Platelet- and plasma-derived factor Va are absolutely essential for thrombin generation catalyzed by the prothrombinase complex, a 1:1 stoichiometric complex of the serine protease factor Xa and the nonenzymatic cofactor, factor Va, assembled on an appropriate membrane surface in the presence of calcium ions. Two whole blood pools of the procofactor, factor V, exist: approximately 75% circulates in the plasma as a single chain inactive molecule, while the other 25% resides in platelet α-granules in a partially proteolytically-activated state. Our laboratory demonstrated that the platelet-derived cofactor originates following endocytosis of plasma-derived factor V by megakaryocytes, the platelet precursor cells, via a two receptor system including an uncharacterized, specific factor V receptor and low density lipoprotein receptor related protein-1. Following endocytosis factor V is physically and functionally modified and trafficked to the platelet α-granule from where it is released upon platelet activation at sites of vascular injury. The first goal of this dissertation was to define how factor V endocytosis changes over the course of megakaryocyte development. Hematopoietic multipotential stem cells were isolated from human umbilical cord blood and subjected to ex vivo differentiation into megakaryocytes. Megakaryocyte differentiation was assessed by flow cytometry using fluorescently-labeled antibodies against megakaryocyte- and platelet-specific markers and factor V directly conjugated to a fluorophore over 12 days. Differentiation was confirmed by a decrease in a stem cell marker (CD34) and an increase in a mature megakaryocyte marker (CD42) and coincident with factor V endocytosis. Live cell imaging verified differentiation and permitted the observation of proplatelet formation, the precursor to circulating platelets. Analogous experiments verified the trafficking of factor V into proplatelet extensions. Factor V is a highly glycosylated protein: potential roles of these glycans may be endocytosis and trafficking by megakaryocytes. We previously demonstrated that factor V endocytosis is mediated by the light chain region of the procofactor. This region of factor V contains three glycans - one high mannose and two complex N-linked glycans. In the second part of this dissertation, a role for the complex N-linked glycans at Asn1675 and Asn2181 of the factor V light chain in factor V endocytosis by megakaryocytes was assessed. Exoglycosidases were used to selectively trim the complex N-linked glycans on human factor V under native conditions. Treatment with neuraminidase removed 100% of the sialic acid residues on the factor V light chain as demonstrated by gel electrophoresis and mass spectrometry. Treatment with β-1,4-galactosidase removed 69% of the galactose residues at Asn1675 and 100% at Asn2181. Glycosidase-treated factor Va behaves similarly to untreated factor Va in thrombin generation assays suggesting that cofactor activity is unaltered by glycan trimming. In addition, glycan removal had no effect on factor V endocytosis by megakaryocyte-like cells. These observations suggest that complex N-linked glycans on the factor V light chain are not important for factor Va cofactor activity or factor V endocytosis by megakaryocyte-like cells, which strongly suggests that they have a role in trafficking.

Les mégacaryocytes sont les précurseurs cellulaires hautement spécialisés qui produisent des plaquettes via des extensions cytoplasmiques appelées proplaquettes. La formation des proplaquettes exige de profonds changements dans l’organisation du cytosquelette: microtubules et actine. Les formines sont une famille de protéines hautement conservées chez les eucaryotes composées de plusieurs domaines qui régulent le remodelage et la dynamique du cytosquelette d'actine et des microtubules. La plupart des formines sont des effecteurs protéiques des Rho-GTPase. DIAPH1, un membre de la famille des formines, est un homologue chez les mammifères du gène diaphanous de la drosophile qui fonctionne comme un effecteur de la petite GTPase Rho et régule le cytosquelette d'actomyosine ainsi que les microtubules. Il contient le domaine de liaison à Rho (Rho-binding domain) dans la partie amino-terminale et deux régions distinctes d’homologie aux formines, FH1 localisée au centre de la protéine et FH2 dans la partie carboxy-terminale. DIAPH1 co-régule le cytosquelette des microtubules et d'actine à travers respectivement ses régions de FH2 et FH1. DIAPH1 est donc un gène candidat idéal dans toutes les fonctions cellulaires qui exigent une coopération entre cytosquelettes d’actine et de microtubules.L'objectif de ce projet de thèse était d’étudier le rôle de DIAPH1 dans la mégacaryopoïèse. A la fin de la maturation des mégacaryocytes, la formation de proplquettes et la migration sont associées à des modifications importantes de la structure du cytosquelette. Nous avons émis l’hypothèse que grâce à la sa double fonction dans la polymérisation de l'actine et la stabilisation des microtubules, DIAPH1 pourrait jouer un rôle essentiel dans les temps terminaux de la différenciation mégacaryocytaire.Nos résultats ont montré qu’au cours de la différenciation mégacaryocytaire, l’expression de DIAPH1 augmente, alors que celles de DIAPH2 et DIAPH3 diminuent, ce qui suggère que DIAPH1 pourrait jouer un rôle plus important que DIAPH2 et DIAPH3 dans les stades tardifs de la différenciation mégacaryocytaire. Les études en immunomarquage montrent que DIAPH1 co-localise avec l’actine F, la tubuline et la myosine IIa en niveau de la membrane plasmique et des proplaquettes. Nous avons étudié la fonction de DIAPH1 par des stratégies d’invalidation (knockdown) et de surexpression d’une forme active de DIAPH1. Les résultats montrent que DIAPH1 est un effecteur important de Rho, pour réguler négativement la formation des proplaquettes en remodelant le cytosquelette d’actine et les microtubules. Le travail antérieur de notre équipe avait montré que Rho-ROCK régulait aussi négativement la formation des proplaquettes, en inhibant l’activation de la myosine IIa. En inhibant simultanément DIAPH1 et ROCK/myosine, nous avons montré que ces deux voies jouent un rôle additif dans la formation des proplaquettes.Ces résultats suggèrent que la coopération entre les voies DIAPH1 et ROCK/myosine est nécessaire pour la formation de structures cellulaire dépendant de l'actomyosine, telles les fibres de stress et l'anneau contractile en agissant à la fois sur le remodelage du cytosquelette et en assurant un équilibre entre l'actomyosine et microtubules. / Megakaryocytes (MKs) are the highly specialized precursor cells that produce platelets via cytoplasm extensions called proplatelets. Proplatelet formation (PPF) requires profound changes in microtubule and actin organization. Formins are a family of highly conserved eukaryotic proteins with multidomains that govern dynamic remodeling of the actin and microtubule cytoskeletons. Most formins are Rho-GTPase effectors proteins. DIAPH1, a member of the formin family, is a mammalian homolog of Drosophila diaphanous gene that works as an effector of the small GTPase Rho and regulates the actomyosin cytoskeleton as well as microtubules. It contains the Rho-binding domain in the N-terminal and two distinct regions of formin homology, FH1 in the center and FH2 in the C-terminus. DIAPH coordinates microtubules and actin cytoskeleton through its FH2 and FH1 regions respectively, making DIAPH an ideal candidate in cell functions that depend closely on the cooperation between the actin and microtubule cytoskeletons.The objective of the project was to decipher the role of DIAPH1 in megakaryopoiesis. At the end of the MK maturation, PPF and MK migration are associated with profound changes in cytoskeleton organization. Due to its dual function in actin polymerization and microtubule stabilization, DIAPH1 was an obvious candidate to play an essential role in PPF and MK migration.Our results showed that DIAPH1 expression increased during MK differentiation, whereas DIAPH2 and DIAPH3 expression decreased, suggesting that DIAPH1 may play a more important role than DIAPH2 and DIAPH3 in the late stages of MK differentiation. Immunostaining showed that DIAPH1 co-localized with F-actin, tubulin and myosin IIa along the plasma membrane and proplatelet. Using a knockdown strategy with shRNA and expression of an active form of DIAPH1, we showed that DIAPH1 is an important effector of Rho that negatively regulates PPF by remodeling actin and microtubule cytoskeletons. A previous work of our team has shown that Rho-ROCK also negatively regulates in PPF by inhibiting myosin IIa activation. By the double inhibition of the DIAPH1 and the ROCK/Myosin pathway, we showed that DIAPH1 and ROCK played additive roles in the negative regulation of PPF. These observations suggest that the cooperation between DIAPH1 and ROCK is required for the formation of cell structures dependent on actomyosin, such as the stress fibers and the contractile ring. Collectively, these results strongly suggest that cooperation of DIAPH1/microtubules and ROCK/Myosin may regulate PPF by modifying the balance between actomyosin and microtubules.