A motif within the N-terminal domain of TSP-1 specifically promotes the proangiogenic activity of endothelial colony-forming cells

Thrombospondin-1 (TSP-1) gives rise to fragments that have both pro- and anti-angiogenic effects in vitro and in vivo. the TSP-HepI peptide (2.3 kDa), located in the N-terminal domain of TSP-1, has proangiogenic effects on endothelial cells. We have previously shown that TSP-1 itself exhibits a dual effect on endothelial colony-forming cells (ECFC) by enhancing their adhesion through its TSP-HepI fragment while reducing their proliferation and differentiation into vascular tubes (tubulogenesis) in vitro. This effect is likely mediated through CD47 binding to the TSP-1 C-terminal domain. Here we investigated the effect of TSP-HepI peptide on the angiogenic properties of ECFC in vitro and in vivo. TSP-HepI peptide potentiated FGF-2-induced neovascularisation by enhancing ECFC chemotaxis and tubulogenesis in a Matrigel plug assay. ECFC exposure to 20 mu g/mL of TSP-HepI peptide for 18 h enhanced cell migration (p < 0.001 versus VEGF exposure), upregulated alpha 6-integrin expression, and enhanced their cell adhesion to activated endothelium under physiological shear stress conditions at levels comparable to those of SDF-1 alpha. the adhesion enhancement appeared to be mediated by the heparan sulfate proteoglycan (HSPG) syndecan-4, as ECFC adhesion was significantly reduced by a syndecan-4-neutralising antibody. ECFC migration and tubulogenesis were stimulated neither by a TSP-HepI peptide with a modified heparin-binding site (S/TSP-HepI) nor when the glycosaminoglycans (GAGS) moieties were removed from the ECFC surface by enzymatic treatment. Ex vivo TSP-HepI priming could potentially serve to enhance the effectiveness of therapeutic neovascularisation with ECFC. (C) 2012 Elsevier Inc. All rights reserved.Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq)Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES)Groupe d'Etude et de Recherches sur l'Hemostase (GEHT)Region Ile-de-France (CORDDIM)Leducq TransAtlantic Network of ExcellenceUniv Estado Rio de Janeiro, Dept Biol Celular, Lab Biol Celula Endotelial & Angiogenese LabAngio, Inst Biol Roberto Alcantara Gomes, BR-20550011 Rio de Janeiro, RJ, BrazilINSERM, U765, Paris, FranceUniv Paris 05, Paris, FranceUniversidade Federal de São Paulo, Escola Paulista Med, Dept Biofis, São Paulo, BrazilUniv Fed Rio de Janeiro, Inst Ciencias Biomed, Rio de Janeiro, RJ, BrazilHop Europeen Georges Pompidou, AP HP, Dept Haematol, Paris, FranceINSERM, Paris Cardiovasc Res Ctr, U970, Paris, FranceUniversidade Federal de São Paulo, Escola Paulista Med, Dept Biofis, São Paulo, BrazilLeducq TransAtlantic Network of Excellence: 04CVD01-LENALeducq TransAtlantic Network of Excellence: 04CVD02 -LINATCNPq: E-26/110.780/2010CAPES: 629/09Web of Scienc

Contribution a l'etude des serine proteases de la coagulation et de la fibrinolyse

SIGLECNRS T Bordereau / INIST-CNRS - Institut de l'Information Scientifique et TechniqueFRFranc

Modulation de la production de thrombine et fibrino-formation dans l'hémostase

Le facteur X de la coagulation (FX) permet la génération de thrombine responsable de la formation d un réseau de fibrine et de l activation des plaquettes qui libèrent de grande quantité de facteur 4 plaquettaire (PF4). La première partie de ce mémoire concerne la caractérisation d un mutant naturel du FX dont nous avons décrypté le défaut fonctionnel expliquant le phénotype du patient. Nous avons par ailleurs préparé et caractérisé cinq dérivés du FX activables par la thrombine. Ces dérivés rétablissent une amplification de la génération de thrombine dans un plasma d hémophile et pourraient constituer un traitement innovant. La seconde partie de ce mémoire porte sur deux fonctions insoupçonnées du PF4. Nous avons dévoilé qu il limite la génération de thrombine en inhibant l activation du facteur V plasmatique et qu il altère profondément la structure du réseau de fibrine. Ces observations apportent un éclairage nouveau sur la formation et la structure du clou plaquettaire initial.Coagulation factor X (FX) participates in the generation of thrombin, which forms the fibrin network, and activates platelets which release platelet factor 4 (PF4) in large quantities. The first part of this report concerns the characterization of a novel mutant of FX, whose functional defect was deciphered by looking at the phenotype of the patient. We also prepared and characterized five recombinant proteins of thrombin-activable FX. These recombinants restored the amplification of thrombin production in blood plasma of haemophiliacs and could constitute an innovative treatment. For the second part, we investigated the characteristics of PF4 and revealed two unsuspected functions. We found that it prevents the generation of thrombin by inhibiting the activation of the plasma-derived factor V and we demonstrated that PF4 profoundly alters the structure of the fibrin network. These observations provide a new insight on the formation and the structure of the initial platelet clot.PARIS-BIUP (751062107) / SudocSudocFranceF

Préparation d'un facteur X* clivable par la thrombine (anti-hémophilique potentiel)

PARIS-BIUP (751062107) / SudocSudocFranceF

Etude des bases moléculaires susceptibles de gouverner la spécificité du FX activé de la coagulation

Le facteur X activé (FXa) est une sérine-protéase de la coagulation sanguine, il joue un rôle central puisque, associé à son cofacteur au sein du complexe prothrombinase, c'est l'unique activateur physiologique de la prothrombine. La thrombine ainsi générée est l'ultime enzyme de la cascade de la coagulation permettant la formation d'un caillot sanguin à même de colmater une brèche vasculaire. L'étude des bases moléculaires susceptibles de gouverner la spécificité du FXa repose sur le modèle de la thrombine dont les relations structure-fonction à l'origine de sa spécificité sont bien établies. Nos travaux montrent que, contrairement à la thrombine, la spécificité du FXa ne repose pas sur la sélectivité de son sillon catalytique. Cette observation nous a donc amené à évaluer l'hypothèse selon laquelle un exosite pourrait moduler l'interaction du FXa avec ses ligands physiologiques. Si effectivement cette hypothèse reste probable nos travaux tendent à montrer que la région topologiquement équivalente à l'exosite 1 de la thrombine sur le FXa ne semble pas jouer ce rôle d'exosite.Activated factor X (FXa) is a key serine protease involved in the blood coagulation cascade. FXa associates to factor Va to form a complex named prothrombinase that activates prothrombin into thrombin. Thrombin, the ultimate enzyme of the coagulation cascade, triggers clot formation able to plug a vascular injury. Our study of the molecular basis that governs the specificity of FXa relied on that of the well-characterized thrombin, used as a model. Results suggest that, contrary to thrombin, FXa specificity mainly originates from outside its catalytic groove. An observation that drove us to evaluate if an exosite could alter interaction of FXa with its physiologic ligands. Extensive study of the region of FXa that is topologically equivalent to the critical exosite 1 of thrombin allowed us to eliminate this potential exosite as a major player.PARIS-BIUP (751062107) / SudocSudocFranceF

Rôle du facteur 4 plaquettaire dans la modulation de l'hémostase

Le facteur 4 plaquettaire (PF4) est un tétramère composé d'unités de 70 acides aminés contenu dans les granules a des plaquettes qui le sécrètent en grande quantité (10 à 25 g/ml) lors de l'activation. L'éventuelle fonction du PF4 dans l'hémostase reste ambiguë car des activités aussi bien pro- qu'anti-coagulantes lui ont été attribuées. Nous avons observé que le PF4 exerce un effet majeur sur la structure du caillot de fibrine. En étudiant la formation d'un caillot de fibrine en turbidimétrie et en microscopie électronique à balayage nous avons montré que le PF4 influence considérablement la polymérisation. Ainsi en se liant à la fibrine, le PF4 oriente l'assemblage des protofibrilles qui forment une nappe imperméable. Nous avons montré que le PF4 inhibait de façon dose-dépendante la génération de thrombine dans un plasma. Nous avons ègalement montré que le PF4 inhibe la formation du complexe prothrombinase en masquant le facteur V à son activateur, la thrombine. De façon surprenante, le PF4 s'est par ailleurs avéré être un puissant inhibiteur de l'agrégation plaquettaire. Nous proposons que le PF4, caractérisé par une très forte densité de charges positives, pourrait entrer en compétition avec la thrombine vis à vis des ligands de son exosite 1.Platelet factor 4 (PF4) is a tetrameric protein composed of four 70-amino-acid subunits that is contained in platelet a-granules. Large amounts of PF4 (up to 10-25 g/ml) are released upon platelet aggregation. The role of PF4 in hemostasis remains elusive as PF4 has been reported to possess procoagulant and anticoagulant activities as well. We have observed that PF4 exerts a major effect on the structure of the fibrin clot. By evaluating the formation of a fibrin clot by turbidimetric methods and by electronic microscopy, we have shown that PF4 has a considerable effect on the fibrin polymerization process. By binding to fibrin, PF4 thus drives the assembly of protofibrils which then form an impermeable network. In addition, we have demonstrated that PF4 dose-dependently inhibits the generation of thrombin in a plasma-based assay. Furthermore, we have shown that PF4 prevents the activation of factor V by thrombin, which results in inhibition of the prothrombinase complex. Surprisingly, PF4 is also a very potent inhibitor of platelet aggregation. We therefore propose that PF4, which contains a high density of positive charges exposed at its surface, might compete with thrombin towards various ligands of thrombin exosite 1, such as factor V, fibrinogen, PAR-1 (...).PARIS-BIUP (751062107) / SudocSudocFranceF

Modulation de l'activité de la thrombine

PARIS-BIUP (751062107) / SudocSudocFranceF

Strategies of neutralization of the direct oral anticoagulants effect review of the literature

International audienceMany neutralizing agents of anticoagulant effect of factor Xa or thrombin inhibitors (xabans and dabigatran, respectively) have been developed since the commercialization of direct oral anticoagulants (DOAC) in 2008. Idarucizumab is a specific antidote of dabigatran commercialised since 2016. An antidote of xabans, andexanet-α, was very recently approved by the Food and Drug Administration (FDA). Other antidotes of DOAC are under pre-clinical or clinical development; the most advanced being the aripazine in addition to γ-thrombine S195A and GDFXa-αM complex. Prothrombin complex concentrates activated or not, are part of the pro-hemostatic agents suggested for DOAC handling in case of haemorrhage or preceeding urgent surgery or invasive procedures. Other pro-hemostatic agents (FXa, FX (a)-C, FVa) are in pre-clinical stage. The efficacy of these different agents in DOAC reversal and mortality reduction is still controversal in the light of the sparse results of in vitro, ex vivo, pre-clinical and clinical studies