Hemostasia del desarrollo: nuevos mecanismos de regulación de la reactividad plaquetaria en neonatos

Las alteraciones en la reactividad plaquetaria se han asociado con el desarrollo de procesos tromboticos y hemorragicos. Los neonatos, especialmente los pretermino, tienen mayor incidencia de hemorragia intracraneal (HIC) que cualquier otro grupo de edad y, aunque su patogenia es multifactorial, la hiporreactividad de sus plaquetas podria ser un factor contribuyente. Los mecanismos geneticos y moleculares que subyacen a la hiporreactividad de las plaquetas neonatales estan pobremente caracterizados, por tanto su estudio tendria una alta relevancia clinica para este grupo de poblacion, pero ademas tambien supondria un modelo idoneo donde investigar los mecanismos reguladores de la reactividad plaquetaria, aun poco caracterizados, e identificar nuevas dianas terapeuticas. El objetivo general de esta Tesis era descubrir nuevos mecanismos moleculares responsables de las diferencias funcionales entre las plaquetas de neonatos (PN) y adultos (PA), lo que se abordo a traves del estudio de 4 objetivos especificos: (1) estudio diferencial de la expresion genica en PN vs. PA, (2) estudio del perfil de microRNA (miRNA) en PN vs. PA y de su posible papel regulador del transcriptoma plaquetario neonatal, (3) evaluacion de las diferencias en adhesion y secrecion en PN vs. PA, y (4) evaluacion de la expresion y funcionalidad de proteinas G y los reguladores de su senalizacion (RGS) en PN. Para ello se aislaron plaquetas de sangre de cordon umbilical de neonatos sanos nacidos a termino, asi como de sangre periferica de un grupo de adultos control sanos. Con RNA procedente de ambos grupos se realizaron dos arrays, uno de expresion genica y otro de miRNA. Las diferencias de expresion en los mRNA y miRNA se validaron en muestras de PN y PA mediante qRT-PCR, realizandose experimentos de transfeccion para las parejas miRNA:mRNA validadas. A continuacion se estudiaron las diferencias de secrecion entre PN y PA mediante qRT-PCR y western blot de elementos del complejo SNARE y de la tubulina-?1. Asi mismo se realizaron experimentos de cinetica de adhesion, estudios morfometricos pre- y post-activacion plaquetaria mediante microscopia de fluorescencia y electronica y evaluacion de las poblaciones de exosomas en plasma de neonatos y adultos. Las diferencias entre PN y PA en los niveles de expresion y/o funcionalidad de proteinas G se estudiaron mediante qRT-PCR, western blot y ensayos de activacion. Los resultados del array de expresion genica revelaron que existian 201 genes diferencialmente expresados entre PN y PA, 162 sobreexpresados y 39 infraexpresados. Entre los mas sobreexpresados en PN se encontraron principalmente: genes que codificaban a proteinas de origen eritroide o asociados con la sintesis y degradacion de proteinas. Para el caso de los infraexpresados en PN se observo predominio de los implicados en transporte/metabolismo de calcio, reorganizacion del citoesqueleto de actina, senalizacion celular y regulacion del ciclo celular. Las rutas mas enriquecidas estaban relacionadas con el ribosoma, el proteasoma y el espliceosoma o la proteolisis mediada por ubiquitinacion entre otras. Los datos del array de miRNA mostraron un total de 200 miRNA diferentemente expresados, implicados en rutas relacionadas con la biologia del desarrollo, la hemostasia y la funcionalidad plaquetaria entre otras. Partiendo del array de expresion genica se seleccionaron 4 genes con relevancia funcional en plaquetas (ADRA2A, GNAZ, RANBP10 y STX11) y para ellos, mediante algoritmos, se seleccionaron 7 miRNA con potencial para regularlos, sin embargo, a pesar de las predicciones in silico los ensayos in vitro no mostraron regulacion de ninguno de los 4 mRNA seleccionados por parte de los miRNA estudiados, debido en parte a las limitaciones del estudio in silico. En cuanto al objetivo 3, se encontro, en PN vs. PA, infraexpresion de alguno de los componentes del complejo SNARE (Stx11) y su regulador (Munc18b) lo que podria justificar en parte los defectos en secrecion observados en las PN. Tambien observamos una reduccion en la adhesion estatica y spreading en PN vs. PA, asi como diferencias morfologicas en las PN tras la activacion y diferencias de tamano en los exosomas derivados de PN con respecto a los de adulto. Por ultimo, los resultados del objetivo 4 mostraron diferencias de expresion de proteinas G y uno de sus reguladores entre PN y PA: las PN expresaron menos G?q, G?z y RGS18 y mas G?s y G?i2. Ademas, las PN tambien mostraron un mayor grado de activacion de G?s. En conclusion, los resultados obtenidos en la presente Tesis identifican, y en algunos casos aclaran, diversos mecanismos moleculares que subyacen a la hiporreactividad de la plaqueta neonatal. Son necesarios estudios futuros para vincular estos mecanismos a procesos patologicos en la poblacion infantil, extrapolarlos a la poblacion adulta y traducirlos en aplicaciones practicas. Alterations in platelet reactivity have been associated with the development of thrombotic and hemorrhagic processes. Neonates, especially preterm infants, have a higher incidence of intracranial hemorrhage (ICH) than any other group of age. Although ICH pathogenesis is multifactorial, hyporeactivity of the neonatal platelets has been considered a contributing factor. The genetic and molecular mechanisms underlying this hyporeactivity are poorly characterized, and their study may have a high clinical relevance for this population group. In addition comparison between neonatal and adult platelets is also an ideal model to investigate the mechanisms regulating platelet reactivity and to identify new therapeutic targets. The general objective of this Thesis was to discover new molecular mechanisms responsible for functional differences between neonatal platelets (NP) and adult platelets (AP). This was addressed through the study of 4 specific objectives: (1) differential study of NP and AP gene expression, (2) study of microRNAs (miRNAs) in NP vs AP and their potential regulatory role of neonatal gene expression, (3) evaluation of differences in adhesion and secretion in NP vs. AP, and (4) evaluation of the expression and functionality of G proteins and their regulators (RGS) in NP. To accomplish these objectives, blood platelets from umbilical cord of term healthy newborns and peripheral blood from a group of healthy adults were isolated. Two arrays were performed with the RNA of these samples, a gene expression array and a miRNA array. Expression differences in mRNAs and miRNAs were validated in NP and AP samples by qRT-PCR. The effect of miRNAs on selected mRNAs was investigated with transfection experiments in order to find relevant functional miRNA:mRNAs pairs. The secretion differences between NP and AP were studied with qRT-PCR and western blot assays of SNARE complex elements and tubulin-?1. Adhesion kinetics experiments and morphometric studies were performed before and after activation, by fluorescence and electron microscopy, respectively. Neonatal and adult plasma exosome populations were also evaluated. Differences between NP and AP levels in expression and/or functionality of G proteins were studied using qRT-PCR, western blot, and activation assays. The results of the gene expression array revealed that there were 201 genes differentially expressed between NP and AP (162 overexpressed and 39 underexpressed). The most overexpressed genes in NP mainly coded for proteins of erythroid origin or were associated with protein synthesis and degradation processes. In the case of the under-expressed genes in NP, we observed a predominance of those involved in calcium transport/metabolism, actin cytoskeletal reorganization, cell signaling, and cell cycle regulation. The most enriched routes were related to ribosome, proteasome, and spliceosome or ubiquitination-mediated proteolysis among others. The miRNA array data showed a total of 200 differentially expressed miRNAs, involved in pathways related to developmental biology, hemostasis, and platelet functionality among others. From the gene expression array, 4 genes relevant for platelet physiology were selected (ADRA2A, GNAZ, RANBP10 y STX11) and, by means of algorithms, 7 miRNAs with the potential to regulate these genes were selected. However, despite the use of a stringent in silico selection, the in vitro assays did not show any regulation of the 4 mRNAs selected by the studied miRNAs, maybe due to the limitations of the in silico study. Regarding objective 3, an underexpression of some of the components of the SNARE complex (Stx11) and its regulator (Munc18b) was found, which could partially justify the secretion defects observed in NP. We also observed a reduction in the static adhesion and spreading in NP vs. AP, as well as morphological differences in NP after activation and differences in size in plasma NP-derived exosomes as compared to adult. Finally, the results of objective 4 showed differences in G proteins expression and one of their regulators between NP and AP: NP showed lower levels of G?q, G?z and RGS18 and higher of G?s and G?i2. In addition, NP also showed a greater degree of G?s activation In conclusion, the results obtained in this Thesis identify, and in some cases clarify, several molecular mechanisms that underlie the hyporeactivity of the NP. Future studies are needed to link these mechanisms to pathological processes in the child population, extrapolate them to the adult population and translate them into practical applications.