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

Analysis of signaling during clot retraction : application to the diagnosis of a defect of primary hemostasis in patients with Lowe syndrome

L’hémostase primaire est un processus permettant la formation d’un clou plaquettaire qui sera stabilisé par un réseau de fibrine. Ce caillot est également consolidé grâce à des phases tardives de l’hémostase primaire résultant des fonctions plaquettaires ; il s’agit principalement de la rétraction qui diminue la taille du caillot afin de le stabiliser. Cette phase est déclenchée par une signalisation « outside-in », consécutive à l’activation de l’intégrine αIIbβ3 et à l’agrégation plaquettaire, et est dépendante d’une réorganisation du cytosquelette. Le premier objectif de ce travail a été d’étudier la signalisation impliquée dans la rétraction, et en particulier l’implication des protéines ROCK, MLCK, Rac-1 et de l’actine dans l’activité de la chaine légère de la myosine (MLC) . MLC est en effet une protéine clé de la réorganisation du cytosquelette. Nous avons mis en évidence une phosphorylation biphasique de MLC dont le deuxième pic, corrélé à la rétraction, est dépendant de Rac1 et de la polymérisation de l’actine. Cette étude a été appliquée à une pathologie, le syndrome de Lowe. Il s’agit d’une maladie génétique rare, également appelée OCRL (Oculo cérébro rénal de Lowe) en référence aux organes majoritairement touchés. Suite à l’observation d’événements hémorragiques per et postopératoires suggérant une instabilité du caillot et l’observation dans une étude précédente d’un temps d’occlusion allongé au PFA100®, nous avons mis en place une étude sur 15 patients et 15 témoins pour lesquels nous avons étudié les différentes phases de l’hémostase primaire. Outre une anomalie et un retard de maturation des mégacaryocytes, nous avons mis en évidence pour la première fois chez ces patients un défaut de la voie « outside-in » responsable d’une anomalie de l’étalement plaquettaire et de la rétraction du caillot. Ce défaut de rétraction, dû à un défaut d’activation de MLC, pourrait être en partie responsable des événements hémorragiques observés chez ces patients.Primary hemostasis is a mechanism allowing platelet clot formation that is thereafter stabilized by a fibrin network. Fibrin clot is also consolidated following post occupancy events, mainly clot retraction that decrease clot size and thus strengthen it. This phase is triggered by « outside-in » signaling. It is consecutive to αIIbβ3 integrin activation and platelet aggregation, dependent on cytoskeleton organization. Our first objective was to investigate signaling events underlying retraction, and particularly the involvement of ROCK, MLCK, Rac-1, and actin in MLC (Myosin Light Chain) phosphorylation. Indeed, MLC, involved in cytoskeleton rearrangement, is a key protein of this mechanism. We described a MLC biphasic phosphorylation profile, which second peak was dependent of Rac1 and actin polymerization. In a second part, we studied clot retraction signaling in patients with the Lowe syndrome. It is a rare genetic disease, caused by absence of OCRL (oculo cerebro renal of Lowe) protein in reference to the majority of affected organs. The rationale of this study was a previous observation of hemorrhagic events during and after surgeries, suggesting clot instability. A thrombopathy was suggested by a closure time lengthening in the PFA-100 system. The study enrolled 15 patients and 15 controls. Besides a defect of megakaryocyte maturation, we described a defect of « outside-in » signaling responsible for spreading and clot retraction abnormality. This retraction defect, caused by a MLC activity defect, could be partly responsible for hemorrhagic events reported in these patients

Etude de la signalisation au cours de la rétraction du caillot (application à l'étude des anomalies de l'hémostase primaire dans le syndrome de Lowe)

L hémostase primaire est un processus permettant la formation d un clou plaquettaire qui sera stabilisé par un réseau de fibrine. Ce caillot est également consolidé grâce à des phases tardives de l hémostase primaire résultant des fonctions plaquettaires ; il s agit principalement de la rétraction qui diminue la taille du caillot afin de le stabiliser. Cette phase est déclenchée par une signalisation outside-in , consécutive à l activation de l intégrine aIIbb3 et à l agrégation plaquettaire, et est dépendante d une réorganisation du cytosquelette. Le premier objectif de ce travail a été d étudier la signalisation impliquée dans la rétraction, et en particulier l implication des protéines ROCK, MLCK, Rac-1 et de l actine dans l activité de la chaine légère de la myosine (MLC) . MLC est en effet une protéine clé de la réorganisation du cytosquelette. Nous avons mis en évidence une phosphorylation biphasique de MLC dont le deuxième pic, corrélé à la rétraction, est dépendant de Rac1 et de la polymérisation de l actine. Cette étude a été appliquée à une pathologie, le syndrome de Lowe. Il s agit d une maladie génétique rare, également appelée OCRL (Oculo cérébro rénal de Lowe) en référence aux organes majoritairement touchés. Suite à l observation d événements hémorragiques per et postopératoires suggérant une instabilité du caillot et l observation dans une étude précédente d un temps d occlusion allongé au PFA100®, nous avons mis en place une étude sur 15 patients et 15 témoins pour lesquels nous avons étudié les différentes phases de l hémostase primaire. Outre une anomalie et un retard de maturation des mégacaryocytes, nous avons mis en évidence pour la première fois chez ces patients un défaut de la voie outside-in responsable d une anomalie de l étalement plaquettaire et de la rétraction du caillot. Ce défaut de rétraction, dû à un défaut d activation de MLC, pourrait être en partie responsable des événements hémorragiques observés chez ces patients.Primary hemostasis is a mechanism allowing platelet clot formation that is thereafter stabilized by a fibrin network. Fibrin clot is also consolidated following post occupancy events, mainly clot retraction that decrease clot size and thus strengthen it. This phase is triggered by outside-in signaling. It is consecutive to aIIbb3 integrin activation and platelet aggregation, dependent on cytoskeleton organization. Our first objective was to investigate signaling events underlying retraction, and particularly the involvement of ROCK, MLCK, Rac-1, and actin in MLC (Myosin Light Chain) phosphorylation. Indeed, MLC, involved in cytoskeleton rearrangement, is a key protein of this mechanism. We described a MLC biphasic phosphorylation profile, which second peak was dependent of Rac1 and actin polymerization. In a second part, we studied clot retraction signaling in patients with the Lowe syndrome. It is a rare genetic disease, caused by absence of OCRL (oculo cerebro renal of Lowe) protein in reference to the majority of affected organs. The rationale of this study was a previous observation of hemorrhagic events during and after surgeries, suggesting clot instability. A thrombopathy was suggested by a closure time lengthening in the PFA-100 system. The study enrolled 15 patients and 15 controls. Besides a defect of megakaryocyte maturation, we described a defect of outside-in signaling responsible for spreading and clot retraction abnormality. This retraction defect, caused by a MLC activity defect, could be partly responsible for hemorrhagic events reported in these patients.PARIS5-Bibliotheque electronique (751069902) / SudocPARIS-BIUM-Bib. électronique (751069903) / SudocSudocFranceF

Bleeding disorders in Lowe syndrome patients: evidence for a link between OCRL mutations and primary haemostasis disorders.

International audienceLowe syndrome (LS) is a rare X-linked disorder caused by mutations in the oculocerebrorenal gene (OCRL), encoding OCRL, a phosphatidylinositol 5-phosphatase with a RhoGAP domain. An abnormal rate of haemorrhagic events was found in a retrospective clinical survey. Herein, we report the results of exploration of haemostasis in six LS patients. All patients had normal coagulation tests but prolonged closure times (CTs) in the PFA-100 system. Healthy donors' blood samples incubated with a RhoA kinase inhibitor had prolonged CTs. This suggests that an aberrant RhoA pathway in platelets contributes to CT prolongation and primary haemostasis disorders in LS

Inhibition of αIIbβ3 Ligand Binding by an αIIb Peptide that Clasps the Hybrid Domain to the βI Domain of β3

Agonist-stimulated platelet activation triggers conformational changes of integrin αIIbβ3, allowing fibrinogen binding and platelet aggregation. We have previously shown that an octapeptide, p1YMESRADR8, corresponding to amino acids 313-320 of the β-ribbon extending from the β-propeller domain of αIIb, acts as a potent inhibitor of platelet aggregation. Here we have performed in silico modelling analysis of the interaction of this peptide with αIIbβ3 in its bent and closed (not swing-out) conformation and show that the peptide is able to act as a substitute for the β-ribbon by forming a clasp restraining the β3 hybrid and βI domains in a closed conformation. The involvement of species-specific residues of the β3 hybrid domain (E356 and K384) and the β1 domain (E297) as well as an intrapeptide bond (pE315-pR317) were confirmed as important for this interaction by mutagenesis studies of αIIbβ3 expressed in CHO cells and native or substituted peptide inhibitory studies on platelet functions. Furthermore, NMR data corroborate the above results. Our findings provide insight into the important functional role of the αIIb β-ribbon in preventing integrin αIIbβ3 head piece opening, and highlight a potential new therapeutic approach to prevent integrin ligand binding

Amino acid sequence alignments.

<p><b>(A)</b> Sequence alignments of the region surrounding amino acids 320, 321 and 353 from the human integrin αIIb subunit and αIIb from other species (upper sequences) or with other human α subunits (lower sequences); <b>(B)</b> Sequence alignments of the regions surrounding amino acids 297, 356 and 384 from the human integrin β3 subunit and β3 from other species (upper sequences) or with other human β subunits (lower sequences). Conserved residues between human αIIb or β3 and other sequences are shown with light shading.</p

Effect of octapeptides on platelet activation.

<p>Washed platelets (2.5x10⁸ platelets/ml) were preincubated with vehicle (NaCl 0.9%) or various peptides (A, B and D: 500 μM) for 5 min. Platelets were then treated with 0.1 U/ml thrombin (activated) or vehicle (resting) as described in “Experimental Procedures”. <b>(A)</b> LIBS (AP5 mAb) expression induced by vehicle, RGDS, ^pYMESRADR, RGDS + ^pYMESRADR, 317-substituted octapeptide (^pY³¹³MESAADR³²⁰), 319-substituted octapeptide (^pY³¹³MESRAAR³²⁰) or 320-substituted octapeptide (^pY³¹³MESRADA³²⁰) on resting or thrombin-activated platelets. AP5 binding was quantified by determining the fluorescence intensity (Geomean UA) of anti-mAb-phycoerythrin binding (n = 6; mean ± SEM). Isotype control binding (NI, first column) was performed in each experiment; <b>(B)</b> Flow cytometry analysis of anti-fibrinogen-FITC, PAC1-FITC, anti-CD62P-PE or isotype control (dashed black lines) antibody binding on resting (black lines) or activated platelets preincubated with vehicle (green lines), ^pYMESRADR octapeptide (red lines), or RGDS (blue lines); <b>(C)</b> Inhibitory effects of octapeptides (^pY³¹³MESRADR³²⁰: black; substituted ^pY³¹³MESRAAR³²⁰: grey or ^pM³¹⁴ESRADRK³²¹: dashed) on human washed platelet aggregation induced by thrombin (0.1 U/ml). Platelets were preincubated with vehicle (peptide 0 μM) or various concentrations of octapeptides and stimulated with thrombin for 5 min. Aggregation is expressed as a percentage of maximal light transmission measured at 5 min. Each point represents the mean (± SEM) of at least 4 experiments, *P<0.05, **P<0.01, ***P< 0,001 versus untreated platelets (peptide 0 μM); <b>(D)</b> Mean fluorescence intensity of PAC1-FITC binding on thrombin-activated platelets preincubated with vehicle, RGDS, ^pYMESRADR or with 317 substituted octapeptide (^pYMESAADR), 319 substituted octapeptide (^pYMESRAAR) or 320 substituted octapeptide (^pYMESRADA).</p

Free solution structures of octapeptides compared to the <i>in silico</i> model of ^pYMESRADR in its inhibitory conformation.

<p><b>(A)</b> Solution structure of native octapeptide ^pYMESRADR determined by NMR; <b>(B)</b><i>In silico</i> model of the octapeptide in its inhibitory conformation shown as sticks (as seen in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0134952#pone.0134952.g003" target="_blank">Fig 3</a>) compared to the NMR ensemble of models of the ^pD319A substituted octapeptide shown as grey wires. Note that the intra-peptide salt-bridge between residues ^pE315- ^pR317 is present in both structures.</p

The β-ribbon YMESRADR sequence clasps αIIb to β3 in the bent-closed conformation.

<p>[LEFT] Two β3 subunit conformations have been observed in different crystal structures: bent-closed (green) (as seen in the structure of αIIbβ3—3FCS) and extended-open (purple) (as seen in the structure of αIIbβ3—1TY3/2VDK) while the αIIb β-propeller (yellow) position remains unchanged. The latch hairpin (blue, YMESRADR motif) is shown in the structure of the αIIb β-propeller; [RIGHT] Detail of the αIIb latch hairpin interaction with the bent-closed β3 (3FCS). The αIIb β-propeller domain (yellow) containing the β-ribbon and the YMESRADR motif (blue) engages the β3 hybrid domain (green). Two salt-bridges are established across the domains αIIb and β3: R320(αIIb)-E297(βI domain from β3) and K321(αIIb)-E356(hybrid domain from β3). An additional salt-bridge can be observed within the β-ribbon E315(αIIb)-R317(αIIb). In the same vicinity the αIIb β-propeller further engages with the β3 βI-domain through Y353(αIIb)-E297(β3). The interactions indicated above suggest that this region of the αIIb β-propeller plays a role in enforcing the positioning of the βI and hybrid domains when β3 is in its bent-closed conformation. The YMESRADR residues are shown as sticks with the carbon atoms coloured cyan, oxygen in red and nitrogen in blue. Residues from β3 involved in inter-subunit interactions are shown as sticks with white carbon atoms.</p