Caractérisation biochimique et fonctionnelle du code tubuline des plaquettes sanguines

Blood platelets are small non-nucleated cellular fragments that ensure hemostasis. They are produced by bone marrow megakaryocytes following extensive microtubule rearrangements that culminate in the formation of a unique circular sub-membranous microtubule array, called marginal band, which supports the typical disc-shaped morphology of circulating platelets. Mechanistically, these microtubular rearrangements are thought to be controlled by a platelet-specific tubulin code. Here, we show that i) the α4A- and β1-tubulin isotypes both cooperate to assemble the marginal band, and that their combined deficiency in mice results in fully spherical platelets devoid of marginal band that are unable to perform hemostasis; ii) mutations in the α8-tubulin isotype can alter platelet and marginal band formation and cause macrothrombocytopenia in humans and; iii) several tubulin post-translational modifications are found in the platelet lineage, with functions that remain to be evaluated. Together, these results shed new light on a very specialized microtubule assembly process with applications in platelet diseases and transfusion.Les plaquettes sanguines sont de petits fragments cellulaires anucléés qui assurent l’hémostase. Elles sont produites par les mégacaryocytes de la moelle osseuse à l’issue d’un processus complexe qui comprend de profonds remaniements du cytosquelette de microtubule. L’un des derniers évènements consiste en la formation d’une structure circulaire et sous-membranaire unique aux mammifères, appelée bande marginale, dont le rôle est d’assurer la forme discoïde typique des plaquettes circulantes. D’un point de vue mécanistique, l’ensemble des remaniements microtubulaires seraient contrôlés par un code tubuline propre au lignage plaquettaire. Dans ce travail, nous montrons que i) les isotypes de tubuline α4A et β1 coopèrent ensemble pour assurer l’assemblage de la bande marginale, et que leur double inactivation chez la souris entraine la formation de plaquettes sphériques dépourvues de bande marginale et incapables d’assurer l’hémostase ; ii) des mutations dans l’isotype α8 perturbent la biogenèse des plaquettes et leur bande marginale, causant des macrothrombopénies chez l’Homme et ; iii) plusieurs modifications post-traductionnelles de tubuline sont retrouvées dans la lignée plaquettaire, dont les rôles restent encore à élucider. Ensemble, nos résultats pourraient avoir des applications directes dans les pathologies plaquettaires et la transfusion

Functional and biochemical characterization of the tubulin code in the platelet lineage

Les plaquettes sanguines sont de petits fragments cellulaires anucléés qui assurent l’hémostase. Elles sont produites par les mégacaryocytes de la moelle osseuse à l’issue d’un processus complexe qui comprend de profonds remaniements du cytosquelette de microtubule. L’un des derniers évènements consiste en la formation d’une structure circulaire et sous-membranaire unique aux mammifères, appelée bande marginale, dont le rôle est d’assurer la forme discoïde typique des plaquettes circulantes. D’un point de vue mécanistique, l’ensemble des remaniements microtubulaires seraient contrôlés par un code tubuline propre au lignage plaquettaire. Dans ce travail, nous montrons que i) les isotypes de tubuline α4A et β1 coopèrent ensemble pour assurer l’assemblage de la bande marginale, et que leur double inactivation chez la souris entraine la formation de plaquettes sphériques dépourvues de bande marginale et incapables d’assurer l’hémostase ; ii) des mutations dans l’isotype α8 perturbent la biogenèse des plaquettes et leur bande marginale, causant des macrothrombopénies chez l’Homme et ; iii) plusieurs modifications post-traductionnelles de tubuline sont retrouvées dans la lignée plaquettaire, dont les rôles restent encore à élucider. Ensemble, nos résultats pourraient avoir des applications directes dans les pathologies plaquettaires et la transfusion.Blood platelets are small non-nucleated cellular fragments that ensure hemostasis. They are produced by bone marrow megakaryocytes following extensive microtubule rearrangements that culminate in the formation of a unique circular sub-membranous microtubule array, called marginal band, which supports the typical disc-shaped morphology of circulating platelets. Mechanistically, these microtubular rearrangements are thought to be controlled by a platelet-specific tubulin code. Here, we show that i) the α4A- and β1-tubulin isotypes both cooperate to assemble the marginal band, and that their combined deficiency in mice results in fully spherical platelets devoid of marginal band that are unable to perform hemostasis; ii) mutations in the α8-tubulin isotype can alter platelet and marginal band formation and cause macrothrombocytopenia in humans and; iii) several tubulin post-translational modifications are found in the platelet lineage, with functions that remain to be evaluated. Together, these results shed new light on a very specialized microtubule assembly process with applications in platelet diseases and transfusion

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

Mutations in the most divergent α‐tubulin isotype, α8‐tubulin, cause defective platelet biogenesis

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J Thromb Haemost

Neutrophils participate in the pathogenesis of thrombosis through the formation of neutrophil extracellular traps (NETs). Thrombosis is the main cause of morbidity and mortality in patients with myeloproliferative neoplasms (MPNs). Recent studies have shown an increase in NET formation (NETosis) both in patients with JAK2V617F neutrophils and in mouse models, and reported the participation of NETosis in the pathophysiology of thrombosis in mice. This study investigated whether JAK2V617F neutrophils are sufficient to promote thrombosis or whether their cooperation with other blood cell types is necessary. NETosis was studied in PF4iCre;Jak2 mice expressing JAK2V617F in all hematopoietic lineages, as occurs in MPNs, and in MRP8Cre;Jak2 mice in which JAK2V617F is expressed only in leukocytes. In PF4iCre;Jak2 mice, an increase in NETosis and spontaneous lung thrombosis abrogated by DNAse administration were observed. The absence of spontaneous NETosis or lung thrombosis in MRP8Cre;Jak2 mice suggested that mutated neutrophils alone are not sufficient to induce thrombosis. Ex vivo experiments demonstrated that JAK2V617F-mutated platelets trigger NETosis by JAK2V617F-mutated neutrophils. Aspirin treatment in PF4iCre;Jak2 mice reduced NETosis and reduced lung thrombosis. In cytoreductive-therapy-free patients with MPN treated with aspirin, plasma NET marker concentrations were lower than that in patients with MPN not treated with aspirin. Our study demonstrates that JAK2V617F neutrophils alone are not sufficient to promote thrombosis; rather, platelets cooperate with neutrophils to promote NETosis in vivo. A new role for aspirin in thrombosis prevention in MPNs was also identified

IL-1β promotes MPN disease initiation by favoring early clonal expansion of JAK2-mutant hematopoietic stem cells

JAK2-V617F is themost frequent somatic mutation causingmyeloproliferative neoplasm(MPN). JAK2-V617F can be found in healthy individuals with clonal hematopoiesis of indeterminate potential (CHIP) with a frequency much higher than the prevalence of MPNs. The factors controlling the conversion of JAK2-V617F CHIP to MPN are largely unknown.We hypothesized that interleukin-1β (IL-1β)-mediated inflammation can favor this progression.We established an experimental system using bone marrow (BM) transplantations from JAK2-V617F and GFP transgenic (VF;GFP) mice that were further crossed with IL-1β-/- or IL-1R1-/- mice. To study the role of IL-1β and its receptor on monoclonal evolution of MPN, we performed competitive BM transplantations at high dilutions with only 1 to 3 hematopoietic stem cells (HSCs) per recipient. Loss of IL-1β in JAK2-mutantHSCs reduced engraftment, restricted clonal expansion, lowered the total numbers of functional HSCs, and decreased the rate of conversion toMPN. Loss of IL-1R1 in the recipients also lowered the conversion to MPN but did not reduce the frequency of engraftment of JAK2-mutant HSCs. Wild-type (WT) recipients transplantedwith VF;GFP BM that developed MPNs had elevated IL-1β levels and reduced frequencies of mesenchymal stromal cells (MSCs). Interestingly, frequencies of MSCs were also reduced in recipients that did not develop MPNs, had only marginally elevated IL-1β levels, and displayed low GFP-chimerism resembling CHIP. Anti-IL-1β antibody preserved high frequencies of MSCs in VF;GFP recipients and reduced the rate of engraftment and the conversion to MPN. Our results identify IL-1β as a potential therapeutic target for preventing the transition from JAK2-V617F CHIP to MPNs.ISSN:2473-9537ISSN:2473-952

Correction: An essential role for α4A-tubulin in platelet biogenesis

International audienceNo abstract availabl

An essential role for α4A-tubulin in platelet biogenesis

International audienceDuring platelet biogenesis, microtubules (MTs) are arranged into submembranous structures (the marginal band) that encircle the cell in a single plane. This unique MT array has no equivalent in any other mammalian cell, and the mechanisms responsible for this particular mode of assembly are not fully understood. One possibility is that platelet MTs are composed of a particular set of tubulin isotypes that carry specific posttranslational modifications. Although β1-tubulin is known to be essential, no equivalent roles of α-tubulin isotypes in platelet formation or function have so far been reported. Here, we identify α4A-tubulin as a predominant α-tubulin isotype in platelets. Similar to β1-tubulin, α4A-tubulin expression is up-regulated during the late stages of megakaryocyte differentiation. Missense mutations in the α4A-tubulin gene cause macrothrombocytopenia in mice and humans. Defects in α4A-tubulin lead to changes in tubulin tyrosination status of the platelet tubulin pool. Ultrastructural defects include reduced numbers and misarranged MT coils in the platelet marginal band. We further observed defects in megakaryocyte maturation and proplatelet formation in Tuba4a-mutant mice. We have, thus, discovered an α-tubulin isotype with specific and essential roles in platelet biogenesis