While some rare and severe forms of platelet function disorders are now well characterized, many common types of platelet function disorders are not yet characterized. My hypothesis was that uncharacterized platelet function disorders that impair platelet function in aggregation and/or dense granule ATP release assays are associated with increased bleeding risk. The main goal of the thesis was to study the phenotype and bleeding risks for uncharacterized platelet function disorders, through analysis of the results from clinical laboratory tests of platelet function and for a detailed analysis of their reported bleeding symptoms. First, I assessed if lumi-aggregometry provides useful diagnostic information on platelet function and can be used to help decide if an individual has a bleeding disorder. Two cohorts of individuals were studied that had dense granule ATP release assessed in response to multiple agonists as part of a work-up for a bleeding disorder. Cohort I was comprised of individuals tested between January 2007 and June 2013 and cohort II was comprised of subjects tested at least twice by this assay prior to September 2015. Among subjects tested more than once for dense granule release defects as part of the work up for a bleeding disorder (cohort I; n=133; cohort II; n=17), normal findings with all tested agonists were often confirmed by the second test (cohort I: 83%; cohort II: 100%), but impaired release with multiple agonists was not often confirmed (cohort I: 34%; cohort II: 54%) and even if it was present, the finding was not predictive of a bleeding disorder. Consequentially, it was recommended that lumi-aggregometry should not be used to diagnose platelet function disorders. Next, I studied the bleeding risks associated with uncharacterized platelet function disorders, by evaluating subjects who had abnormal findings by validated assays, namely subjects who had defective aggregation responses to two or more agonists and/or dense granule deficiency. Bleeding history was evaluated using the International Society for Thrombosis and Haemostasis bleeding assessment tool (ISTH BAT) and the likelihood for bleeding symptoms/ problems, was estimated using odds ratios (OR) collected using the clinical history assessment tool - platelet (CHAT-P) for all affected subjects, a subgroup family with a mutation RUNX1, unaffected family v members and general population controls. Individuals with platelet function disorders (n=29) and the affected members of the family with the RUNX1 mutation (n=6) had elevated ISTH BAT scores (median: 9; range:0-18 and median: 8.5, range 4-15, respectively) and an increased risk of abnormal bruising (OR 15-65 and 11-67), nosebleeds (OR 23-40 and 19-121), menorrhagia (OR 6.5-29) and excessive bleeding after trauma or dental/surgical procedures (OR 9.5-44 and 15-77 ) and wound healing problems (OR 13 and 38) compared to general population control (n=60) and unaffected (n=12) family members. Overall, the platelet function disorders in the study present with a significantly increased risk of mild, rather than severe bleeding problems. These findings are important for individuals and healthcare providers to promote evidence-based care of common uncharacterized inherited platelet function disorders for individuals with RUNX1 mutations, dense granule deficiency and/or impaired aggregation responses. / Thesis / Master of Science (MSc) / Platelets are small blood cells that help stop bleeding. People who have platelets that do not work properly are more likely to bleed. Determining who has platelet problems can be challenging as there are limitations to diagnostic tests for these conditions. Additionally, the risks for bleeding in individuals with platelet problems are unknown. We looked at individuals with bleeding problems and found that a recommended test to assess platelet dense granule release, called lumi-aggregometry, wasn’t able to reliably identify persons with bleeding problems. Based on this, we recommend that lumi-aggregometry should not be used to diagnose platelet function disorders. We also found that individuals with uncharacterized platelet function disorders have increased risks for wound healing problems and experiencing bruising, nosebleeds, menorrhagia, and excessive bleeding after dental or surgical procedures. These risks are common among other mild bleeding disorders and will be important to differentiate bleeding risk from other platelet disorders.

京都大学 / 新制・課程博士 / 博士(医学) / 甲第25175号 / 医博第5061号 / 新制||医||1071(附属図書館) / 京都大学大学院医学研究科医学専攻 / (主査)教授 江藤 浩之, 教授 長尾 美紀, 教授 大鶴 繁 / 学位規則第4条第1項該当 / Doctor of Agricultural Science / Kyoto University / DFAM

Diss. (sammanfattning) Stockholm : Karolinska institutet, 2005. / Härtill 4 uppsatser.

INTRODUÇÃO: Os Inibidores de Bombas de Prótons (IBP´s) são comumente prescritos a pacientes em uso de dupla antiagregação plaquetária (DAP) com ácido acetilsalicílico (AAS) e clopidogrel. Entretanto, esta classe de medicamentos, especialmente o omeprazol, tem sido associada à redução da potência antiplaquetária do clopidogrel, levando em muitos casos ao uso de ranitidina como alternativa. MÉTODOS: Foram analisados pacientes com doença arterial coronária (DAC) estável em uso de AAS 100 mg uma vez ao dia. A agregabilidade plaquetária foi medida no momento basal e após uma semana de terapia com clopidogrel na dose de 75 mg uma vez ao dia. Após essa fase inicial, os participantes foram randomizados de modo duplo-cego e duplo-mascarado para omeprazol 20 mg duas vezes dia ou ranitidina 150 mg duas vezes ao dia, sendo os testes de agregação plaquetária novamente repetidos após uma semana. A agregabilidade foi avaliada com a utilização dos seguintes métodos: VerifyNow P2Y12® (Accumetrics - San Diego, CA, EUA, meta principal do estudo), utilizando-se Unidades de Reatividade ao P2Y12 (\"P2Y12 Reactivity Units\" - PRU) e Inibição Percentual da Agregabilidade (IPA) na descrição da agregabilidade; agregometria de sangue total (AST) por bioimpedância utilizando os reagentes ADP e colágeno, sendo a agregabilidade medida em Ohms; \"Platelet Function Analyser\" 100® (Siemens Healthcare Diagnostics®, Newark, Delaware, EUA) utilizando o cartucho de colágeno/ADP, com a agregabilidade avaliada pelo tempo de fechamento do orifício em segundos. Além disso, foi feita dosagem de tromboxano B2 (TXB2) sérico na última visita a fim de se avaliar o efeito do AAS. RESULTADOS: Oitenta e cinco pacientes foram incluídos na análise final, sendo 41 no grupo omeprazol e 44 no grupo ranitidina. Houve redução significativa da IPA após o acréscimo de omeprazol (de 26,3 ± 32,9% para 17,4 ± 33,1%; P = 0,025), enquanto o grupo ranitidina não demonstrou modificação significativa (de 32,6 ± 28,9% para 30,1 ± 31,3%; P = 0,310). Levando-se em conta o valor em PRU, houve um aumento numérico porém não significativo estatisticamente no grupo omeprazol (de 159,73 ± 83,06 para 173,54 ± 72,29; P = 0,116) enquanto no grupo ranitidina houve uma diferença muito pequena (de 153,61 ± 70,12 to 158,77 ± 76,37; P = 0,440). Em relação aos demais testes de agregabilidade e à dosagem de TXB2 sérico, não houve alterações significativas em qualquer um dos grupos. CONCLUSÃO: A ranitidina não influenciou o efeito antiplaquetário do clopidogrel, ao contrário do omeprazol, que reduziu a atividade antiplaquetária do medicamento. Esses achados podem ter um importante impacto na tomada de decisão quanto ao protetor gástrico a ser utilizado em pacientes submetidos a DAP com AAS e clopidogrel. / BACKGROUND: Proton-pump inhibitors (PPIs) are often prescribed to patients taking dual antiplatelet therapy (DAPT) with acetylsalicylic acid (ASA) and clopidogrel. However, this class of medication, especially omeprazole, has been associated with a reduction of clopidogrel efficacy, leading many to substitute omeprazole with ranitidine. METHODS: The present study analyzed patients with stable coronary artery disease (CAD) in use of ASA 100 mg daily. Platelet aggregability was measured at baseline and after one week of clopidogrel 75 mg daily. Then, the subjects were randomized, in a double-blinded, doubledummy fashion, to omeprazole 20 mg twice a day or ranitidine 150 mg twice a day. After one more week, aggregability tests were repeated. Platelet aggregability was evaluated by the following methods: VerifyNow P2Y12TM (Accumetrics - San Diego, California, USA, main endpoint of the study), with aggregability depicted as percent Inhibition of Platelet Aggregation (IPA) and as P2Y12 Reactivity Units (PRU); whole blood aggregometry by bioimpendance using ADP and collagen with aggregability measured in Ohms; and Platelet Function Analyser 100TM (Siemens Healthcare Diagnostics, Newark, Delaware, USA) using collagen/ADP cartridge with aggregability measured in time to closure in seconds. Besides that, serum thromboxane B2 dosage was done on the last visit to evaluate ASA effect. RESULTS: Eighty-five patients were included in final analysis (41 in the omeprazole group and 44 in the ranitidine group). IPA was significantly decreased after addition of omeprazole (from 26.3% ± 32.9 to 17.4% ± 33,1; P = 0.025), with no significant changes being observed in the ranitidine group (from 32.6% ± 28.9 to 30.1% ± 31.3; P = 0.310). When taking into account PRU values, there was a numerical, but statistically non-significant increase in the omeprazole group (from 159.73 ± 83.06 to 173.54 ± 72.29; P = 0.116), with a very slight difference in the ranitidine group (from 153.61 ± 70.12 to 158.77 ± 76.37; P = 0.44). There were no significant changes taking into account other aggregability tests and serum thromboxane B2 dosage. CONCLUSION: In patients with stable CAD, ranitidine did not influence clopidogrel antiplatelet activity, in contrast to omeprazole, which reduced antiplatelet drug effect. These findings may have a great impact in clinical decision making regarding gastrointestinal prophylaxis choice in patients taking DAPT with ASA and clopidogrel

En Si Wang. / Thesis (Ph.D.)--Chinese University of Hong Kong, 1994. / Includes bibliographical references (leaves 126-140 (2nd gp.)). / Acknowledgements --- p.i / Contents --- p.ii / Abstract --- p.iv / List of Acronyms and Abbreviations --- p.vi / introduction --- p.1 / Chapter I. --- "Platelet Activating Factor (PAF)´ؤPast, Present, and Future" --- p.1 / Chapter I-1. --- What is PAF? --- p.1 / Chapter I-2. --- Biochemistry of PAF --- p.2 / Chapter I-2-1. --- Metabolic Cycle of PAF --- p.3 / Chapter I-2-1-A. --- Biosynthesis of PAF --- p.4 / Chapter I-2-1 -B. --- Inactivation of PAF --- p.6 / Chapter I-2-2. --- Role of Endogenous PAF in Cell --- p.7 / Chapter I-3. --- Chemistry of PAF --- p.8 / Chapter I-4. --- Pathobiology of PAF --- p.9 / Chapter II. --- PAF Receptor --- p.10 / Chapter II-1. --- Presence and Characteristics of PAF Receptor --- p.10 / Chapter II-l-l. --- Solubilization of PAF Receptor --- p.10 / Chapter II-1-2. --- G-Protein Involvement --- p.11 / Chapter II-1-3. --- Species Differences --- p.11 / Chapter II-1-4. --- Multiple Conformational States of PAF Receptor --- p.12 / Chapter II-1-5. --- PAF Receptor Heterogeneity --- p.12 / Chapter II-2. --- Putative Conformation of PAF Membrane Binding Sites --- p.13 / Chapter II-3. --- Recent Progress in PAF Receptor Research --- p.15 / Chapter III. --- PAF Receptor Antagonist --- p.18 / Chapter III-1. --- Classification of PAF Antagonists --- p.18 / Chapter III-2. --- Inhibition Types of PAF Receptor Antagonists --- p.19 / Chapter III-2-1. --- Nonspecific Inhibition of the Effects of PAF --- p.21 / Chapter III-2-2. --- Specific Inhibition of PAF --- p.22 / Chapter III-3. --- Recent Progress in PAF Receptor Antagonist Research --- p.22 / Chapter IV. --- Pharmacology and Syntheses of Spiro-Ether Structural Units --- p.26 / Chapter IV-1. --- Natural Products Containing Spiro-Ether and Related Structural Units --- p.30 / Chapter IV-1-1. --- Labdane Diterpenoids Containing Spiro-Ether Structural Units --- p.30 / Chapter IV-1-2. --- Leucodrin and Related Derivatives --- p.32 / Chapter IV-2. --- Synthetic Methods of Spiro-Ethers and Related Derivatives --- p.34 / Chapter V. --- Aim of the Present Work --- p.45 / RESULTS AND DISCUSSION --- p.47 / Chapter I. --- Isolation and Structure Elucidation of Prehispanolone (1) and Preleoheterin (3) --- p.47 / Chapter I-1. --- Material and Isolation --- p.47 / Chapter I-2. --- Structure Elucidation of Prehispanolone (1) and Preleoheterin (3) --- p.47 / Chapter II. --- Synthesis of Model Compounds --- p.53 / Chapter II-l. --- "Synthesis of 2-Methyl-1,7-dioxaspiro[4.4]nonane (137)" --- p.53 / Chapter II-2. --- "Synthesis of 2,2-Dimethyl-l,7-dioxaspiro[4.4]nonane (139)" --- p.68 / Chapter II-3. --- "Synthesis of 2,2-Diphenyl-1,7-dioxaspiro[4.4]nonane (141) and 2,2-Diphenyl-l,7-dioxaspiro[4.4]non-8-ene (142)" --- p.72 / Chapter III. --- "Partial Synthesis of 13R, 14,15-Dihydroprehispanolone (5)，13S,14,15-Di- hydroprehispanolone (135) and prehispanolone (1)" --- p.76 / CONCLUSION --- p.89 / EXPERIMENTAL SECTION --- p.91 / REFERENCES --- p.126 / APPENDIX --- p.141

A adesão de plaquetas nas paredes dos vasos sanguíneos e subsequente agregação são eventos cruciais tanto na hemorragia e quanto na trombose. A hiperagregação (agregação excessiva) das plaquetas pode causar a formação de um trombo e a posterior oclusão dos vasos sanguíneos levando a um processo isquêmico. A terapia antiplaquetária com ácido acetilsalicílico reduz em até 25% o risco de infartos do miocárdio não-fatais, acidentes vasculares cerebrais isquêmicos ou mortes de causa vascular em pacientes de alto risco, independentemente do sexo ou idade. Para avaliar laboratorialmente a eficácia dessa terapêutica, o método mais utilizado é o teste de agregação plaquetária, que pode ser feito em duas metodologias, a turbidimétrica e a por impedância elétrica. Com esse cenário, o objetivo do estudo é comparar essas duas metodologias para a monitorização do tratamento. Para isso foram utilizadas amostras de sangue de 30 pacientes adultos (média de 42 anos) de ambos os sexos que fazem uso regular do fármaco e analisadas através das duas metodologias, tendo seus resultados analisados e comparados. Os resultados do estudo mostraram pouca diferença estatística significante (p<0,05) entre os dois métodos nos principais agentes estimulantes, indicando que o método de impedância elétrica pode ser utilizado em rotina laboratorial em substituição, ou em complementação, com a metodologia turbidimétrica para monitorar a terapia antiplaquetária a base de ácido acetilsalicílico / The adhesion of platelets in the blood vessel wall and subsequent aggregation are crucial events in both bleeding and thrombosis. The hyperaggregation (excessive aggregation) of platelets can cause the formation of a thrombus and subsequent occlusion of blood vessels leading to an ischemic process. Antiplatelet therapy with acetylsalicylic acyd reduces by 25% the risk of myocardial infarctions, non-fatal strokes or deaths from vascular causes in patients at high risk, regardless of sex or age. To laboratory evaluation of the effectiveness of this therapy, the method most used is the platelet aggregation test, which can be done in two methods, the turbidimetric and electrical impedance. With this scenario, the objective of the study is to compare these two methodologies for monitoring the treatment. For such purpose, blood samples from 30 adult patients (mean 42 years) of both sexes who make regular use of the drug was analyzed using the two methodologies, and their results was analyzed and compared. The studys results showed little difference statistically significant (p <0.05) between the two methods with the main stimulant agents, indicating that the method of electrical impedance can be used in routine laboratory instead or complementing the methodology of turbidimetric for monitor antiplatelet therapy by acetylsalicylic acyd

Conselho Nacional de Desenvolvimento CientÃfico e TecnolÃgico / Operculina macrocarpa (L.) Urb. (âjalapa brasileiraâ) (Convolvulaceae) à uma espÃcie comum do Nordeste brasileiro, sendo popularmente utilizada pelas suas propriedades laxativa, purgativa e âdepurativaâ. A tintura do tubÃrculo de jalapa, matÃria-prima majoritÃria, ao lado da tintura de Convolvulus scammonia (TCS) compÃem o fitoterÃpico Aguardente Alemà(AAL) ou Tintura de Jalapa Composta, indicada como antitrombÃtico na medicina popular. O objetivo do presente trabalho foi investigar o potencial antiagregante plaquetÃrio e anticoagulante de O. macrocarpa em plasma humano, incluindo estudo de bioprospecÃÃo e determinaÃÃo do possÃvel mecanismo de aÃÃo. A avaliaÃÃo da atividade antiagregante plaquetÃria das drogas testes (tintura e fraÃÃes de O. macrocarpa, TCS e AAL) em plasma humano rico em plaquetas (PRP) foi mensurada por mÃtodo turbidimÃtrico, sendo a agregaÃÃo induzida por vÃrios agonistas como difosfato de adenosina (ADP), epinefrina (EPI), Ãcido araquidÃnico (AA), colÃgeno (COL) ou trombina (TROM). Ao contrÃrio da TCS e da AAL, tanto a tintura de O. macrocarpa (TOM/precipitado(P) e sobrenadante(S)) quanto uma das fraÃÃes orgÃnicas obtidas da planta (TOM/1-F4) mostraram atividade antiagregante plaquetÃria, onde a TOM/1-F4 (100 Âg/mL) apresentou efeito comparÃvel ao Ãcido acetilsalicÃlico (AAS). CaracterizaÃÃo fÃsico-quÃmica (CLAE e espectrofotometria) da TOM/PS (teor de resinas 1,38g%) e da TOM/1-F4 permitiu a identificaÃÃo de Ãcidos fenÃlicos (clorogÃnico, gÃlico e cafÃico), bem como a determinaÃÃo do teor de fenÃis totais da TOM (0,14g%). O efeito antiagregante plaquetÃrio da TOM/PS e da TOM/1F-4 parece resultar de vÃrias aÃÃes intracelulares. Contudo, a TOM/PS na presenÃa do AAS, L-ARG, ODQ, pentoxifilina (PTX) ou ticlopidina (TIC) teve seu efeito antiagregante plaquetÃrio modificado principalmente pela TIC, sugerindo um papel importante dos receptores purinÃrgicos na bioatividade da TOM/PS. A TOM/PS apresentou atividade antioxidante determinada atravÃs do teste do DPPH. AlÃm disso, a TOM/PS e a TOM/1F-4 nÃo mostraram aÃÃo anticoagulante em plasma humano, mas a TOM/PS aumentou o tempo de sangramento em camundongos. Foi observada uma toxicidade relativa da TOM/PS em neutrÃfilo humano. Dessa forma, o presente estudo comprovou, de maneira inÃdita, o potencial antiagregante plaquetÃrio da tintura e da fraÃÃo orgÃnica de jalapa, que estÃo pelo menos em parte relacionados à presenÃa de fenÃis, particularmente Ãcidos fenÃlicos, e resinas na planta. / Operculina macrocarpa (L.) Urb ("Brazilian jalapa") (Convolvulaceae) is a common species of the Brazilian Northeast. It is popularly used because of its laxative and purgative properties. The tincture of Operculina macrocarpa tubers (major constituent) and Convolvulus scammonia composes the raw materials of Aguardente AlemÃÂ, herbal medicine, referred to as antithrombotic in folk medicine. The objective of this study was to investigate the antiplatelet and anticoagulant potential for Operculina macrocarpa in human plasma, including the bioprospection study and determination of possible mechanism of action. The evaluation of antiplatelet activity of drugs (tincture and fractions of O. macrocarpa, tincture of C. scammonia and AAL) in human platelet-rich plasma (PRP) was measured by the turbidimetric method where the aggregation was induced by the addition of agonists (adenosine diphosphate (ADP), epinephrine (EPI), arachidonic acid (AA) collagen (COL) or thrombin (TROM). Unlike tincture of C. scammonia and AAL, both O. macrocarpa tincture (TOM/preciptade (P) and supernatant (S)) and one of the organic fractions obtained from O. macrocarpa (TOM/1-F4) showed antiplatelet activity in human plasma where TOM/1-F4 (100 Âg/mL) presented comparable effect to AAS. Physicochemical characterization (HPLC and spectrophotometer) of TOM/PS (content of resins 1,38g%) and TOM/1-F4 allowed the identification of phenolic acids (chlorogenic, galic and caffeic) as well as the determination of total phenols of TOM (0,14g%). The antiplatelet effect of TOM/PS and TOM/1-F4 seems to result from many intracellular actions. However, TOM/PS in presence of AAS, L-ARG, ODQ, pentoxifilin (PTX) or ticlopidine (TIC) had its antiplatelet effect modified mainly because of TIC, suggesting an important role of purinergic receptors in TOM/PS. TOM/PS presented antioxidant activity determined by DPPH test. Furthermore, TOM/PS and TOM/1-F4 did not showed anticoagulant action in human plasma but TOM/PS increased the bleeding time in mice. It was observed a relative toxicity of TOM/PS in human neutrophil. Therefore, this study demonstrated, as never before, the antiplatelet potencial of tincture and the organic fraction of "jalapa" and that at least related to the presence of phenols, particulary phenolic acids, and resins in plant.

INTRODUÇÃO: Os Inibidores de Bombas de Prótons (IBP´s) são comumente prescritos a pacientes em uso de dupla antiagregação plaquetária (DAP) com ácido acetilsalicílico (AAS) e clopidogrel. Entretanto, esta classe de medicamentos, especialmente o omeprazol, tem sido associada à redução da potência antiplaquetária do clopidogrel, levando em muitos casos ao uso de ranitidina como alternativa. MÉTODOS: Foram analisados pacientes com doença arterial coronária (DAC) estável em uso de AAS 100 mg uma vez ao dia. A agregabilidade plaquetária foi medida no momento basal e após uma semana de terapia com clopidogrel na dose de 75 mg uma vez ao dia. Após essa fase inicial, os participantes foram randomizados de modo duplo-cego e duplo-mascarado para omeprazol 20 mg duas vezes dia ou ranitidina 150 mg duas vezes ao dia, sendo os testes de agregação plaquetária novamente repetidos após uma semana. A agregabilidade foi avaliada com a utilização dos seguintes métodos: VerifyNow P2Y12® (Accumetrics - San Diego, CA, EUA, meta principal do estudo), utilizando-se Unidades de Reatividade ao P2Y12 (\"P2Y12 Reactivity Units\" - PRU) e Inibição Percentual da Agregabilidade (IPA) na descrição da agregabilidade; agregometria de sangue total (AST) por bioimpedância utilizando os reagentes ADP e colágeno, sendo a agregabilidade medida em Ohms; \"Platelet Function Analyser\" 100® (Siemens Healthcare Diagnostics®, Newark, Delaware, EUA) utilizando o cartucho de colágeno/ADP, com a agregabilidade avaliada pelo tempo de fechamento do orifício em segundos. Além disso, foi feita dosagem de tromboxano B2 (TXB2) sérico na última visita a fim de se avaliar o efeito do AAS. RESULTADOS: Oitenta e cinco pacientes foram incluídos na análise final, sendo 41 no grupo omeprazol e 44 no grupo ranitidina. Houve redução significativa da IPA após o acréscimo de omeprazol (de 26,3 ± 32,9% para 17,4 ± 33,1%; P = 0,025), enquanto o grupo ranitidina não demonstrou modificação significativa (de 32,6 ± 28,9% para 30,1 ± 31,3%; P = 0,310). Levando-se em conta o valor em PRU, houve um aumento numérico porém não significativo estatisticamente no grupo omeprazol (de 159,73 ± 83,06 para 173,54 ± 72,29; P = 0,116) enquanto no grupo ranitidina houve uma diferença muito pequena (de 153,61 ± 70,12 to 158,77 ± 76,37; P = 0,440). Em relação aos demais testes de agregabilidade e à dosagem de TXB2 sérico, não houve alterações significativas em qualquer um dos grupos. CONCLUSÃO: A ranitidina não influenciou o efeito antiplaquetário do clopidogrel, ao contrário do omeprazol, que reduziu a atividade antiplaquetária do medicamento. Esses achados podem ter um importante impacto na tomada de decisão quanto ao protetor gástrico a ser utilizado em pacientes submetidos a DAP com AAS e clopidogrel. / BACKGROUND: Proton-pump inhibitors (PPIs) are often prescribed to patients taking dual antiplatelet therapy (DAPT) with acetylsalicylic acid (ASA) and clopidogrel. However, this class of medication, especially omeprazole, has been associated with a reduction of clopidogrel efficacy, leading many to substitute omeprazole with ranitidine. METHODS: The present study analyzed patients with stable coronary artery disease (CAD) in use of ASA 100 mg daily. Platelet aggregability was measured at baseline and after one week of clopidogrel 75 mg daily. Then, the subjects were randomized, in a double-blinded, doubledummy fashion, to omeprazole 20 mg twice a day or ranitidine 150 mg twice a day. After one more week, aggregability tests were repeated. Platelet aggregability was evaluated by the following methods: VerifyNow P2Y12TM (Accumetrics - San Diego, California, USA, main endpoint of the study), with aggregability depicted as percent Inhibition of Platelet Aggregation (IPA) and as P2Y12 Reactivity Units (PRU); whole blood aggregometry by bioimpendance using ADP and collagen with aggregability measured in Ohms; and Platelet Function Analyser 100TM (Siemens Healthcare Diagnostics, Newark, Delaware, USA) using collagen/ADP cartridge with aggregability measured in time to closure in seconds. Besides that, serum thromboxane B2 dosage was done on the last visit to evaluate ASA effect. RESULTS: Eighty-five patients were included in final analysis (41 in the omeprazole group and 44 in the ranitidine group). IPA was significantly decreased after addition of omeprazole (from 26.3% ± 32.9 to 17.4% ± 33,1; P = 0.025), with no significant changes being observed in the ranitidine group (from 32.6% ± 28.9 to 30.1% ± 31.3; P = 0.310). When taking into account PRU values, there was a numerical, but statistically non-significant increase in the omeprazole group (from 159.73 ± 83.06 to 173.54 ± 72.29; P = 0.116), with a very slight difference in the ranitidine group (from 153.61 ± 70.12 to 158.77 ± 76.37; P = 0.44). There were no significant changes taking into account other aggregability tests and serum thromboxane B2 dosage. CONCLUSION: In patients with stable CAD, ranitidine did not influence clopidogrel antiplatelet activity, in contrast to omeprazole, which reduced antiplatelet drug effect. These findings may have a great impact in clinical decision making regarding gastrointestinal prophylaxis choice in patients taking DAPT with ASA and clopidogrel

Submitted by Rosivalda Pereira (mrs.pereira@ufma.br) on 2017-05-22T16:51:30Z No. of bitstreams: 1 SamiraAbdala.pdf: 3051742 bytes, checksum: 816c5c802400bcf5a75deb6a99de0617 (MD5) / Made available in DSpace on 2017-05-22T16:51:30Z (GMT). No. of bitstreams: 1 SamiraAbdala.pdf: 3051742 bytes, checksum: 816c5c802400bcf5a75deb6a99de0617 (MD5) Previous issue date: 2017-04-07 / Fundação de Amparo à Pesquisa e ao Desenvolvimento Científico e Tecnológico do Maranhão (FAPEMA) / Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPQ) / Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES) / Platelets, the blood cells involved in maintaining hemostasis, play a key role in the development of acute ischemic coronary, cerebrovascular events and are critically involved in the thrombosis process. In response to vascular injury, changes in blood flow or chemical stimuli, platelets trigger three functional mechanisms: adhesion, activation and aggregation. After platelet capture, a rapid stabilization of adhesion is required for thrombus formation to occur. Platelet activation results from conformational changes dependent of the protein disulfide isomerase (PDI), so that it has recently been proposed as a molecular target in platelet antiaggregant activity. The use of plant species rich in phenolic compounds as a source of bioactive substances is a promising strategy for the development of new therapeutic alternatives for thromboembolic diseases. Previously, we have shown that Syzygium cumini (L.) Skeels leaf contains multiple polyphenols, which support its use for antiplatelet purposes. Therefore, this study sought to evaluate the effects of polyphenol-rich extract (PESc) from S. cumini leaf on platelet activation and aggregation, as well as on PDI reductase activity. Platelet-rich plasma from healthy volunteers (n=5) was incubated with PESc (10-1000 μg/mL), for 25 min, before activation with ADP, thrombin or PMA. To analyze PESc effect on integrin αIIbβ3 activation, flow citometry protocols were conducted in washed platelets pre-treated with PESc (10-1000μg/mL) and activated with thrombin before tagging with PAC–1 antibody. Finally, PESc (0.1-100 μg/mL) effects on PDI reductase activity were assessed in absence or presence of polyphenolic standards gallic acid, myricetin and quercetin. PESc dose-dependently inhibited platelet aggregation despite the agonist used, even though lower agonist concentration potentiated PESc inhibitory effects to a maximal 77% inhibition at 2.5 μM ADP. Similarly, PESc dose-dependently reduced the proportion of activated αIIbβ3 molecules per platelet up to one third of control at 1000 μg/mL. These effects correlated with the strong inhibitory action of PESc on PDI activity, an effect synergically augmented in presence of standards. Therefore, our data show that PESc reduces platelet aggregation and activation, probably through PDI inhibition, strengthening its prominent antiplatelet activity. / As plaquetas, células sanguíneas envolvidas na manutenção da hemostase, exercem uma função essencial no desenvolvimento de eventos isquêmicos agudos coronarianos, cerebrovasculares e estão criticamente envolvidas no processo de formação da trombose. Em resposta à lesão vascular, às alterações no fluxo sanguíneo ou a estímulos químicos, as plaquetas desencadeiam três mecanismos funcionais: adesão, ativação e agregação. Após a captura da plaqueta, uma rápida estabilização da adesão é necessária para que ocorra a formação do trombo. A ativação plaquetária resulta de alterações conformacionais dependentes da proteína dissulfeto isomerase (PDI), de tal modo, que recentemente foi proposto o seu uso como alvo molecular na atividade antiagregante plaquetária. O uso de espécies vegetais ricas em compostos fenólicos como fonte de substâncias bioativas apresenta-se como uma estratégia promissora para o desenvolvimento de novas alternativas terapêuticas das doenças tromboembólicas. Anteriormente, temos mostrado que as folhas de Syzygium cumini (L.) Skeels contém múltiplos polifenóis, que apoiam a sua utilização para fins antiplaquetários. Portanto, este estudo procurou avaliar os efeitos do extrato rico em polifenóis (ERP) da folha de S. cumini sobre a ativação e agregação plaquetária, bem como, sobre a atividade redutase da PDI. Para tanto, plasma rico em plaquetas de voluntários saudáveis foi incubado com ERP (10 - 1000μg/mL), durante 25 min, antes da ativação com ADP, trombina ou PMA. Para analisar o efeito de ERP sobre a ativação da integrina αIIbβ3, os protocolos de citometria de fluxo foram conduzidos em plaquetas lavadas pré-tratadas com ERP (10 -1000 μg/mL) e ativadas com trombina antes da marcação com anticorpo PAC-1. Finalmente, os efeitos de ERP (0,1-100 μg/mL) na atividade redutase da PDI foram avaliados na ausência ou presença de padrões polifenólicos de ácido gálico, miricetina e quercetina. ERP inibiu a agregação plaquetária dependente da dose apesar do agonista utilizado, embora uma menor concentração de agonistas potencializasse os efeitos inibitórios de ERP até uma inibição máxima de 77% a 2,5 μM de ADP. De modo semelhante, o ERP reduziu a dose proporcionalmente a proporção de moléculas de αIIbβ3 ativadas por plaquetas até um terço do controle a 1000μg/mL. Estes efeitos correlacionaram-se com a forte ação inibitória de ERP na atividade da PDI, um efeito sinergicamente aumentado na presença de padrões. Portanto, nossos dados mostram que o ERP reduz a agregação e ativação plaquetária, provavelmente através da inibição da PDI, fortalecendo sua proeminente atividade antiplaquetária.

Submitted by Rosivalda Pereira (mrs.pereira@ufma.br) on 2017-06-14T18:35:57Z No. of bitstreams: 1 HiranReisSousa.pdf: 2489901 bytes, checksum: 8a82807f0600af87559439a496bd0d2a (MD5) / Made available in DSpace on 2017-06-14T18:35:57Z (GMT). No. of bitstreams: 1 HiranReisSousa.pdf: 2489901 bytes, checksum: 8a82807f0600af87559439a496bd0d2a (MD5) Previous issue date: 2016-12-06 / Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES) / Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPQ) / Fundação de Amparo à Pesquisa e ao Desenvolvimento Científico e Tecnológico do Maranhão (FAPEMA) / Recent researches have emphasized the importance of redox mechanisms for platelet function modulation. The platelet surface contains a large variety of integrin receptors and other molecules presenting functional thiol groups in their structures, which are potential targets for redox regulation. Among these various thiol-containing proteins, integrin αIIbβ3 stands out for being the convergence path of platelet activation induced by various agonists. Activation of αIIbβ3 integrin is catalyzed by protein disulfide isomerase (PDI) through an essential conformational change leading to the exposure of fibrinogen-binding site. Thus, PDI has been shown to be an important target for the development of antiplatelet drugs. In recent years, many studies have described substances from plan (DE A. PAES et al., 2011), as well as synthetics that are capable of inhibiting PDI. In a previous study of our research group has shown that the synthetic peptide CxxC, which contains the redox motif of PDI in its original sequence CGHC, inhibited reductase activity of this enzyme, effect not observed with AxxA peptide, whose cysteines were replaced with alanine and Scr peptide, which contains the same aminoacids from CxxC peptide, but under random sequence. It has been also demonstrated that CxxC peptide was the only to reduce by 30% ADP-induced aggregation (5μM) in platelet rich plasma, an effect apparently mediated by the association of CxxC and PDI at platelet surface. Thus, in this work, we further assessed the effects of CxxC and its control peptides on platelet aggregation. Washed human platelets were incubated with CxxC peptide at concentrations of 3, 6 and 10 μM, resulting in a dose-dependent inhibition of maximum aggregation activated by thrombin (0.02 U/mL) at 25, 60 and 74%, respectively with IC50 of 6.13 ± 1.09 μM. The presence of control peptides did not produce any inhibitory effect. CxxC peptide also reduced the activation of αIIbβ3 integrin at platelet surface, but did not affect the expression of the markers CD 62-P and CD 63. Control peptides did not alter the expression of these markers. Analysis by mass spectrometry of the interaction of recombinant human PDI with the peptide showed that only CxxC peptide associated with the redox Cys400 of a’ motif of PDI, which has been considered essential for platelet aggregation. Together, these results demonstrate that CxxC peptide reduces platelet aggregation by association with PDI and can be further used as a model for the development of new antithrombotic drugs. / Investigações recentes têm enfatizado a importância de mecanismos redox na modulação da função plaquetária. A superfície da plaqueta contém grande variedade de integrinas e outras moléculas receptoras que possuem tióis funcionais em sua estrutura, os quais são alvos potenciais de regulação redox. Dentre estas várias proteínas tiólicas, a integrina αIIbβ3 destaca-se por ser a via de convergência da ativação plaquetária induzida por diversos agonistas. A ativação da integrina αIIbβ3 é catalisada pela proteína dissulfeto isomerase (PDI), essencial à mudança de conformação que leva à exposição do sitio de ligação ao fibrinogênio. Sendo assim, a PDI tem se mostrado como um alvo importante para o desenvolvimento de fármacos reguladores da agregação plaquetária. Nos últimos anos, diversos estudos têm descrito substâncias de origem vegetal, animal e sintéticas que são capazes de inibir a PDI. Em trabalho do nosso grupo de pesquisa (DE A. PAES et al., 2011), demonstrou que o peptídeo sintético CxxC, o qual contém o motivo redox da PDI na sua sequência original CGHC, inibiu a atividade redutase desta enzima; efeito não observado com os peptídeos AxxA, que possui as cisteínas substituídas por alanina e Scr, peptídeo controle contendo os mesmos aminoácidos do peptídeo CxxC, porém com sequência aleatória sem formação de ditiol. Demonstrou-se, também, que apenas o peptídeo CxxC reduziu em 30% a agregação induzida por ADP (5M) em plasma rico em plaquetas, efeito aparentemente mediado pela associação do CxxC com a PDI na superfície plaquetária. Sendo assim, neste trabalho continuamos a avaliação dos efeitos do peptídeo CxxC e seus controles sobre a agregação plaquetária. Para tanto, incubamos lavado de plaquetas humanas com o peptídeo CxxC nas concentrações de 3, 6 e 10 μM, resultando em inibição concentração-dependente da agregação ativada por trombina (0,02 U/mL) em 25, 60 e 74 %, respectivamente, com IC50 de 6,13 ± 1,09 μM. A presença dos peptídeos controle não produziu quaisquer efeitos inibitórios. O peptídeo CxxC reduziu a ativação da integrina αIIbβ3 na superfície da plaqueta, porém não impactou a expressão dos antígenos CD 62-P e CD 63. Os peptídeos controle não alteraram a expressão desses marcadores. A análise por espectrometria de massas da interação da PDI recombinante humana com os peptídeos, mostrou que apenas o peptídeo CxxC associa-se com a Cys400 do motivo redox a’ da hPDI, o qual tem sido considerado fundamental para a agregação plaquetária. Em conjunto, estes resultados demonstram que o peptídeo CxxC reduz a agregação plaquetária via associação com a PDI, podendo ser empregado como modelo para o desenvolvimento de fármacos novos antitrombogênicos.