Cytoskeletal-based mechanisms differently regulate <i>in vivo</i> and <i>in vitro</i> proplatelet formation

Platelets are produced by bone marrow megakaryocytes through cytoplasmic protrusions, named native proplatelets (nPPT), into blood vessels. Proplatelets also refer to protrusions observed in megakaryocyte culture (cPPT) that are morphologically different. Contrary to cPPT, the mechanisms of nPPT formation are poorly understood. We show here in living mice that nPPT elongation is in equilibrium between protrusive and retraction forces mediated by myosin-IIA. We also found, using WT and β1-tubulin-deficient mice, that microtubule behavior differs between cPPT and nPPT, being absolutely required in vitro, while less critical in vivo. Remarkably, microtubule depolymerization in myosin-deficient mice did not affect nPPT elongation. We then calculated that blood Stokes'forces may be sufficient to promote nPPT extension, independently of myosin and microtubules. Together, we propose a new mechanism for nPPT extension that might explain contradictions between severely affected cPPT production and moderate platelet count defects in some patients and animal models

Etude du rôle de la GPIbb dans la thrombopoïèse et les fonctions plaquettaires

L'hémostase est un processus physiologique intervenant dans le maintien de l'intégrité des vaisseaux. Un dérèglement de ce mécanisme favorise les accidents thrombotiques ou entraîne des problèmes hémorragiques graves. L'hémostase primaire fait intervenir les plaquettes et permet l'arrêt des saignements dans les vaisseaux de petits calibres suite à une lésion. En présence de flux sanguins élevés (artères, microvaisseaux), l'adhésion est principalement assurée par le complexe glycoprotéique GPIb-V-IX, récepteur du facteur de Willebrand. L'importance de ce récepteur est attestée par l'existence d'un syndrome hémorragique grave, le syndrome de Bernard-Soulier, provoqué par l'absence ou l'anomalie d'une des sous-unités du complexe, caractérisé par un temps de saignement allongé, une thrombopénie et des plaquettes de grande taille. L'objectif de ce travail de thèse a été d'évaluer in vivo et in vitro le rôle de la GPIbb, membre du complexe GPIb-V-IX, dans les fonctions plaquettaires et la thrombopoïèse.Deux lignées de souris transgéniques ont été produites par recombinaison homologue: une lignée de souris knock-out où le gène codant pour la GPIbb a été invalidé et une lignée de souris knock-in où la GPIbb a été délétée de son domaine intracellulaire. Ces modèles de souris reproduisent un phénotype de type Bernard-Soulier et nous permettront d'étudier plus précisément le rôle de la GPIbb et de son domaine intracellulaire dans les fonctions plaquettaires et dans les mécanismes de libération des plaquettes.Un autre aspect du travail a été d'évaluer, in vitro, la fonction de la GPIbb. Trois mutations, retrouvées chez des patients, ont été reproduites par transfection dans des lignées de cellules d'ovaire de hamster (CHO). Ces études nous ont permis démontrer le rôle clé de la GPIbb dans la biosynthèse du complexe GPIb-IX et de préciser les domaines structuraux essentiels à l'expression de ce dernier.The glycoprotein (GP) Ib-V-IX complex is a specialized multisubunit receptor abundantly expressed at the surface of platelets and whose physiological role is to ensure normal haemostasis. This property is best illustrated by the existence of a severe bleeding tendency in patients with the rare Bernard-Soulier syndrome resulting from a genetic defect of the GPIb-V-IX complex. Increased bleeding is mainly due to the lack of platelet adhesion to VWF mediated by the complex, resulting in defective platelet plug formation at sites of vessel wall injury. These patients additionally have platelets with enlarged size and decreased numbers in the circulation. The purpose of this work was to evaluate the role, in vivo and in vitro, of the GPIb-V-IX complex, and more particularly the role of GPIb within this complex, in platelet function and in platelet production.For this study, two genetically modified mouse models were engineered. A Bernard-Soulier-like knock-out strain was generated by inactivating the GPIbb gene (GPIbb KO) and a knock-in strain was created by interrupting the sequence coding for the GPIbb intracellular domain (GPIbbC). The GPIbbKO mice exhibited the Bernard-Soulier phenotype, which is thrombocytopenia, giant platelets and a severely prolonged bleeding time. The further perspectives of this work will be to evaluate the function of GPIbb in haemostatic function and in artertial thrombosis.Another aspect of this work was to evaluate, in vitro, the function of the GPIbb subunit by transfecting mutated GPIb-IX complex into CHO cells. Three mutations from patients were reproduced and analysed. This study underlined the key role of the GPIbb in the biosynthesis process and precised the structural domains essential for the complex expression. Overall, these results have provided direct evidence for the involvement of the GPIbb in platelet function and the transgenic mice will proved useful to directly evaluate the role of this subunit in in vivo thrombosis.STRASBOURG-Sc. et Techniques (674822102) / SudocSudocFranceF

On the Way to in vitro Platelet Production

The severely decreased platelet counts (10–30. 103 platelets/μL) frequently observed in patients undergoing chemotherapy, radiation treatment, or organ transplantation are associated with life-threatening increased bleeding risks. To circumvent these risks, platelet transfusion remains the treatment of choice, despite some limitations which include a limited shelf-life, storage-related deterioration, the development of alloantibodies in recipients and the transmission of infectious diseases. A sustained demand has evolved in recent years for controlled blood products, free of infectious, inflammatory, and immune risks. As a consequence, the challenge for blood centers in the near future will be to ensure an adequate supply of blood platelets, which calls for a reassessment of our transfusion models. To meet this challenge, many laboratories are now turning their research efforts toward the in vitro and customized production of blood platelets. In recent years, there has been a major enthusiasm for the cultured platelet production, as illustrated by the number of reviews that have appeared in recent years. The focus of the present review is to critically asses the arguments put forward in support of the culture of platelets for transfusion purposes. In light of this, we will recapitulate the main advances in this quickly evolving field, while noting the technical limitations to overcome to make cultured platelet a transfusional alternative

Glycoprotein V : the unsolved GPV puzzle

Glycoprotein V (GPV) is a highly expressed 82 KDa platelet surface transmembrane protein which is loosely attached to the GPIb-IX complex. Despite remaining questions concerning its function, GPV presents several unique features which have repercussions in hematology, atherothrombosis, immunology and transfusion. GPV is specifically expressed in platelets and megakaryocytes and is an ideal marker and reporter gene for the late stages of megakaryopoiesis. The ectodomain of GPV can be released by a number of proteases, namely thrombin, elastase and ADAM10 and 17. Although it was originally proposed as a thrombin receptor, this hypothesis was abandoned since thrombin activation was preserved after blockade of GPV cleavage and in Gp5 knockout mice. The combined potential of GPV to reflect the direct action of thrombin, platelet exposure to strong agonists and inflammatory conditions has led one to evaluate its utility as a marker in the context of atherothrombosis. Increased plasma levels of soluble GPV have notably been recorded in myocardial infarction, stroke and venous thromboembolism. It is also highly valued in transfusion to monitor platelet storage lesions. GPV presents several polymorphisms, which are a possible source of alloantibodies, while autoantibodies have been frequently detected in immune thrombocytopenia. The real biological function of this glycoprotein nevertheless remains an enigma, despite the respectively decreased and increased responses to low concentrations of collagen and thrombin observed in Gp5 knockout mice. Current studies are exploring its role in modulating general or VWF-induced platelet signaling, which could bear relevance in thrombosis and platelet clearance

Hirudin and heparin enable efficient megakaryocyte differentiation of mouse bone marrow progenitors.

International audienceHematopoietic progenitors from murine fetal liver efficiently differentiate in culture into proplatelet-producing megakaryocytes and have proved valuable to study platelet biogenesis. In contrast, megakaryocyte maturation is far less efficient in cultured bone marrow progenitors, which hampers studies in adult animals. It is shown here that addition of hirudin to media containing thrombopoietin and serum yielded a proportion of proplatelet-forming megakaryocytes similar to that in fetal liver cultures (approximately 50%) with well developed extensions and increased the release of platelet particles in the media. The effect of hirudin was maximal at 100U/ml, and was more pronounced when it was added in the early stages of differentiation. Hirugen, which targets the thrombin anion binding exosite I, and argatroban, a selective active site blocker, also promoted proplatelet formation albeit less efficiently than hirudin. Heparin, an indirect thrombin blocker, and OTR1500, a stable heparin-like synthetic glycosaminoglycan generated proplatelets at levels comparable to hirudin. Heparin with low affinity for antithrombin was equally as effective as standard heparin, which indicates antithrombin independent effects. Use of hirudin and heparin compounds should lead to improved culture conditions and facilitate studies of platelet biogenesis in adult mice

Importance of environmental stiffness for megakaryocyte differentiation and proplatelet formation.

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Dual role of IL-21 in megakaryopoiesis and platelet homeostasis: Modulation of platelet homeostasis by IL-21

International audienceGene profiling studies have indicated that in vitro differentiated human megakaryocytes express the receptor for IL-21 (IL-21R), an immunostimulatory cytokine associated with inflammatory disorders and currently under evaluation in cancer therapy. The aim of this study was to investigate whether IL-21 modulates megakaryopoiesis. We first checked the expression of IL-21 receptor on human BM and in vitro differentiated megakaryocytes. Then, we investigated the effect of IL-21 on the in vitro differentiation of human blood CD34+ progenitors into megakaryocytes. Finally, we analyzed the consequences of hydrodynamic transfection-mediated transient expression of IL-21, on megakaryopoiesis and thrombopoiesis in mice. The IL-21Rα chain was expressed in human BM megakaryocytes and was progressively induced during in vitro differentiation of human peripheral CD34+ progenitors, while the signal transducing γ chain was down-regulated. Consistently, the STAT3 phosphorylation induced by IL-21 diminished during the later stages of megakaryocytic differentiation. In vitro, IL-21 increased the number of CFU-MKs generated from CD34+ cells and the number of megakaryocytes differentiated from CD34+ progenitors in a JAK3- and STAT3-dependent manner. Forced expression of IL-21 in mice increased the density of bi-potent MK progenitors and BM megakaryocytes, and the platelet generation, but increased platelet clearance and consequently resulting in reduced blood platelet counts. Our work suggests that IL-21 regulates megakaryocyte development and platelet homeostasis. Thus IL-21 may link immune responses to physiological or pathological platelet-dependent processes

miR-204-5p and Platelet Function Regulation: Insight into a Mechanism Mediated by CDC42 and GPIIbIIIa

Background: Several platelet-derived microRNAs are associated with platelet reactivity (PR) and clinical outcome in cardiovascular patients. We previously showed an association between miR-204-5p and PR in stable cardiovascular patients, but data on functional mechanisms are lacking. Aims: To validate miR-204-5p as a regulator of PR in platelet-like structures (PLS) derived from human megakaryocytes and to address mechanistic issues. Methods: Human hematopoietic stem cells were differentiated into megakaryocytes, enabling the transfection of miR-204-5p and the recovery of subsequent PLS. The morphology of transfected megakaryocytes and PLS was characterized using flow cytometry and microscopy. The functional impact of miR-204-5p was assessed using a flow assay, the quantification of the activated form of the GPIIbIIIa receptor, and a fibrinogen-binding assay. Quantitative polymerase chain reaction and western blot were used to evaluate the impact of miR-204-5p on a validated target, CDC42. The impact of CDC42 modulation was investigated using a silencing strategy. Results: miR-204-5p transfection induced cytoskeletal changes in megakaryocytes associated with the retracted protrusion of proPLS, but it had no impact on the number of PLS released. Functional assays showed that the PLS produced by megakaryocytes transfected with miR-204-5p were more reactive than controls. This phenotype is mediated by the regulation of GPIIbIIIa expression, a key contributor in platelet-fibrinogen interaction. Similar results were obtained after CDC42 silencing, suggesting that miR-204-5p regulates PR, at least in part, via CDC42 downregulation. Conclusion: We functionally validated miR-204-5p as a regulator of the PR that occurs through CDC42 downregulation and regulation of fibrinogen receptor expression.</p