Linker regions and flexibility around the metalloprotease domain account for conformational activation of ADAMTS-13.

BACKGROUND: Recently, conformational activation of ADAMTS-13 was identified. This mechanism showed the evolution from a condensed conformation, in which the proximal MDTCS and distal T2-CUB2 domains are in close contact with each other, to an activated, open structure due to binding with von Willebrand factor (VWF). OBJECTIVES: Identification of cryptic epitope/exosite exposure after conformational activation and of sites of flexibility in ADAMTS-13. METHODS: The activating effect of 25 anti-T2-CUB2 antibodies was studied in the FRETS-VWF73 and the vortex assay. Cryptic epitope/exosite exposure was determined with ELISA and VWF binding assay. The molecular basis for flexibility was hypothesized through rapid automatic detection and alignment of repeats (RADAR) analysis, tested with ELISA using deletion variants and visualized using electron microscopy. RESULTS: Eleven activating anti-ADAMTS-13 antibodies, directed against the T5-CUB2 domains, were identified in the FRETS-VWF73 assay. RADAR analysis identified three linker regions in the distal domains. Interestingly, identification of an antibody recognizing a cryptic epitope in the metalloprotease domain confirmed the contribution of these linker regions to conformational activation of the enzyme. The proof of flexibility around both the T2 and metalloprotease domains, as shown by by electron microscopy, further supported this contribution. In addition, cryptic epitope exposure was identified in the distal domains, because activating anti-T2-CUB2 antibodies increased the binding to folded VWF up to ~3-fold. CONCLUSION: Conformational activation of ADAMTS-13 leads to cryptic epitope/exosite exposure in both proximal and distal domains, subsequently inducing increased activity. Furthermore, three linker regions in the distal domains are responsible for flexibility and enable the interaction between the proximal and the T8-CUB2 domains.

The novel ADAMTS13-p.D187H mutation impairs ADAMTS13 activity and secretion and contributes to thrombotic thrombocytopenic purpura in mice.

BACKGROUND: Congenital thrombotic thrombocytopenic purpura (TTP) is characterized by mutations in the ADAMTS13 gene, which either impair protein secretion or influence ADAMTS13 (A Disintegrin-like And Metalloprotease domain with ThromboSpondin type-1 motif, member 13) activity. Phenotypic consequences of these mutations have not yet been evaluated in animal models for TTP. OBJECTIVES: To identify the in vitro effect of a novel ADAMTS13 mutation and to investigate whether this mutation induces TTP in vivo. METHODS: All 29 ADAMTS13 exons with exon-intron boundaries of a patient with pregnancy-onset TTP were sequenced. Wild-type and mutant ADAMTS13 proteins were both transiently and stably expressed in human embryonic kidney cells, and their activity was evaluated in vitro using fluorescence resonance energy transfer and flow assays. Molecular dynamics simulations were performed to study Ca(2+) stability. Adamts13(-/-) mice were hydrodynamically injected with wild-type and mutant expression plasmids and triggered with recombinant human von Willebrand factor. RESULTS: We identified a novel heterozygous c.559G>C mutation in exon 6 of the proposita's ADAMTS13 gene. This mutation resulted in a p.Asp187His substitution (p.D187H), which was located in the high affinity Ca(2+) -binding site in the metalloprotease domain of ADAMTS13. The homozygous p.D187H mutation down-regulated ADAMTS13 activity in vitro. Impaired proteolytic activity was linked to unstable Ca(2+) binding as visualized using a molecular dynamics simulation. In addition, the p.D187H mutation affects protein secretion in vitro. In Adamts13(-/-) mice, the homozygous p.D187H mutation reduced ADAMTS13 secretion and activity and contributed to TTP when these mice were triggered with recombinant human von Willebrand factor. CONCLUSIONS: Our data indicate that the p.D187H mutation impairs ADAMTS13 activity and secretion and is responsible for TTP onset in mice.

Preventable but neglected: rickets in an informal settlement, Nairobi, Kenya.

SETTING: The primary care clinics of Médecins Sans Frontières within the informal settlement of Kibera, Nairobi, Kenya. OBJECTIVE: To describe the demographic and clinical characteristics of children clinically diagnosed with rickets from September 2012 to October 2013. DESIGN: Descriptive retrospective case review of diagnosis and treatment course with vitamin D and calcium using routine programme data. RESULTS: Of the 82 children who met the clinical diagnosis of rickets, 57% were male, with a median age of 12 months and 14 months for females. Children with rickets were found to have ⩽3 hours/week sunlight exposure for 71% of the children and malnutrition in 39%. Clinical findings on presentation revealed gross motor developmental delays in 44%. The loss to follow-up rate during treatment was 40%. CONCLUSIONS: This study found that rickets is a common clinical presentation among children living in the informal settlement of Kibera and that there are likely multiple factors within that environment contributing to this condition. As rickets is a simply and inexpensively preventable non-communicable disease, we suggest that routine vitamin D supplementation be formally recommended by the World Health Organization for well-child care in Africa, especially in the contexts of informal settlements.

Contexte : Le dispensaire de soins de santé primaire de Médecins sans Frontières au cœur du bidonville de Kibera, Nairobi, Kenya.Objectif : Décrire les caractéristiques démographiques et cliniques d'enfants ayant eu un diagnostic clinique de rachitisme de septembre 2012 à octobre 2013.Schéma : Revue descriptive et rétrospective du diagnostic et du traitement par vitamine D et calcium à travers les données des programmes de routine.Résultats : Sur 82 enfants répondant au diagnostic clinique de rachitisme, 57% étaient des garçons d'un âge médian de 12 mois tandis que l'âge médian des filles était de 14 mois. L'exposition au soleil déclarée par la famille était de ⩽3 heures par semaine pour 71% des enfants et 39% présentaient une malnutrition. Les constatations cliniques à l'arrivée ont mis en évidence un retard de développement moteur marqué chez 44% des enfants. Le taux de perdus de vue pendant le traitement a été de 40%.Conclusion : Cette étude a constaté que le rachitisme était une affection fréquente parmi les enfants vivant dans le bidonville de Kibera et que de nombreux facteurs de l'environnement de Kibera y contribuaient vraisemblablement. Comme le rachitisme est une maladie non transmissible qui peut bénéficier d'une prévention simple et peu coûteuse, nous suggérons qu'une supplémentation en vitamine D soit formellement recommandée par l'Organisation Mondiale de la Santé dans les soins de santé aux enfants en Afrique, surtout dans le contexte de bidonvilles.

Marco de referencia: Los consultorios de atención primaria dirigidos por Médecins Sans Frontières en el asentamiento informal de Kibera, en Nairobi, Kenia.Objetivo: Describir las características clínicas y demográficas de los niños con diagnóstico clínico de raquitismo entre septiembre del 2012 y octubre del 2013.Método: Fue este un análisis descriptivo de casos, en el cual se evaluaron retrospectivamente el diagnóstico y la evolución del tratamiento con vitamina D y calcio, a partir de los datos corrientes del programa.Resultados: De los 82 niños que cumplían con los criterios diagnósticos de raquitismo, el 57% era de sexo masculino, con una mediana de edad de 12 meses y la mediana de edad de las niñas fue 14 meses. En el 71% los casos de raquitismo se encontró que la exposición directa al sol era de ⩽3 horas por semana y el 39% presentaba desnutrición. El examen físico en el momento de la consulta demostró un grave retraso del desarrollo motor en el 44% de los niños. Durante el seguimiento se perdió el 40% de los casos.Conclusiones: El presente estudio puso en evidencia que el raquitismo es una enfermedad frecuente en los niños que acuden a la consulta y viven en el asentamiento informal de Kibera; existen múltiples factores en el medio ambiente que contribuyen a esta situación. Dado que el raquitismo es una enfermedad no transmisible cuya prevención es sencilla y de bajo costo, se propone que la Organización Mundial de la Salud recomiende formalmente el aporte complementario sistemático de vitamina D en la consulta del niño sano en África, sobre todo en los entornos de asentamientos no estructurados.

Antiretroviral treatment uptake and attrition among HIV-positive patients with tuberculosis in Kibera, Kenya.

Using data of human immunodeficiency virus-positive patients with tuberculosis from three primary care clinics in Kibera slums, Nairobi, Kenya, we report on the proportion that started antiretroviral treatment (ART) and attrition (deaths, lost to follow-up and stopped treatment) before and while on ART. Of 427 ART eligible patients, enrolled between January 2004 and December 2008, 70% started ART, 19% were lost to attrition and 11% had not initiated ART. Of those who started ART, 14% were lost to attrition, making a cumulative pre-ART and ART attrition of 33%. ART uptake among patients with TB was relatively good, but programme attrition was high and needs urgent addressing.

Demographic characteristics and opportunistic diseases associated with attrition during preparation for antiretroviral therapy in primary health centres in Kibera, Kenya.

Using routine data from HIV-positive adult patients eligible for antiretroviral therapy (ART), we report on routinely collected demographic characteristics and opportunistic diseases associated with pre-ART attrition (deaths and loss to follow-up). Among 2471 ART eligible patients, enrolled between January 2005 and November 2008, 446 (18%) were lost to attrition pre-ART. Adjusted risk factors significantly associated with pre-ART attrition included age <35 years (Odds Ratio, OR 1.4, 95% Confidence Interval, CI 1.1-1.8), severe malnutrition (OR 1.5, 95% CI 1.1-2.0), active pulmonary tuberculosis (OR 1.6, 95% CI 1.1-2.4), severe bacterial infections including severe bacterial pneumonia (OR 1.9, 95% CI 1.2-2.8) and prolonged unexplained fever (>1 month), (OR 2.6, 95% CI 1.3-5.2). This study highlights a number of clinical markers associated with pre-ART attrition that could serve as 'pointers' or screening tools to identify patients who merit fast-tracking onto ART and/or closer clinical attention and follow-up.

Identification of a VWF peptide antagonist that blocks platelet adhesion under high shear conditions by selectively inhibiting the VWF-collagen interaction.

BACKGROUND: Because the collagen-VWF-GPIb/IX/V axis plays an important role in thrombus formation, it represents a promising target for development of new antithrombotic agents. OBJECTIVES: We used phage display to identify potential small peptides that interfere with the VWF-collagen binding and might serve as lead products for the development of possible oral antithrombotic compounds. METHODS: A random linear heptamer peptide library was used to select VWF-binding peptides. RESULTS: We identified a phage clone, displaying the YDPWTPS sequence, further referred to as L7-phage, that bound to VWF in a specific and a dose-dependent manner. This L7-phage specifically inhibited the VWF-collagen interaction under both static and flow conditions. Epitope mapping using deletion mutants of VWF revealed that the L7-phage does not bind to the known collagen-binding A3 domain within VWF, but to the more carboxyterminal situated C domain. This inhibition was not due to steric hindrance of the A3 domain-collagen interaction by the L7-phage. Indeed, a tetrabranched multi-antigen peptide (MAP) presenting four copies of the peptide, but not the scrambled MAP, also inhibited VWF-collagen interaction under conditions of high shear stress at a concentration of 148 nmol L(-1). CONCLUSIONS: Based on these results, we conclude that we have identified the first peptide antagonist that binds to the VWF C domain and by this specifically inhibits the VWF binding to collagen, suppressing platelet adhesion and aggregation under high shear conditions. As a consequence, this peptide and its future derivates are potentially interesting antithrombotic agents.