Genome-wide association studies to identify loci responsible for platelet function and cardiovascular disease susceptibility have repeatedly identified polymorphisms linked to a gene encoding platelet endothelium aggregation receptor 1 (PEAR1), an "orphan" cell surface receptor that is activated to stabilize platelet aggregates. To investigate how PEAR1 signaling is initiated, we sought to identify its extracellular ligand by creating a protein microarray representing the secretome and receptor repertoire of the human platelet. Using an avid soluble recombinant PEAR1 protein and a systematic screening assay designed to detect extracellular interactions, we identified the high affinity immunoglobulin E (IgE) receptor subunit α (FcεR1α) as a PEAR1 ligand. FcεR1α and PEAR1 directly interacted through their membrane-proximal Ig-like and 13th epidermal growth factor domains with a relatively strong affinity (KD ∼ 30 nm). Precomplexing FcεR1α with IgE potently inhibited the FcεR1α-PEAR1 interaction, and this was relieved by the anti-IgE therapeutic omalizumab. Oligomerized FcεR1α potentiated platelet aggregation and led to PEAR1 phosphorylation, an effect that was also inhibited by IgE. These findings demonstrate how a protein microarray resource can be used to gain important insight into the function of platelet receptors and provide a mechanistic basis for the initiation of PEAR1 signaling in platelet aggregation.
La maladie de Willebrand de type 2B (VWD-type 2B) se caracterise par des mutations « gain de fonction » touchant le facteur de Willebrand (VWF) et augmentant son affinite pour le recepteur plaquettaire GPIbα, entrainant une augmentation de la clairance des plaquettes et une pertes des multimeres de VWF de haut poids moceculaire (HPM). L’evolution sur le plan clinique de la VWD-type 2B est largement determinee par la mutation. Le phenotype hemorragique de la maladie est souvent explique par : (i) l’absence de multimeres HPM de VWF, (ii) l’indisponibilite des recepteurs GPIbα, constitutivement lies au VWF mute, et (iii) la thrombocytopenie, moderee a severe, souvent observee chez ces patients. Cette derniere resulte elle-meme d’une perturbation de la production de plaquettes et/ou de leur integration dans des complexes circulants associant des plaquettes et du VWF. Des etudes recentes ont montre que le VWF mute associe a la VWD-type 2B est susceptible d’entrainer une thrombopathie, laquelle pourrait expliquer, du moins en partie, la tendance hemorragique des patients. La diversite des genotypes et des phenotypes representes dans la VWD-type 2B, ainsi que la variabilite des manifestations clinicobiologiques de la maladie – entre individus, mais aussi chez un sujet donne –, lesquelles sont fonction du contexte physiopathologique (grossesse, chirurgie, inflammation, etc.), se traduisent par la multiplicite des attitudes therapeutiques, qui vont de la simple administration de desmopressine a celle de VWF ou aux transfusions de plaquettes. Une meilleure comprehension des mecanismes responsables de la tendance hemorragique observee dans la maladie permettrait de mieux la prendre en charge.
Abstract Blood platelets are essential for controlling hemostasis. They are released by megakaryocytes (MKs) located in the bone marrow, upon extension of cytoplasmic protrusions into the lumen of bone marrow sinusoids. Their number increases in postpulmonary capillaries, suggesting a role for oxygen gradient in thrombopoiesis (ie, platelet biogenesis). In this study, we show that initiation of thrombopoiesis from human mature MKs was enhanced under hyperoxia or during pro-oxidant treatments, whereas antioxidants dampened it. Quenching mitochondrial reactive oxygen species (mtROS) with MitoTEMPO decreased thrombopoiesis, whereas genetically enhancing mtROS by deacetylation-null sirtuin-3 expression increased it. Blocking cytosolic ROS production by NOX inhibitors had no impact. Classification according to the cell roundness index delineated 3 stages of thrombopoiesis in mature MKs. Early-stage round MKs exhibited the highest index, which correlated with low mtROS levels, a mitochondrial tubular network, and the mitochondrial recruitment of the fission activator Drp1. Intermediate MKs at the onset of thrombopoiesis showed high mtROS levels and small, well-delineated mitochondria. Terminal MKs showed the lowest roundness index and long proplatelet extensions. Inhibiting Drp1-dependent mitochondrial fission of mature MKs by Mdivi-1 favored a tubular mitochondrial network and lowered both mtROS levels and intermediate MKs proportion, whereas enhancing Drp1 activity genetically had opposite effects. Reciprocally, quenching mtROS limited mitochondrial fission in round MKs. These data demonstrate a functional coupling between ROS and mitochondrial fission in MKs, which is crucial for the onset of thrombopoiesis. They provide new molecular cues that control initiation of platelet biogenesis and may help elucidate some unexplained thrombocytopenia.
