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Le facteur VIII (FVIII) est une glycoprotéine de la coagulation plasmatique, co-facteur du facteur IX activé (FIXa). Son métabolisme dépend de multiples facteurs limitant ou favorisant sa survie ou sa fonction. L’objectif de ce travail était d’analyser les différents paramètres pouvant influencer le taux de FVIII circulant et sa fonction pro-coagulante. Il a compris 4 volets : 1) Nous avons montré que la rétention intra-cellulaire connue du FVIII était en grande partie liée à son agrégation et sa dégradation par les 2 voies protéasomale et lysosomale. 2) Nous avons analysé les mutations du gène F8 responsables de l’hémophilie A dans une large cohorte de patients. Leur analyse bio-informatique a permis de démontrer leur caractère délétère pour le FVIII. L’influence de ces mutations sur la survenue d’allo-anticorps anti-FVIII a été étudiée et stratifiée, en association avec l’origine ethnique et la recherche d’antécédents familiaux d’nticorps. 3) La recherche des facteurs influençant les taux de FVIII chez les conductrices d’hémophilie A a mis en évidence des déterminants majeurs : l’existence d’une maladie génétique additionnelle responsable d’un déficit en FVIII, le taux de facteur Willebrand et l’inactivation non aléatoire du chromosome X ; et des déterminants secondaires : l’âge, la sévérité de l’hémophilie, le polymorphisme D1241E du gène F8 et 5 nouveaux polymorphismes de la LRP1 localisés dans ses sites de liaison pour le FVIII. 4) Nous avons analysé 8 FVIII recombinant mutés in vitro en alanine dans la région 1808-1818 du FVIII. Les études antérieures n’analysant que la chaîne légère, ont montré que cette région était la plus affine pour le FIXa. Nous démontrons ici que la région 1808-1818 ne paraît pas si essentielle à la fonction du FVIIIcar au sein de la molécule entière, son affinité diminue et ses mutations n’altèrent que très modérément l’activité du FVIII. / The factor VIII (FVIII) is a glucoprotein of the coagulation, being the cofactor of the activated factor IX (FIXa). Its metabolism depends on various limiting factors or enhancing its survey or function. The objective of this research’s work was to analyse different parameters that could influence the plasma FVIII level and its pro-coagulant function. It included 4 parts : 1) We showed that the known intra-cellular retention of FVIII was mainly due to its aggregation and degradation following both proteasomal and lysosomal pathways. 2) We analysed FVIII gene mutations responsible for hemophilia A in a large patients cohort. The bio-informatic analysis demonstrated its deleterious consequence. The influence of these mutations on the anti-FVIII antibodies occurrence was stratified, in association with ethnicity and familial antecedent of inhibitor. 3) The research of factors influencing FVIII levels in hemophilia A carriers showed : i) major determinants such as the presence of an additional genetic disease characterised by a FVIII deficiency, the factor Willebrad’s level and the non-random inactivation of the X chromosome; and ii) minor determinants : age, severity of hemophilia, the polymorphism D1241E of FVIII gene, and 5 new polymorphisms of LRP1 located in its binding site for FVIII.4) We analysed 8 recombinant FVIII with in vitro created mutations in its 1808-1818 region. Previous studies that analysed only the FVIII light chain, have shown that this region constituted the more affine binding site of FVIII for FIXa. We demonstrated here that the 1808-1818 region is not as essential as it was reported because within the entire molecule, its affinity decreases and mutations affecting it do alter mildly the FVIII activity.

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.

Le facteur Willebrand (VWF) est une proteine multimerique qui a une sensibilite unique aux forces de cisaillement et aux variations hemodynamiques du flux sanguin comme celles rencontrees lors d’utilisation de dispositifs cardiovasculaires tels qu’un remplacement valvulaire aortique transcatheter (TAVI) ou un assistance circulatoire mecanique a flux continu (ACM-FC). Des travaux anterieurs nous ont permis de mettre en evidence une secretion endotheliale declenchee par les modifications du flux liees a l’utilisation de ces dispositifs.Dans la première partie de la these, nous avons choisi d’etudier le role de la pulsatilite arterielle dans la reponse endotheliale a l’aide de plusieurs modeles animaux porcins d’ACM-FC pour isoler le role de la pulsatilite dans un environnement a forces de cisaillement elevees et constantes. Nous avons observe dans un modele dose-reponse la relation entre le niveau de pulsatilite et la multimerisation du VWF et dans un modele en cross-over le caractere dynamique du relargage endothelial en reponse a des variations aigues de pulsatilite.Ces resultats nous ont permis de conceptualiser dans la deuxième partie l'utilisation du VWF dans l’evaluation de la severite des fuites paravalvulaires (FPV) post-procedure TAVI. Deux cohortes de 183 et 201 patients ont permis de demontrer l’excellente capacite diagnostique de l’analyse multimerique du VWF avec une sensibilite, une specificite et une valeur predictive negative de respectivement 92.3%, 94.9%, et 98.7%. Le test de diagnostic rapide TO-ADP (temps d’occlusion a l’ADP) donnait des resultats equivalents pour un seuil > 180 sec.Enfin dans la troisième partie de la these nous avons concu le design d’un essai clinique permettant d’evaluer la valeur ajoutee de l’utilisation de ce test de diagnostic rapide TO-ADP en salle de catheterisme pour l’amelioration des resultats proceduraux et cliniques des procedures TAVI. / Willebrand factor (VWF) is a multimeric protein that has a unique sensitivity to shear forces and hemodynamic variations in blood flow such as those encountered when using cardiovascular devices such as transcatheter aortic valve replacement (TAVI) or continuous flow mechanical circulatory assistance (CF-CAM).

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