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Abstract Background: Deficiency of Von Willebrand Factor Cleaving Protease (VWFCP) has been implicated as the cause of Thrombotic Thrombocytopenic Purpura (TTP). TTP is a lifethreatening disease characterised by microangiopathic thrombosis due to accumulation of Ultralarge Von Willebrand Factor (ULVWF) multimers. The clinical features of TTP include microangiopathic haemolysis and thrombocytopenia. TTP is being seen with increased frequency in the context of HIV. However, in the context of HIV infection, cytopenias are often multifactorial in nature and levels of VWFCP in HIV-related thrombocytopenia have not specifically been assessed. This study assessed VWFCP activity in the setting of patients with HIV and thrombocytopenia in the absence of TTP, in order to determine the utility of a VWFCP assay in the diagnosis of HIV-related TTP. Acquired VWFCP deficiency is generally assumed to be due to the presence of autoantibody inhibitors to the enzyme, but limited data are available regarding VWFCP activity in HIV positive TTP patients. There is also currently no assay available for measuring VWFCP activity in our laboratory. Aim of Study: To establish a practical assay for VWFCP activity for routine use in our laboratory. The rapid collagen binding assay, based on the ELISA method of Rick, et al., 2002, was chosen. This was initially used to measure VWFCP activity in patients with HIV with and without thrombocytopenia (of any cause except TTP), in order to ascertain whether assessment of VWFCP activity is likely to be of value in facilitating early diagnosis of HIV related TTP. The ELISA assay was performed to establish cut-off values for VWFCP in HIV negative controls and two HIV positive groups (HIV thrombocytopenia / low platelets and HIV normal platelets). Depending on the outcome of this, the assay could then be performed to assess VWFCP activity in HIV positive patients with TTP. Methods: The rapid collagen binding assay for VWFCP activity was established and optimised for routine use in our laboratory. The cut-off values for percentage Residual Collagen Binding Activity (RCBA) in both HIV negative and HIV positive groups were identified. The assay could then be used to assess VWFCP activity in 20 HIV positive patients with TTP at the time of presentation. In patients with reduced VWFCP activity, patient plasma was mixed with normal pool plasma in a 50:50 mix, to assess for the presence of inhibitors. Correlation of VWFCP activity, inhibitors and other laboratory and clinical parameters were performed. Results: The cut-off values for percentage RCBA in both HIV negative (<37.12%) and HIV positive (<51.51%) patients were established. The % RCBA for the HIV negative control group was statistically significantly different from the HIV positive group with normal platelets (p=0.0001) and from the HIV positive group with low platelets (p=0.0006). The cut-off value in the two HIV positive patient groups was higher than for HIV negative control patients, indicating mildly reduced VWFCP enzyme activity in HIV positive patients (regardless of the platelet count), in the absence of TTP. However, no significant difference in the cut-off value was noted between HIV positive patients with low platelet counts versus HIV positive patients with normal platelet counts (p=0.7783). The assay could therefore be used in HIV positive patients with TTP. VWFCP activity was assessed in twenty HIV positive patients with TTP. Two groups of HIV positive patients with TTP were identified based on VWFCP activity. Six patients (30%) had normal (one borderline) VWFCP activity (RCBA <51.51%), while the remaining 14 patients had severely reduced VWFCP levels (RCBA >90%). Of the patients with reduced VWFCP activity, only 5 patients had a detectable inhibitor, while an inhibitor was not detected in the remaining 8 patients. Conclusion: The rapid collagen binding ELISA assay is a cost effective semi-quantitative assay for the assessment of VWFCP activity. VWFCP activity in HIV positive patients appears to be slightly lower, however is not related to the platelet count. This suggests a slight baseline deficiency of VWFCP in the setting of HIV. The baseline VWFCP cut-off value in HIV allowed assessment of HIV positive patients with TTP. The results suggest heterogeneity of VWFCP activity in HIV-related TTP. A negative result (normal VWFCP activity) does not exclude TTP in patients with HIV-related TTP and other pathogenic factors may therefore be involved.

Thesis (M.Sc.)--University of Alberta, 2009. / A thesis submitted to the Faculty of Graduate Studies and Research in partial fulfillment of the requirements for the degree of Master of Science in Experimental Medicine, Department of Medicine. Title from pdf file main screen (viewed on January 17, 2010). Includes bibliographical references.

Hydroxyethyl starches (HES) are the most routinely used synthetic colloids during fluid resuscitation and have reported effects on coagulation. The overall goal of the investigation in this thesis was to evaluate the effects of tetrastarch administration on hemodynamic, laboratory, and hemostatic variables in healthy dogs and dogs with systemic inflammation. The objectives were to compare hemodynamic and laboratory variables in dogs receiving an isotonic crystalloid (0.9% NaCl) or tetrastarch during health and after induction of systemic inflammation; to compare the hemostatic effects of an isotonic crystalloid (0.9% NaCl) and synthetic colloid (tetrastarch) in healthy dogs and dogs with induced systemic inflammation; to compare two different protocols for TEG® activation and to determine the correlation between TEG® variables and traditional coagulation test results. Sixteen adult purpose-bred Beagles were randomized into one of two groups receiving fluid resuscitation with either 40 mL/kg IV isotonic crystalloid (0.9% NaCl) or synthetic colloid (tetrastarch) after administration of lipopolysaccharide (LPS; 5 μg/kg, IV) or an equal volume of placebo (0.9% NaCl, IV). Blood samples, for analysis, were collected at 0, 1, 2, 4, and 24 hours from the time of fluid resuscitation. After a 14-day washout period, the study was repeated such that dogs received the opposite treatment (LPS or placebo) and the same resuscitation fluid. Resuscitation with equal volumes of 0.9% NaCl and tetrastarch caused similar changes in hemodynamic and laboratory variables in dogs with LPS-induced systemic inflammation; however, larger increases in HR and blood pressure were seen within the first 2 hours following tetrastarch administration compared to 0.9% NaCl. Tetrastarch administration increased COP in all dogs, despite a decrease in TS. Tetrastarch bolus administration to dogs with LPS-induced systemic inflammation also resulted in a transient hypocoagulability characterized by a prolonged PTT, decreased clot formation speed and clot strength, and acquired type 1 von Willebrand disease. Considering the limited additional benefit of tetrastarch administration on hemodynamic variables demonstrated, as well as the transient adverse hemostatic effects of tetrastarch administration, the increased cost associated with the use of tetrastarch likely negates its use as a first line treatment during fluid resuscitation in dogs. / Pet Trust Fund

Thesis (Ph. D.)--University of Virginia, 2008. / Title from title page. Includes bibliographical references. Also available online through Digital Dissertations.

Cysteine rich with EGF-like domains 2 (Creld2) is a novel endoplasmic reticulum (ER) resident molecular chaperone that has been recently implicated in the ER stress signalling response (ERSS) and the unfolded protein response (UPR). Global transcriptomic data derived from in vivo mouse models of rare chondrodysplasias; Multiple Epiphyseal Dysplasia (MED Matn3 p.V194D) and Metaphyseal chondrodysplasia type Schmid (MCDS Col10a1 p.N617K), identified a significant upregulation in Creld2 expression in mutant chondrocytes. These chondrodysplasias share a common disease signature consisting of aberrant folding of a matrix component often as a result of inappropriate alignment of intramolecular disulphide bonds. This in turn culminates in toxic protein aggregation, intracellular retention mutant polypeptides and a classical ER stress response. The aim of this study was to further analyse the function of Creld2 in cartilage development and chondrodysplasias in which endochondral bone growth is perturbed. Protein disulphide isomerases (PDIAs) were amongst the most up-regulated genes in the MED and MCDS mouse models, consistent with the prolonged exposure of normally 'buried' cysteine residues. This led to the hypothesis that Creld2 was functioning as a novel PDI-like oxidoreductase to assist in the correct folding and maturation of aggregated misfolded polypeptide chains through REDOX regulated thiol disulphide exchange. A series of Creld2-CXXA substrate trapping mutants were generated in order to determine whether Creld2 possessed inherent isomerase activity. Here potential substrates interacting with Creld2 were 'trapped' as mixed disulphide intermediates, then isolated by immunoprecipitation and identified by mass spectrometry analysis. It was demonstrated that Creld2 possessed a catalytic active CXXC motif in its N-terminus that enabled the molecular chaperone to participate in REDOX regulated thiol disulphide exchange with at least 20 potential substrates including; laminin (alpha3,β3,γ2), thrombospondin 1, integrin alpha3 and type VI collagen. There was also numerous co-chaperones and foldases thought to be part of a specialised protein-protein interactome (PPI) for folding nascent polypeptides translocating the ER lumen. Moreover, co-immunoprecipitation experiments supported a protein-protein interaction between Creld2 and mutant matrilin-3, thereby inferring a potential chondro-protective role in resolving non-native disulphide bonded aggregates in MED. An established biochemical approach was employed to test the hypothesis that all MATN3-MED disease causing mutations have a generic cellular response to the β-sheet V194D mutation, consisting of intracellular retention, protein aggregation and ER stress induction. Several missense mutations were selected for analyses which encompassed a spectrum of disease severity and included examples of both β-sheet and alpha helical mutations. It was possible to define a reliable and reproducible assay for categorising MATN3 missense mutations into pathological or benign based on these basic parameters. This study was extended further to determine whether there were common pathological mechanisms behind MED and Bethlem myopathy (BM) caused by missense mutations in von Willebrand Factor A domain (vWF-A) containing proteins (matrilin-3 and type VI collagen respectively). We chose to compare and contrast the effects of an archetypal MATN3-MED causing mutation (R121W) with the equivalent COL6A2-BM causing mutation (R876H). These mutations compromised protein folding and maturation, resulting in the familiar disease profile of intracellular retention, protein aggregation and an ER stress response in an artificial overexpression system. However, the mutant C2 domain was efficiently targeted for degradation whilst mutant matrilin-3 vWF-A domain appeared to be resistant to these molecular processes.Molecular genetics was employed to study the role of Creld2 in vivo. Creld2-/- null mice (both global and conditional) were generated to directly examine the role of Creld2 in endochondral bone growth. Global knock-out mice were viable with no overt phenotype at birth. However, female Creld2-/- null mice showed a significant reduction in body weight and tibia bone length at 3 weeks of age. A cartilage specific knock-out was generated to determine whether these skeletal abnormalities were attributed to a systemic or a direct effect on cartilage development. [Creld2Flox/Flox Col2Cre (+)] demonstrated a severe chondrodysplasia with significantly reduced body weight and long bone growth compared to control littermates. Morphological and histochemical analysis of mutant growth plates revealed gross disorganisation of the chondrocyte columns with extensive regions of hypocellularity. These pathological features were confirmed to be the result of reduced chondrocyte proliferation and increased/spatially dysregulated apoptosis throughout all zones of differentiation. Taken together, these data provide evidence that Creld2 possesses isomerase activity and exhibits distinct substrate specificity. Furthermore, Creld2 has a fundamental role in post-natal cartilage development and chondrocyte differentiation in the growth plate.

Made available in DSpace on 2015-06-17T19:34:48Z (GMT). No. of bitstreams: 0 Previous issue date: 2014-07-29. Added 1 bitstream(s) on 2015-06-18T12:47:02Z : No. of bitstreams: 1 000831074.pdf: 615076 bytes, checksum: c97e8862ed17121a7d6c2ddd7dfb24e1 (MD5) / Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP) / O fator VIII (FVIII), o fator de von Willebrand (FvW) e o fibrinogênio são de suma importância na coagulação sanguínea, com diferentes funções fisiológicas. Por conter altas concentrações destes fatores a transfusão de crioprecipitado é uma terapia utilizada principalmente em pacientes que apresentam Doença de von Willebrand, Hemofilia A (deficiência do FVIII), ou pacientes que sofrem de hipo ou disfibrinogenemia. Este hemocomponente é um precipitado obtido após o descongelamento parcial (entre 1 e 6°C) do plasma fresco congelado, e também é conhecido como fator anti-hemofílico. Estudos têm demonstrado que o protocolo de congelamento e armazenamento do crioprecipitado afeta a qualidade do produto e a viabilidade destes fatores. Com o objetivo de avaliar a viabilidade do crioprecipitado canino em diferentes protocolos de congelamento e armazenamento foram avaliados dois grupos compostos de 10 unidades de crioprecipitado canino (n=20). Após a centrifugação das bolsas de sangue, o plasma fresco foi congelado a -80ºC (grupo I) e a -20ºC (grupo II). Vinte e quatro horas após o congelamento das bolsas, estas foram submetidas ao procedimento de extração do crioprecipitado. Os crioprecipitados das bolsas dos dois grupos foram submetidos à determinação do TP, TTPA, FVIII, FvW e fibrinogênio, no momento zero e após seis meses de estocagem. Para a realização das coletas, foram utilizadas bolsas sanguíneas triplas de plástico, com anticoagulante CPDA-1, sendo a bolsa principal com capacidade para 450 mL de sangue total (JP Indústria Farmacêutica®). Após o crioprecipitado devidamente pronto, uma alíquota de aproximadamente 5 mL da bolsa de crioprecipitado foi separada em criotubos para análise da amostra pré-estocagem e seis meses pós estocagem. As amostras obtidas em cada momento foram congeladas à -80ºC até o momento do processamento. Os resultados mostraram um decréscimo significativo dos fatores e ... / The factor VIII (FVIII), the von Willebrand factor (vWF) and the fibrinogen are extremely important to the blood clotting process, with various physiological functions. Because it contains high concentrations of these factors and fibrinogen, transfusing cryoprecipitate is a therapy mainly used in patients who have von Willebrand disease, Hemophilia A (FVIII deficiency), or who suffer from hypo/dysfibrinogenemia. This hemocomponent is a precipitate obtained after the partial thawing process (between 1 and 6ºC) of fresh frozen plasma, and which is also known as the anti-hemophilic factor. Studies have demonstrated that the cryoprecipitate freezing and storage protocol affects the product quality as well as these factors viability. In order to evaluate the canine cryoprecipitate viability in different freezing and storage protocols, two groups containing 10 units of canine cryoprecipitate (n=20) were evaluated. Following the blood centrifugation, the fresh plasma was frozen at -80ºC (group I) and at -20ºC (group II). Twenty-four hours after freezing the blood bags, they were submitted to the cryoprecipitate extraction procedure. The cryoprecipitate from both groups of blood bags were submitted to the TP, TTPA, FVIII, FvW and fibrinogen determination process, at time zero and after six months of storage. During the collections, triple plastic blood bags were used, along with the anticoagulant CPDA-1, being the main bag capacity of 450 mL of whole blood (JP Indústria Farmacêutica®). After having the cryoprecipitate properly ready, an approximately 5 mL aliquot of cryoprecipitate was separated into cryovials to be analysed pre-storage and six months after storage. However, there was no significant difference between treatments, demonstrating that the difference in initial freezing temperature did not influence the decrease of the factors after six months storage at -20°C / FAPESP: 2012/13677-6

Orientador: Regina Kiomi Takahira / Banca: Luiz Henrique de Araújo Machado / Banca: Simone Gonçalves Rodrigues Gomes / Resumo: O fator VIII (FVIII), o fator de von Willebrand (FvW) e o fibrinogênio são de suma importância na coagulação sanguínea, com diferentes funções fisiológicas. Por conter altas concentrações destes fatores a transfusão de crioprecipitado é uma terapia utilizada principalmente em pacientes que apresentam Doença de von Willebrand, Hemofilia A (deficiência do FVIII), ou pacientes que sofrem de hipo ou disfibrinogenemia. Este hemocomponente é um precipitado obtido após o descongelamento parcial (entre 1 e 6°C) do plasma fresco congelado, e também é conhecido como fator anti-hemofílico. Estudos têm demonstrado que o protocolo de congelamento e armazenamento do crioprecipitado afeta a qualidade do produto e a viabilidade destes fatores. Com o objetivo de avaliar a viabilidade do crioprecipitado canino em diferentes protocolos de congelamento e armazenamento foram avaliados dois grupos compostos de 10 unidades de crioprecipitado canino (n=20). Após a centrifugação das bolsas de sangue, o plasma fresco foi congelado a -80ºC (grupo I) e a -20ºC (grupo II). Vinte e quatro horas após o congelamento das bolsas, estas foram submetidas ao procedimento de extração do crioprecipitado. Os crioprecipitados das bolsas dos dois grupos foram submetidos à determinação do TP, TTPA, FVIII, FvW e fibrinogênio, no momento zero e após seis meses de estocagem. Para a realização das coletas, foram utilizadas bolsas sanguíneas triplas de plástico, com anticoagulante CPDA-1, sendo a bolsa principal com capacidade para 450 mL de sangue total (JP Indústria Farmacêutica®). Após o crioprecipitado devidamente pronto, uma alíquota de aproximadamente 5 mL da bolsa de crioprecipitado foi separada em criotubos para análise da amostra pré-estocagem e seis meses pós estocagem. As amostras obtidas em cada momento foram congeladas à -80ºC até o momento do processamento. Os resultados mostraram um decréscimo significativo dos fatores e ... / Abstract: The factor VIII (FVIII), the von Willebrand factor (vWF) and the fibrinogen are extremely important to the blood clotting process, with various physiological functions. Because it contains high concentrations of these factors and fibrinogen, transfusing cryoprecipitate is a therapy mainly used in patients who have von Willebrand disease, Hemophilia A (FVIII deficiency), or who suffer from hypo/dysfibrinogenemia. This hemocomponent is a precipitate obtained after the partial thawing process (between 1 and 6ºC) of fresh frozen plasma, and which is also known as the anti-hemophilic factor. Studies have demonstrated that the cryoprecipitate freezing and storage protocol affects the product quality as well as these factors viability. In order to evaluate the canine cryoprecipitate viability in different freezing and storage protocols, two groups containing 10 units of canine cryoprecipitate (n=20) were evaluated. Following the blood centrifugation, the fresh plasma was frozen at -80ºC (group I) and at -20ºC (group II). Twenty-four hours after freezing the blood bags, they were submitted to the cryoprecipitate extraction procedure. The cryoprecipitate from both groups of blood bags were submitted to the TP, TTPA, FVIII, FvW and fibrinogen determination process, at time zero and after six months of storage. During the collections, triple plastic blood bags were used, along with the anticoagulant CPDA-1, being the main bag capacity of 450 mL of whole blood (JP Indústria Farmacêutica®). After having the cryoprecipitate properly ready, an approximately 5 mL aliquot of cryoprecipitate was separated into cryovials to be analysed pre-storage and six months after storage. However, there was no significant difference between treatments, demonstrating that the difference in initial freezing temperature did not influence the decrease of the factors after six months storage at -20°C / Mestre

Orientador: Joyce Maria Annichino-Bizzacchi / Dissertação (mestrado) - Universidade Estadual de Campinas, Faculdade de Ciências Médicas / Made available in DSpace on 2018-08-20T16:00:11Z (GMT). No. of bitstreams: 1 Fonseca_BrunadeMoraesMazetto_M.pdf: 657024 bytes, checksum: 715789e040378f27c5de278879f4535b (MD5) Previous issue date: 2011 / Resumo: Níveis elevados de marcadores inflamatórios e fatores de coagulação têm sido relacionados com a patogênese do TEV. Particularmente, a relação inversa entre o FVW e a atividade da ADAMTS13 já foi previamente descrita em pacientes com trombose arterial. Níveis de FVW também mostraram-se elevados durante processos inflamatórios e portanto, poderiam desempenhar um papel de ligação entre inflamação e coagulação nos pacientes com TEV. Objetivo: Avaliar a atividade da ADAMTS13 e do FVW e sua associação com marcadores inflamatórios e evolução clínica pós-trombótica em pacientes com TEV. Pacientes e Métodos: Setenta e sete pacientes com TEV, entre sete meses e seis anos após o episódio agudo, atendidos no Hemocentro de Campinas - UNICAMP foram incluídos neste estudo e 77 indivíduos normais foram selecionados como controles, pareados por idade, gênero, etnia e grupo sanguíneo. A atividade da ADAMTS13 e do FVW foram avaliados pela ligação do FVW ao colágeno, o dímero-D por turbidimetria, a PCR por nefelometria, TNF-'alfa', IL-6, IL-8, antígeno do FVW e da ADAMTS13 foram determinados por ELISA. A presença de trombo residual foi avaliada por ultrassom com Doppler e a SPT através da escala Villalta. Resultados: Trinta pacientes (39%) tiveram TEV causado por fatores de risco transitórios, especialmente pelo uso de anticoncepcional e 47 pacientes tiveram TVE espontâneo. A atividade inflamatória estava aumentada nos pacientes em comparação aos controles, demonstrada pelo aumento significativo dos níveis séricos de TNF-'alfa' and IL-6 nos primeiros (mediana= 2,25 vs 1,59pg/mL, P?0,001; 1,16 vs 0,98pg/ml, P=0,013, respectivamente). Os níveis de IL-8 e PCR foram similares entre os 2 grupos (mediana= 18,3 vs 18,27pg/mL, P=0,47; 0.21 vs 0,17mg/dL, P=0,29, respectivamente). Trinta e dois pacientes (42,8%) foram definidos como tendo um aumento da atividade coagulante, expressa pelo dímero-D > 0,55mg/dL. Nesse grupo de pacientes todos os marcadores inflamatórios como TNF-'alfa', IL-6, IL-8 and PCR, estavam significativamente aumentados quando comparados aos pacientes com dímero-D ? 0,55 mg/L (P=0,0057; 0,001; 0,0093 e 0,0075; respectivamente). A presença de SPT e trombo residual não foram associados ao aumento da atividade coagulante. A atividade da ADAMTS13 e os níveis séricos de IL-8 estavam aumentados em pacientes com SPT quando comparados aos pacientes sem SPT. Todos os marcadores inflamatórios e parâmetros da coagulação estudados foram similares em pacientes independentemente da presença do trombo residual. Conclusão: Este estudo sugere que exista atividade inflamatória e procoagulante nos pacientes mesmo após o episódio agudo do TEV, que, entretanto, não se mostrou estar relacionada com a persistência das seqüelas clínicas e radiológicas da TVP. Além disso, o aumento do FVW nos pacientes corrobora a hipótese de ativação crônica da inflamação. Neste contexto, o aumento observado da ADAMTS 13 poderia ser compensatório frente ao aumento crônico do FVW e poderia inclusive atuar com um mecanismo protetor contra a atividade pró-trombótica observada nestes pacientes / Abstract: Introduction: Increased levels of inflammatory markers and clotting factors have been related to the pathogenesis of VTE. Particularly, the inverse relation between VWF and ADAMTS13 activity has been previously described in patients with arterial thrombosis. VWF levels are also known to be increased during inflammatory processes and therefore could play a role linking the inflammatory and coagulation systems activities in patients with VTE. Objective: To evaluate the activity of ADAMTS13 and VWF in patients with VTE and its association with inflammatory markers and clinical outcome of post-thrombotic syndrome. Patients and methods: Seventy-seven patients with VTE, 7 months to six years after the acute episode, attended at the Hemocentro of Campinas - UNICAMP, were included in this study and 77 normal subjects were selected as controls, matched by gender, age, ethnicity and ABO blood group. The activity of ADAMTS 13 was performed by VWF collagen binding, D-dímer by turbidimetry, CRP by nephelometry , and TNF-'alpha', IL-6 and IL-8, VWF and ADAMTS13 antigen by ELISA. The presence of RVO was investigated by duplex examination and PTS by Villalta scale. Results: Thirty patients (39%) had VTE caused by transient risk factors, mainly the use of oral contraceptives, and 47 patients had spontaneous VTE. Serum levels of TNF-'alpha' and IL-6 were significantly increased in patients when compared to controls (median= 2.25 vs 1.59pg/mL, P?0.001; 1.16 vs 0.98pg/ml, P=0.013, respectively) whereas levels of IL-8 and CRP were similar among the groups (median= 18.3 vs 18.27pg/mL, p=0.47; 0.21 vs 0.17mg/dL, P=0.29, respectively). Thirty-two patients (42,8%) had D-dimer > 0.55 mg/L and were defined as having increased coagulation activity. Inflammatory markers, such as TNF-'alpha', IL-6, IL-8 and CRP, were significantly higher in those patients, comparing to patients with D-dimer ? 0.55 mg/L (P=0.0057, 0.001, 0.0093 and 0.0075, respectively). The presence of PTS or RVO were not associated with increased inflammatory or coagulation activity. Only ADAMTS13-CBA and plasma levels of IL-8 were higher in patients with PTS comparing to patients without PTS. All inflammatory markers and coagulation parameters studied were similar in patients regardless the presence of RVO. Conclusion: Our findings suggest that there is an inflammatory and pro-coagulant activity in patients even after the acute episode of VTE, however, these activities were not related to the persistence of clinical and radiological sequels of DVT. Moreover, the increasing levels of VWF, observed in patients, support the hypothesis that the inflammation is chronically activated. In this context, the increasing levels of ADAMTS13, also observed in patients, could be explained as a compensatory mechanism and maybe act as a protection against pro-thrombotic activity seen in these patients / Mestrado / Biologia Estrutural, Celular, Molecular e do Desenvolvimento / Mestre em Fisiopatologia Médica

Protéolyse du facteur von Willebrand et cardiopathies à forces de cisaillement élevées: nouvelles approches diagnostiques et thérapeutiques Dans la première partie de ce travail, nous mettons en évidence l’intérêt d’une immunothérapie spécifique à base d’anticorps monoclonal pour la prévention de la dégradation du facteur von Willebrand (VWF) sous assistance circulatoire mécanique à flux continu. Via un anticorps monoclonal murin ciblant le domaine D4 du VWF et inhibant partiellement l’interaction VWF-ADAMTS13, une inhibition partielle de la dégradation du VWF est observée sur sang total dans un modèle ex-vivo d’assistance circulatoire mécanique.Dans la seconde partie, nous avons étudié l’influence de soudaines variations de l’intensité des forces de cisaillement sur la multimérisation du VWF dans 3 modèles in-vivo: un modèle lapin de sténose de l’aorte ascendante, à l’initiation d’une assistance ventriculaire gauche par une pompe à flux continu chez des patients en insuffisance cardiaque terminale et lors d’un remplacement valvulaire aortique par voie percutané chez des patients avec un rétrécissement aortique sévère. Les variations observées du profil multimérique sont très dynamiques survenant quelques minutes après les modifications des conditions de flux. Notre étude met ainsi en évidence une nouvelle application potentielle du VWF comme biomarqueur d’anomalies de flux dans les cardiopathies à forces de cisaillement élevées. Un monitoring en temps réel du VWF pourrait notamment avoir un intérêt en cardiologie interventionnelle pour les techniques percutanées utilisées pour le traitement du rétrécissement aortique.La dernière partie de ce travail porte sur le développement d’un test ELISA pour le diagnostic des formes acquises ou constitutionnelles de maladie de Willebrand secondaire à une protéolyse excessive du VWF par l’ADAMTS13. Ce test pourrait constituer une alternative intéressante aux actuelles méthodes électrophorétiques pour le diagnostic et la prise en charge de ces pathologies hémorragiques. / In the first part of the thesis, we describe a novel approach based on antibody-based therapy to prevent the acquired von Willebrand factor (VWF) degradation observed in continuous-flow mechanical circulatory assist device therapy. Via a murine monoclonal antibody directed against VWF D4 domain and thus interfering with VWF-ADAMTS13 binding, a partial inhibition of VWF degradation is observed in whole blood using an ex vivo circulatory assist device model. In the second part of the thesis, we investigated the relationship between acute changes in shear stress and variations in VWF multimeric profile in three distincts models in vivo: in a rabbit aortic banding model, in end-stage heart failure patients at initiation of continuous-flow ventricular assist device therapy and in severe aortic stenosis patients undergoing percutaneous aortic valve procedures. Variations in VWF multimeric profile in those settings are highly dynamic occuring within minutes after changes in shear stress status. Our study highlights that VWF could be used as a biomarker of blood flow in high shear cardiovascular disorders. A bedside VWF-monitoring could be of clinical interest in interventional cardiology for percutaneous aortic valve procedures used in severe aortic stenosis.The last part of the thesis focused on the development of an ELISA-based diagnosis of constitutive or acquired VWF disorders associated with an increased ADAMTS13-mediated VWF proteolysis. Such assay might represent an attractive alternative to electrophoresis-based assays in the diagnosis and management of such bleeding disorders.