Elastin-Derived Peptides Are New Regulators of Thrombosis

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The Innate Immune Response in Eisenia Fetida to Microbial Challenges

The common earthworm, Eisenia fetida, exhibits a rudimentary immune system. The earthworm needs cellular and chemical responses against a constant microbial exposure from its natural environment. Some cellular and chemical responses are found in the coelomic fluid and have been shown to demonstrate anti-microbial characteristics. This project uses microscopy and modified staining techniques to differentiate and categorize the cellular components found in the coelomic fluid. Following a microbial challenge by Klebsiella pneumoniae, an inflammatory response was initiated. Six groups of earthworms were injected with 0.05 ml of 1.0 x 106 cfu /ml K. pneumoniae on day one and tested over a period of five days. A group of three worms was shocked each day for the next five days to cause the coelomic fluid and cells to pass through the body wall. The coelomic fluid was placed directly on glass slides, dried and stained with a modified Wright’s stain using a wash buffer solution with a pH of 6.3. The stained cells were differentiated into four categories. Total cell counts were determined. The data indicated a marked proliferation in total cell counts in comparison to the control worms. This trend of increasing total cell counts continued over the five days. The percent ages of the four types of coelomic cells from the differential remained constant. Cells were photographed and documented for comparisons. Additional studies are ongoing to determine how long the Eisenia fetida take to remove Klebsiella pneumoniae from the coelomic cavity

Relevance of platelet desialylation and thrombocytopenia in type 2B von Willebrand disease: preclinical and clinical evidence

Patients with type 2B von Willebrand disease (vWD) (caused by gain-of-function mutations in the gene coding for von Willebrand factor) display bleeding to a variable extent and, in some cases, thrombocytopenia. There are several underlying causes of thrombocytopenia in type 2B vWD. It was recently suggested that desialylation-mediated platelet clearance leads to thrombocytopenia in this disease. However, this hypothesis has not been tested in vivo. The relationship between platelet desialylation and the platelet count was probed in 36 patients with type 2B von Willebrand disease (p.R1306Q, p.R1341Q, and p.V1316M mutations) and in a mouse model carrying the severe p.V1316M mutation (the 2B mouse). We observed abnormally high elevated levels of platelet desialylation in both patients with the p.V1316M mutation and the 2B mice. In vitro, we demonstrated that 2B p.V1316M/von Willebrand factor induced more desialylation of normal platelets than wild-type von Willebrand factor did. Furthermore, we found that N-glycans were desialylated and we identified αIIb and β3 as desialylation targets. Treatment of 2B mice with sialidase inhibitors (which correct platelet desialylation) was not associated with the recovery of a normal platelet count. Lastly, we demonstrated that a critical platelet desialylation threshold (not achieved in either 2B patients or 2B mice) was required to induce thrombocytopenia in vivo. In conclusion, in type 2B vWD, platelet desialylation has a minor role and is not sufficient to mediate thrombocytopenia

Role of elastin peptides in type 2 diabetes and thrombosis, and functionality and biochemical characterization of Neuraminidase-1, subunit of elastin receptor complex

L’élastine est la protéine de la matrice extracellulaire (MEC) responsable des propriétés de résilience et d'élasticité des tissus élastiques. Durant le vieillissement, les protéines de la MEC vasculaire sont exposées à des réactions délétères qui altèrent leurs propriétés structurales et fonctionnelles. Une des caractéristiques principales des protéines de la MEC est leur longue demi-vie, associée à un renouvellement très lent, comme pour l'élastine. Ainsi, tout dommage survenant sur l'élastine est essentiellement irréparable. La fragmentation des fibres élastiques génère des peptides d’élastine (EDP) bioactifs capables de modifier le comportement des cellules environnantes en se liant au complexe récepteur de l’élastine (CRE), composé de trois sous-unités dont la neuraminidase-1 (Neu-1), sous-unité catalytique du CRE. Cette thèse a consisté en l'étude, chez la souris, du rôle des EDP dans le développement du diabète de type 2 et dans la thrombose, deux pathologies vasculaires liées à l'âge, et s'est également focalisée sur la sous-unité Neu-1 du CRE. Dans un premier temps, nous avons montré que les EDP favorisent le développement d’une insulinorésistance et d’un diabète de type 2. Cet effet implique l'interaction de Neu-1 avec la sous-unité β du récepteur à l'insuline qui diminue son niveau de sialylation altérant ses voies de signalisation. Dans un second temps, nous avons identifié un mécanisme d'action des EDP à deux niveaux (matriciel et plaquettaire) et mis en évidence la présence d'un CRE fonctionnel dans les plaquettes régulant la thrombose. Enfin, nous avons étudié la topologie membranaire de Neu-1 par différentes approches technologiques et identifié un domaine transmembranaire potentiel jouant un rôle important pour sa dimérisation et son activité sialidase. En conclusion, les EDP sont des acteurs clefs du remodelage vasculaire physiopathologique et des pathologies vasculaires associées et de contribuer à faire avancer nos connaissances sur l'organisation de Neu-1 à la membrane plasmique.Elastin is the extracellular matrix (ECM) protein responsible for resilience and elasticity of tissues such as arteries. During ageing, vascular ECM proteins are subjected to deleterious reactions that alter their structural and functional properties (addition reactions, proteolysis). One of the main features of ECM proteins is their long half-life, associated with a low, or even, inexistent turnover. This is the case for elastin with an estimated half-life at 70 years. Therefore, any damage occurring on elastin will be mostly irreparable. Fragmentation of elastic fibers produces bioactive elastin-derived peptides (EDP) able to modify the behavior of surrounding cells by binding to the elastin receptor complex (ERC). This receptor is composed of three subunits, among which neuraminidase-1 (Neu-1) is the catalytic subunit. The aim of this thesis was to study, in mice, the role of EDP in the development of type 2 diabetes and in thrombosis, two age-related vascular diseases, and to focus on the Neu-1 subunit of the ERC. In a first time, we have shown that EDP promote the development of insulin resistance and type 2 diabetes. This effect involves Neu-1 interaction with the β subunit of the insulin receptor and leads to its reduced sialylation level and signaling. In a second time, we have demonstrated that EDP are regulators of thrombosis. We identified a two-level mechanism (matrix and platelet) and the presence of a functional ERC in platelets. Finally, we have studied the membrane topology of Neu-1 by different biophysical, biochemical and molecular biology approaches, and identified a potential transmembrane domain involved in the dimerization and sialidase activity of Neu-1. In conclusion, this thesis consolidates the concept that EDP are crucial actors of pathophysiological vascular remodeling and related vascular diseases, and expands our knowledge on the plasma membrane organization of Neu-1

Role of elastin peptides in type 2 diabetes and thrombosis, and functionality and biochemical characterization of Neuraminidase-1, subunit of elastin receptor complex

L’élastine est la protéine de la matrice extracellulaire (MEC) responsable des propriétés de résilience et d'élasticité des tissus élastiques. Durant le vieillissement, les protéines de la MEC vasculaire sont exposées à des réactions délétères qui altèrent leurs propriétés structurales et fonctionnelles. Une des caractéristiques principales des protéines de la MEC est leur longue demi-vie, associée à un renouvellement très lent, comme pour l'élastine. Ainsi, tout dommage survenant sur l'élastine est essentiellement irréparable. La fragmentation des fibres élastiques génère des peptides d’élastine (EDP) bioactifs capables de modifier le comportement des cellules environnantes en se liant au complexe récepteur de l’élastine (CRE), composé de trois sous-unités dont la neuraminidase-1 (Neu-1), sous-unité catalytique du CRE. Cette thèse a consisté en l'étude, chez la souris, du rôle des EDP dans le développement du diabète de type 2 et dans la thrombose, deux pathologies vasculaires liées à l'âge, et s'est également focalisée sur la sous-unité Neu-1 du CRE. Dans un premier temps, nous avons montré que les EDP favorisent le développement d’une insulinorésistance et d’un diabète de type 2. Cet effet implique l'interaction de Neu-1 avec la sous-unité β du récepteur à l'insuline qui diminue son niveau de sialylation altérant ses voies de signalisation. Dans un second temps, nous avons identifié un mécanisme d'action des EDP à deux niveaux (matriciel et plaquettaire) et mis en évidence la présence d'un CRE fonctionnel dans les plaquettes régulant la thrombose. Enfin, nous avons étudié la topologie membranaire de Neu-1 par différentes approches technologiques et identifié un domaine transmembranaire potentiel jouant un rôle important pour sa dimérisation et son activité sialidase. En conclusion, les EDP sont des acteurs clefs du remodelage vasculaire physiopathologique et des pathologies vasculaires associées et de contribuer à faire avancer nos connaissances sur l'organisation de Neu-1 à la membrane plasmique.Elastin is the extracellular matrix (ECM) protein responsible for resilience and elasticity of tissues such as arteries. During ageing, vascular ECM proteins are subjected to deleterious reactions that alter their structural and functional properties (addition reactions, proteolysis). One of the main features of ECM proteins is their long half-life, associated with a low, or even, inexistent turnover. This is the case for elastin with an estimated half-life at 70 years. Therefore, any damage occurring on elastin will be mostly irreparable. Fragmentation of elastic fibers produces bioactive elastin-derived peptides (EDP) able to modify the behavior of surrounding cells by binding to the elastin receptor complex (ERC). This receptor is composed of three subunits, among which neuraminidase-1 (Neu-1) is the catalytic subunit. The aim of this thesis was to study, in mice, the role of EDP in the development of type 2 diabetes and in thrombosis, two age-related vascular diseases, and to focus on the Neu-1 subunit of the ERC. In a first time, we have shown that EDP promote the development of insulin resistance and type 2 diabetes. This effect involves Neu-1 interaction with the β subunit of the insulin receptor and leads to its reduced sialylation level and signaling. In a second time, we have demonstrated that EDP are regulators of thrombosis. We identified a two-level mechanism (matrix and platelet) and the presence of a functional ERC in platelets. Finally, we have studied the membrane topology of Neu-1 by different biophysical, biochemical and molecular biology approaches, and identified a potential transmembrane domain involved in the dimerization and sialidase activity of Neu-1. In conclusion, this thesis consolidates the concept that EDP are crucial actors of pathophysiological vascular remodeling and related vascular diseases, and expands our knowledge on the plasma membrane organization of Neu-1

HXG-106

Aerial Photograph of the greater Mankato Area, Mankato, Minnesota from 1959.https://cornerstone.lib.mnsu.edu/aerial_1959_HXG/1094/thumbnail.jp

A factor VIII–nanobody fusion protein forming an ultrastable complex with VWF: effect on clearance and antibody formation

International audienceVon Willebrand factor (VWF) modulates factor VIII (FVIII) clearance and the anti-FVIII immune response. Despite the high affinity that defines the FVIII/VWF interaction, association/dissociation kinetics dictates 2% to 5% FVIII being present as free protein. To avoid free FVIII when studying the FVIII-VWF complex in vivo, we designed a FVIII-nanobody fusion protein, with the nanobody part being directed against VWF. This fusion protein, designated FVIII-KB013bv, had a 25-fold higher affinity compared with B-domainless FVIII (BDD-FVIII) for VWF. In vitro analysis revealed full cofactor activity in 1-stage clotting and chromogenic assays (activity/antigen ratio 1.0 ± 0.3 and 1.1 ± 0.3, respectively). In vivo, FVIII-013bv displayed a twofold increased mean residence time compared with BDD-FVIII (3.0 hours vs 1.6 hours). In a tail clip–bleeding assay performed 24 hours after FVIII infusion, blood loss was significantly reduced in mice receiving FVIII-KB013bv vs BDD-FVIII (15 ± 7 μL vs 194 ± 146 μL; P = .0043). Unexpectedly, when examining anti-FVIII antibody formation in FVIII-deficient mice, the immune-response toward FVIII-KB013bv was significantly reduced compared with BDD-FVIII (1/8 vs 14/16 mice produced anti-FVIII antibodies after treatment with FVIII-KB013bv and BDD-FVIII, respectively). Our data show that a stabilized interaction between FVIII and VWF is associated with a prolonged survival of FVIII and a reduced immune response against FVIII

A hemophilia A mouse model for the in vivo assessment of emicizumab function

International audienceThe bispecific antibody emicizumab is increasingly used for hemophilia A treatment. However, its specificity for human factors IX and X (FIX and FX) has limited its in vivo functional analysis to primate models of acquired hemophilia. Here, we describe a novel mouse model that allows emicizumab function to be examined. Briefly, FVIII-deficient mice received IV emicizumab 24 hours before tail-clip bleeding was performed. A second infusion with human FIX and FX, administered 5 minutes before bleeding, generated consistent levels of emicizumab (0.7-19 mg/dL for 0.5-10 mg/kg doses) and of both FIX and FX (85 and 101 U/dL, respectively, after dosing at 100 U/kg). Plasma from these mice display FVIII-like activity in assays (diluted activated partial thromboplastin time and thrombin generation), similar to human samples containing emicizumab. Emicizumab doses of 1.5 mg/kg and higher significantly reduced blood loss in a tail-clip-bleeding model using FVIII-deficient mice. However, reduction was incomplete compared with mice treated with human FVIII concentrate, and no difference in efficacy between doses was observed. From this model, we deducted FVIII-like activity from emicizumab that corresponded to a dose of 4.5 U of FVIII per kilogram (ie, 9.0 U/dL). Interestingly, combined with a low FVIII dose (5 U/kg), emicizumab provided enough additive activity to allow complete bleeding arrest. This model could be useful for further in vivo analysis of emicizumab

Development and characterization of single‐domain antibodies neutralizing protease nexin‐1 as tools to increase thrombin generation: Single-domain antibodies against protease nexin-1

International audienceBackground: Protease nexin-1 (PN-1) is a member of the serine protease inhibitor (Serpin)-family, with thrombin as its main target. Current polyclonal and monoclonal antibodies against PN-1 frequently cross-react with Plasminogen activator inhibitor-1 (PAI-1), a structurally and functionally homologous Serpin. Objectives: Here, we aimed to develop inhibitory single-domain antibodies (VHHs) that show specific binding to both human (hPN-1) and murine (mPN-1) PN-1. Methods: PN-1-binding VHHs were isolated via phage-display using llama-derived or synthetic VHH-libraries. Following bacterial expression, purified VHHs were analyzed in binding and activity assays.Results and Conclusions: By using a llama-derived library, 2 PN-1 specific VHHs were obtained (KB-PN1-01 & KB-PN1-02). Despite their specificity, none displayed inhibitory activity towards hPN-1 or mPN-1. From the synthetic library, 4 VHHs (H12, B11, F06, A08) could be isolated that combined efficient binding to both hPN-1 and mPN-1 with negligible binding to PAI-1. Of these, B11, F06 and A08 were able to fully restore thrombin activity by blocking PN-1. As monovalent VHH, IC50-values for hPN-1 were 50±10 nM, 290±30 and 960±390 nM, for B11, F06 and A08, respectively, and 1580±240 nM, 560±130 nM and 2880±770 nM for mPN-1. The inhibitory potential was improved 4- to 7-fold when bivalent VHHs were engineered. Importantly, all VHHs could block PN-1 activity in plasma as well as PN-1 released from activated platelets, one of the main sources of PN-1 during hemostasis.In conclusion, we report the generation of inhibitory anti-PN-1 antibodies using a specific approach to avoid cross-reactivity with the homologous Serpin PAI-1

Targeting protease nexin-1, a natural anticoagulant serpin, to control bleeding and improve hemostasis in hemophilia

International audienceHemophilia A and B, diseases caused by the lack of factor VIII (FVIII) and factor IX (FIX) respectively, lead to insufficient thrombin production, and therefore to bleeding. New therapeutic strategies for hemophilia treatment that do not rely on clotting factor replacement, but imply the neutralization of natural anticoagulant proteins, have recently emerged. We propose an innovative approach consisting of targeting a natural and potent thrombin inhibitor, expressed by platelets, called protease nexin-1 (PN-1). By using the calibrated automated thrombin generation assay, we showed that a PN-1-neutralizing antibody could significantly shorten the thrombin burst in response to tissue factor in platelet-rich plasma (PRP) from patients with mild or moderate hemophilia. In contrast, in PRP from patients with severe hemophilia, PN-1 neutralization did not improve thrombin generation. However, after collagen-induced platelet activation, PN-1 deficiency in F8-/-mice or PN-1 blocking in patients with severe disease led to a significantly improved thrombin production in PRP, underlining the regulatory role of PN-1 released from platelet granules. In various bleeding models, F8-/-/PN-1-/- mice displayed significantly reduced blood loss and bleeding time compared with F8-/-mice. Moreover, platelet recruitment and fibrin(ogen) accumulation were significantly higher in F8-/-/PN-1-/- mice than in F8-/-mice in the ferric chloride-induced mesenteric vessel injury model. Thromboelastometry studies showed enhanced clot stability and lengthened clot lysis time in blood from F8-/-/PN-1-/- and from patients with hemophilia A incubated with a PN-1-neutralizing antibody compared with their respective controls. Our study thus provides proof of concept that PN-1 neutralization can be a novel approach for future clinical care in hemophilia