 2.5 --- WST-1 assay --- p.43 / Chapter 2.6 --- Trypan blue exclusion --- p.43 / Chapter 2.7 --- Cell cycle analysis --- p.44 / Chapter 2.8 --- Apoptosis analysis --- p.44 / Chapter 2.9 --- In vitro tube formation assay --- p.45 / Chapter 2.10 --- SCID-rab mice model --- p.45 / Chapter 2.10.1 --- Construction of SCID-rab mice --- p.45 / Chapter 2.10.2 --- Establishment and monitoring of myeloma in SCID-rab mice --- p.46 / Chapter 2.10.3 --- Enzyme-linked immunosorbent assay (ELISA) --- p.46 / Chapter 2.10.4 --- PF4 treatment --- p.47 / Chapter 2.10.5 --- Immunohistochemistry --- p.48 / Chapter 2.11 --- Protein/DNA arrays --- p.49 / Chapter 2.12 --- Electrophoretic mobility shift assay (EMSA) --- p.50 / Chapter 2.13 --- Luciferase reporter assay --- p.52 / Chapter 2.14 --- Western blotting --- p.53 / Chapter 2.15 --- RNA extraction --- p.54 / Chapter 2.16 --- Real-time Polymerase Chain Reaction (Real-time PCR) --- p.54 / Chapter 2.17 --- Nuclear transfection --- p.55 / Chapter 2.18 --- Statistical analysis --- p.55 / Chapter Chapter3 --- The role of PF4 in MM: in vitro studies --- p.58 / Chapter 3.1 --- Results --- p.58 / Chapter 3.1.1 --- PF4 inhibited growth of human MM cell lines --- p.58 / Chapter 3.1.2 --- PF4 did not cause cell cycle arrest --- p.59 / Chapter 3.1.3 --- PF4 induced apoptosis of myeloma cell lines --- p.63 / Chapter 3.1.4 --- PF4 caused cell apoptosis in primary MM cells cultured in vitro --- p.64 / Chapter 3.1.5 --- PF4 suppressed MMECs growth --- p.69 / Chapter 3.1.6 --- PF4 suppressed MMECs tube formation --- p.69 / Chapter 3.2 --- Discussion --- p.73 / Chapter 3.2.1 --- Negative regulation of PF4 in MM cells growth in vitro --- p.73 / Chapter 3.2.2 --- PF4 induces apoptosis in MM cell lines and primary MM cells --- p.74 / Chapter 3.2.3 --- PF4 inhibits angiogenesis in MM in vitro --- p.76 / Chapter 3.3 --- Summary --- p.79 / Chapter Chapter4 --- The role ofPF4 in MM tumorigenesis: in vivo studies --- p.82 / Chapter 4.1 --- Results --- p.82 / Chapter 4.1.1 --- PF4 inhibited MM tumor growth and prolonged survival in subcutaneous matrigel xenograft model --- p.82 / Chapter 4.1.2 --- PF4 inhibited MM tumor growth and prolonged survival in SCID-rab mouse model --- p.85 / Chapter 4.1.3 --- PF4 reduced human MM cell proliferation, angiogenesis and induced apoptosis in SCID-rab mice --- p.88 / Chapter 4.2 --- Discussion --- p.91 / Chapter 4.2.1 --- PF4 inhibited human tumor growth in subcutaneous matrigel xenograft mouse model --- p.91 / Chapter 4.2.2 --- SCID-rab mouse model was successfully established and PF4 inhibited human MM turnor growth in this model --- p.92 / Chapter 4.2.3 --- PF4 inhibited human MM cell proliferation, angiogenesis and induced apoptosis in SCID-rab mice --- p.95 / Chapter 4.3 --- Summary --- p.96 / Chapter Chapter 5 --- The molecular mechanisms of PF4 in MM tumorigenesis --- p.98 / Chapter 5.1 --- Results --- p.98 / Chapter 5.1.1 --- ProteinlDNA array hybridization and Quantification of protein/DNA array spots --- p.98 / Chapter 5.1.2 --- PF4 suppressed DNA binding and transcriptional activity of STAT3 --- p.102 / Chapter 5.1.3 --- PF4 inhibited constitutive STAT3 phosphorylation in MM cells --- p.104 / Chapter 5.1.4 --- PF4 inhibited IL-6-induced STAT3 activation --- p.105 / Chapter 5.1.5 --- PF4 suppressed STAT3 regulated gene expression --- p.107 / Chapter 5.1.6 --- Enforced expression of constitutively active STAT3 rescued cells from PF4-induced apoptosis --- p.109 / Chapter 5.1.7 --- PF4 induced the expression of SOCS3 --- p.111 / Chapter 5.1.8 --- PF4-induced inhibition of STAT3 activation was reversed by gene silencing of SOCS3 --- p.111 / Chapter 5.1.9 --- PF4 inhibited nuclear accumulation of STAT3 and induced expression of SOCS3 in vivo --- p.114 / Chapter 5.2 --- Discussion --- p.115 / Chapter 5.2.1 --- PF4 regulated several TFs --- p.115 / Chapter 5.2.2 --- PF4 inhibited constitutive activation of STAT3 --- p.118 / Chapter 5.2.3 --- PF4 inhibited IL-6 induced activation of STAT3 --- p.120 / Chapter 5.2.4 --- PF4 suppressed STAT3 regulated gene expression --- p.121 / Chapter 5.2.5 --- PF4 induced the expression of SOCS3 --- p.124 / Chapter 5.3 --- Summary --- p.125 / Chapter Chapter 6 --- Conclusion and future studies --- p.128 / Chapter 6.1 --- Conclusion --- p.128 / Chapter 6.2 --- Future studies --- p.135 / Appendices --- p.137 / References list --- p.139

Estudo descritivo, correlacional, de delineamento longitudinal, com 78 pacientes que iniciaram anticoagulante oral (ACO) entre abril de 2008 a junho de 2009 em três serviços de saúde de um município do oeste do Paraná. Os objetivos foram avaliar a adesão medicamentosa e comparar a qualidade de vida relacionada à saúde (QVRS) e o estado global de saúde no início e com seis meses de tratamento. Os dados foram coletados por entrevistas individuais com instrumentos específicos para adesão farmacológica (Medida de Adesão ao Tratamento), QVRS (Medical Outomes Survey Short form - SF-36; Duke Anticoagulation Satisfaction Scale DASS), todos validados para o uso no Brasil, e o estado global de saúde (Escala Visual analógica EVA). Foram realizados testes de comparação de médias (Teste t de Student pareado e para amostras independentes), de correlação (coeficiente de correlação de Pearson) e de regressão linear múltipla. O nível de significância foi 0,05. Entre os sujeitos, 53,8% eram mulheres, com idade média de 56,8 anos, casados (71,8%), com baixa escolaridade e 48,7% não desempenhavam atividades remuneradas. As principais indicações para o uso do ACO foram fibrilação atrial (34,6%) e prótese cardíaca mecânica (26,9%) e o ACO mais usado foi a varfarina sódica (91%). Os resultados apontaram que após seis meses, apenas dois participantes foram classificados como não aderentes ao tratamento com ACO e que, no geral, houve melhora na QVRS avaliada por ambos os instrumentos. A avaliação pelo SF-36 mostrou que as diferenças entre os oito domínios foram estatisticamente significantes, exceto para saúde mental. Entretanto, as comparações das médias dos domínios do DASS foram estatisticamente significantes apenas para os domínios Impacto psicológico negativo e Impacto psicológico positivo. O estado global de saúde avaliado pela EVA apresentou valores médios que aumentaram da primeira para segunda avaliação, de 74 para 83, respectivamente, em um intervalo possível de zero a 100. Considerando como variável resposta a medida do DASS total, um modelo de regressão linear multivariada composto pelas variáveis idade, escolaridade, número de medicamentos em uso, indicação para o ACO, dosagem semanal do ACO, Saúde mental (domínio do SF-36), Vitalidade (domínio do SF-36) e intervalo terapêutico explicaram 39,3% da variância da medida da QVRS. Neste modelo, as variáveis com maiores valores de coeficiente beta () e estatisticamente significantes foram: idade (= - 0,317; p=0,017), número de medicamentos usados pelo indivíduo (= -0,353; p=0,005) e saúde mental (= -0,364; p=0,032). Um segundo modelo de regressão linear multivariada foi feito tendo como variável resposta a medida do estado global de saúde. As variáveis explanatórias foram: escolaridade, número de medicamentos em uso, Vitalidade, Saúde mental, Aspectos emocionais e intervalo terapêutico que explicaram 40,4% da variância desta medida. Os resultados obtidos podem subsidiar a prática dos profissionais da saúde na prevenção de fatores que possam afetar à adesão ao medicamento e a qualidade de vida dos usuários de ACO. / A descriptive, correlational design of longitudinal, with 78 patients who initiated oral anticoagulant taking (OAC) within the months of April, 2008 and June, 2009 in three health care services from a municipality of the state of Parana. The aims of this study were to evaluate the medication adherence and compare the health-related quality of life (HRQL) and the global health status in its beginning and within six months of treatment. The datas were all collected through individual interviews making use of specific instruments for pharmacological adherence (Means of Adherence to Treatment), QVRS (Medical Outcomes Survey Short form - SF-36; Duke Anticoagulation Satisfaction Scale DASS), which ones are validated to use in Brazil, and the global health status (Visual Analog Scale VAS).Comparison of average tests were applied (Students test t for paired and independent samples), of correlation (Pearsons correlation test) and of multiple linear regression. The significance level was set at 0,05. Among the subjects, 53,8% were women, at the average age of 56.8, married (71.8%), with low education and 48,7% did not performed any paid job. The main indications to the use of OAC were atrial fibrillation (34,6%) and mechanical cardiac prosthesis (26,9%) and the most used OAC was the warfarin sodium (91%). The results pointed out that after six months, only two participants were classified as not-adherent to treatment with OAC and that, by and large, there was improvement in the HRQL evaluated by both instruments. The evaluation with SF-36 showed that the differences among the eight domains were statistically significant, except for mental health. However, the average comparisons of domains of the DASS were statistically significant only to the negative psychological impact and positive psychological impact domains.The global health status evaluated by VAS presented average score increase from the first to the second evaluation, from 74 to 83, respectively, in a possible interval from zero to 100. Considering it as a variable response to the measurement of the total DASS, a model of linear regression multivariate made up by age variables, education, number of chemicals in use, indication to the OAC, weekly dose of OAC, mental health (domain of SF-36), Vitality (domain of SF-36) and interval therapy explained 39,3% of the variability of the measurement of HRQL. In this model, the variables with higher beta () coefficient scores and statistically significant, were: age (= -0,317; p=0,017), number of chemicals taken by the individual (= -0,353; p=0,005) and mental health (= -0,364; p=0,032).A second model of linear multivariate regression was done, taking into account as a variable response to the measurement of global state of health. The explanatory variables were: education, number of chemicals in use, Vitality, Mental health, Emotional functioning and interval therapy explained 40,4% of the variability of this measurement. The results obtained may subside the practice of healthcare professionals in the prevention of factors that may affect the adherence to the medication and the health-related quality of life of OAC users.

This thesis combines five published research papers investigating methods of improving the safety and control of oral anticoagulation, with the use of computer assisted dosage and the standardisation of the International Normalised Ratio (INR). The INR is a conventional measurement derived from the time it takes blood of a patient to form a clot and is used to monitor the effects of widely used oral anticoagulants such as warfarin for the prevention of stroke and other related disorders. The first paper investigates whether the use of computer-assisted programs was as safe and effective as medical staff manual dosage in the prevention of bleeding or thrombotic complications during oral anticoagulant treatment. This was an international multi-centre randomised study conducted by the European Action on Anticoagulation (EAA) investigating the clinical benefit of two computer programs, PARMA 5 (Italy) and DAWN AC (UK). Composite clinical events were reduced by 7.6% using computer programs, though not achieving statistical significance (p=0.1), showing computer programs to be not dissimilar to medical staff dosage. The second paper recommends guidelines for screening safety and effectiveness of other marketed computer programs based on the results of the EAA study. A process for a candidate computer program to achieve non-inferiority relative to the medical staff dosage arm from the EAA study is explained. The third paper introduces a modified approach to the 'Direct INR' method for the standardisation of INR termed the 'Prothrombin Time/INR Line' (PT/INR). This was directly compared to the local International Sensitivity Index (ISI) calibration procedure originally approved by the World Health Organisation and later by the United States Food and Drug Administration (FDA). Using manually certified lyophilised plasmas tested by specialist centres, the PT/INR Line using a set of 5 calibrant plasmas to establish a fitted line to estimate local INR was shown to be as effective as the FDA procedure. The fourth paper investigates the PT/INR Line further by using simulated sets of calibrant plasmas across the therapeutic range of 2.0-4.5 INR and determining the PT/INR Line. Local INR of five validation plasmas, certified by 3 centres using the manual PT technique, was determined using the estimated PT/INR Lines and compared with local ISI calibration. Using 4 or 5 calibrant plasmas to determine the PT/INR Line was shown to be as accurate as local ISI calibrations for reliable local INR.The fifth and final paper assessed INR variability and control in oral anticoagulant therapy using a method termed the Variance Growth Rate (VGR), and compared its predictive ability of adverse events with the Time in Target INR range (TIR), the conventional method used in evaluating the quality of oral anticoagulant therapy. The VGR method was shown to be a better predictor of adverse bleeding or thrombotic episodes in the short term period prior to an event (3 and 6 months) compared with TIR.