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Down syndrome is the most common chromosomal abnormality in humans. Patients with Down syndrome have hematologic disorders, including mild to moderate thrombocytopenia. In case of Down syndrome, thrombocytopenia is not associated with bleeding, and it remains poorly characterized regarding molecular mechanisms. We investigated the effects of overexpression of Dyrk1A, an important factor contributing to some major Down syndrome phenotypes, on platelet number and bleeding in mice. Mice overexpressing Dyrk1A have a decrease in platelet number by 20%. However, bleeding time was found to be reduced by 50%. The thrombocytopenia and the decreased bleeding time observed were not associated to an abnormal platelet receptors expression, to a defect of platelet activation by ADP, thrombin or convulxin, to the presence of activated platelets in the circulation or to an abnormal half‐life of the platelets. To propose molecular mechanisms explaining this discrepancy, we performed a network analysis of Dyrk1A interactome and demonstrated that Dyrk1A, fibronectin and fibrinogen interact indirectly through two distinct clusters of proteins. Moreover, in mice overexpressing Dyrk1A, increased plasma fibronectin and fibrinogen levels were found, linked to an increase of the hepatic fibrinogen production. Our results indicate that overexpression of Dyrk1A in mice induces decreased bleeding consistent with increased plasma fibronectin and fibrinogen levels, revealing a new role of Dyrk1A depending on its indirect interaction with these two proteins. [ABSTRACT FROM AUTHOR]

Résumé: Le contenu en acide sialique à la surface des plaquettes décroît au cours de la vie de la plaquette. Cette élimination correspond au mécanisme de désialylation qui implique des enzymes, les sialidases. Cette évolution se traduit par l'exposition de résidus de β-galactose, un sucre greffé sur les chaînes glycanes avant l'acide sialique. Ces résidus ainsi exposés suite à la désialylation, forment des antigènes de sénescence qui entraînent la clairance des plaquettes et sont des régulateurs de leur durée de vie dans la circulation. Certaines thrombopénies peuvent être causées par des altérations des gènes régulant la synthèse et le transfert de l'acide sialique, ou par des mécanismes physiopathologiques acquis qui exacerbent la désialylation. Dans cette revue, nous exposerons les données de la littérature aussi bien fondamentales que cliniques mettant en lumière le rôle important de la sialylation plaquettaire dans la régulation du compte plaquettaire. Nous aborderons également la possibilité de mesurer l'exposition du β-galactose à la surface des plaquettes et son intérêt en clinique afin d'identifier les patients thrombopéniques qui pourraient bénéficier d'un traitement – encore expérimental – inhibant les sialidases. The sialic acid content on the platelet surface decreases during their life. This elimination corresponds to the mechanism of desialylation, which involves sialidases. The absence of sialic acid exposes β-galactose residues, which are considered to be sénescence antigens. Genes regulating the synthesis and the transfer of sialic acid and also mechanisms that exacerbate desialylation are responsible of thrombocytopenia. In this review, we will present both basic and clinical data from the literature highlighting the important role of platelet sialylation. We will also discuss the possibility of measuring platelet β-galactose exposure from a clinical point of view in order to identify patients with certain form of thrombocytopenias which could benefit from a still experimental treatment inhibiting sialidases. [ABSTRACT FROM AUTHOR]

A novel missense mutation in a leucine-rich repeat of GPIb in a Bernard-Soulier variant reduces shear-dependent adherence on von Willebrand factor (A) Platelets and megakaryocytes (MK) morphology on May-Grünwald-Giemsa (MGG) stained blood and bone marrow smears for patient and control. Botrocetin-induced platelet adhesion on immobilized human VWF was performed using washed platelets and visualized by differential interference contrast microscopy. Chinese hamster ovary (CHO) cells stably expressing GPIb -IX were co-transfected with either wild-type GPIb (CHO IX) or with mutated GPIb (CHO L139P IX). [Extracted from the article]

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The circulatory system is walled off by different cell types, including vascular mural cells and podocytes. The interaction and interplay between endothelial cells (ECs) and mural cells, such as vascular smooth muscle cells or pericytes, play a pivotal role in vascular biology. Endoglin is an RGD-containing counter-receptor for β1 integrins and is highly expressed by ECs during angiogenesis. We find that the adhesion between vascular ECs and mural cells is enhanced by integrin activators and inhibited upon suppression of membrane endoglin or β1-integrin, as well as by addition of soluble endoglin (SolEng), anti-integrin α5β1 antibody or an RGD peptide. Analysis of different endoglin mutants, allowed the mapping of the endoglin RGD motif as involved in the adhesion process. In Eng mice, a model for hereditary hemorrhagic telangectasia type 1, endoglin haploinsufficiency induces a pericyte-dependent increase in vascular permeability. Also, transgenic mice overexpressing SolEng, an animal model for preeclampsia, show podocyturia, suggesting that SolEng is responsible for podocytes detachment from glomerular capillaries. These results suggest a critical role for endoglin in integrin-mediated adhesion of mural cells and provide a better understanding on the mechanisms of vessel maturation in normal physiology as well as in pathologies such as preeclampsia or hereditary hemorrhagic telangiectasia. [ABSTRACT FROM AUTHOR]

Thrombocytopenia and increased platelet clearance observed in von Willebrand disease-type 2B (VWD-2B) may be explained by platelet apoptosis triggered by the constitutive binding of VWF to its receptor, glycoprotein Ib (GPIb). Apoptosis was assessed in platelets from two patients with a severe VWD-2B mutation VWF/p.V1316M and from mice transiently expressing VWF/p.V1316M. We now report that the VWD-2B mutation VWF/p.V1316M which binds spontaneously to its receptor GPIbα does not induce apoptosis. In 2 unrelated patients (P1 and P2) exhibiting different VWF plasma levels (70% and 36%, respectively, compared with normal pooled human plasma given as 100%), inner transmembrane depolarization of mitochondria, characteristic of apoptotic events was undetectable in platelets, whether washed or in whole blood. No or a moderate phosphatidyl serine (PS) exposure as measured by annexin-V staining was observed for P1 and P2, respectively. Expression of pro-apoptotic proteins Bak and Bax, and caspase-3 activity were similar to control platelets. In the VWD-2B mouse model expressing high levels of mVWF/p.V1316M (423%), similar to what is found in inflammatory pathologies, no significant difference was observed between mice expressing mVWF/WT and mVWF/p.V1316M. These results strongly argue against apoptosis as a mechanism for the thrombocytopenia of severe VWD-2B exhibiting the VWF/p.V1316M mutation. [ABSTRACT FROM AUTHOR]

Aims: Platelet endothelial aggregation receptor-1 (PEAR1) is a cell membrane protein, expressed on platelets and endothelial cells (ECs). PEAR1 sustains aIIbb3 activation in aggregating platelets and attenuates megakaryopoiesis via controlling the degree of Akt phosphorylation. Its role in EC biology is unknown. The aim of this study was to determine the expression of PEAR1 in the human endothelium of various tissues and to investigate its role in ECs in vitro and in angiogenesis, using Pear1-/- mice. Methods and results: PEAR1 is present on the membrane and on filo- and lamellipodia of human cultured ECs, and its expression coincides with CD31 in various tissues. PEAR1 expression is variable in ECs of different origin. Lentiviral knockdown of PEAR1 in cultured ECs doubled EC proliferation and significantly stimulated EC migration, in turn enhancing in vitro tube formation on matrigel through the Akt/PTEN-dependent p21/CDC2 pathway. Even when physiological blood vessel formation was unaffected in Pear1-/- mice, neoangiogenesis in these mice was significantly increased both in a hind limb ischaemia ligation model [4.7-fold increase in capillary density in the ligated limb of Pear1-/- mice compared with ligated limbs in wild-type (WT) mice] and in a skin wound-healing model, resulting in a two-fold faster wound closure in Pear1-/- mice compared with WT littermates. Conclusion: We established an inverse correlation between endothelial PEAR1 expression and vascular assembly both in vitro and in vivo. These findings identify PEAR1 as a novel modifier of neoangiogenesis. [ABSTRACT FROM AUTHOR]

Hematopoietic stem cells (HSC) differentiate into megakaryocytes (MK), whose function is to release platelets. Attempts to improve in vitro platelet production have been hampered by the low amplification of MK. Providing HSC with an optimal three-dimensional (3D) architecture may favor MK differentiation by mimicking some crucial functions of the bone marrow structure. To this aim, porous hydrogel scaffolds were used to study MK differentiation from HSC as well as platelet production. Flow cytometry, qPCR and perfusion studies showed that 3D was suitable for longer kinetics of CD34+ cell proliferation and for delayed megakaryocytic differentiation far beyond the limited shelf-life observed in liquid culture but also increased production of functional platelets. We provide evidence that these 3D effects were related to 1) persistence of MK progenitors and precursors and 2) prolongation of expression of EKLF and c-myb transcription factors involved in early MK differentiation. In addition, presence of abundant mature MK with increased ploidy and impressive cytoskeleton elongations was in line with expression of NF-E2 transcription factor involved in late MK differentiation. Platelets produced in flow conditions were functional as shown by integrin αIIbβ3 activation following addition of exogenous agonists. This study demonstrates that spatial organization and biological cues synergize to improve MK differentiation and platelet production. Thus, 3D environment constitutes a powerful tool for unraveling the physiological mechanisms of megakaryopoiesis and thrombopoiesis in the bone marrow environment, potentially leading to an improved amplification of MK and platelet production. [ABSTRACT FROM AUTHOR]

Endoglin (Eng) is an endothelial cell (EC) transmembrane glycoprotein involved in adhesion and angiogenesis. Eng mutations result in vessel abnormalities as observed in hereditary hemorrhagic telangiectasia of type 1. The role of Eng was investigated in endothelial functions and permeability under inflammatory conditions, focusing on the actin dynamic signaling pathway. Endothelial Colony-Forming Cells (ECFC) from human cord blood and mouse lung/aortic EC (MLEC, MAEC) from Eng+/+ and Eng+/− mice were used. ECFC silenced for Eng with Eng-siRNA and ctr-siRNA were used to test tubulogenesis and permeability +/− TNFα and +/− LIM kinase inhibitors (LIMKi). In silico modeling of TNFα–Eng interactions was carried out from PDB IDs 5HZW and 5HZV. Calcium ions (Ca2+) flux was studied by Oregon Green 488 in epifluorescence microscopy. Levels of cofilin phosphorylation and tubulin post-translational modifications were evaluated by Western blot. F-actin and actin–tubulin distribution/co-localization were evaluated in cells by confocal microscopy. Eng silencing in ECFCs resulted in a decrease of cell sprouting by 50 ± 15% (p < 0.05) and an increase in pseudo-tube width (41 ± 4.5%; p < 0.001) compared to control. Upon TNFα stimulation, ECFC Eng–siRNA displayed a significant higher permeability compared to ctr-siRNA (p < 0.01), which is associated to a higher Ca2+ mobilization (p < 0.01). Computational analysis suggested that Eng mitigated TNFα activity. F-actin polymerization was significantly increased in ECFC Eng-siRNA, MAEC+/−, and MLEC+/− compared to controls (p < 0.001, p < 0.01, and p < 0.01, respectively) as well as actin/tubulin distribution (p < 0.01). Furthermore, the inactive form of cofilin (P-cofilin at Ser3) was significantly decreased by 36.7 ± 4.8% in ECFC Eng-siRNA compared to ctr-siRNA (p < 0.001). Interestingly, LIMKi reproduced the absence of Eng on TNFα-induced ECFC-increased permeability. Our data suggest that Eng plays a critical role in the homeostasis regulation of endothelial cells under inflammatory conditions (TNFα), and loss of Eng influences ECFC-related permeability through the LIMK/cofilin/actin rearrangement-signaling pathway. [ABSTRACT FROM AUTHOR]

The genetic causes of congenital hypothyroidism due to thyroid dysgenesis (TD) remain largely unknown. We identified three novel TUBB1 gene mutations that co‐segregated with TD in three distinct families leading to 1.1% of TUBB1 mutations in TD study cohort. TUBB1 (Tubulin, Beta 1 Class VI) encodes for a member of the β‐tubulin protein family. TUBB1 gene is expressed in the developing and adult thyroid in humans and mice. All three TUBB1 mutations lead to non‐functional α/β‐tubulin dimers that cannot be incorporated into microtubules. In mice, Tubb1 knock‐out disrupted microtubule integrity by preventing β1‐tubulin incorporation and impaired thyroid migration and thyroid hormone secretion. In addition, TUBB1 mutations caused the formation of macroplatelets and hyperaggregation of human platelets after stimulation by low doses of agonists. Our data highlight unexpected roles for β1‐tubulin in thyroid development and in platelet physiology. Finally, these findings expand the spectrum of the rare paediatric diseases related to mutations in tubulin‐coding genes and provide new insights into the genetic background and mechanisms involved in congenital hypothyroidism and thyroid dysgenesis. Synopsis: Whole‐exome sequencing in a consanguineous family with congenital hypothyroidism (CH) revealed a novel homozygous mutation in TUBB1, encoding for a member of beta‐tubulins, essential for microtubule organisation. Until now, TUBB1 mutations were only known to be involved in platelet disorders. Two more mutations were identified in two distinct families with thyroid dysgenesis (TD) and abnormal platelet physiology, reinforcing the link between TUBB1 mutations and thyroid disease.TUBB1 is expressed in the developing and adult thyroid in humans and mice.Tubb1−/− mice have large platelets and show hypothyroidism with early thyroid progenitors proliferation defects, altered thyroid migration and failure of thyroid hormone synthesis.Platelet findings concerned basal activation and increased aggregation.These results expand our knowledge on genetic background of CH and TD. Whole‐exome sequencing in a consanguineous family with Congenital Hypothyroidism (CH) revealed a novel homozygous mutation in TUBB1, encoding for a member of beta‐tubulins, essential for microtubule organisation. Until now, TUBB1 mutations were only known to be involved in platelet disorders. [ABSTRACT FROM AUTHOR]