Le traitement des patients avec une coronarographie après un SCA est l'aspirine et les thiénopyridines. La réponse aux thiénopyridines est variable, cette variabilité, multifactorielle, a des répercutions cliniques. Leur efficacité a été évaluée sur la réduction de la survenue d'évènements cliniques et peu sur le risque d'hémorragie qui est un effet indésirable majeur. Les plaquettes, jouent un rôle dans l'athérosclérose et les SCA notamment par le CD40L.J'ai étudié les facteurs plaquettaires conditionnant le risque hémorragique chez ces patients et apporté un éclairage sur des fonctions plaquettaires peu connues comme l'inflammation. Les génotypes du cytochrome P450 CYP2C19*2 et *17 ont une influence sur la réponse plaquettaire aux thiénopyridines et il existe une relation entre les complications hémorragiques et la réactivité plaquettaire.Une très faible réactivité plaquettaire (VASP<10%) est un facteur prédictif du risque hémorragique et les valeurs de VASP < 10 % sont plus fréquentes chez les patients traités par prasugrel. Nous avons ensuite ciblé un marqueur de l'état inflammatoire plaquettaire, le CD40L. Sa libération plaquettaire dépend de la voie du P2Y12, son expression, elle, dépend moins de cette voie. Une faible expression du CD40L est associée à des évènements hémorragiques chez les patients traités par thiénopyridines.Ainsi le déterminisme génétique de l'efficacité du traitement par thiénopyridines a un impact sur le risque hémorragique et d'autres paramètres plaquettaires influencent ce risque indépendamment de l'inhibition de l'agrégation plaquettaire. Le CD40L, serait un lien entre l'inflammation et l'équilibre saignement/thrombose. / Aspirin and thienopyridine are the therapy for patients with percutaneous coronary intervention after ACS. The level of platelet inhibition by thienopyridine varies between patients, this variability, multifactorial, is associated with adverse clinical outcomes. Treatment efficacy was evaluated mainly on the association between poor thienopyridine response and thrombotic events but less on the principal side effect: bleeding complications. Platelet play a key role in atherosclerosis and thrombosis, notably via CD40L.I studied platelet factors that influence the bleeding risk in these patients and brought a new highlight on platelet function less known such as inflammation.P450 cytochrome genetic variants (2C19*2 and 2C19*17) influence platelet response to thienopyridines. There is a relation between platelet reactivity and bleeding events. A very low on-treatment platelet reactivity (VASP<10 %) is a predictor of bleeding and is mainly observed with prasugrel treatment. We then focussed on a marker of platelet inflammatory status, CD40L. Its release by platelets depends on P2Y12 signalling, whereas its surface expression is less dependent on this signalling pathway. A low platelet-CD40L surface expression is associated with bleeding events in these patients We show that genetic background on thienopyridine treatment efficacy is related to bleeding risk and that other platelet parameters influence the bleeding risk independently of platelet aggregation inhibition. Thus, a molecule of inflammation, CD40L, would be a link between inflammation and bleeding/thrombosis equilibrium.

Orientador: Sisi Marcondes Paschoal / Dissertação (mestrado) - Universidade Estadual de Campinas, Faculdade de Ciências Médicas / Made available in DSpace on 2018-08-26T18:31:06Z (GMT). No. of bitstreams: 1 Bonfitto_PedroHenriqueLeite_M.pdf: 791921 bytes, checksum: 967c29bbcdf8e6df7bd3a2d919b84111 (MD5) Previous issue date: 2015 / Resumo: As plaquetas são importantes células na inflamação, entretanto, os trabalhos que estudam as citocinas na reatividade plaquetária são raros. O objetivo do presente trabalho foi estudar os efeitos do fator de necrose tumoral-alfa (TNF-?) em plaquetas. Ensaios de agregação foram realizados incubando-se plaquetas com crescentes concentrações de TNF-? (1 - 3000 pg/ml) por diferentes intervalos de tempo (5 - 60 min), na ausência ou presença do antagonista não seletivo dos receptores TNFR1 e TNFR2, o R7050. Também foi estudado o efeito do TNF-? na viabilidade plaquetária utilizando-se o MTT. O efeito do TNF-? na mobilização de Ca2+ em plaquetas foi investigado através de ensaios de fluorescência utilizando-se o fluo-3-AM; os ensaios de western blotting foram realizados para o estudo da ativação da enzima c-Src e do receptor de fibrinogênio. Finalmente, foram determinados os níveis intraplaquetários de AMPc e GMPc por ELISA. O TNF-? inibiu a agregação plaquetária induzida por ADP ou trombina de forma dependente da concentração da citocina e do tempo de incubação. O efeito inibitório máximo do TNF-? na agregação induzida por ADP (5 ?M) foi obtido com a concentração de 300 pg/ml por um tempo de incubação de 30 min (90 ± 7% de inibição), o qual foi significativamente prevenido pela pré-incubação das plaquetas com o R7050. A viabilidade plaquetária não foi modificada pela incubação por 60 min com o TNF-? (30 e 3000 pg/ml). A incubação de plaquetas com TNF-? (300 pg/ml, 30 min) reduziu em 53% o aumento da concentração de Ca2+ total causado pela adição de trombina (200 mU/ml). A queda da concentração de Ca2+ citosólica plaquetária causada pelo TNF-? foi em decorrência da diminuição em 1,8 e 3,4 vezes da mobilização interna do íon e do influxo do mesmo, respectivamente. O TNF-? reduziu em 60% os níveis de AMPc em plaquetas ativadas com ADP. Por outro lado, o TNF-? aumentou significativamente os níveis de GMPc em plaquetas ativadas por ADP (aumento de 51%). A pré-incubação de plaquetas com o inibidor da guanilil ciclase ODQ não reduziu o efeito inibitório do TNF-? na agregação induzida por ADP. Os ensaios de western blotting mostraram que o TNF-? reduziu significativamente a fosforilação do resíduo de Tyr416 da c-Src em plaquetas ativadas. Da mesma forma, o TNF-? reduziu em 37% a fosforilação do resíduo de Tyr773 da subunidade ?3 da integrina ?IIb?3 (receptor de fibrinogênio) em plaquetas ativadas por ADP. Portanto concluímos que o TNF-? inibe a agregação plaquetária via receptores TNFR1 e/ou TNFR2, sem reduzir a viabilidade das plaquetas. O efeito inibitório do TNF-? na agregação é acompanhado pela redução de Ca2+ citosólico e inibição de c-Src e do receptor de fibrinogênio em plaquetas, sendo estes independentes de AMPc ou GMPc / Abstract: Platelets have been described as important cells in inflammation; however, the effects of cytokines on platelet reactivity are rarely studied. The objective of the present work was to investigate the effects of the tumor necrosis factor-alpha (TNF-?) in platelets. Aggregation assays were carried out incubating platelets with increasing TNF-? concentrations (1 - 3000 pg/ml) for different intervals of times (5 - 60 min), in the absence or in presence of the non-selective antagonist of TNFR1 and TNFR2, R7050. Effect of TNF-? on platelet viability was determined using MTT. The effect of TNF-? on the Ca2+ mobilization in platelets was investigated through fluorescence assays using fluo-3AM and Western blotting assays were carried out to determine the activation of c-Src and the fibrinogen receptor. Finally, the cAMP and cGMP levels in platelets were determined by ELISA. TNF-? dose- and time-dependently inhibited ADP or thrombin-induced platelet aggregation. The inhibitory effect of TNF-? on ADP(5 ?M)-induced platelet aggregation was maximum in a concentration of 300 pg/ml incubated with platelets for 30 min (90 ± 7% of inhibition), which was significantly prevented by the incubation of platelets with R7050. Platelet viability was not modified by TNF-? (30 and 3000 pg/ml) incubated for 5 to 60 min. Incubation of platelets with TNF-? (300 pg/ml, 30 min) reduced the increased total Ca2+ concentration induced by thrombin (200 mU/ml) by 53%. Decreasing Ca2+ internal mobilization (1,8 fold) and decreasing in external Ca2+ influx (3,4 fold) led to a reduction of total cytosolic Ca2+ in TNF-? activated platelets. TNF-? reduced the cAMP levels in ADP-activated platelets by 60%. On the other hand, TNF-? significantly increased cGMP levels in ADP-activated platelets (51% increase). Pre-incubation of platelets with the guanylyl cyclase inhibitor ODQ did not modify the inhibitory effect of TNF-? on ADP-induced platelet aggregation. Western blotting analysis showed that TNF-? significantly reduced phosphorylation on Tyr416 of c-Src in activated platelets. Similarly, TNF-? reduced by 37% the Tyr773 phosphorylation of ?3 subunit of ?IIb?3 integrin (fibrinogen receptor) in ADP-activated platelets. Therefore, our results show that TNF-? inhibits platelet aggregation via TNFR1 and/or TNFR2 receptors, without affecting platelet viability. The inhibitory effect of TNF-? on aggregation is accompanied by a reduction in cytosolic Ca2+ and the inhibition of c-Src and fibrinogen receptor activation, which are cAMP and cGMP-independent effects / Mestrado / Farmacologia / Mestre em Farmacologia

Indiana University-Purdue University Indianapolis (IUPUI) / Platelet-activating Factor (PAF) stimulates various cell types by the activation of the G-protein coupled PAF-receptor (PAFR). Systemic PAFR activation induces an acute pro-inflammatory response, as well as delayed systemic immunosuppressive effects in vivo. De novo enzymatic PAF synthesis and degradation are closely regulated, but oxidative stressors, such as UVB, and cigarette smoke, can generate PAF-like species via the oxidation of membrane lipids in an unregulated process. Mast cells (MCs) and the PAFR have been shown to be necessary to mediate the resulting systemic immune suppression from oxidative stressors. The work herein implicates pro-oxidative chemotherapeutics, such as melphalan and etoposide, in mediating augmentation in tumor growth by inducing the generation of PAFR agonists via the oxidation of membrane lipids. This work also demonstrates the role of MCs and MC-released mediators in PAFR systemic immunosuppression. Through a contact hypersensitivity (CHS) model, the MC PAFR was found to be necessary and sufficient for PAF to mediate systemic immunosuppression. Additionally, activation of the MC PAFR seems to induce MC histamine and prostaglandin E2 release. Furthermore, by transplanting histamine- or COX-2-deficient MCs into MC-deficient mice, MC-derived histamine and prostaglandin release were found to be necessary for PAF to induce systemic immunosuppression. Lastly, we have evidence to suggest that prostaglandin release modulates MC migration to draining lymph nodes, a process necessary to promote immunosuppression. These studies fit with the hypothesis that MC PAFR activation mediates PAFR systemic immunosuppression in part by histamine and prostaglandin release.

No description available.

Aufgrund der verfügbaren Literatur und Daten ist nicht erwiesen, dass eine prophylaktische Thrombozytentransfusion nach myeloablativer Chemotherapie notwendig oder für den Patienten vorteilhaft ist. Die im Verlauf der Jahre immer weiter gesenkten Schwellenwerte zur prophylaktischen Thrombozytentransfusion legten nahe, die Möglichkeit zu überprüfen, auf eine prophylaktische Substitution ganz zu verzichten und nur im Falle relevanter Blutungen zu transfundieren. Mit der hier ausgewerteten Studie liegen erstmals Daten aus einer multizentrischen, prospektiv randomisierten Studie zum Vergleich einer prophylaktischen mit einer therapeutischen Transfusionsstrategie für Thrombozyten nach autologer Stammzelltransplantation vor: es wurde eine prophylaktische Thrombozytentransfusion bei Thrombozytenwerten ≤ 10/nl mit einer neuen Transfusionsstrategie (Substitution nur bei relevanter Blutung oder definierten Risikosituationen) verglichen. Mit der experimentellen, therapeutischen Transfusionsstrategie für Thrombozyten kann eine Reduktion der Thrombozytentransfusionen um ca. 50% im Vergleich zu dem etablierten prophylaktischen Transfusionsregime erreicht werden: bei den hier untersuchten 92 Patienten wurden im experimentellen Arm für 47 Patienten nur 37 Thrombozytenkonzentrate benötigt, für die 45 prophylaktisch behandelten Patienten wurden insgesamt 71 Thrombozytenkonzentrate verbraucht. Die experimentelle therapeutische Transfusionsstrategie für Thrombozyten führte zu keiner statistisch signifikanten Zunahme von Blutungskomplikationen; auch bei der Anzahl der benötigten Erythrozytentransfusionen gab es keine signifikanten Unterschiede; Nebenwirkungen der Transfusionen, Dauer der Thrombopenie und Anzahl der Tage im Krankenhaus waren ebenso nicht signifikant unterschiedlich. Das Risiko, während der Beobachtungszeit (Chemotherapie und autologe Stammzelltransplantation bis zur Regeneration der Thrombozytenwerte), eine Blutung zu erleiden, lag insgesamt bei 14.1%; im experimentellen Arm lag das Risiko bei 19.2%, bei den prophylaktisch substituierten Patienten bei 8.9%; dieser Unterschied war statistisch nicht signifikant, ohnehin traten bei den beobachteten Patienten nur milde, klinisch wenig bedeutsame Blutungen des WHO – Schweregrades &amp;lt; 3 auf, es kam zu keinen blutungsassoziierten Todesfällen Bei klinisch stabilen Patienten und sorgfältiger Überwachung ist ein therapeutisches Transfusionsregime für Thrombozyten nach autologer Stammzelltransplantation praktikabel und sicher anwendbar, die Sicherheit dieses Vorgehens bei Patienten nach autologer Stammzelltransplantation wird mit der vorliegenden randomisierten Studie belegt. Eine therapeutische Thrombozytentransfusionsstrategie ist vermutlich bei einer Vielzahl weiterer hämato-onkologischer Patienten bzw. Krankheitsbilder ausreichend und kann unter signifikanter Einsparung kostbarer Thrombozytenkonzentrate bedrohliche Blutungen ebenso aufhalten oder verhindern wie ein prophylaktisches Regime.

Hemostasis, thrombosis, and inflammation are tightly interconnected processes which may give rise to thrombo-inflammation, involved in infectious and non-infectious acute and chronic diseases, including cardiovascular diseases (CVD). Traditionally, due to its hemostatic role, blood coagulation is isolated from the inflammation, and its critical contribution in the progressing CVD is underrated, until the full occlusion of a critical vessel occurs. Underlying vascular injury exposes extracellular matrix to deposit platelets and inflammatory cells. Platelets being key effector cells, bridge all the three key processes (hemostasis, thrombosis, and inflammation) associated with thrombo-inflammation. Under physiological conditions, platelets remain in an inert state despite the proximity to the endothelium and other cells which are decorated with glycosaminoglycan (GAG)-rich glycocalyx (GAGs). A pathological insult to the endothelium results in an imbalanced blood coagulation system hallmarked by increased thrombin generation due to losses of anticoagulant and cytoprotective mechanisms, i.e., the endothelial GAGs enhancing antithrombin, tissue factor pathwayinhibitor (TFPI) and thrombomodulin-protein C system. Moreover, the loss of GAGs promotes the release of mediators, such as von Willebrand factor (VWF), platelet factor 4 (PF4), and P-selectin, both locally on vascular surfaces and to circulation, further enhancing the adhesion of platelets to the affected sites. Platelet-neutrophil interaction and formation of neutrophil extracellular traps foster thrombo-inflammatory mechanisms exacerbating the cardiovascular disease course. Therefore, therapies which not only target the clotting mechanisms but simultaneously or independently convey potent cytoprotective effects hemming the inflammatory mechanisms are expected to provide clinical benefits. In this regard, we review the cytoprotective protease activated protein C (aPC) and its strong anti-inflammatory effects thereby preventing the ensuing thrombotic complications in CVD. Furthermore, restoring GAGlike vasculo-protection, such as providing heparin-proteoglycan mimetics to improve regulation of platelet and coagulation activity and to suppress of endothelial perturbance and leukocyte-derived pro-inflammatory cytokines, may provide a path to alleviate thrombo-inflammatory disorders in the future. The vascular tissue-modeled heparin proteoglycan mimic, antiplatelet and anticoagulant compound (APAC), dual antiplatelet and anticoagulant, is an injury-targeting and locally acting arterial antithrombotic which downplays collagen- and thrombin-induced and complement-induced activation and protects from organ injury.

No description available.

<p> Inherited bleeding disorders can be caused by mutations affecting platelet, coagulation, or fibrinolytic proteins. Quebec platelet disorder (QPD) is a rare, autosomal dominant disorder associated with increased expression of the fibrinolytic enzyme urokinase plasminogen activator (uPA) in platelets. Individuals with QPD experience delayed-onset bleeding after hemostatic challenges that is attenuated with fibrinolytic inhibitor therapy. The aims of this thesis were to: 1) determine if increased platelet uPA contributes to QPD clot lysis in vitro; 2) investigate whether QPD individuals have increased urinary uPA, as some individuals experience hematuria; and 3) map the genetic locus of QPD, and look for the putative mutation. Studies of clot lysis indicated that QPD platelets induce a gain-of-function defect in fibrinolysis when platelets are incorporated into clots. This suggests that accelerated fibrinolysis may contribute to QPD bleeding. Studies of urinary uPA in QPD showed that uPA is not increased, indicating that hematuria in QPD is likely a consequence of increased platelet uPA. This finding also suggests that uPA overexpression in QPD may be megakaryocyte-specific. Linkage studies showed that QPD is strongly linked to a 2 megabase region on chromosome 10 that harbors the uPA gene, PLA U. No mutations in PLA U or its regulatory regions were identified; however, a common haplotype for a 32.5 kilobase region around PLA U, including inheritance of a rare, linked polymorphism, suggests this is the most likely locus for QPD. mRNA studies in QPD platelets showed that QPD selectively increases expression of the linked PLAU allele, without similar increases in megakaryocyte progenitors or in saliva. These findings implicate a cis-mutation near PLA U as the cause of QPD. This thesis provides novel insights on the fibrinolytic abnormality in QPD blood, and on the QPD genetic locus. which will be important for identifying the precise mutation that converts normally prohemostatic platelets to profibrinolytic cells. </p> / Thesis / Doctor of Philosophy (PhD)

No description available.

Background: Platelets provide the initial haemostatic plug at sites of vascular injury. They also participate in pathological thrombosis that leads to myocardial infarction, stroke and peripheral vascular disease. The outcome of an acute myocardial infarction depends not only on the formation and stability of an occlusive thrombus, but also on the efficacy of the endogenous thrombolytic process, which allows reperfusion of the infarct related artery and prevents recurrent ischaemic episodes. Various platelet function tests are available to measure the thrombogenic potential of an individual, but the sensitivity of these tests remain questionable as most of these tests use citrated blood and measure response to a particular agonist. Endogenous thrombolysis has been a neglected entity, and its beneficial effects on cardiovascular outcomes has not been studied in depth in the past, possibly as until recently there has been no available technique to measure spontaneous thrombolytic activity in native blood. The Global Thrombosis Test (GTT) is a new point of care tests that allows us to measure time to thrombus formation (Occlusion time: OT) using native blood, avoiding the use of agonists and making the test results more physiological. The GTT also measures the time to lyse this formed thrombi without use of any lytic agents (Lysis time: LT), allowing us to measure the patient’s endogenous thrombolytic potential. Aim: Our aim in this study was to detect patients who are at risk of future thrombotic events despite dual antiplatelet therapy, either due to prothrombotic tendency or due to impaired endogenous thrombolysis, and to determine if these two parameters were correlated. Methods: GTT was used to assess the thrombotic and thrombolytic activity in healthy volunteers, and in different patient populations. 100 healthy volunteers were tested using the GTT, and a normal range was established. 300 patients admitted to hospital with a diagnosis of acute coronary syndrome (ACS) were included in the study, and tested using the GTT after they had been stabilized on dual antiplatelet therapy (Aspirin and Clopidogrel). All these patients were followed up for a year, to determine if their baseline GTT results were a predictor of recurrent cardiac events. The primary endpoint of the study was major adverse cardiovascular events (MACE), which was a composite of cardiovascular death, nonfatal myocardial infarction, or stroke at 12 months. Results: All results were analysed using statistical package SPSS version 16.0 (SPSS Inc., Chicago, Illinois). The 100 healthy volunteers were all non-smokers, and were not taking any medications. There were 55 males and 45 females, and mean age was 38±11 years (range 22-76, IQR 11). OT was normally distributed with mean OT 377.80s, and using mean ± 2SD, we derived a normal range of 185-569s (200-550s). LT demonstrated a skewed distribution with values ranging between 457 – 2934s. Using log transformation, a normal range of 592 – 1923 (600- 2000s) was established for LT. OT and LT were both prolonged in ACS patients compared to normal volunteers (p< 0.001). No association was observed between OT and risk of major adverse cardiovascular events. LT was noted to be a significant and independent predictor of MACE in a multivariate model adjusted for cardiovascular risk factors. LT ≥ 3000 s was the optimal cutoff value for predicting 6 month MACE [hazard ratio (HR): 2.48, 95% CI: 1.2-4.8, P= 0.008] and cardiovascular death [HR: 4.04, 95% CI : 1.3-12.0, P= 0.012 ] and 12 month MACE [HR:1.9, 95% CI: 1.04- 3.5,P= 0.03] and cardiovascular death [HR: 3.9,95% CI: 1.34-11.9, P= 0.013 ]. LT ≥ 3000 s was observed in 23% of ACS patients. Conclusions: Our study suggests that endogenous thrombolytic activity based on lysis of platelet rich thrombi can be assessed by the point of care GTT assay, which can help in identification of ACS patients at high risk of future cardiac events. Prolongation of OT may be explained by the antiplatelet effects of Aspirin and Clopidogrel, as both these drugs prolong time to thrombus formation and hence increase OT. Further large studies are required to study factors which can reduce thrombogenic potential, and improve endogenous thrombolytic activity, which can be monitored using the GTT to improve cardiovascular outcomes.