Дисертації з теми "Thrombotic risk"

Thrombosis is common in Behçet’s Syndrome (BS), and there is a clear need both for a better understanding of causation and for biomarkers to enable thrombotic risk assessment. I set out to determine whether plasma microparticles (MP), particularly tissue factor expressing (TF+ MP) were increased in BS. Additionally, I investigated whether MP expressing tissue factor pathway inhibitor (TFPI) were also evident. Finally, I investigated whether these MP had a functional consequence role. MP were prepared from 88 BS patients and 72 healthy controls. The BS group contained 21 patients with a history of thrombosis (Th+) and 67 without (Th-). MP were identified by size and annexin V binding using flow cytometry, and were further analysed with antibodies to surface antigens. Assays used to assess the function of MP were an in-house coagulation lysis assay, calibrated automated thrombin generation assay and Factor Xa generation assay. Total MP numbers were increased in BS compared to HC, as were MP expressing TF and TFPI (all p < 0.001). Amongst BS patients, the Th+ group had increased total and TF positive MP (both p < 0.001) compared to the Th- group, but a lower proportion of TFPI positive MPs (p < 0.05). Consequently, the ratio of TFPI to TF MP counts (TFPI/TF) was significantly lower in Th+ versus Th- BS patients (p < 0.001), with no history of clinical thrombosis in BS patients with a TFPI/TF MP ratio > 0.7. Functional assays revealed no correlation with MP count. I conclude that MP expressing TF are increased in BS, more so in patients with thrombosis. An imbalance between microparticulate TF and TFPI may be pathophysiologically important for thrombosis in BS and may allow improved identification and appropriate treatment of thrombotic risk.

Oxidative damage has been suggested to play a key role in the pathogenesis of atherosclerosis and other cardiovascular disease. Increased free radical production induced by oxidative stress can oxidise low density lipoproteins, activates platelets, induces endothelial dysfunction and disturbs glucose transport by consuming endogenous antioxidants. Using a combination, of in vitro and in vivo experimental models, the primary aims of the studies undertaken for this thesis were to examine whether different antioxidants could negate risk factors leading to thrombosis, atherosclerosis and other cardiovascular diseases. The studies utilised the mechanisms involved in platelet activity and glucose uptake by skeletal muscle myotubes. The first study determined if olive leaf extract would attenuate platelet activity in healthy human subjects. Blood samples (n=11) were treated with five different concentrations of extract of Olea europaea L. leaves ranging from 5.4£gg/mL to 54£gg/mL. A significant reduction in platelet activity (pless than0.001) and ATP release from platelets (p=0.02) was observed with 54£gg/mL olive leaf extract. The next crossover study compared the effect of exercise and antioxidant supplementation on platelet function between trained and sedentary individuals. An acute bout of 1 hour exercise (sub maximal cycling at 70% of VO2max) was used to induce oxidative stress in 8 trained and 8 sedentary male subjects, before and after one week supplementation with 236 mg/day of cocoa polyphenols. Baseline platelet count and ATP release increased significantly (pless than0.05) after exercise in all subjects. Baseline platelet numbers in the trained were higher than in the sedentary (235¡Ó37 vs. 208¡Ó34 x109/L, p less than 0.05), whereas platelet activation in trained subjects was lower than sedentary individuals (51¡Ó6 vs. 59¡Ó5%, p less than0.05). Seven days of cocoa polyphenol supplementation did not change platelet activity compared to the placebo group. The third study determined the effect of 5 weeks of either 100mg/day £^-Tocopherol (n=14), 200mg/d £^-Tocopherol (n=13) or placebo (n=12) on platelet function, lipid profile and the inflammatory marker C-reactive protein. Blood £^-tocopherol concentrations increased significantly (pless than0.05) relative to dose. Both doses attenuated platelet activation (pless than0.05). LDL cholesterol, platelet aggregation and mean platelet volume were decreased by 100mg/d £^-tocopherol (all pless than0.05). The final study determined the effect of glucose oxidase induced oxidative stress and £^-tocopherol treatment on glucose transport and insulin signalling in cultured rat L6 muscle cells. One hour treatment with 100mU/mL glucose oxidase significantly decreased glucose uptake both with and without 100nM insulin stimulation (pless than0.05). Pre-treatment with 100ƒÝM and 200ƒÝM £^-tocopherol partially protected cells from the effect of glucose oxidase, whereas 200ƒÝM £^-tocopherol restored both basal and insulin stimulated glucose transport to control levels. Glucose oxidase-induced oxidative stress did not impair basal or insulin stimulated phosphorylation of Akt or AS160, but 200ƒÝM £^-tocopherol improved insulin-stimulated phosphorylation of these proteins. In summary, the results from the studies undertaken for this thesis provide evidence that antioxidant supplementation maintains normal platelet function, exerts a positive effect on blood lipid profile and improves glucose uptake in normal healthy asymptomatic population as well as under conditions of induced oxidative stress. Antioxidants including foods rich in cocoa, olive and gamma tocopherol have the potential to combat oxidative stress induced risk factors leading to cardiovascular diseases.

End stage renal disease (ESRD) patients have an excess cardiovascular risk, above that predicted by traditional risk factor models. Despite the advances in both Cardiovascular disease (CVD) management and renal replacement therapy (RRT), there still is a major burden of cardiovascular mortality and morbidity in the chronic kidney disease (CKD) population. Declining renal function itself represents a continuum of cardiovascular risk and in those individuals who survive to reach ESRD, the risk of suffering a cardiac event is uncomfortably and unacceptably high. Pro-thrombotic status may contribute to this increased risk. Global thrombotic status assessment, including measurement of occlusion time (OT) the time taken to form an occlusive platelet rich thrombus and thrombolytic status (time taken to lyse such thrombus) as assessed by measuring Lysis Time (LT), may identify vulnerable patients. The aim of this study was to assess overall thrombotic status in ESRD and relate this to cardiovascular and peripheral thrombotic risk. Small sub studies were also planned to establish the effect of RRT modality on the thrombotic status.

The haemostatic response comprises the interaction of coagulation factors and peripheral blood cells which play a vital role in maintaining vascular integrity. This study begins with the finding that plasma from premature MI subjects has increased endogenous thrombotic potential linked to higher levels of circulating Tissue Factor (TF), suggesting these individuals may have a hypercoagulable phenotype. This study seeks to further understand how cellular interaction, in particular platelet-monocyte interaction, modulates the haemostatic response. Throughout the study, TF and Tissue Factor Pathway Inhibitor (TFPI) were studied as representing the procoagulant and anticoagulant response, respectively. Study of the effects of activated platelets on monocytes found that whereas direct platelet or platelet-microparticle adhesion to monocytes, and release of platelet soluble mediators induced monocyte gene expression of TF, induction of TFPI was driven solely by platelet soluble mediators. Extension of these studies using gene expression microarray technology found that whereas activation of monocytes via PSGL-1 generates a pro-angiogenic expression profile, platelet soluble mediators significantly enhanced this profile, enabling monocytes to interact with ECM components involved in the wound healing environment of the thrombus, and additionally induced anti-inflammatory, and anti-atherothrombotic genes. Whether cells within a thrombus act simply as structural and secretory components, or play a more active role involving gene expression is unclear, therefore gene expression array analysis was carried out on thrombi generated in vitro. Genes demonstrating significant time-dependent increases included those encoding chemotactic proteins (IL8, CCL2, CXCL1, CXCL2), cell adhesion (ITGAV, ITGA5, ITGB1), regulation of coagulation (THBD, PLAU, SERPINE1), wound-healing (ENDG, SPP1, LAMB3), and regulatory transcription factors (FOS, EGR1, PPARG). Whereas initiation of thrombosis is driven by plasma proteins and facilitated by the platelet surface, this study provides evidence that thrombus resolution may be driven by changes in gene expression within the thrombus that regulate the haemostatic response, thrombus growth, and facilitate wound-healing. These findings could have implications for individuals at risk of plaque rupture, where variation in gene expression may affect not just the formation of an occlusive thrombus but also the rate of resolution.

Gilbert’s syndrome (GS) is a common genetic condition associated with mildly elevated unconjugated bilirubin (UCB) concentrations and increased protection against development of cardiovascular disease (CVD). Mutation in the uridine diphosphate glucuronosyltransferase (UGT1A1) gene causing a reduction in UGT1A1 enzyme activity that is responsible for conjugation and elimination of UCB, is considered as the cause for GS. Numerous studies have shown that elevated levels of UCB are negatively associated with risk of developing CVD. While, several in vitro and in vivo trials have shown that UCB at physiological concentrations can improve endothelial function, lipid profile and reduce vascular inflammation, thereby imparting its cardiovascular protection. Cardiovascular disease is a major cause of death globally, an estimated 17.3 million CVD related deaths are reported annually by the World Health Organization (WHO), making it the largest cause of human mortality. Furthermore, several studies have predicted that these numbers would continue rising in the coming years, due to factors such as aging, life style choices and environmental changes.
Thesis (PhD Doctorate)
Doctor of Philosophy (PhD)
School of Medical Science
Griffith Health
Full Text

A left ventricular thrombus develops in approximately 40% of patients following an anterior myocardial infarction. Embolization from these thrombi has been regarded as the most important cause of stroke following a myocardial infarction. The occurrence and characteristics of left ventricular thrombi and stroke after anterior myocardial infarction may, however, have changed after the introduction of aspirin and thrombolytics as standard therapy. The occurrence of left ventricular thrombi was examined in 99 patients with an acute anterior myocardial infarction, 74 of whom were treated with streptokinase. Thrombi were equally common in the thrombolysis group (46%, 95% confidence interval [Cl], 35-57%) as in the non-thrombolysis group (40%, 95% Cl, 21-59%). The risk of thrombus formation was related to the degree of left ventricular segmental dysfunction. Using serial echocardiographic examinations, the formation and resolution of thrombi was found to be highly dynamic. The majority of thrombi diagnosed during the hospital stay had resolved at follow-up one month later, irrespective of treatment with streptokinase or anticoagulants. The development of new thrombi was, however, observed at every follow-up examination interval. One-hundred-and-twenty-four patients suffering a stroke within 28 days of an acute myocardial infarction were identified in the northern Sweden MONICA stroke registry between 1985 and 1994. The overall event rate of ischemic myocardial infarction-related stroke was 1.07%. The risk of a stroke was highest duringt he first 5 days after the infarction. Only approximately half the strokes were preceded by an anterior myocardial infarction. In a case-control analysis, atrial fibrillation (chronic or new onset), ST elevation and a history of a previous stroke were found to be independent predictors of stroke. There was a long-term trend towards a lower incidence and event rate for myocardial infarction-related stroke. Clinical stroke characteristics were examined in 103 patients with a first-ever stroke within 28 days of a myocardial infarction and compared with stroke characteristics in 206 control subjects without a recent myocardial infarction. The sudden onset of neurological symptoms, an impairment of consciousness, a progression in neurological deficits and a stroke of the total anterior circulation infarction subclass were more common in cases than in controls. The risk of a recurrent stroke during one year of follow-up was not influenced by a recent myocardial infarction, but patients who had suffered a myocardial infarction had markedly higher mortality. To conclude, thrombolytic treatment does not reduce the occurrence of left ventricular thrombi after a myocardial infarction. The risk of thrombus formation is related to the extent of the myocardial injury. The development and resolution of thrombi is a highly dynamic process. There is a long-term trend towards a lower incidence and event rate of ischemic stroke after a myocardial infarction. Although the clinical stroke characteristics differ, they are not specific enough to differentiate between patients with and without a recent myocardial infarction.

S. 1-84: sammanfattning, s. 85-136: 5 uppsatser

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La contraception hormonale combinée (CHC) est la contraception la plus utilisée en France. La maladie veineuse thromboembolique (MVTE), constitue le principal effet délétère de ces CHC. Des recommandations de bonnes pratiques sont publiées pour guider les prescripteurs. La meilleure compréhension des modifications biologiques associées aux différents types de CHC, les caractéristiques cliniques des femmes ayant eu une MVTE, la place de la recherche d’antécédents familiaux de MVTE (AFVTE) et d’une thrombophilie biologique avant la prescription d’une CHC constituent des pistes de recherche qui permettrait potentiellement d’optimiser la balance bénéfice-risque des CHC. Enfin l’impact de l’utilisation d’une contraception hormonale après un 1er épisode de MVTE reste peu évalué. Ce travail de thèse a été réalisé à la fois à l’aide de données biologiques d’utilisatrices de contraceptions hormonales (Etude EDGAR) et aussi à partir des données de l’étude de cohorte française COREVE (COntraception and REcurrent Venous Event). Cette étude a inclus 3121 femmes de moins de 45 ans au moment de leur 1er épisode de MVTE. Nous nous sommes particulièrement intéressés à l’analyse des caractéristiques de ces femmes en fonction du type de contraception utilisée, à la prévalence des facteurs de risque vasculaires et notamment les AFVTE. La fréquence d’épisode de MVTE associé à une prescription inadaptée de CHC, variait ainsi de 8.8 à 25.9 %. Par ailleurs, à l’aide d’une méthodologie de type cas versus cas, l’interaction entre l’utilisation d’une CHC et la présence d’une mutation du facteur V Leiden sur le risque de MVTE diffère significativement en fonction du progestatif combiné des CHC
Combined hormonal contraception (CHC) is the most widely used contraception in France in which venous thrombosis embolism (VTE) is the main deleterious effect. Best practice recommendations are published in order to guide prescribers.The better understanding of the biological changes associated with different types of CHC, the clinical characteristics of women with VTE, the place of family history of VTE (FHVTE) and for biological thrombophilia before prescribing a CHC constitute research paths that could potentially optimize the risk-benefit balance of CHCs. Finally, the impact of hormonal contraception use after a first episode of VTE remains rarely evaluated.This work was carried out both using biological data from hormonal contraception users (EDGAR study) and also using data from the French cohort study COREVE (COntraception and REcurrent Venous Event).This study included 3121 women under 45 at the time of their 1st episode of VTE. We were particularly interested in analyzing the characteristics of these women according to the type of contraception used, the prevalence of vascular risk factors and especially the FHVTE.The frequency of VTE episode associated with an inadequate CHC prescription varied from 8.8 to 25.9%. Moreover, using a case-only methodology, the interaction between the use of CHC and the presence of a mutation of the factor V Leiden on the risk of VTE differs significantly depending on the progestin combined of the CHCs

L’analyse de la causalité, qui consiste à déterminer si la prise d’un médicament est la cause de la survenue d’un événement, est la problématique centrale de la pharmacovigilance et de la pharmaco-épidémiologie.La première partie de ce travail aborde l’étude de la causalité au plan individuel, au travers des méthodes d’imputabilité. Nous avons d’abord comparé une méthode d’imputabilité récemment développée, la méthode logistique, et la méthode d’imputabilité officiellement utilisée en France à un jugement consensuel d’experts pris comme référence. Les résultats montrent que la méthode française d’imputabilité tend à sous-coter la responsabilité du médicament (faible sensibilité) alors que la méthode logistique tend à la surestimer (faible spécificité). Par la suite, une nouvelle version de la méthode française d’imputabilité visant à améliorer sa sensibilité et son pouvoir discriminant a été proposée. Le travail de validation portant sur cette méthode réactualisée montre une amélioration de sa sensibilité et des résultats se rapprochant plus du jugement consensuel d’experts. Pour la méthode logistique, les critères d’imputabilité et leurs poids ont été réévalués sur un échantillon plus important d’observations que celui ayant servi à la pondération initiale. La validité de cette nouvelle version et celle de l’un des algorithmes les plus couramment utilisés en pharmacovigilance, la méthode Naranjo, ont été comparées à un jugement consensuel d’experts. Les résultats concernant la validité interne et les qualités prédictives de la méthode Naranjo ne sont pas satisfaisants alors que la méthode logistique présente une spécificité améliorée ainsi qu’une bonne sensibilité et valeurs prédictives. Cette dernière méthode présente donc des caractéristiques qui devraient améliorer l’évaluation de la responsabilité des médicaments dans la survenue des événements indésirables. La mise en place de méthode d’imputabilité spécifique à une classe thérapeutique et/ou à un type d’événement indésirable pourrait aussi améliorer l’évaluation des événements indésirables. Nous proposons une grille d’imputabilité adaptée aux accidents hémorragiques sous antithrombotique. Dans la deuxième partie de cette thèse, l’analyse épidémiologique de la causalité est abordée en proposant deux méthodes : l’analyse populationnelle des cas individuels, en particulier leur délai de survenue après exposition médicamenteuse, et l’approche cas-population. Bien que beaucoup moins robustes que les méthodes classiques, elles sont testées sur des problématiques réelles de pharmacovigilance et les résultats montrent qu’elles peuvent être utiles pour une première exploration d’une association causale potentielle. En conclusion, ce travail méthodologique pourrait aider à mieux évaluer la responsabilité des médicaments dans la survenue d’événements indésirables après leurs autorisations de mise sur le marché
The analysis of causality, which consists of determining if drug intake is the cause of the event occurrence, is the central issue of pharmacovigilance and pharmacoepidemiology. The first part of this work deals with the study of causality assessment methods at the level of individual cases. We first compared the recently developed logistic causality assessment method and the method officially used in France, to consensusual expert judgement taking as a reference. The results showed that the French causality assessment method tended to underestimate the responsibility of the drug (low sensitivity) whereas the logistic method tended to overestimate it (low specificity). Subsequently a new version of the French causality assessment method aiming to improve its sensitivity and discriminating power was proposed. The validation phase of this updated method showed improved sensitivity and a performance closer to consensual expert judgement. For the logistic method, the criteria of causality assessment and their weights were re-evaluated on a larger sample of drug-event pairs that had been used in the initial weighting. The validity of this method and that of one of the most commonly used algorithms in pharmacovigilance, the Naranjo method, were compared to consensual expert judgement. Results concerning the internal validity and the predictive qualities of the Naranjo method were not satisfactory while the logistic method presented an improved specificity and good sensitivity and predictive values. The logistic method now presents characteristics that should improve the assessment of drug responsibility in the occurrence of adverse events. The implementation of causality assessment method specific to a therapeutic class and / or to a type of adverse event could also improve the assessment of adverse events. We proposed a scale adapted to hemorrhages with antithrombotics and derived from the French causality assessment method. In the second part of this thesis, the epidemiological analysis of causality was tackled by proposing two methods: the populational analysis of individual cases, in particular their time to onset after drug exposure, and the case-population approach. Although less robust than the conventional methods, these were tested on real problems of pharmacovigilance and the results indicate that they may be useful for an initial exploration of a potential causal association. In conclusion, this methodological work could help to better assess drug causality in the occurrence of adverse event in post maketing surveillance

Budd-Chiari syndrome (BCS) i.e. thrombosis in the hepatic veins and/or inferior vena cava, and portal vein thrombosis (PVT) are rare disorders. Epidemiological data are scarce and previous reports have been from highly specialised referral centres. The aims of the thesis were: (i) to investigate the epidemiology, clinical features and survival of Swedish patients with BCS or PVT, and to (ii) determine common underlying risk factors i.e. thrombophilic factors and genetic markers of myeloproliferative disorders (MPD). In the first two papers we retrospectively reviewed the medical records of all BCS (1986-2003) and PVT (1995-2004) patients identified by searching the computerized patient registers of 11 hospitals including all university hospitals and liver transplantation units. In the following two papers we excluded patients with malignancy and included new cases diagnosed during the years 2004-2009; blood samples were collected and compared with controls and other patient groups. A total of 43 patients with BCS were identified (median age 40 years, 24 women). The mean agestandardised incidence and prevalence rates were 0.8 per million per year and 1.4 per million inhabitants respectively. Two or more risk factors were present in 44%. The overall transplantationfree survival at 1, 5 and 10 years was 47%, 28% and 17% respectively. 173 patients (median age 57 years, 93 men) with portal vein thrombosis were identified. The incidence and prevalence rates were 0.7 per 100 000 per year and 3.7 per 100 000 inhabitants, respectively. In the absence of cirrhosis and malignancy, being the most common risk factors, the survival at 1 year and 5 years was 92% and 76%, respectively. We observed an increased plasma level of the procoagulant factor VIII in BCS (mean 1.63 kIE/L), PVT without cirrhosis (1.87 kIE/L), PVT with cirrhosis (1.97 kIE/L), deep vein thrombosis (1.41 kIE/L) and cirrhosis patients alone (2.22 kIE/L), all p <0.001 compared to healthy controls (1.04 kIE/L). Elevated factor VIII levels were found in 50% of BCS and in 85% of PVT patients without previously identified prothrombotic risk factors. The somatic JAK2 V617F-mutation, a marker of MPD, was present in 63% of BCS and 14% of PVT patients. The frequency of the germline JAK2 46/1 haplotype was significantly higher in BCS (53%) and PVT (36%) patients compared to controls (27%) (p=0.02). However, the enrichment was only observed in JAK2 V617F positive patients. Conclusions: The incidence and prevalence rates of BCS in Sweden were calculated to be 0.8/million inhabitants per year and 1.4/million inhabitants, respectively. The rates of PVT were higher; 0.7/100 000 inhabitants per year and 3.7/100 000 inhabitants, respectively. In BCS the transplantation-free survival was poor, whereas in PVT the survival was variable and highly dependent on the presence of underlying disease. Concurrent prothrombotic risk factors are common in both disorders. High plasma levels of procoagulant factor VIII was observed in a majority of idiopathic BCS and PVT. The prevalence of the somatic JAK2 V617F mutation was high in our cohort and associated with the presence of a germline JAK2 46/1 haplotype.

Includes bibliographical references
Includes abstract.
The evidence suggests an association between HIV, TB and DVT. There are no studies of this link in the Southern African setting, where the incidence of both of these conditions (HIV and TB) is high. We therefore undertook a study to define the incidence of HIV and TB in patients with confirmed DVT in this setting. The aim of this study is to describe the incidence of HIV, TB and the more commonly accepted risk factors in patients with confirmed DVT in a South African public hospital.

Objectifs Les patients hospitalisés en réanimation pour un sepsis sont à haut risque d’accidents thrombotiques (AT) intéressant l’ensemble des circulations (grande, coronaire, petite). Nous souhaitons étudier ce risque thrombotique lors du sepsis (i) au sein de la grande circulation chez les patients présentant une fibrillation atriale de novo (FAN), (ii) au sein de la circulation coronaire chez les patients présentant un infarctus du myocarde (IDM) et (iii) au sein de la petite circulation chez les patients COVID-19 sévère. Par ailleurs, si le risque d’AT pose la question de l’intérêt de la thromboprophylaxie à dose croissante, l’évaluation du risque hémorragique en regard devra être systématique afin d’établir la balance bénéfice/risque d’un tel traitement. Méthodes Nous avons investigué le risque d’événements cardio-vasculaires majeurs (caractérisation et stratification) incluant les ATs, les hémorragies sévères, et le décès au sein de trois populations de patients septiques présentant une FAN, un IDM, ou une COVID-19 sévère par l’étude (i) de marqueurs tels que la dysfonction de l’auricule gauche à l’échocardiographie trans-oesophagienne (ETO) et la troponine cardiaque, et (ii) des scores de risque thrombotique et hémorragique utilisés chez les patients de cardiologie. Nous avons mené une enquête de pratique sur la gestion du risque thrombotique chez les patients ayant une FA lors d’un sepsis. Enfin nous avons effectué deux essais thérapeutiques : l’essai CAFS (Control Atrial Fibrillation Sepsis) de supériorité multicentrique randomisé, contrôlé comparant trois stratégies usuelles de prévention du risque hémodynamique chez les patients en choc septique présentant une FAN (en cours d’inclusion), et l’essai ANTICOVID (ANTIcoagulation in patients with hypoxemic COVID-19 pneumonia) comparant trois stratégies de traitement anticoagulant avec escalade de dose chez des patients présentant une pneumonie hypoxémiante COVID-19. Résultats Le risque d’évènements cardio-vasculaires majeures est élevé au cours d’un sepsis. Chez les patients en FAN, les approches cardiologiques de stratification des risques thrombotiques (anomalies ETO, score CHA2DS2-VASc) et hémorragiques (score HAS-BLED) semblent limitées. Une approche individualisée basée sur l’ETO et le score CHA2DS2-VASc pourrait néanmoins être intéressante. Ce travail a également mieux caractérisé le risque de formation d’un thrombus intra-cardiaque (absence de thrombus dans les 48 h suivant le début de la FA, prévalence rare de la sidération post cardioversion de l’auricule gauche). Enfin, nous avons confirmé l’hétérogénéité de prise en charge des risques hémodynamiques et thrombotiques justifiant la réalisation d’essais thérapeutiques. Chez les patients ayant un IDM au cours d’un sepsis, les approches cardiologiques usuelles de stratification des risques thrombotiques (scores GRACE et TIMI) semblent également limitées. En pratique usuelle, une stratégie invasive avec revascularisation coronaire précoce est très rarement effectuée. Chez les patients explorés par angiographie coronaire, l’incidence d’une coronaropathie obstructive est importante. Chez les patients présentant une pneumonie hypoxémiante COVID-19, le traitement anticoagulant préventif à dose forte, comparé au traitement anticoagulant préventif à dose standard, a été associé à un meilleur bénéfice clinique net, en raison d’une diminution du risque de thrombose et d'un faible risque hémorragique. Le traitement anticoagulant curatif n'a pas apporté de bénéfice supplémentaire. Conclusions Sur une base physiopathologique pro-thrombotique inhérente au sepsis, ce travail a permis (i) de mieux caractériser certaines situations à haut risque thrombotique (FAN, IDM, COVID sévère), (ii) élaborer des stratégies individuelles thérapeutiques de prévention du risque thrombotique (COVID-19), et (iii) poser les bases de futurs essais au sein de populations spécifiques à très haut risque thrombotique
Objectives Patients admitted to intensive care units with sepsis are at high risk of thrombotic events (TEs) throughout the circulatory systems (systemic, coronary, and pulmonary). We aimed to investigate the thrombotic risk during sepsis (i) within the systemic circulation in patients with new-onset atrial fibrillation (NOAF), (ii) within the coronary circulation in patients with acute myocardial infarction (MI), and (iii) within the pulmonary circulation in patients with severe COVID-19. Furthermore, while the risk of TE raises the question of whether thromboprophylaxis doses should be escalated, assessment of associated bleeding risk should be systematic in order to establish the benefit/risk balance of such treatment. Methods We investigated the risk of major cardiovascular events (risk characterization and stratification), including AT, major bleeding and death in three populations of septic patients with NOAF, MI or severe COVID-19 by studying (i) markers such as left atrial dysfunction on transesophageal echocardiography (TEE) and cardiac troponin, and (ii) thrombotic and hemorrhagic risk scores used in cardiology patients. We conducted a practice survey on thrombotic risk management in patients with de NOAF during sepsis. Finally, we carried out two therapeutic trials: the CAFS (Control Atrial Fibrillation Sepsis) multicenter, randomized, controlled superiority trial comparing three usual strategies to prevent hemodynamic risk with NOAF during septic shock (currently being included), and the ANTICOVID (ANTIcoagulation in patients with hypoxemic COVID-19 pneumonia) multicenter, randomized, controlled superiority trial comparing three anticoagulation strategies with dose escalation in patients with hypoxemic COVID-19 pneumonia Results Our work confirmed the high risk of major cardiovascular events during sepsis. In patients with NOAF, cardiological approaches to thrombotic (TEE abnormalities, CHA2DS2-VASc score) and hemorrhagic (HAS-BLED score) risk stratification seem limited. An individualized approach with TEE based on the CHA2DS2-VASc score could nevertheless be of interest. This work also better characterized the risk of intra-cardiac thrombus formation (absence of thrombus within 48 h of AF onset, low prevalence of post-cardioversion left atrial stunning). Finally, we confirmed the heterogeneity of hemodynamic and thrombotic risks management, calling for randomized trials. In patients with MI during sepsis, cardiological approaches to thrombotic risk stratification (GRACE and TIMI scores) also appear limited. In usual practice, an invasive strategy involving early coronary revascularization is very uncommon. In patients investigated using coronary angiography, the incidence of obstructive coronary artery disease is high. In patients with hypoxemic COVID-19 pneumonia, high-dose prophylactic anticoagulation, provided a better net clinical benefit driven by a 4-fold reduction in de novo thrombosis rate with no increase in major bleeding compared with standard-dose prophylactic anticoagulation. Also, therapeutic anticoagulation did not provide additional benefit in comparison with high-dose prophylactic anticoagulation. Conclusions On the basis of the common pro-thrombotic pathophysiology described in septic conditions, our work has made it possible to (i) better characterize clinical situations at particularly high thrombotic risk (NOAF, MI, severe COVID-19 infection), (ii) develop individual therapeutic strategies for thrombotic risk prevention (COVID-19), and (iii) establish the basis for subsequent trials in specific intensive care populations at very high thrombotic risk

Background: Up to 40% of trauma patients die during the first 24 hours after injury due to massive hemorrhage. In patients who survive this critical time period, no information is available on rates of delayed major bleeding or factors associated with delayed major bleeding. Methods: A retrospective chart review of 150 critically ill adult trauma patients was used to determine the incidence of delayed major bleeding events. Cox proportional hazards multivariate analysis was performed to assess for risk factors associated with delayed major bleeding events. The anticipated rate of delayed major bleeding events was 10%. Results: The incidence of delayed major bleeding in this cohort of critically ill trauma patients was 44%. Predictors that were statistically significantly associated with delayed major bleeding included: male gender, pre-injury use of the antiplatelet agents aspirin and/or clopidogrel, presence of intracranial bleeding, higher injury severity scores, requirement of massive transfusion, and low pH values. Use of anticoagulant prophylaxis was not associated with delayed major bleeding. Conclusion: The rate of delayed major bleeding was higher than estimated. Larger retrospective and prospective cohorts are needed to confirm these findings.

Venous thromboembolism (VTE) is a common disorder which causes significant morbidity and mortality. Upper extremity deep vein thrombosis(UEDVT) is a relatively understudied subtype of VTE which is commonly associated with central venous catheters, cancer, and thrombophilia. The goal of this project was to better characterize the risk of UEDVT and to design and execute a pilot study that will demonstrate the efficacy of a strategy preventing the occurrence of VTE in a high-risk population for UEDVT. This M.Sc project, was conducted in three parts. Chapter 1 of the thesis outlines a systematic review of the literature which assessed the risk of VTE in UEDVT patients by search for and including data from studies with patients with prospectively enrolled symptomatic UEDVT. Chapter 2 describes the development and final protocol of the TRIM-Line pilot study, a randomized open-label study comparing 90 days of rivaroxaban 10mg po daily against the current standard of care (observation) in patients with active cancer and central venous catheters, two known risk factors for VTE. Finally in Chapter 3 the TRIM-Line study was executed as a pilot trial involving The Ottawa Hospital and the Juravinski Cancer Centre located in Hamilton. The study was conducted from March 2019 until February 2020. 105 patients underwent randomization at the two Canadian centres. The study met its prespecified feasibility endpoint average enrolment rate of 7.5 per month (95% CI:4.56, 10.44) at the coordinating Ottawa Hospital site and 2.0 per month (95% CI:0.87, 3.13) for the Juravinski Cancer Centre site. The randomized controlled trial met its enrollment targets and demonstrated that a full scale randomized controlled trial on the topic of prevention of cancer associated venous thromboembolism is feasible.

Chronic kidney disease (CKD) affects more than 30 million adults in the U.S. and is strongly associated with cardiovascular events and mortality. Venous thromboembolism (VTE) is the third leading vascular disease, affects up to 900,000 Americans each year and contributes to as many as 100,000 deaths annually. The relationship of CKD and VTE has been described in patients receiving dialysis, kidney transplants recipients and in nephrotic syndrome, however, data supporting the association of VTE in mild to moderate CKD is conflicted. The overall goal of this research was to study the association of CKD and VTE and to understand the mechanisms of this association. To accomplish this goal we studied participants of the Reasons for Geographic and Racial Differences in Stroke (REGARDS) Study, a nationally representative cohort of 30,239 blacks and whites in the U.S.. The first chapter provides a review of the state-of-the science on CKD and VTE and potential mechanisms for this association. We focus on factor VIII as a potential mediator of VTE risk in CKD by reviewing the biochemistry and epidemiology linking factor VIII and CKD. In Chapter 2, we use a cohort study design and a competing risk analysis to determine the risk of VTE with albuminuria (ACR) and with various equations for estimated glomerular filtration rate (eGFR). There was no association of ACR and VTE and the risk of VTE was similar among eGFR equations. Compared to a normal eGFR (>90 ml/min/1.73m2), eGFR < 45 ml/min/1.73m2 was associated with a two-fold risk of VTE. The association of eGFR and unprovoked VTE was similar to the association with provoked VTE. The population attributable fraction of CKD (eGFR<60 ml/min/1.73m2) was modest at 5%. In Chapter 3, we utilize a case-cohort study to determine if biomarkers of inflammation (C-reactive protein) and procoagulation (Factor VIII and D-dimer) attenuate the risk of VTE in CKD. These biomarkers were higher in lower kidney function and were also strongly associated with VTE. Adjustment for factor VIII fully attenuated the risk of VTE in CKD, thus factor VIII is a potential mediator of the association of CKD and VTE. We assessed whether lifestyle factors and medications mitigate the risk of VTE in those with and without CKD. Exercise frequency and use of statins were associated with reduced risk of VTE in the presence and absence of CKD, but normal BMI was associated with reduced VTE risk only in those without CKD. We conclude that CKD is a risk factor for VTE, and findings shed light on the mechanisms of this association. Interventions that might lower VTE risk in CKD patients include exercise and statin therapy, but not weight loss. Factor VIII is a potential mediator of VTE in CKD and deserves further study. We suggest several avenues for future research to explore the relationship of Factor VIII and CKD.

Advanced liver disease is characterized by profound hemostatic alterations that can lead both to bleeding or to thrombotic complications. While pro-hemorrhagic alterations are present including thrombocytopenia and reduced plasmatic levels of coagulation factors, pro-thrombotic abnormalities such as decrease in anti-coagulant proteins antithrombin, Proteins C and S, increase in prothrombotic Factor VIII, and von Willebrand factor are also present. The most frequent site of thrombosis in cirrhotic patients is the portal vein, consequence of an interplay of factors including altered hemostasis and venous stasis. The endothelium, the third component of the thrombosis triad, however, probably plays an important role in the genesis of in situ thrombosis within the portal vein. In the present study, endothelium form portal and cava veins were analyzed in cirrhotic patients, and compared to that of non-cirrhotic subjects, in order to determine the possible role of local alterations in the development of thrombosis. The immunofluorescence study of the main endothelial anticoagulant protein, thrombomodulin, revealed decreased presence of this component in the endothelium of the portal vein with respect to the vena cava in cirrhosis patients. On the other hand, the immunohistochemical analysis of pro-coagulant Factor VIII revealed that this endothelial protein is present uninterruptedly lining the lumen of portal vein and vena cava of both cirrhosis patients and non-cirrhotic subjects, without showing any differences between them. Diminished thrombomodulin may hamper the endothelium’s anticoagulant properties, which, in the presence of conserved Factor VIII, may lead to the development of thrombosis. The thrombosis of the portal vein represents an important milestone in the natural history of patients with cirrhosis, often increasing morbidity before and mortality after liver transplantation. Obtainment of recanalization through anticoagulation is therefore paramount, and in the present study, an analysis was performed regarding factors that may have an impact on efficacy of anticoagulation with low molecular weight heparin in cirrhotic patients with this complication. Anticoagulation with low molecular weight heparin was demonstrated to be a valid strategy for achieving portal vein recanalization, with a response rate of 65.2%, including complete recanalization in 24 of the 46 treated patients, after a mean of 4.5 months (±3.1 months) of anticoagulation. Whereas the hemostatic status of patients did not correlate with the response to anticoagulation, the interval between thrombus onset and start of therapy was the only predictive factor of therapeutic efficacy. Specifically, thrombus age at diagnosis (1.9 ± 1.2 months vs 6.3 ± 4.5 months in the recanalization group and in the non-recanalization group, respectively, p<.001), and the interval between thrombus onset and start of anticoagulation (3.2 ± 1.7 months vs 7.78 ± 4.5 months in the recanalization group and in the non-recanalization group, respectively, p<.002) were the principal determinants of therapeutic efficacy. This underlines the importance of prompt diagnosis and start of therapy to increase the probability of successful anticoagulant therapy. Although in cirrhosis the low levels of antithrombin, which is necessary for the action of heparins, could theoretically hamper the anticoagulant effect, the clinical efficacy of anticoagulant therapy with low molecular weight heparin has been herein demonstrated. The anticoagulant effect of low molecular weight heparin was then tested in vitro using the thrombin generation assay, and concentrations within the therapeutic range achieved reduction of endogenous thrombin potential notwithstanding the marked reduction in antithrombin levels that were present in plasma from cirrhotic patients and the low plasma anti-Xa activity determined in vitro. In particular, patients with Child Pugh C cirrhosis were characterized by antithrombin levels which were as low as those of subjects with the prothrombotic condition of genetic antithrombin deficit (42±14% versus 52±4%, respectively, p=.06). At low molecular weight heparin 0.35 UI/mL concentration in vitro, anti-Xa activity was significantly lower in Child Pugh B and Child Pugh C patients as compared to controls (p<0.001), as well as in patients with congenital AT defect as compared to controls (p<0.001). Despite low levels of antithrombin and anti-Xa activity, patients with cirrhosis showed a greater anticoagulant effect of low molecular weight heparin, with a mean endogenous thrombin potential reduction of 72.6±11% (p=0.02 versus controls). This increased susceptibility of cirrhosis patients with advanced stages of the disease may therefore actually warrant dose reduction of anticoagulation.
La cirrosi epatica avanzata è caratterizzata da alterazioni emostatiche importanti che possono portare a complicanze emorragiche o trombotiche. Nonostante siano presenti alterazioni pro-emorragiche come trombocitopenia e ridotti livelli dei fattori della coagulazione, sono presenti anche anormalità pro-trombotiche come la diminuzione di proteine anti-coagulanti, quali antitrombina, Proteina C ed S, ed incremento del Fattore VIII e Fattore von Willebrand . Il sito più frequente di trombosi in pazienti cirrotici è la vena porta, risultato di vari fattori quali le alterazioni emostatiche sistemiche così come la stasi venosa locale. Tuttavia, l’endotelio, il terzo componente del triade trombotica, probabilmente gioca un ruolo importante nella genesi della trombosi in situ della vena porta. Nel presente studio, è stato analizzato l’endotelio della vena porta e comparato a quello della vena cava in pazienti cirrotici e non, per determinare il possibile ruolo delle alterazioni locali nello sviluppo della trombosi. Come principale proteina endoteliale anticoagulante, è stata studiata la trombomodulina tramite immunofluorescenza, rivelandone una ridotta presenza nell’endotelio della vena porta rispetto a quello della vena cava nei pazienti cirrotici. D’altro canto, l’analisi immunoistochimica del Fattore VIII, con proprietà pro-coagulanti, ha rivelato che questa proteina endoteliale è presente in maniera continua e costante lungo il lumen della vena porta e della vena cava sia nei pazienti cirrotici che non. La diminuzione della trombomodulina può dannegiare le proprietà anticoagulanti dell’endotelio che, in presenza del Fattore VIII preservato, può portare allo sviluppo della trombosi. La trombosi della vena porta rappresenta una complicanza rilevante nella storia naturale dei pazienti cirrotici, causando frequentemente un aumento della morbilità prima e della mortalità dopo il trapianto epatico. L’ottenimento della ricanalizzazione tramite terapia anticoagulante è perciò importante, e nel presente studio è stata fatta un’analisi dei fattori che possono avere un impatto sull’efficacia della terapia con eparina a basso peso molecolare in pazienti cirrotici con questa complicanza. Si è dimostrato che l’anticoagulazione con eparina a basso peso molecolare è una strategia valida per la ricanalizzazione della vena porta, con un tasso di risposta del 65.2%, includendo ripermeazione completa in 24 dei 46 pazienti trattati, dopo una media di 4.5 mesi (±3.1 mesi) di anticoagulazione. Nonostante lo status emostatico dei pazienti non correlava con la risposta all’anticoagulazione, l’intervallo tra lo sviluppo del trombo e l’inizio della terapia è stato l’unico fattore predittivo dell’efficacia terapeutica. Specificamente, l’età del trombo alla diagnosi (1.9 ± 1.2 mesi vs 6.3 ± 4.5 mesi, rispettivamente, p<.001) e l’intervallo tra lo sviluppo del trombo e l’inizio della terapia anticoagulante (3.2 ± 1.7 mesi vs 7.78 ± 4.5 mesi nel gruppo che ha ottenuto ricanalizzazione e nel gruppo che non ha ottenuto ricanalizzazione, rispettivamente, p<.002) sono stati i principali determinanti dell’efficacia terapeutica. Questo sottolinea l’importanza di una diagnosi precoce e di un opportuno inizio della terapia, per incrementare la probabilità di successo del trattamento anticoagulante. Benché i livelli bassi di antitrombina, necessaria per l’azione dell’eparina, verificatesi in cirrosi possano teoricamente diminuire l’effetto anticoagulante, in questo studio si è dimostrata l’efficacia clinica dell’anticoagulazione con eparina a basso peso molecolare. L’effetto anticoagulante dell’eparina a basso peso molecolare è stato esplorato in vitro utilizzando il test della trombino generazione, e concentrazioni di eparina dentro il range terapeutico sono state in grado di ridurre la generazione della trombina, nonostante la spiccata riduzione nei livelli plasmatici di antitrombina e i bassi livelli di anti-Xa determinati in vitro. In particolare, i pazienti in classe C di Child Pugh si sono caratterizzati da livelli di antitrombina bassi quanto quelli presenti in pazienti con la condizione protrombotica di deficit genetico di questa proteina (42±14% vs 52±4%, rispettivamente, p=.06). Alla concentrazione in vitro di 0.35 UI/mL di eparina a basso peso molecolare, l’attività anti-Xa è stata significativamente più bassa in pazienti in classi di Child Pugh B e C rispetto ai controlli (p<.001), così come in pazienti con difetto genetico dell’antitrombina rispetto ai controlli (p<.001). Nonostante i ridotti livelli di attività anti-Xa, i pazienti cirrotici hanno dimostrato un maggiore effetto anticoagulante dell’eparina a basso peso molecolare, con una riduzione del potenziale endogeno di trombina di 72.6±11% (p=0.02 vs i controlli). Data l’incrementata suscettibilità dei pazienti cirrotici in stadi avanzati della malattia epatica, potrebbe essere necessaria la riduzione della dose di anticoagulazione.

We carried out the first genome-wide linkage analysis in British families for VWF levels. ABO and VWF loci were specifically examined. The results showed that VWF levels are highly heritable 42% ( P>0.000001) with age and C-reactive protein (CRP) the main covariates. The ABO locus was strongly associated with VWF levels ( P=2x10-18), but linkage was modest (LOD = 1.6). VWF gene marker showed no significant association or linkage with phenotype. Genome-wide linkage analysis, conditioned on age and ABO genotypes, revealed regions with potential linkage. Highlighted regions were on chromosomes 2 and 3 (LOD = 1.78 & 1.08 respectively) and two areas on chromosome 12 (LOD = 1.5 & 1.3). Inflammatory biomarkers concentrations were also investigated. Heritabilities of CRP and tumour necrosis factor-alpha (TNF-alpha) were significantly high 38% and 47% respectively, while interleukin-6 was not heritable. VWF levels showed significant genetic correlation with CRP. As ABO group has a major role in determining VWF levels, if the VWF stroke association is causal, blood groups should be associated with stroke (Mendelian Randomisation). ABO genotype frequencies in stroke patients were investigated by a case-control study (503 objectively diagnosed cases and 327 controls). Non-O groups were not significantly over-represented in stroke cases (OR 1.1, CI95 0.83--1.47). Monte Carlo method, taking into account ABO effect on VWF levels, showed no association between ABO genotypes and stroke risk. This was reinforced by the findings of the systematic review we conducted on ABO groups and stroke. In contrast to results obtained from venous TE systematic review, where almost a two fold increased risk was found with non-O groups vs. O group.

Objetivo: Avaliar a incidência de tromboembolismo e as condições clínicolaboratoriais associadas em pacientes pediátricos internados em um hospital terciário. Pacientes e métodos: Estudo retrospectivo onde foram revisados os prontuários de todas as 6140 crianças de 0 à 13 anos que internaram entre fevereiro de 2007 à janeiro de 2009. Foram identificadas 31 crianças que tiveram 34 casos de tromboembolismo confirmado por diagnóstico de imagem, e estas foram incluídas no banco de dados para análise. Resultados: A incidência de tromboembolismo foi de 43,3 (intervalo de confiança de 95%: 30,0-60,4) para cada 10.000 internações. Vinte e seis (83,9%) crianças tinham condições clínicas associadas, sendo que 11 (35,4%) tinham apenas 1 condição associada, 5 (16,1%) tinham 2 condições associadas, 6 (19,3%) tinham 3 condições associadas, 4 (12,9%) tinham 4 condições associadas e 5 (16,1%) não tinham condições associadas. As frequências das principais condições clínico-laboratoriais foram: sepse, 41,9%; trombofilias, 35,5%; cardiopatia acianótica, 29,9%; e cateter central, 22,5%. Conclusão: O presente estudo evidenciou uma incidência de tromboembolismo muito mais alta do que a descrita na literatura, provavelmente devido ao estudo ter sido realizado em um hospital terciário, com maior complexidade e complicações associadas mostrando a importância de considerar este diagnóstico. Observamos também uma alta taxa de trombofilias nos pacientes que tiveram tromboembolismo, salientando que a associação de fatores de risco e predisposição genética ou fatores intrínsecos devem ser considerada no diagnostico e manejo destes pacientes.
Objective: To assess the incidence of thromboembolism and associated clinical conditions and laboratory abnormalities in a sample of pediatric patients admitted to a tertiary referral center. Methods: This was a retrospective chart review study. The medical records of all 6140 children between the ages of 0 and 13 years admitted to the study facility from February 2007 through January 2009 were reviewed. A total of 34 cases of imaging-confirmed thromboembolism were identified in 31 children and included for analysis. Results: The incidence of thromboembolism was 43.3 (95%CI, 30.0–60.4) per 10,000 admissions. Twenty-six children (83.9%) had associated clinical conditions: 11 (35.4%) had only 1 associated condition, 5 (16.1%) had 2 associated conditions, 6 (19.3%) had 3 associated conditions, 4 (12.9%) had 4 associated conditions, and 5 (16.1%) had no associated conditions. The main associated conditions were sepsis (41.9%), thrombophilia (35.5%), acyanotic heart disease (29.9%), and central venous catheterization (22.5%). Conclusion: In the sample studied herein, the incidence of thromboembolism was much higher than that reported in the literature, most likely due to the study setting (tertiary referral hospital) and its implications (greater case complexity and frequency of complications), stressing the importance of considering the diagnosis. Furthermore, among patients who developed thromboembolism, there was a high rate of thrombophilia, highlighting the importance of the association between risk factors and genetic predispositions or intrinsic factors in the diagnosis and management of these patients.

Background: Venous thromboembolism (VTE), comprising the conditions of deep vein thrombosis (DVT) and pulmonary embolism (PE), is a common acute cardiovascular event associated with increased long-term morbidity, functional disability, all-cause mortality, and high rates of recurrence. Major advances in identification, prophylaxis, and treatment over the past 3-decades have likely changed its clinical epidemiology. However, there are little published data describing contemporary, population-based, trends in VTE prevention and management. Objectives: To examine recent trends in the epidemiology of clinically recognized VTE and assess the risk of recurrence after a first acute episode of VTE. Methods: We used population-based surveillance to monitor trends in acute VTE among residents of the Worcester, Massachusetts, metropolitan statistical area (WMSA) from 1985 through 2009, including in-hospital and ambulatory settings. Results: Among 5,025 WMSA residents diagnosed with acute PE and/or lower-extremity DVT between 1985 and 2009 (mean age = 65 years), 46% were men and 95% were white. Age- and sex-adjusted annual event rates (per 100, 000) of clinically recognized acute first-time and recurrent VTE was 142 overall, increasing from 112 in 1985/86 to 168 in 2009, due primarily to increases in PE occurrence. During this period, non-invasive diagnostic VTE testing increased, vi while treatment shifted from the in-hospital (chiefly with warfarin and unfractionated heparin) to out-patient setting (chiefly with low-molecular-weight heparins and newer anticoagulants). Among those with community-presenting first-time VTE, subsequent 3-year cumulative event rates of key outcomes decreased from 1999 to 2009, including all-cause mortality (41% to 26%), major bleeding episodes (12% to 6%), and recurrent VTE (17% to 9%). Active-cancer (with or without chemotherapy), a hypercoagulable state, varicose vein stripping, and Inferior vena cava filter placement were independent predictors of recurrence during short- (3-month) and long-term (3-year) follow-up after a first acute episode of VTE. We developed risk score calculators for VTE recurrence based on a 3-month prognostic model for all patients and separately for patients without active cancer. Conclusions: Despite advances in identification, prophylaxis, and treatment between 1985 and 2009, the disease burden from VTE in residents of central Massachusetts remains high, with increasing annual events. Declines in the frequency of major adverse outcomes between 1999 and 2009 were reassuring. Still, mortality, major bleeding, and recurrence rates remained high, suggesting opportunities for improved prevention and treatment. Clinicians may be able to use the identified predictors of recurrence and risk score calculators to estimate the risk of VTE recurrence and tailor outpatient treatments to individual patients.

Background: Venous thromboembolism (VTE), comprising the conditions of deep vein thrombosis (DVT) and pulmonary embolism (PE), is a common acute cardiovascular event associated with increased long-term morbidity, functional disability, all-cause mortality, and high rates of recurrence. Major advances in identification, prophylaxis, and treatment over the past 3-decades have likely changed its clinical epidemiology. However, there are little published data describing contemporary, population-based, trends in VTE prevention and management. Objectives: To examine recent trends in the epidemiology of clinically recognized VTE and assess the risk of recurrence after a first acute episode of VTE. Methods: We used population-based surveillance to monitor trends in acute VTE among residents of the Worcester, Massachusetts, metropolitan statistical area (WMSA) from 1985 through 2009, including in-hospital and ambulatory settings. Results: Among 5,025 WMSA residents diagnosed with acute PE and/or lower-extremity DVT between 1985 and 2009 (mean age = 65 years), 46% were men and 95% were white. Age- and sex-adjusted annual event rates (per 100, 000) of clinically recognized acute first-time and recurrent VTE was 142 overall, increasing from 112 in 1985/86 to 168 in 2009, due primarily to increases in PE occurrence. During this period, non-invasive diagnostic VTE testing increased, vi while treatment shifted from the in-hospital (chiefly with warfarin and unfractionated heparin) to out-patient setting (chiefly with low-molecular-weight heparins and newer anticoagulants). Among those with community-presenting first-time VTE, subsequent 3-year cumulative event rates of key outcomes decreased from 1999 to 2009, including all-cause mortality (41% to 26%), major bleeding episodes (12% to 6%), and recurrent VTE (17% to 9%). Active-cancer (with or without chemotherapy), a hypercoagulable state, varicose vein stripping, and Inferior vena cava filter placement were independent predictors of recurrence during short- (3-month) and long-term (3-year) follow-up after a first acute episode of VTE. We developed risk score calculators for VTE recurrence based on a 3-month prognostic model for all patients and separately for patients without active cancer. Conclusions: Despite advances in identification, prophylaxis, and treatment between 1985 and 2009, the disease burden from VTE in residents of central Massachusetts remains high, with increasing annual events. Declines in the frequency of major adverse outcomes between 1999 and 2009 were reassuring. Still, mortality, major bleeding, and recurrence rates remained high, suggesting opportunities for improved prevention and treatment. Clinicians may be able to use the identified predictors of recurrence and risk score calculators to estimate the risk of VTE recurrence and tailor outpatient treatments to individual patients.

Rationnel: Bien qu'elles constituent la majorité des thromboses veineuses des membres inférieurs, les thromboses veineuses profondes (TVP) distale et les thromboses veineuses superficielles (TVS) ont été peu étudiées et leur significativité clinique et leur prise en charge sont débattues.Méthodes: Cette thèse collige les résultats des travaux effectués par J.P. Galanaud sur les TVP distales et les TVS à partir des études épidémiologiques OPTIMEV, POST et RIETE.Résultats commentés: TVP distale: La TVP distale n'a pas le même profil de facteur de risque que la TVP proximale. Sa mortalité associée à court terme est plus faible que celle des TVP proximales mais supérieure à celle de témoins confirmant qu'il s'agit d'une entité cliniquement significative. Les différences de profil de population et de complications entre ces deux types de TVP suggèrent que le rapport bénéfice/risque du traitement anticoagulant est différent. Il n'est donc pas légitime d'extrapoler les résultats des essais des TVP proximales aux TVP distales. Des essais spécifiques sont donc nécessaires.TVS: En cas de TVS le risque de TVP concomitante est élevé. Un examen écho-doppler doit être réalisé et devra au moins explorer l'ensemble du réseau profond du membre inférieur affecté. Sexe masculin et antécédents de TVP/Embolie pulmonaire constituent des facteurs prédictifs indépendants de récidive. Si certaines TVS peuvent être traitées avec succès sans traitement anticoagulant, celles associées à un cancer ou à une atteinte saphéno-fémorale sont à haut risque de récidive y compris après un traitement anticoagulant curatif
Background: Though they represent the majority of all lower limbs thromboses, isolated distal deep-vein thrombosis (DVT) (without symptomatic pulmonary embolism (PE)) and isolated superficial vein thrombosis (SVT) (without DVT or PE) have been poorly studied. Their clinical significance and management are under debate.Methods: Data from epidemiological multicenter prospective studies OPTIMEV, POST, RIETEResults and comments: Isolated distal DVT: Distal and proximal DVTs exhibit a different risk factor profile, the latter being more associated with chronic risk factors. Three-month mortality of distal DVT patient is lower than that of proximal DVT ones but is higher than that of controls. This evidences that distal DVT is a clinically significant finding. Differences in population profile and outcomes suggests that the benefit/risk ratio of anticoagulant treatment is not similar. Data from proximal DVT clinical trials should no longer be extrapolated to distal DVT.Isolated SVT: In case of SVT the risk of concomitant DVT is high. A compression ultrasonographic exam should be performed and at least explore the whole deep venous system of the affected limb. Male gender and history of DVT/Pulmonary embolism are independent predicators of recurrence. Some SVT can be safely treated without anticoagulants. On contrary, in patients with cancer or a sapheno-femoral junction involvement, the risk of deep venous recurrence is high even upon full therapeutic dose of anticoagulants

L'objectif de notre première étude était d'identifier les déterminants génétiques et environnementaux du risque de maladie thromboembolique veineuse (MTEV) chez les femmes sous contraceptifs oraux combinés (COC). Après ajustement pour les facteurs confondants, les principaux déterminants environnementaux de la MTEV étaient le tabagisme (OR = 1,65) et un indice de masse corporelle supérieur à 35 kg.m2 (OR = 3,46). En outre, la thrombophilie héréditaire sévère (OR = 2,13) et les groupes sanguins non-O (OR = 1,98). Nous avons confirmé que l’histoire familiale au premier degré de MTEV prédit mal la thrombophilie. En conclusion, cette étude confirme l'influence du tabagisme et de l'obésité et pour la première fois l'impact du groupe sanguin ABO sur le risque de MTEV chez les femmes sous COC. Elle confirme également la faible sensibilité de l'histoire familiale de MTEV pour dépister les thrombophilies héréditaires.Le but de la deuxième étude était d'étudier, chez les utilisatrices de COC, l'impact des polymorphismes génétiques nouvellement identifiés par les études pangénomiques associés au risque de MTEV dans la population générale. Neuf polymorphismes situés sur les gènes KNG1, F11, F5, F2, PROCR, FGG, TSPAN15 et SLC44A2 ont été génotypés dans un échantillon de 766 cas et 464 témoins dans le cadre de l’étude PILGRIM. Seul le polymorphisme rs2289252 situé sur le F11 était significativement associé au risque de MTEV. La présence de l’allèle rs2289252-A du F11 était associée à un risque accru de MTEV (OR =1,6). En outre, la combinaison de l’allèle rs2289252-A et du groupe sanguin non-O, était associée à un risque d’OR de 4
The aim of our first study was to identify the genetic and environmental determinants of venous thromboembolism (VTE) risk in a large sample of women using combined oral contraceptives (COC). A total of 968 women with a personal history of VTE during COC use were compared with 874 women under COC, but no personal history of VTE. After adjustment for confounding factors, the main environmental determinants of VTE were smoking odds ratio (OR = 1.65) and a body mass index greater than 35 kg.m-2 (OR = 3.46). In addition, severe hereditary thrombophilia (OR = 2.13) and non-O blood groups (OR = 1.98) have been shown to be important genetic risk factors for VTE under COC. First-degree family history of VTE predicts thrombophilia poorly. In conclusion, this study confirms the influence of smoking and obesity and for the first time the impact of ABO blood group on the risk of VTE in women under COC It also confirms the low sensitivity of the family history of VTE to detect hereditary thrombophilia. The purpose of the second study was to study, in COC users, the impact of newly identified genetic polymorphisms by genome-wide as associated with the risk of VTE in the general population. Nine polymorphisms on the KNG1, F11, F5, F2, PROCR, FGG, TSPAN15 and SLC44A2 genes were genotyped in a sample of 766 patients and 464 controls in the PILGRIM study. Only the rs2289252 polymorphism on the F11 was significantly associated with the risk of VTE. The presence of the F11 rs2289252-A allele was associated with an increased risk of VTE (OR = 1.6). In addition, the combination of the rs2289252-A allele and the non-O blood group was associated with an OR risk of 4

El tromboembolisme venós (TEV) és una malaltia complexa. És conseqüència de la interacció de factors ambientals i genètics que molts cops es reflecteixen en els anomenats fenotips intermediaris. Aquests, s’associen per definició al risc de trombosi i per això tenen una gran utilitat tant diagnòstica com en la busca de factors causals. La forma d’estudiar l’hemostàsia ha variat i, avui en dia, es parla del model cel·lular. El model cel·lular proporciona una visió més àmplia dels processos de l’hemostàsia projectant les múltiples interaccions entre els elements que hi intervenen. En aquest model, les cèl·lules no actuen només com una superfície fosfolipídica que serveix de suport als diferents factors, sinó que tenen un paper actiu. Això fa que les cèl·lules sanguínies siguin objectius on focalitzar la busca de nous fenotips implicats en les malalties de l’hemostàsia. El principal objectiu d’aquesta tesi ha estat la caracterització de nous fenotips eritrocitaris, plaquetaris i leucocitaris per descriure la seva associació amb el risc de TEV. Els estudis realitzats s’han desenvolupat en l’àmbit de dos projectes: Genetic Analysis of Idiopathic Thrombophilia 2 (GAIT-2) un estudi de 35 famílies, i el projecte Riesgo de Enfermedad TROmboembólica Venosa (RETROVE), un estudi de casos i controls no emparentats amb 400 casos amb TEV i 400 controls. L’estudi de la sèrie eritrocitària a la població GAIT-2 ha conduit cap a la identificació de 5 fenotips intermediaris genèticament correlacionats amb la trombosi: Hematòcrit, l’amplitud de distribució eritrocitària (RDW), índex de fluorescència reticulocitària (IRF), el receptor soluble de la transferrina i el ferro sèric. Aquests genotips poden utilitzar-se per a la recerca de gens relacionats amb la trombosi. L’estudi de la funció plaquetària en pacients amb trombosi (projecte RETROVE) mitjançant dispositius habitualment utilitzats per a l’estudi de malalties hemorràgiques, com el PFA-100®, ens ha permès demostrar que el risc de TEV és superior en aquells pacients amb temps d’obturació curts. Aquest fet podria indicar que l’adhesió plaquetària tindria un paper en la formació i probablement en la propagació del trombus. I finalment l’estudi de la població leucocitària fet dins el projecte RETROVE, ens ha permès identificar la xifra de monòcits superior a 0,7x109/L com un factor de risc de TEV, fet que concorda amb estudis previs. I per altra banda, s’ha observat que els neutròfils dels pacients amb antecedents de TEV, tenen major capacitat per a fer NETosi, fet que s’observa amb un increment dels marcadors plasmàtics de trampes de neutròfils (NETs). Aquests marcadors poden ser utilitzats com a fenotips intermediaris de trombosi, però, a més, en un futur, poden arribar a ser dianes terapèutiques antitrombòtiques.
Venous thromboembolism (VTE) is a complex disease. It is the result of the interaction between the environment and genes. This interaction is manifested by “intermediary phenotypes”. These intermediary phenotypes are useful for estimating the risk of developing the disease. The prevailing view of hemostasis has changed and nowadays, there is another point of view based on a model in which coagulation is regulated by properties of cell surfaces: The Cell-based Model of Hemostasis. This model provides a wide vision of the hemostatic process and reflects the multiple pathways of hemostasis in vivo. The clearer understanding of the role of cells will allow us to answer some unanswered questions in hemostasis. The main objective of this thesis has been the characterization of new erythrocyte, platelet and leucocyte phenotypes that will allow us to describe the association between blood cells and VTE risk. The studies included in this thesis were performed in the context of two projects: the Genetic Analysis of Idiopathic Thrombophilia 2 (GAIT-2) project, a study of 35 families and 935 individuals, and the project Riesgo de Enfermedad TROmboembólica Venosa (RETROVE), a case-control study of 400 unrelated cases with VTE and 400 controls. Using the extended pedigrees of GAIT-2 project, we have identified 5 intermediate erythrocyte-related phenotypes: Hematocrit, red cell distribution width (RDW), index reticulocyte fluorescence (IRF), soluble transferrin receptor and serum iron, that are genetically correlated with VTE. They can be used in the research for thrombosis –related genes. The study of platelet function in patients with thrombosis (in the RETROVE project) using devices that are frequently used for the study of hemorrhagic diseases, like PFA-100®, has allowed us to demonstrate that the risk of VTE is higher in patients with shortest closure times. This fact, supports the relevance of platelet adhesion in thrombus formation and probably, in their perpetuation. And finally, the study of leucocyte counts in the RETROVE project, have allowed us, in one hand, to identify that monocyte counts over 0,7x109/L are statistically associated with previous VTE. Our findings are in line with previous studies that reported the same association. In the other hand, we have observed that circulating neutrophils in patients with VTE are prone to undergo NETosis, which is associated with enhanced expression of neutrophil extracellular traps (NETs) biomarkers. These biomarkers can be used as intermediary phenotypes for VTE, and, in the future, they can be therapeutic targets.

L'objet de cette thèse est l'étude de la génétique des coronaropathies dans le contexte du diabète de type I et de la génétique de la thrombose veineuse ainsi que de l'interaction entre ces facteurs génétiques et la fonction rénale. Le but de ces travaux et d'apporter de nouveaux éléments permettant de caractériser le rôle de la fonction rénale dans le développement des maladies cardiovasculaires. À l'aide d'une approche GWAS je cherche à identifier des marqueurs génétiques associés au risque de coronaropathie dans un groupe cas-témoin composé de patients atteints de diabète de type I et d'étudier l'effet de l'interaction entre ces marqueurs génétiques et la néphropathie diabétique sur le risque de coronaropathie. Ensuite, j'étudie le rôle de la fonction rénale dans le risque de thrombose veineuse en rassemblant des marqueurs génétiques de la fonction rénale dans un score polygénique. Ces travaux ont permis de mettre en évidence des gènes candidats associés au risque de coronaropathie chez les patients diabétiques de type I qui sont impliqués dans le développement du rein et la fonction rénale et l'association entre le score polygénique et le risque de thrombose veineuse a permis de renforcer l'hypothèse de la fonction rénale en tant que facteur de risque de la thrombose veineuse. Les résultats obtenus dans ces travaux doivent être confirmés mais dans l'ensemble ils montrent que la fonction rénale joue un rôle dans le développement des maladies cardiovasculaires
This thesis deals with the study of genetic factors of coronary artery diseases as complications of type I diabetes, genetics factors of venous thrombosis and the interactions between these genetic factors and renal function. This work aims to add new pieces of knowledge concerning the role of renal function in the development of cardiovascular diseases. From a GWAS approach I aim to identify genetic markers associated with coronary artery disease within a group of type I diabetic patients a study the effect of the interaction between these markers and diabetic nephropathy. Then, I will study the link between renal function and venous thrombosis using a polygenetic risk score composed from genetic markers of renal function. This work highlighted new candidate genes associated with coronary artery disease among type I diabetic patients that are involved in kidney development and renal function. The association between the polygenetic risk score and renal function strengthened the hypothesis of renal function as a risk factor for venous thrombosis. The obtained results must be confirmed but they globally demonstrate that renal function plays a role in the development of cardiovascular diseases

[Truncated abstract] Cardiac surgery involving cardiopulmonary bypass is a complex procedure that results in significant changes to blood coagulation, fibrinolytic biochemistry, platelet number and function, and the vasculature. These are due to pharmacological agents which are administered, haemodilution and contact of the blood with artificial surfaces. Consequently there are significant risks of thrombosis and haemorrhage associated with this procedure. The research presented in this thesis utilises in vitro, in vivo, and a novel ex vivo model to investigate the nature of the haemostatic defect induced by cardiopulmonary bypass. The components studied include the drugs heparin, protamine sulphate, and aprotinin, different types of bypass circuitry (including heparin bonded circuits) and procedures such as acute normovolaemic haemodilution. Patient variables, such as Factor V Leiden, are also studied. Each of these components is assessed for the effects on a number of laboratory measures of haemostasis including activated partial thromboplastin time, prothrombin time, activated protein C ratio, antithrombin concentration, heparin concentration, thrombin-antithrombin complex formation, prothrombin fragment 1+2 formation, markers of platelet surface activation and secretion, activated clotting time, haemoglobin concentration and coagulation factor assays.

Tromboza danas, u većini razvijenih zemalja, predstavlja vodeći uzrok obolevanja i umiranja. Poslednjih godina veoma aktuelna su istraživanja venskog tromboembolizma, obzirom da je incidenca ovog oboljenja 2/1000 osoba godišnje, a njegov razvoj posledica udruženog delovanja više genetskih i stečenih faktora rizika. Što preciznije prepoznavanje i sagledavanje što većeg broja ovih faktora osnovni je cilj u borbi, kako protiv prve epizode venske tromboze, tako i protiv recidiva ove bolesti. Brojni faktori rizika već su prepoznati kao sastavne karike patofiziološkog lanca venskog trombotskog procesa, ali je evidentno da otkrića mnogih od njih tek predstoje. Među najaktulenijim istraživanjima na ovom polju nalazi se i ispitivanje uloge poremećaja fibrinoliznog mehanizma u venskoj tromboembolijskoj bolesti. Iako su već pruženi dokazi da suprimirana fibrinolizna aktivnost povećava rizik od nastanka ovog oboljenja, još uvek postoje brojna otvorena pitanja, koja se pre svega odnose na ulogu pojedinačnih činilaca fibrinoliznog mehanizma u venskoj trombozi, kao i na globalnu ulogu fibrinoliznog mehanizma u različitim tipovima i lokalizacijama venske trombotske bolesti. Pored toga, ispitivanje uticaja pojedinih genskih mutacija na pojadinačne činioce fibrinoliznog mehanizma, njegovu globalnu funkcionalnost i posredno na rizik za nastanak venske tromboze, takođe zaokuplja pažnju stručne javnosti, obzirom na nekonzistentnost rezultata dobijenih studijama koje se bave ovom problematikom. Cilj ovoga istraživanja je ispitivanje kako globalne funkcionalnosti fibrinoliznog mehanizma, tako i njegovih pojedinačnih činilaca, kod bolesnika sa različitim tipovima i lokalizacijama venske tromboze i poređenje ovih parametara sa njihovim vrednostima u zdravoj populaciji. Pored toga, cilj istraživanja je i ispitivanje zastupljenosti 4G/5G PAI-1 polimorfizma kod bolesnika sa venskom trombozom u poređenju sa zdravim osobama. Ispitivanu grupu je sačinjavalo 100 bolesnika koji su doživeli trombozu dubokih vena a kontrolnu grupu je činilo 100 zdravih ispitanika, koji nikada nisu imali trombozni incident. Iz ispitivanja su isključene: osobe sa prethodno dokazanim poremećajem hemostaznog mehanizma, osobe koje uzimaju lekove za koje se zna da mogu imati uticaja na hemostazni mehanizam, osobe koje su imale akutnu bolest u momentu uzorkovanja krvi ili 6 nedelja pre toga, osobe sa malignitetom, trudnice, osobe sa težim duševnim bolestima, bolestima jetre i bubrega, autoimunim bolestima, ispitanici koji su odbili da potpišu pristanak informisanog ispitanika. Kao test za procenu globalne funkcionalnosti fibrinoliznog mehanizma korišteno je euglobulinsko vreme lize koaguluma, dok su od pojedinačnih činilaca određivani: tkivni aktivator plazminogena (t-PA) i trombinom aktivišući fibrinolizni inhibitor (TAFI) - ELISA metodom, kao i inhibitor aktivatora plazminogena-1 (PAI-1) - metodom hromogenog substrata. Genetskim ispitivanjem je utvrđivano prisustvo PAI-1 4G/5G genskog polimorfizma. Prema rezultatima istraživanja kod 56% bolesnika bila je prisutna spontana venska tromboza, dok je 44% njih imalo trombozu provociranu jednim od priznatih faktora rizika. U odnosu na lokalizaciju venskog tromboznog procesa proksimalna venska tromboza bila je prisutna kod 63% bolesnika, izolovana distalna venska tromboza kod 29% bolesnika, a atipično lokalizovana venska tromboza kod 8% bolesnika. Posmatrajući zastupljenost pojedinih faktora rizika uočili smo da je značajno viši procenat osoba sa hipertenzijom bio prisutan u grupi bolesnika sa primarnom trombozom dubokih vena u odnosu na grupu bolesnika sa provociranom trombozom dubokih vena (61% vs.16%; p=0.000). Što se funkcionalnosti fibrinoliznog mehanizma tiče, prema našim rezultatima bolesnici koji su doživeli trombozu dubokih vena imaju značajno duže vreme lize koaguluma, odnosno suprimiranu funkcionalnost fibrinolize u poređenju sa zdravim kontrolama (204.34±51.24 vs. 185.62±42.30; p=0.011), a kada posmatramo podgrupe bolesnika u odnosu na lokalizaciju i vrstu venske tromboze uočavamo da podgrupa bolesnika sa izolovanom distalnom venskom trombozom ima značajno duže euglobulinsko vreme lize koaguluma u odnosu na kontrolnu grupu (218.32±41.12 vs.185.62±42.30: p=0.001), kao i bolesnici koji su imali provociranu vensku trombozu u poređenju sa kontrolama (208.18±48.53 vs. 185.62±42.30; p=0.018). Ispitivanjem pojedinačnih komponenti fibrinoliznog mehanizma došli smo do rezultata da bolesnici koji su doživeli venski trombozni incident imaju značajno više koncentracije TAFI u poređenju sa osobama koje nikada nisu imale vensku trombozu (19.70 ng/ml ± 5.17 vs.17.13 ng/ml ± 4.25; p=0.001). Poređenjem bolesnika sa provociranom trombozom dubokih vena i kontrolnih ispitanika uočili smo da bolesnici iz ove podgrupe imaju značajno više vrednosti plazminogena u poređenju sa zdravim osobama (127.14 % ± 27.73 vs.117.09 % ± 24.49; p= 0.044), kao i značajno više koncentracije t-PA (20.02 ng/ml ± 11.07 vs. 16.78 ng/ml ± 8.08; p=0.042). Što se tiče TAFI, bolesnici sa distalnom trombozom dubokih vena u poređenju sa kontrolama (20.72 ng/ml ± 4.96 vs.17.13 ng/ml ± 4.25; p=0.001), kao i bolesnici sa proksimalnom trombozom dubokih vena u poređenju sa kontrolama (19.37 ng/ml ± 5.33 vs.17.13 ng/ml ± 4.25; p=0.013) imaju značajno više koncentracije TAFI. Koncentracija ovog inhibitora fibrinoliznog procesa značajno je veća i kod bolesnika sa provociranom trombozom dubokih vena u poređenju s zdravim osobama (19.93 ng/ml ± 3.97 vs.17.13 ng/ml ± 4.25; p=0.000), kao i kod bolesnika sa primarnom trombozom dubokih vena u poređenju sa zdravim ispitanicima (19.53 ng/ml ± 5.97 vs.17.13 ng/ml ± 4.25; p=0.023). Što se genetskih analiza tiče, u okviru grupe bolesnika imali smo 25% homozigotnih i 58% heterozigotnih nosilaca mutacije gena za PAI-1, dok 17% bolesnika nije imalo pomenutu gensku mutaciju. U okviru kontrolne grupe pak, bilo je 30% homozigotnih i 56% heterozigotnih nosilaca mutacije a 14% ispitanika nije imalo mutaciju. Nije uočena značajna razlika u zastupljenosti 4G/4G genotipa između bolesnika sa različitim lokalizacijama venskog trombotskog procesa (distalna DVT 29% vs. proksimalna DVT 21% vs. DVT retke lokalizacije 12%; p=0.501), kao ni u zastupljenosti ovoga genotipa kod provocirane i spontane tromboze dubokih vena (27% vs. 23%; p=0.642), niti kod izolovane tromboze dubokih vena u poređenju sa plućnom tromboembolijom (25% vs. 33%; p=0.735). Procena rizika za nastanak venske tromboze u odnosu na postojanje poremećaja globalne funkcionalnosti fibrinoliznog mehanizma, u odnosu na patološke koncentracije pojedinih komponenti fibrinoliznog mehanizma, kao i u odnosu na postojanje 4G/4G mutacije u genu za PAI-1, pokazala je da suprimirana funkcionalnost fibrinoliznog mehanizma trostruko povećava rizik za nastanak tromboze dubokih vena (OR 3.02; CI 1.26-7.22), povišen nivo PAI-1 nema uticaja na rizik od nastanka tromboze dubokih vena, na šta ukazuje OR od 0.86 sa CI 0.59-1.25, povišen nivo t-PA antigena ne utiče na rizik od nastanka tromboze dubokih vena (OR 1.53; CI 0.79-2.94), ali povišena koncentracija TAFI više od dvostruko povećava ovaj rizik (OR 2.25; CI 1.16-4.35). Prema našim rezultatima PAI-1 4G/4G genotip nema uticaja na rizik od nastanaka venske tromboze, što potvrđuje OR koji iznosi 0.57 (0.27-1.20). Na osnovu dobijenih rezultata zaključujemo da bolesnici sa trombozom dubokih vena imaju suprimiranu funkcionalnost fibrinoliznog mehanizma u poređenju sa zdravim osobama, da je nivo t-PA antigena, kao i plazminogena značajno viši kod bolesnika sa provociranom venskom trombozom nego kod zdravih osoba, da nema razlike u koncentraciji PAI-1 između bolesnika sa venskom trombozom i zdravih osoba, ali da bolesnici sa trombozom dubokih vena, bez obzira na njenu lokalizaciju ili vrstu imaju značajno više nivoe TAFI u poređenju sa zdravim ispitanicima. Pored toga možemo zaključiti da ne postoji razlika u zastupljenosti 4G/5G polimorfizma između bolesnika sa venskom trombozom i zdravih ispitanika. Konačno, možemo reći da na osnovu naših rezultata možemo zaključiti da suprimirana funkcionalnost fibrinoliznog mehanizma trostruko povećava rizik od nastanka tromboze dubokih vena, a povišen nivo TAFI-a dvostruko povećava ovaj rizik, dok 4G/5G PAI-1 polimorfizam nema uticaja na rizik za nastanak venskog tromboembolizma.
Thrombosis is nowadays leading cause of morbidity and mortality worldwide. Lately, studies dealing with venous thromboembolism are very actual, since incidence of this disease is 2/1000 persons per year and its development is consequence of joint action of many different inherited and acquired risk factors. Precise recognition and understanding as many of those factors as possible represents imperative in fight against the first episode of venous thrombosis, and also against the recurrence of the disease. Numerous risk factors have been already recognized as constituent links of pathophysiological chain of venous thrombotic process, but it is also clear that the discovery of many of them are yet to come. Investigations of the role of fibrinolytic mechanism disorders in venous thrombosis are topical in the field. Although, we have some evidences that suppressed fibrinolytic activity increases the risk of this disease, still there are many open issues, especially those dealing with the role of individual factors of fibrinolytic mechanism in venous thrombosis, and with the role of global fibrinolytic function in different types and localizations of venous thrombotic disease. Further, investigation of the effects of gene mutations on individual fibrinolytic mechanism components, its global functionality and indirectly to the risk of venous thrombosis, also attracts the attention of experts, given the inconsistency of results obtained from studies dealing with this issue. The aim of this study was to evaluate fibrinolytic mechanism global functionality, as well as functionality of its integral individual components in patients with different venous thrombosis types and localizations, and to compare them with those of the healthy persons. In addition, the aim was to evaluate presence of 4G/5G PAI-1 polymorphism in patients with venous thrombosis compared with healthy subjects. The case group consisted of 100 patients with deep vein thrombosis and the control group consisted of 100 healthy subjects who had never had thrombotic incident. Exclusion criteria were: documented haemostatic disease, taking drugs proven to affect fibrinolytic function, acute illness within 6 weeks before blood sampling, malignancy, pregnancy, severe mental illness, kidney or liver diseases, autoimmune diseases, examinee refusal to sign the informed consent. We used euglobulin cloth lysis time test as test for global fibrinolytic mechanism function estimation, and also determined: t-PA and TAFI concentrations using ELISA method and PAI-1 concentrations using chromogenic substrate method. The presence of PAI-1 4G/5G gene polymorphism was determined by genetic testing. According to results 56% of patients had unprovoked and 44% had provoked venous thrombosis. Proximal venous thrombosis was present in 63% of cases, distal venous thrombosis in 29% of cases and atypical venous thrombosis in 8% of them. Significantly higher frequency of hypertension was present in patients with primary deep vein thrombosis than in the group of patients with provoked deep vein thrombosis (61% vs. 16%, p = 0.000). Patients who have experienced deep vein thrombosis had a significantly longer clot lysis time, and suppressed fibrinolysis function compared with healthy controls (204.34 ± 51.24 vs. 185.62 ± 42.30, p = 0.011). Also, this parameter was significantly longer in patients with isolated distal deep vein thrombosis compared with healthy controls (218.32±41.12 vs. 185.62±42.30: p=0.001), such as in patients with provoked venous thrombosis compared with controls (208.18±48.53 vs. 185.62±42.30; p=0.018). Patients with venous thrombosis had significantly higher TAFI concentrations in comparison with healthy volunteers (19.70 ng/ml ± 5.17 vs. 17.13 ng/ml ± 4.25; p=0.001). Patients with provoked venous thrombosis had significantly higher concentrations of plasminogen (127.14 % ± 27.73 vs. 117.09 % ± 24.49; p= 0.044) and t-PA (20.02 ng/ml ± 11.07 vs. 16.78 ng/ml ± 8.08; p=0.042), in comparison with controls. Regarding TAFI, we noticed that patients with isolated distal deep vein thrombosis have higher values of this parameter compered with healthy people (20.72 ng/ml ± 4.96 vs. 17.13 ng/ml ± 4.25; p=0.001), such as patients with proximal deep vein thrombosis (19.37 ng/ml ± 5.33 vs. 17.13 ng/ml ± 4.25; p=0.013). The same was obtained when compared patients with provoked venous thrombosis and controls (19.93 ng/ml ± 3.97 vs. 17.13 ng/ml ± 4.25; p=0.000), and patients with unprovoked venous thrombosis and controls (19.53 ng/ml ± 5.97 vs. 17.13 ng/ml ± 4.25; p=0.023). As far as genetic analysis, in the group of patients we had 25% homozygous and 58% heterozygous carriers of PAI-1 gene mutation, whereas 17% of patients did't have this mutation. In controls, we had 30% homozygous and 56% heterozygous carriers of mutation and 14% of those without mutation. There was no significant difference in the frequency of 4G/4G genotype between patients with different localization of venous thrombotic process (distal DVT 29% vs. proximal DVT 21% vs. rare localization DVT 12%, p = 0.501), as well as the representation of this genotype in provoked and unprovoked deep vein thrombosis (27% vs. 23%, p = 0.642), or in isolated deep vein thrombosis compared to pulmonary thromboembolism (25% vs. 33%, p = 0.735). Finaly, our results show that suppressed fibrinolytic functionality threefold increases risk of venous thrombosis (OR 3.02, CI 1.26-7.22), elevated levels of PAI-1 have no effect on the risk of deep vein thrombosis, as evidenced by OR of 0.86 with CI 0.59-1.25, elevated levels of t-PA antigen do not affect the risk of deep venous thrombosis (OR 1.53; CI 0.79-2.94), but increased concentration of TAFI increases more than twice this risk (OR 2.25; CI 1.16-4.35). PAI-1 4G/4G genotype does not affect venous thrombotic risk (OR 0.57; CI 0.27-1.20). Based on these results, we conclude that patients with deep vein thrombosis have suppressed fibrinolytic mechanism functionality compared to healthy subjects, the levels of t-PA antigen and plasminogen are significantly higher in patients with provoked venous thrombosis than in healthy subjects, there is no difference in PAI-1 concentration in patients with venous thrombosis and healthy persons, but the patients with deep vein thrombosis, regardless of its localisation or the type have a significantly higher level of TAFI as compared with healthy subjects. In addition, we can conclude that there is no difference in the prevalence of 4G/5G polymorphism in patients with venous thrombosis and healthy persons. Finally, we can say that suppressed fibrinolytic mechanism functionality threefold increases risk of deep vein thrombosis, elevated level of TAFI-a double increases this risk, while PAI-1 4G/5G polymorphism has no influence on the risk of venous thromboembolism.

Les néoplasies myéloprolifératives (NMP) sont des hémopathies myéloïdes clonales, chroniques et prolifératives. Les plus fréquentes sont la polyglobulie de Vaquez et la thrombocytémie essentielle. Elles s’accompagnent de risques importants de thrombose (artérielles et veineuses) et de transformation en pathologies plus agressives (myélofibrose secondaire et leucémie aigüe). Les thromboses peuvent être la situation diagnostique de ces maladies, ou survenir au cours de la prise en charge. Le sujet de cette thèse est d’étudier la relation clinique entre NMP et thrombose. Dans un contexte de survenue de thrombose veineuse idiopathique, sans antécédent NMP, nous nous sommes intéressés à la recherche de mutation clonale chez les patients comme moyen diagnostique d’une NMP. Nous avons ainsi exploité la cohorte EDITH du CIC en prenant les patients ayant expérimentés un puis une récurrence thrombotique. A l’inverse, nous avons constitué une base de données (OBENE) des patients pris en charge pour une NMP au CHRU de Brest.Nous avons ensuite exploité cette base, en analysant la fréquence et l’impact des arythmies cardiaques auriculaires, la balance bénéfice-risque à l’utilisation des NACO, l’impact des statines sur la réduction du risque de thrombose ainsi que la fréquence et l’impact de la nonadhérence aux traitements dans les PV et TE.NMP et thromboses sont liées, il est donc nécessaire d’approfondir les connaissances de leur physiopathologie spécifique pour améliorer la prévention et le traitement des épisodes. Cette thèse amène quelques réponses àcertaines questions mais elle est surtout le point de départ de réflexion commune entre les praticiens et biologistes intéressés par ces domaines
The myeloproliferative neoplasms (MPN) are clonal myeloid, chronic and proliferative disorders. The most frequent are polycythemia vera and essential thrombocythemia. The more frequent complications are thromboses (arterial and venous) and phenotypic evolutions (secondary myelofibrosis and acute leukemia). Thromboses can be a situation of diagnosis or observed during the followup of a MPN. This thesis is focused on the clinical link between MPN and thromboses.In a context of idiopathic venous thromboses (first event or recurrence), without medical history of MPN, we have tested patients for the most frequent MPN clonal mutations. So, we have used the informations and patients of the dedicated EDITH cohort.On the other hand, we have constituted a MPN database (OBENE) of the patients diagnosed for MPN in our Hospitalcentre. By this way, we have analysed the frequency and impact of atrial arrhythmias, the benefit-risk balance of the use of DOAC, the impact of statins to reduce the thrombotic risk and the frequency and impact of the treatment nonadherence in this population.MPN and thromboses are linked, so it is necessary to increase our knowledge of their physiopathology to improve prevention and treatment of the events. This thesis brings some answers to some questions but, she is almost the starting point of common reflexion between clinicians and biologists interested in these domains

Увод: Бројне генске мутације су у основи наследних склоности ка повећаном згрушавању крви (наследних тромбофилија). Најчешће испитиване наследне тромбофилије су: недостатак антитромбина, недостатак протеина Ц, недостатак протеина С, мутација проакцелерина FV Leiden и мутација протромбина FII G 20210 А. Тромбоза дубоких вена (ТДВ) често се јавља у општој популацији а код великог броја оболелих долази до поновне појаве болести. Стога је важно утврђивање значајности параметара који доприносе ризику за настанак ТДВ. Циљ: Утврђивање учесталости тромбофилних мутација код болесника са венским тромбоемболизмом и код здравих особа у популацији Војводине, испитивање повезаности тромбофилних мутација са локализацијом тромбозе и појавом плућне емболије, утврђивање разлике у времену појављивања ТДВ између оболелих са присуством појединачне и удружене тромбофилије, утврђивање учесталости удружене тромбофилије код оболелих и здравих особа и њен утицај на понављање тромбозног процеса у односу на појединачне тромбофилије и на основу добијених резултата утврђивање у којим случајевима је потребно извршити генетичко испитивање тромбофилије ради одређивања врсте и трајања антитромбозне терапије. Материјал и методе: Испитано је 175 оболелих особа са потврђеном ТДВ и 115 клинички и лабораторијски здравих особа које никад нису доживеле тромбозни инцидент и које су одабране тако да се контролна група поклапа са групом оболелих по полу. Активност антитромбина, протеина Ц и С, резистенција на активирани протеин Ц, ниво фактора VIII и ниво фибриногена одређивани су коагулационим тестовима а присуство мутација FV Leiden, FII G 20210 А, MTHFRС 677 Т и MTHFRА 1298 С испитивано је методом алелске дискриминације на Real-Time PCR инструменту. Оболелима је мерен и крвни притисак и одређиван липидни статус у случајевима сумње на поремећај нивоа липида у крви. Добијени подаци су нумерички обрађивани стандардним процедурама дескриптивне статистике и статистичке анализе. Резултати: Постојање наследне тромбофилије је утврђено код 57,14% оболелих са ТДВ и 48,21% здравих особа. Код укупно 16,57% оболелих испитаника утврђен је недостатак барем једног од природних инхибитора коагулације: код 4,0% недостатак антитромбина, код 1,71% недостатак протеина Ц, код 10,29% недостатак протеина С и код 0,57% истовремено постојање недостатка протеина Ц и протеина С. Постојање резистенције на активирани протеин Ц утврђено је код 30,38% оболелих. Постојање FV Leiden мутације утврђено је код 21,14% оболелих, код 16,0% у хетерозиготном а код 5,14% у хомозиготном облику и код 10,42% испитаника контролне групе у хетерозиготном облику док хомозиготних носилаца није било. Постојање FIIG 20210 А мутације утврђено је код 7,43% оболелих, код 4,0% у хетерозиготном облику а код 3,43% у хомозиготном облику и код 8,18% испитаника контролне групе, код 6,36% у хетерозиготном облику а код 1,82% у хомозиготном облику. Постојање мутације MTHFR С 677 Т је утврђено код 50,86% оболелих, код 42,86% у хетерозиготном облику а код 18,0% у хомозиготном облику и код 62,62% испитаника контролне групе, код 43,93% у хетерозиготном облику а код 18,69% у хомозиготном облику. Постојање мутације MTHFR А 1298 С утврђено је код 44,94% оболелих и 25,37% здравих испитаника са MTHFR С 677 Т мутацијом. Присуство фактора VIII у повишеној концентрацији утврђено је код 17,71% оболелих а присуство повишених вредности концентрац ије фибриногена код 9,81% оболелих. Код укупно 84,57% оболелих испитаника утврђено је постојање барем једне тромбофилије. Закључци: Између постојања недостатака природних инхибитора коагулације, постојања FV Leiden мутације у хомозиготном облику, као и постојања удружене наследне тромбофилије и настанка ТДВ утврђено је постојање статистички значајне повезаности. Недостатак неког од природних инхибитора повезан је са највећим ризиком за настанак ТДВ. Између постојања FIIG 20210 А мутације, у хомозиготном или хетерозиготном облику, постојања FV Leiden мутације у хетерозиготном облику, као и постојања MTHFRC677 T мутације, самостално или истовремено са MTHFRА 1298 C мутацијом и настанка ТДВ није утврђено постојање статистички значајне повезаности. Између присуства FIIG 20210 А мутације или FV Leiden мутације и MTHFRC677 T мутације у хомозиготном облику, као и удружене наследне тромбофилије и појаве ТДВ у трудноћи или пуерперијуму утврђено је постојање статистички значајне повезаности. Између постојања наследне тромбофилије, појединачне или удружене, и клиничког испољавања венске тромбоемболијске болести код оболелих са ТДВ није утврђено постојање статистички значајне повезаности. Између постојања наследне тромбофилије, појединачне или удружене, и старости у време настанка ТДВ није утврђено постојање статистички значајне повезаности. Између присуства MTHFRC677 T мутације и поновљеног тромбозног процеса утврђено је постојање статистички значајне повезаности. Између присуства FIIG 20210 А мутације и FV Leiden мутације, у хомозиготном или хетерозиготном облику, удружене наследне тромбофилије и поновљеног тромбозног процеса није утврђено постојање статистички значајне повезаности. Између гојазности и повишеног крвног притиска и настанка ТДВ утврђено је постојање статистички значајне повезаности. Између хиперлипопротеинемије и настанка ТДВ није утврђено постојање статистички значајне повезаности. На основу добијених резултата можемо закључити да се одлука о испитивању наследне тромбофилије доноси узимајући у обзир старост, пол и присуство пролазних или трајних фактора ризика.
Uvod: Brojne genske mutacije su u osnovi naslednih sklonosti ka povećanom zgrušavanju krvi (naslednih trombofilija). Najčešće ispitivane nasledne trombofilije su: nedostatak antitrombina, nedostatak proteina C, nedostatak proteina S, mutacija proakcelerina FV Leiden i mutacija protrombina FII G 20210 A. Tromboza dubokih vena (TDV) često se javlja u opštoj populaciji a kod velikog broja obolelih dolazi do ponovne pojave bolesti. Stoga je važno utvrđivanje značajnosti parametara koji doprinose riziku za nastanak TDV. Cilj: Utvrđivanje učestalosti trombofilnih mutacija kod bolesnika sa venskim tromboembolizmom i kod zdravih osoba u populaciji Vojvodine, ispitivanje povezanosti trombofilnih mutacija sa lokalizacijom tromboze i pojavom plućne embolije, utvrđivanje razlike u vremenu pojavljivanja TDV između obolelih sa prisustvom pojedinačne i udružene trombofilije, utvrđivanje učestalosti udružene trombofilije kod obolelih i zdravih osoba i njen uticaj na ponavljanje tromboznog procesa u odnosu na pojedinačne trombofilije i na osnovu dobijenih rezultata utvrđivanje u kojim slučajevima je potrebno izvršiti genetičko ispitivanje trombofilije radi određivanja vrste i trajanja antitrombozne terapije. Materijal i metode: Ispitano je 175 obolelih osoba sa potvrđenom TDV i 115 klinički i laboratorijski zdravih osoba koje nikad nisu doživele trombozni incident i koje su odabrane tako da se kontrolna grupa poklapa sa grupom obolelih po polu. Aktivnost antitrombina, proteina C i S, rezistencija na aktivirani protein C, nivo faktora VIII i nivo fibrinogena određivani su koagulacionim testovima a prisustvo mutacija FV Leiden, FII G 20210 A, MTHFRS 677 T i MTHFRA 1298 S ispitivano je metodom alelske diskriminacije na Real-Time PCR instrumentu. Obolelima je meren i krvni pritisak i određivan lipidni status u slučajevima sumnje na poremećaj nivoa lipida u krvi. Dobijeni podaci su numerički obrađivani standardnim procedurama deskriptivne statistike i statističke analize. Rezultati: Postojanje nasledne trombofilije je utvrđeno kod 57,14% obolelih sa TDV i 48,21% zdravih osoba. Kod ukupno 16,57% obolelih ispitanika utvrđen je nedostatak barem jednog od prirodnih inhibitora koagulacije: kod 4,0% nedostatak antitrombina, kod 1,71% nedostatak proteina C, kod 10,29% nedostatak proteina S i kod 0,57% istovremeno postojanje nedostatka proteina C i proteina S. Postojanje rezistencije na aktivirani protein C utvrđeno je kod 30,38% obolelih. Postojanje FV Leiden mutacije utvrđeno je kod 21,14% obolelih, kod 16,0% u heterozigotnom a kod 5,14% u homozigotnom obliku i kod 10,42% ispitanika kontrolne grupe u heterozigotnom obliku dok homozigotnih nosilaca nije bilo. Postojanje FIIG 20210 A mutacije utvrđeno je kod 7,43% obolelih, kod 4,0% u heterozigotnom obliku a kod 3,43% u homozigotnom obliku i kod 8,18% ispitanika kontrolne grupe, kod 6,36% u heterozigotnom obliku a kod 1,82% u homozigotnom obliku. Postojanje mutacije MTHFR S 677 T je utvrđeno kod 50,86% obolelih, kod 42,86% u heterozigotnom obliku a kod 18,0% u homozigotnom obliku i kod 62,62% ispitanika kontrolne grupe, kod 43,93% u heterozigotnom obliku a kod 18,69% u homozigotnom obliku. Postojanje mutacije MTHFR A 1298 S utvrđeno je kod 44,94% obolelih i 25,37% zdravih ispitanika sa MTHFR S 677 T mutacijom. Prisustvo faktora VIII u povišenoj koncentraciji utvrđeno je kod 17,71% obolelih a prisustvo povišenih vrednosti koncentrac ije fibrinogena kod 9,81% obolelih. Kod ukupno 84,57% obolelih ispitanika utvrđeno je postojanje barem jedne trombofilije. Zaključci: Između postojanja nedostataka prirodnih inhibitora koagulacije, postojanja FV Leiden mutacije u homozigotnom obliku, kao i postojanja udružene nasledne trombofilije i nastanka TDV utvrđeno je postojanje statistički značajne povezanosti. Nedostatak nekog od prirodnih inhibitora povezan je sa najvećim rizikom za nastanak TDV. Između postojanja FIIG 20210 A mutacije, u homozigotnom ili heterozigotnom obliku, postojanja FV Leiden mutacije u heterozigotnom obliku, kao i postojanja MTHFRC677 T mutacije, samostalno ili istovremeno sa MTHFRA 1298 C mutacijom i nastanka TDV nije utvrđeno postojanje statistički značajne povezanosti. Između prisustva FIIG 20210 A mutacije ili FV Leiden mutacije i MTHFRC677 T mutacije u homozigotnom obliku, kao i udružene nasledne trombofilije i pojave TDV u trudnoći ili puerperijumu utvrđeno je postojanje statistički značajne povezanosti. Između postojanja nasledne trombofilije, pojedinačne ili udružene, i kliničkog ispoljavanja venske tromboembolijske bolesti kod obolelih sa TDV nije utvrđeno postojanje statistički značajne povezanosti. Između postojanja nasledne trombofilije, pojedinačne ili udružene, i starosti u vreme nastanka TDV nije utvrđeno postojanje statistički značajne povezanosti. Između prisustva MTHFRC677 T mutacije i ponovljenog tromboznog procesa utvrđeno je postojanje statistički značajne povezanosti. Između prisustva FIIG 20210 A mutacije i FV Leiden mutacije, u homozigotnom ili heterozigotnom obliku, udružene nasledne trombofilije i ponovljenog tromboznog procesa nije utvrđeno postojanje statistički značajne povezanosti. Između gojaznosti i povišenog krvnog pritiska i nastanka TDV utvrđeno je postojanje statistički značajne povezanosti. Između hiperlipoproteinemije i nastanka TDV nije utvrđeno postojanje statistički značajne povezanosti. Na osnovu dobijenih rezultata možemo zaključiti da se odluka o ispitivanju nasledne trombofilije donosi uzimajući u obzir starost, pol i prisustvo prolaznih ili trajnih faktora rizika.
Introduction: Numerous mutations are involved in genetic basis of an increased tendency for blood to clot (inherited thrombophilia). Most commonly investigated are: antithrombin deficiency, protein C deficiency, protein S deficiency, proaccelerin gene mutation FV Leiden and prothrombin gene mutation FIIG 20210 А. Deep vein thrombosis (DVT) is common in the general population and many patients experience a recurrence of the disease. It is, therefore, important to determine the significance of parameters contributing to the increased risk for DVT development. Objective: The aim of this study was to determine frequencies of thrombophilic mutations in patients with venous thromboembolism and healthy subjects in population of Vojvodina, to evaluate the association between thrombophilic mutations and thrombosis localization including pulmonary embolism, to determine the difference in age of DVT occurrence between patients with single and combined thrombophilic abnormalities, to determine frequencies of combined thrombophilic abnormalities in patients and healthy subjects and their influence on DVT recurrence compared to single thrombophilic abnormalities and on the basis of the results to determine when genetic testing is needed in order to determine the type and duration of anticoagulation therapy. Materials and methods: The study included 175 patients with documented DVT and sex-matched group of 115 healthy subjects without clinical or laboratory abnormalities, never having experienced DVT. Antithrombin, protein C and protein S activity; activated protein C resistance, factor VIII and fibrinogen levels are determined using coagulation tests, while for FV Leiden, FII G 20210 А, MTHFR C 677 T and MTHFR A 1298C mutation detection allelic discrimination assays on Real-Time PCR instrument are performed. Blood pressure is measured on all patients and in case of suspected plasma lipid levels elevation lipid profile is determined. The data obtained were subject to descriptive statistic and statistical analysis. Results: The total incidence of inherited thrombophilia was 57,14% in patients and 48,21% in healthy subjects. Total frequency of natural coagulation inhibitor deficiencies in patients was 16,57%: antithrombin deficiency 4,0%, protein C deficiency 1,71%, protein S deficiency 10,29% and combined protein C and protein S deficiency 0,57%. The incidence of activated protein C resistance in patients was 30,38%. The incidence of FV Leiden mutation was 21,14% in patients, 16,0% were heterozygous and 5,14% were homozygous, and 10,42% in healthy subjects, all of which were heterozygous. The incidence of FIIG 20210 А mutation was 7,43% in patients, 4,0% were heterozygous and 3,43% were homozygous, and 8,18% in healthy subjects, 6,36% were heterozygous and 1,82% were homozygous. The incidence of MTHFR C 677 T mutation was 50,86% in patients, 42,86% were heterozygous and 18,0% were homozygous, and 62,62% in healthy subjects, 43,93% were heterozygous and 18,69% were homozygous. The incidence of MTHFR A 1298 C mutation was 44,94% in patients with MTHFR C 677 T mutation and 25,37% in healthy subjects MTHFR C 677 T mutation. At least one thrombophilic abnormalitiy was present in 84,57% patients. Conclusions: The association between natural coagulation inhibitor deficiencies, FV Leiden mutation in homozygous form and combined thrombophilia and DVT development was statistically significant. Natural coagulation inhibitor deficiencies were associated with greatest risk of DVT development. The association between FII G 20210 А mutation, in homozygous or heterozygous form, FV Leiden mutation in heterozygous form, MTHFR C 677 T mutation alone or combined with MTHFR A 1298 C mutation and DVT development was not statistically significant. The association between FII G 20210 А mutation, FV Leiden mutation and MTHFR C 677 T mutation in homozygous form and DVT development during pregnancy and puerperium was statistically significant. The association between single or combined inherited thrombophilia and thrombosis localization including pulmonary embolism was not statistically significant. The association between single or combined inherited thrombophilia and the age of DVT occurrence was not statistically significant. The association between MTHFR C 677 T mutation and DVT recurrence was statistically significant. The association between FII G 20210 А mutation and FV Leiden mutation, in homozygous or heterozygous form, combined thrombophilia and DVT recurrence was not statistically significant. The association between obesity and high blood pressure and DVT development was statistically significant. The association between hyperlipoproteinemia and DVT development was not statistically significant. Based on the results we can conclude that decision to perform inherited thrombophilia testing should be based upon age, gender and the presence of transient or permanent risk factors.

碩士
靜宜大學
食品營養學系
99
Hyperglycemia-induced oxidative damage plays an important role in thrombogenic complications in diabetic patients. Research showed that astaxanthin is a natural food additive with superior anti-inflammatory and antioxidative capacity, however, its effects on diabetic macroangiopathy is unknown. Therefore, the purpose of this study was to investigate the effect of astaxanthin on thrombogenic risk in STZ-induced diabetic rats. Sixty Sprague-Dawley rats were randomly assigned into four experimental groups, including healthy control, diabetic control, diabetic rats with low dose (5 mg/kg BW) of astaxanthin, diabetic rats with high dose (15 mg/kg BW) of astaxanthin. After 8 weeks of feeding period, concentration of fasting glucose, insulin, lipid profile (TG、TC), antioxidative markers (MDA、ROS、GSH), inflammatory markers (CRP、IL-6、TNF-α、MCP-1), coagulation factor VII (FVII), anticoagulation factor (AT-III、PC), plasminogen activator inhibitor-1 (PAI-1) and von Willebrand factor (vWF) were measured. The results of this study showed that plasma concentration of MDA、ROS and CRP, renal IL-6、TNF-α and MCP-1, concentration of vWF and PAI-1 was significantly decreased in diabetic rats fed with astaxanthin. In addition, renal GSH was significantly increased, and the activity of FVII was significantly decreased in high-dosage astaxanthin fed diabetic rats, when compared to diabetic control rats. In conclusion, feeding high-dosage astaxanthin to diabetic rats may inhibit hyperglycemia-induced oxidative stress. Astaxanthin feeding, by inhibiting pro-inflammatory cytokines secretions, also ameliorates chronic inflammation, hypercoagulability, endothelial damage, and hence decrease the risk of developing thrombogenic complications.

碩士
靜宜大學
食品營養學系
102
Macrovascular complications are the major causes of death among diabetic patients, and hyperglycemia-induced oxidation and subsequent chronic inflammation play an important role in the etiology of diabetic macroangiopathy. Astaxanthin (AX) is one of the carotenoids with excellent anti-inflammatory and antioxidative capacity; therefore, the purpose of this study was to investigate the effect of astaxanthin and astaxanthin formula (AXF) on thrombogenic risk in type 2 diabetic patients. One hundred and three type 2 diabetic patients were recruited from Cheng Ching Hospital in Taichung city. After exclusion of unqualified patients, all subjects were randomly assigned into three treatment groups including placebo group (P), AXF (AX 6 mg + vitamin E 150 mg + Tocotrienol 45 mg/day) group and AX (AX: 14 mg/day) group, respectively. After two month’s supplementation, plasma concentration of AX, fasting blood glucose (FBG), HbA1c, lipid profiles, markers of hepatic and renal function, oxidative and inflammatory markers, coagulation and anti-coagulation factors were measured. The results showed that plasma levels of AX increased by supplementation in a dose-dependent manner. Plasma concentration of HbA1c, CRP and vWF was significantly decreased in AXF group. Plasma concentration of HbA1c, TG and TC, GPT, inflammatory cytokines of TNF-α, CRP and vWF was significantly decreased in AX group. In addition, plasma concentration of coagulation factor VII and PAI-1 was significantly decreased, and AT-III level was significantly increased in AX group. In conclusion, AX supplementation for two months may have beneficial effects on type 2 DM patients in ameliorating hyperglycemia, hyperlipidemia and liver malfunction. AX supplementation also decreases oxidation stress and inhibits chronic inflammation, hence ameliorates endothelial damage and thrombogenic risk in type 2 DM patients.

La trombosi portale è una occlusione parziale o totale del tronco portale e/o dei rami portali intraepatici associata o meno a trombosi dei vasi venosi affluenti (vena splenica e mesenterica superiore). E’ un evento che complica non di rado il decorso della cirrosi epatica. Varie sono le cause di trombosi portale sia in corso di cirrosi epatica sia in caso di soggetti non cirrotici. Per quanto riguarda la situazione specifica del paziente affetto da cirrosi epatica, l’insorgenza di trombosi portale può essere secondaria a manovre terapeutiche invasive come la sclerosi delle varici esofagee. In altri casi la presenza di un epatocarcinoma potrebbe determinare la comparsa di trombosi portale qualora la lesione focale si trovi in prossimità del vaso portale, ovvero la lesione stessa venga sottoposta ad alcolizzazioni, termoablazioni e/o ad chemioembolizzazioni. Abbiamo voluto valutare prospetticamente in una coorte di pazienti cirrotici senza Trombosi Portale (TP) e senza epatocarcinoma (HCC) all’arruolamento: • L’incidenza di nuovi casi di TP • I fattori di rischio della TP • L’influenza della TP incidente • sulla storia clinica (ascite, sanguinamento da varici esofagee, encefalopatia, etc) • L’influenza della TP incidente sulla sopravvivenza di pazienti cirrotici. Nel nostro Centro è stato eseguito uno screening di 292 pazienti consecutivi senza trombosi portale, sottoposti ad una ecografia bidimensionale, doppler ed ecocolor – doppler ogni sei mesi. In questo gruppo di pazienti è stata riscontrata una prevalenza della trombosi portale pari al 23,3 % (68 pazienti). Nel 22 % la TP è risultata essere completa e nel 78 % incompleta. Dei 292 pazienti osservati, 129 pazienti senza trombosi portale, senza epatocarcinoma all’arruolamento e con almeno 6 mesi di follow-up, sono stati osservati per valutare l’incidenza della trombosi portale e gli eventuali fattori di rischio ad essa associati. Ad ogni controllo ecografico sono stati valutati: • Presenza, localizzazione ed entità della trombosi portale • Presenza, numerosità e dimensioni di noduli sospetti per epatocarcinoma • Presenza ed entità dell’ascite • Presenza, dimensioni e flusso nelle vene paraombelicali In occasione di ogni controllo ecografico: • E’ stato somministrato un questionario su pregressi (nei sei mesi precedenti) episodi di ascite, sanguinamento da varici esofagee, sclerosi o legatura di varici, encefalopatia epatica, assunzione di farmaci (ponendo particolare attenzione all’uso dei beta-bloccanti); • Sono stati rilevati i dati biochimici per la stadiazione della epatopatia (C-P) In caso di decesso sono state rilevate data e cause. L’incidenza cumulativa di trombosi portale (PVT) nel nostro campione di pazienti cirrotici è risultata del 18,6 %. Nei pazienti che hanno sviluppato un epatocarcinoma nel corso del follow-up l’incidenza di una trombosi portale è risultata maggiore, sebbene in modo non statisticamente significativo, rispetto ai pazienti senza trombosi portale incidente. I pazienti che assumevano beta-bloccanti all’inizio del follow-up hanno sviluppato una maggiore incidenza di trombosi portale, in modo statisticamente significativo, rispetto ai pazienti che non assumevano beta-bloccanti. I pazienti che hanno sviluppato una trombosi portale nel follow -up, hanno presentato una significativa maggiore incidenza di: • Sanguinamento da varici esofagee • Scompenso ascitico • Encefalopatia epatica La sopravvivenza in pazienti con PVT incidente è risultata significativamente inferiore nel gruppo di pazienti che hanno sviluppato una trombosi portale incidente rispetto al gruppo dei pazienti senza trombosi portale.

博士
國立臺灣大學
藥理學研究所
105
The life-threatening thrombocytopenia, a common side effect of clinically available αIIbβ3 antagonists (e.g. abciximab, eptifibatide, and tirofiban), is associated with drug-dependent antibodies that recognize conformation-altered integrin αIIbβ3. Therefore, development of new antagonists with less tendency in causing bleeding is highly warranted. In these reports, we found that two disintegrins, TMV-2 and TMV-7, purified from snake venom of Trimeresurus mucrosquamatus, exhibit different antithrombotic properties. TMV-7 endows with a binding motif toward αIIb β-propeller domain of αIIbβ3, different from that of TMV-2, with advantages in that TMV-7 neither primed the platelets to bind ligand nor caused conformational change of αIIbβ3 identified by ligand-induced binding site (LIBS) mAb AP5. In contrast to eptifibatide and TMV-2, co-treatment of TMV-7 with AP2 did not induce FcγRIIa-mediated platelet aggregation and downstream activation cascade. Both TMV-2 and TMV-7 efficaciously prevented occlusive thrombosis in vivo. Notably, either eptifibatide or TMV-2 caused severe thrombocytopenia mediated by FcγRIIa, prolonged tail bleeding time in vivo, and repressed human whole blood coagulation-indexes, whereas TMV-7 did not impair the hemostatic capacity. These findings highlight the αIIbβ3 antagonist TMV-7 with minimal conformational effects may potentially help resolve the major problem associated with the current integrin antagonists that at doses where they exhibit high potency they also increase the risk of hemorrhage. Emerging evidence suggests that selective inhibition of outside-in signaling has the potential to have potent antithrombotic effects without causing bleeding. Our study also reveals that TMV-7 exhibits different binding epitope of αIIbβ3 from that of 7E3 and TMV-2, with advantages in that TMV-7 selectively inhibits Gα13 binding to integrin β3 without inhibiting talin binding to β3 in human and mouse platelets. Furthermore, TMV-2 and TMV-7 diminished activation of protein kinase c-Src and stimulated RhoA, consequently inhibiting platelet spreading. TMV-2 inhibited thrombin-induced clot retraction of platelet-rich plasma whereas TMV-7 did not. Notably, TMV-2 significantly prolonged the tail bleeding time and suppressed coagulation indexes as monitored by thromboelastography while TMV-7 did not as they were administered at efficacious antithrombotic doses. The study identifies the lead αIIbβ3 antagonist TMV-7 with unique binding epitopes efficaciously prevents thrombosis without affecting physiological hemostasis. In order to design an optimal anti-thrombotic agent with better safety profile, we mutated the RGD-domain of trimucrin from ARGDNP to AKGDRR, and found that this trimucrin AKGDRR mutant (RR) exhibits higher potency in inhibiting platelet aggregation and its safety index is raised to 70-time higher than TMV-7. RR prevented thrombosis by a mechanism similar to TMV-7 that decelerates αIIbβ3 ligation without causing conformational change and inducing little exposure of LIBs epitope on αIIbβ3, thus RR does not trigger FcγRII-mediated platelet aggregation. Furthermore, RR selectively inhibits Gα13-binding without affecting talin-binding to β3 in human thrombin-activated platelets. At efficacious antithrombotic doses, both TMV-7 and RR had little effect on tail-bleeding time even given at higher dose (i.e., 2.5 μg/g, 20-fold higher), indicating that they are efficacious antithrombotic αIIbβ3 antagonists with a greater safety profile than the current αIIbβ3 antagonists. Further examining the molecular interaction between TMV-7 and its derivative may provide valuable information for development of new anti-thrombotic with a better safety profile.

M. Tech. Biomedical Sciences.
Aims to assess the prevalence rates of hyperlipidaemia, hyperglycaemia, elevated homocysteine levels, elevated FVII levels and high blood pressure, as risk parameters of arterial thrombosis, in a rural black community in the Limpopo Province South Africa.