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Mice lacking the immunoreceptor tyrosine-based inhibition motif-containing co-inhibitory receptor G6b-B (Mpig6b, G6b knockout, KO) are born with a complex megakaryocyte (MK) per platelet phenotype, characterized by severe macrothrombocytopenia, expansion of the MK population, and focal myelofibrosis in the bone marrow and spleen. Platelets are almost completely devoid of the glycoprotein VI (GPVI)-FcRγ-chain collagen receptor complex, have reduced collagen integrin α2β1, elevated Syk tyrosine kinase activity, and a subset has increased surface immunoglobulins. A similar phenotype was recently reported in patients with null and loss-of-function mutations in MPIG6B. To better understand the cause and treatment of this pathology, we used pharmacological- and genetic-based approaches to rescue platelet counts and function in G6b KO mice. Intravenous immunoglobulin resulted in a transient partial recovery of platelet counts, whereas immune deficiency did not affect platelet counts or receptor expression in G6b KO mice. Syk loss-of-function (R41A) rescued macrothrombocytopenia, GPVI and α2β1 expression in G6b KO mice, whereas treatment with the Syk kinase inhibitor BI1002494 partially rescued platelet count but had no effect on GPVI and α2β1 expression or bleeding. The Src family kinase inhibitor dasatinib was not beneficial in G6b KO mice. In contrast, treatment with the thrombopoietin mimetic romiplostim rescued thrombocytopenia, GPVI expression, and platelet reactivity to collagen, suggesting that it may be a promising therapeutic option for patients lacking functional G6b-B. Intriguingly, GPVI and α2β1 expression were significantly downregulated in romiplostim-treated wild-type mice, whereas GPVI was upregulated in romiplostim-treated G6b KO mice, suggesting a cell intrinsic feedback mechanism that autoregulates platelet reactivity depending on physiological needs.

While loss-of-function variants in the WAS gene are associated with Wiskott-Aldrich syndrome and lead to microthrombocytopenia, gain-of-function variants of WAS are associated with X-linked neutropenia (XLN) and the absence of microthrombocytopenia. Only a few XLN families have been reported so far, and their platelet phenotype was not described in detail. To date, no renal involvement was described in XLN. In the present study, we report exome sequencing of individuals from 3 generations of a family with a dominant disease combining neutropenia, macrothrombocytopenia, and renal failure. We identified a heterozygous missense gain-of-function variant in the WAS gene (c.881T>C, p.I294T) that segregates with the disease and is already known to cause XLN. There was no pathogenic variant in MYH9, TUBB1, or ACTN1. This is the first report of a WAS gain-of-function variant associated with both the hematological phenotype of XLN (neutropenia, macrothrombocytopenia) and renal disease (proteinuria, renal failure) with glomerular tip lesion hyalinosis and actin condensations in effaced podocytes foot processes.

Transfusion-related acute lung injury (TRALI) remains a major cause of transfusion-related fatalities. The mechanism of human antibody-mediated TRALI, especially the involvement of the Fcγ receptors, is not clearly established. Contrary to mice, human platelets are unique in their expression of the FcγRIIA/CD32A receptor, suggesting that our understanding of the pathogenesis of antibody-mediated TRALI is partial, as the current murine models incompletely recapitulate the human immunology. We evaluated the role of FcγRIIA/CD32A in TRALI using a humanized mouse model expressing the FcγRIIA/CD32A receptor. When challenged with a recombinant chimeric human immunoglobulin G1/mouse anti–major histocompatibility complex class I monoclonal antibody, these mice exhibited exacerbated alveolar edema and higher mortality compared with wild-type (WT) mice. Unlike in WT mice, monocytes/macrophages in CD32A+ mice were accessory for TRALI initiation, indicating the decisive contribution of another cell type. Platelet activation was dramatically increased in CD32A+ animals, resulting in their increased consumption and massive release of their granule contents. Platelet depletion prevented the exacerbation of TRALI in CD32A+ mice but did not affect TRALI in WT animals. By blocking platelet serotonin uptake with fluoxetine, we showed that the severity of TRALI in CD32A+ mice resulted from the serotonin released by the activated platelets. Furthermore, inhibition of 5-hydroxytryptamine 2A serotonin receptor with sarpogrelate, before or after the induction of TRALI, abolished the aggravation of lung edema in CD32A+ mice. Our findings show that platelet FcγRIIA/CD32A activation exacerbates antibody-mediated TRALI and provide a rationale for designing prophylactic and therapeutic strategies targeting the serotonin pathway to attenuate TRALI in patients.

Objective Integrins are key regulators of various platelet functions. The pathophysiological importance of most platelet integrins has been investigated, with the exception of α5β1, a receptor for fibronectin. The aim of this study was to characterize the role of α5β1 in megakaryopoiesis, platelet function, and to determine its importance in hemostasis and arterial thrombosis. Approach and Results We generated a mouse strain deficient for integrin α5β1 on megakaryocytes and platelets (PF4Cre-α5−/−). PF4Cre-α5−/− mice were viable, fertile, and presented no apparent signs of abnormality. Megakaryopoiesis appears unaltered as evidence by a normal megakaryocyte morphology and development, which is in agreement with a normal platelet count. Expression of the main platelet receptors and the response of PF4Cre-α5−/− platelets to a series of agonists were all completely normal. Adhesion and aggregation of PF4Cre-α5−/− platelets under shear flow on fibrinogen, laminin, or von Willebrand factor were unimpaired. In contrast, PF4Cre-α5−/− platelets displayed a marked decrease in adhesion, activation, and aggregation on fibrillar cellular fibronectin and collagen. PF4Cre-α5−/− mice presented no defect in a tail-bleeding time assay and no increase in inflammatory bleeding in a reverse passive Arthus model and a lipopolysaccharide pulmonary inflammation model. Finally, no defects were observed in three distinct experimental models of arterial thrombosis based on ferric chloride-induced injury of the carotid artery, mechanical injury of the abdominal aorta, or laser-induced injury of mesenteric vessels. Conclusion In summary, this study shows that platelet integrin α5β1 is a key receptor for fibrillar cellular fibronectin but is dispensable in hemostasis and arterial thrombosis.

Alloimmunization against platelets remains a potentially serious adverse transfusion event. Alloantibodies produced by the recipient, mainly directed against human leukocyte antigen class I donor antigens, can compromise the therapeutic efficacy of subsequent transfusions, and may lead to refractoriness. Because the mechanism of anti-HLA alloantibody formation is poorly understood, this study aimed to identify the cells involved in the platelet immune response by focusing on the spleen, the main organ that orchestrates this alloimmune response. In the spleen, transfused allogeneic platelets are located in the marginal zone and interact with marginal zone B (MZB) cells, a specialized B-cell population implicated in the capture and follicular delivery of blood-borne antigens. To study the involvement of MZB cells in alloantibody production, we used a murine model reproducing major histocompatibility complex incompatibility between a donor (H2b) and recipient (H2d) that occurs during platelet transfusion. Following weekly H2b platelet transfusions, recipient H2d mice produced anti-H2b immunoglobulin G, which induced a refractory state upon subsequent transfusions. Specific immunodepletion of MZB cells or their displacement from the marginal zone to the B-cell follicles by treatment with an S1P1 antagonist before each transfusion prevented significant alloantibody formation. Under these conditions, transfused platelets were still circulating after 24 hours, whereas they were rapidly removed from circulation in alloimmunized mice. The identification of MZB cells as key players in the platelet alloimmune response opens up new perspectives for minimizing platelet alloimmunization and avoiding the associated refractory state in frequently transfused patients.

Severe COVID-19 has been associated with a high rate of thrombotic events but also of bleeding events, particularly when the level of prophylactic anticoagulation was increased. Data on the contribution of platelets to these thrombotic events are discordant between reports, while the involvement of platelets in bleeding events has never been investigated. The objective of the present study was to assess platelet function during the first week of ICU hospitalization in patients with severe COVID-19 pneumonia. A total of 35 patients were prospectively included and blood samples were drawn on day (D) 0, D2 and D7. COVID-19 pneumonia was severe with a median PaO

Background Blood platelets are anucleate cell fragments that prevent bleeding and minimize blood vessel injury. They are formed from the cytoplasm of megakaryocytes located in the bone marrow. For successful platelet production, megakaryocyte fragments must pass through the sinusoid endothelial barrier by a cell biology process unique to these giant cells as compared with erythrocytes and leukocytes. Currently, the mechanisms by which megakaryocytes interact and progress through the endothelial cells are not understood, resulting in a significant gap in our knowledge of platelet production. Objective The aim of this study was to investigate how megakaryocytes interact and progress through the endothelial cells of mouse bone marrow sinusoids. Methods We used a combination of fluorescence, electron, and three-dimensional microscopy to characterize the cellular events between megakaryocytes and endothelial cells. Results We identified protrusive, F-actin-based podosome-like structures, called in vivo-MK podosomes, which initiate the formation of pores through endothelial cells. These structures present a collective and spatial organization through their interconnection via a contractile network of actomyosin, essential to regulate the endothelial openings. This ensures proper passage of megakaryocyte-derived processes into the blood circulation to promote thrombopoiesis. Conclusion This study provides novel insight into the in vivo function of podosomes of megakaryocytes with critical importance to platelet production.

Blood flow is a major regulator of hemostasis and arterial thrombosis. The current view is that low and intermediate flows occur in intact healthy vessels, whereas high shear levels (>2000 s−1) are reached in stenosed arteries, notably during thrombosis. To date, the shear rates occurring at the edge of a lesion in an otherwise healthy vessel are nevertheless unknown. The aim of this work was to measure the shear rates prevailing in wounds in a context relevant to hemostasis. Three models of vessel puncture and transection were developed and characterized for a study that was implemented in mice and humans. Doppler probe measurements supplemented by a computational model revealed that shear rates at the edge of a wound reached high values, with medians of 22 000 s−1, 25 000 s−1, and 7000 s−1 after puncture of the murine carotid artery, aorta, or saphenous vein, respectively. Similar shear levels were observed after transection of the mouse spermatic artery. These results were confirmed in a human venous puncture model, where shear rates in a catheter implanted in the cubital vein reached 2000 to 27 000 s−1. In all models, the high shear conditions were accompanied by elevated levels of elongational flow exceeding 1000 s−1. In the puncture model, the shear rates decreased steeply with increasing injury size. This phenomenon could be explained by the low hydrodynamic resistance of the injuries as compared with that of the downstream vessel network. These findings show that high shear rates (>3000 s−1) are relevant to hemostasis and not exclusive to arterial thrombosis.

Introduction The specific role of platelets during sepsis is not yet fully understood, probably related to the paradox of platelets being potentially beneficial but also deleterious via their thrombotic functions. Objective To evaluate the impact of thrombocytopenia on septic shock in mice and to investigate whether transfusion of fresh washed platelets, either fully functional or with impaired hemostatic properties, might have beneficial effects. Methods Septic shock was induced by cecal ligation and puncture (CLP). Experimental depletion of circulating platelets was induced with a rat anti-mouse GPIbα monoclonal antibody. Transfusion of either WT washed platelets, platelets treated with the antiplatelet drugs acetylsalicylic acid (ASA) and clopidogrel, or GPIIbIIIa-deficient washed platelets treated with ASA and clopidogrel was performed 4 h after CLP surgery. Results Depletion of circulating platelets negatively affected septic shock, worsening systemic inflammation, coagulopathy, organ damage and mortality, raising the question of whether a higher platelet count could be protective. Transfusion of fully functional platelets or platelets with combined treatment with ASA and clopidogrel, with or without additional GPIIbIIIa deficiency, afforded an immediate return of circulating platelet counts to their initial values before surgery. However, transfusion of each of the three types of platelets did not prevent arterial hypotension, inflammatory response, coagulopathy and organ damage during septic shock. Conclusion Depletion of circulating platelets negatively affects septic shock, while transfusion of washed platelets has no significant beneficial effect in mice.

Background In the panel of genes commonly associated with inherited macrothrombocytopenia, an important fraction encodes key cytoskeletal proteins such as tubulin isotypes, the building blocks of microtubules. Macrothrombocytopenia causing mutations have been identified in the TUBB1 and TUBA4A genes, emphasizing their importance in the formation of platelets and their marginal band, a unique microtubule ring-like structure that supports the platelet typical disc-shaped morphology. This raised the hypothesis that other tubulin isotypes normally expressed in platelets could play a similar role in their formation. Objectives To assess whether tubulin isotype genes other than TUBA4A and TUBB1 could be implicated in inherited macrothrombocytopenia. Methods We used high throughput sequencing to screen a cohort of 448 French blood donors with mild thrombocytopenia for mutations in a panel of selected genes known or suspected to be involved in platelet biogenesis. Results We identified six distinct novel mutations in TUBA8, which encodes the most-divergent α-tubulin, as the causative determinant of macrothrombocytopenia and platelet marginal band defects. Functionally, all TUBA8 mutations were found to fully or partially inhibit the incorporation of the mutated α8-tubulin in the microtubule network. Conclusion This study provides strong support for a key role of multiple tubulin genes in platelet biogenesis by discovering variants in a tubulin gene that was previously not known to be important for platelets.

Electron microscopy (EM) has a long history in megakaryocyte (MK) cellular biology. This chapter shows how the electron microscope, since its first appearance almost 90 years ago, has occupied center stage in the studies of MK morphology and function. It describes some of the more productive EM techniques that have shaped our understanding of the physiology of thrombopoiesis. These include the standard transmission and scanning EM techniques as well as the new imaging methods, correlative microscopy and volume EM which provide information on the 3D organization of MKs on different scales: single organelles, whole cells and tissues. For each technique, we list the advantages and limitations, the resolution that can be achieved, the technical difficulties and the applications in MK biology.

Laboratory tests of platelet function are instrumental in studying platelet physiology and inherited or acquired platelet abnormalities. Light transmission aggregometry, developed in the early 1960s, is still considered the gold standard for the identification and diagnosis of platelet function disorders. Since then, novel techniques have been developed, including flow‐based assays and flow cytometry. In this tutorial, we describe the basic methodologies for the preparation of citrated platelet‐rich plasma and washed platelet suspensions and discuss their respective advantages and limitations as well as important factors to consider to perform high‐quality tests of platelet function. In addition, the methodologies of the main platelet function tests (light transmission aggregation, flow‐based assays, and flow cytometric assays) are described, and their respective strengths and limitations are discussed to assess various aspects of platelet biology.

L’allo-immunisation plaquettaire post-transfusionnelle est une complication potentiellement serieuse de la transfusion. En effet les allo-anticorps produits, principalement diriges contre les antigenes HLA I, peuvent conduire a un etat refractaire (ER) qui compromet le succes de la transfusion de concentres plaquettaires. La rate orchestre la reponse alloimmune mais les cellules spleniques initiant ce processus ne sont pas precisement identifiees. Parmi les splenocytes, les cellules B de la zone marginale (MZB) sont connues pour reagir rapidement aux antigenes sanguins. L’objectif de cette etude est d’evaluer l’implication des MZB dans l’allo-immunisation plaquettaire et l’ER associe. Nous avons developpe un modele murin d’allo-immunisation qui repose sur une multi-transfusion de plaquettes d’haplotype H2b dans une souris H2d. Ce protocole mene a la production d’allo-anticorps antiplaquettaires (anti-H2b) mis en evidence par cytometrie en flux. La transfusion de plaquettes H2b dans les souris alloimmunisees conduit a leur elimination de la circulation en moins de 2 h, traduisant un ER. Dans ce modele, l’elimination des MZB par immunodepletion entraine une diminution de l’ordre de 50 % de la production d’allo-anticorps. La delocalisation des MZB suite a un traitement au FTY720, un antagoniste des S1P1, conduit egalement a une reduction significative de la production d’allo-anticorps. Lorsque les MZB sont absentes ou delocalisees, les plaquettes transfusees circulent encore 24 h apres la transfusion, indiquant un role majeur des MZB dans la prevention de l’ER. En conclusion, nos resultats montrent que les MZB favorisent la production d’allo-anticorps impliques dans les ER.

L’impact d’un traitement Intercept sur la capacite hemostatique plaquettaire reste debattu. L’etude par viscoelastometrie (VE) quantifie les proprietes mecaniques plaquettaires dans un caillot de sang total. Objectifs Etude fonctionnelle plaquettaire par VE en condition thrombocytopenique creee in vitro puis application a l’etude ex vivo de concentres plaquettaires (EFFIPAP : plaquettes traitees Intercept vs. non traitees ; transfusion pour thrombocytopenie d’une hemopathie maligne – Garban, 2018). Methodes Avec VE-ROTEM, la composante plaquettaire est obtenue par la difference EXTEM – FIBTEM des elasticites maximales (MCE) derivees du parametre MCF (Solomon, 2015). Du sang de 20 donneurs sains consentants est fractionne pour reconstitution autologue : culot erythrocytaire avec plasma et plaquettes lavees afin d’obtenir des numerations plaquettaires (NP) normales et basses, jusqu’a 20 G/L. Des prelevements sont effectues avant et apres transfusion : etude ancillaire EFFIPAP-bio (Dijon, Grenoble, Lille). Resultats (1) Avec la relation etablie entre MCE et NP un intervalle de reference est defini sur l’etendue des NP ( Fig. 1 ). L’incubation avec aspirine a un effet faible mais statistiquement significatif : l’approche est sensible a un defaut fonctionnel mineur sur l’etendue des NP. (2) Aucune difference des MCE entre les types de concentres n’est trouvee, en prenant en compte la NP ( Figure 1 , Figure 2 ). Les rendements transfusionnels sont en accord avec ceux de EFFIPAP. Conclusion L’etude fonctionnelle plaquettaire par VE (resultat exprime en MCE) est faisable en condition thrombocytopenique. Les resultats d’EFFIPAP-bio n’indiquent pas une alteration fonctionnelle due a Intercept.

BACKGROUND The production of platelet concentrates (PCs) is evolving, and their survival capacity needs in vivo evaluation. This requires that the transfused platelets (PLTs) be distinguished from those of the recipient. Labeling at various biotin (Bio) densities allows one to concurrently trace multiple PLT populations, as reported for red blood cells. STUDY DESIGN AND METHODS A method is described to label human PLTs at two densities of Bio for future clinical trials. Injectable-grade PLTs were prepared in a sterile environment, using injectable-grade buffers and good manufacturing practices (GMP)-grade Sulfo-NHS-Biotin. Sulfo-NHS-Biotin concentrations were chosen to maintain PLT integrity and avoid potential alloimmunization while enabling the detection of circulating BioPLTs. The impact of biotinylation on human PLT recirculation was evaluated in vivo in a severe immunodeficient mouse model using ex vivo flow cytometry. RESULTS BioPLTs labeled with 1.2 or 10 μg/ml Sulfo-NHS-Biotin displayed normal ultrastructure and retained aggregation and secretion capacity and normal expression of the main surface glycoproteins. The procedure avoided detrimental PLT activation or apoptosis signals. Transfused human BioPLT populations could be distinguished from one another and from unlabeled circulating mouse PLTs, and their survival was comparable to that of unlabeled human PLTs in the mouse model. CONCLUSIONS Provided low Sulfo-NHS-Biotin concentrations (

Background Deterioration in quality of platelet concentrates (PCs) during storage results from the appearance of storage lesions affecting the hemostatic functions and posttransfusion survival of platelets. These lesions depend on the preparation and pathogen inactivation methods used, duration of storage, and platelet additive solutions (PASs) present in storage bags. Methods We investigated the effects of citrate contained in third-generation PAS (PAS-III) on storage lesions in buffy-coat PCs with or without photochemical (amotosalen-ultraviolet A) treatment over 7 days. Results Platelet counts were conserved in all groups during storage, as was platelet swirling without appearance of macroscopic aggregates. Glycoprotein (GP) IIbIIIa and GPVI expression remained stable, whereas GPIbα declined similarly in all groups during storage. Removal of citrate from PAS-III, resulting in global reduction of citrate from 11 to 5 mM, led to a significant decrease in glucose consumption, which largely countered a modest deleterious effect of photochemical treatment. Citrate reduction also resulted in decreased lactate generation and better maintenance of pH during storage, while photochemical treatment had no impact on these parameters. Moreover, citrate-free storage significantly reduced exposure of P-selectin and the apoptosis signal phosphatidylserine, thereby abolishing the activating effect of photochemical treatment on both parameters. Citrate reduction benefited platelet aggregation to various agonists up to Day 7, whereas PCT had no impact on these responses. Conclusion Removal of citrate from PAS-III has a beneficial impact on platelet metabolism, spontaneous activation, and apoptosis, and improves platelet aggregation, irrespective of photochemical treatment, which should allow transfusion of platelets with better and longer-lasting functional properties.

During vertebrate, definitive hematopoietic stem cells (HSCs) are first generated in the aorta-gonad-mesonephros (AGM) region of the posterior embryo.1 We previously showed that the angiotensin-converting enzyme (ACE) is a cell-surface marker of human HSCs in both adult bone marrow (BM)2 and all hematopoietic niches developmentduring human ontogeny.3 In the embryo, ACE identifies HSCs associated with the ventral side of the aortic endothelium (in the AGM region) and of vitelline and umbilical arteries, both in humans3 and mice.4 ACE is a key component of the renin-angiotensin system (RAS), catalyzing the production of angiotensin II (AngII),5 which we have recently shown to be involved in hematopoietic emergence during ontogeny.4 ACE overexpression, leading to AngII increase, has also been reported in the BM of patients with acute myeloid leukemia (AML), influencing malignancy6,7 ; however, its mechanisms of regulation are still unknown.8 The caudal-related homeobox gene 2 (CDX2) encodes an important transcription factor involved in tissue expansion and patterning of the posterior embryo.9,10 At midgestation, it becomes limited to the presumptive mid-/hindgut endoderm and maintains selectively in the adult intestinal epithelium11 where it has tumor-suppressor activity.12-14 Conversely, although CDX2 is not expressed in adult HSCs, ectopic expression occurs in 80% of acute leukemia, which is considered a driver of leukemogenesis.15-17 Here, we investigated the ACE and CDX2 expression patterns during human and mouse hematopoietic development, as well as in AML, and we conducted functional assays supporting that CDX2 participates in ACE regulation.

Background\ud \ud Thrombocytopenia has a variety of different etiologies, both acquired and hereditary. Inherited thrombocytopenia may be associated with other symptoms (syndromic forms) or may be strictly isolated. To date, only about half of all the familial forms of thrombocytopenia have been accounted for in terms of well‐defined genetic abnormalities. However, data are limited on the nature and frequency of the underlying causative genetic variants in individuals with mild isolated nonsyndromic thrombocytopenia.\ud Study Design and Methods\ud \ud Thirteen known or candidate genes for isolated thrombocytopenia were included in a gene panel analysis in which targeted next‐generation sequencing was performed on 448 French blood donors with mild isolated nonsyndromic thrombocytopenia.\ud Results\ud \ud A total of 68 rare variants, including missense, splice site, frameshift, nonsense, and in‐frame variants (all heterozygous) were identified in 11 of the 13 genes screened. Twenty‐nine percent (N = 20) of the variants detected were absent from both the French Exome Project and gnomAD exome databases. Using stringent criteria and an unbiased approach, we classified seven predicted loss‐of‐function variants (three in ITGA2B and four in TUBB1) and four missense variants (one in GP1BA, two in ITGB3 and one in ACTN1) as being pathogenic or likely pathogenic. Altogether, they were found in 13 members (approx. 3%) of our studied cohort.\ud Conclusion\ud \ud We present the results of gene panel sequencing of known and candidate thrombocytopenia genes in mild isolated nonsyndromic thrombocytopenia. Pathogenic and likely pathogenic variants in five known thrombocytopenia genes were identified, accounting for approximately 3% of individuals with the condition.

BACKGROUND Among labile blood products, platelet concentrates (PCs) are the leading cause of hypersensitivity transfusion reactions (HTRs). These reactions often lead to interruption of PC transfusion and can result in a prolonged transfusion process leading to significant morbidity and use of premedication and close monitoring for patients with a history of allergic transfusion reactions. The French hemovigilance database is one of the largest standardized databases providing information on HTRs following administration of labile blood products. In this study, we analyzed this database to assess the relative risk of HTR for each type of PC. STUDY DESIGN AND METHODS HTRs following PC transfusion were retrospectively extracted from the e-Fit Hemovigilance database of the French National Agency for Medicines and Health Products Safety (ANSM). Frequencies were calculated using the number of specific PCs transfused. RESULTS Between 2008 and 2014, the overall estimated incidence of HTRs following PC administration was calculated at 232 HTRs per 100,000 PCs transfused. The rate of HTRs was significantly higher with apheresis PC (337/100,000) than with buffy-coat PC (94/100,000). Platelets in additive solutions (PAS) were associated with a significantly lower frequency of HTRs when compared with PCs in native plasma. Amotosalen/UVA- PCs (APCs and BCPCs) which are always in PAS in France, exhibited the lowest frequency of HTRs when compared with their corresponding PCs in native plasma or in PAS (p < 10-7 in all comparisons). CONCLUSION Our results showed that the type of PC and its processing may have an impact on the risk of HTR.

It took approximately 40 years from the seminal identification of adenosine diphosphate (ADP) as the factor R, an agent derived from red blood cells inducing platelet adhesion to glass, to the completion of the repertoire of its receptors on platelets and its importance in haemostasis and thrombosis. ADP, either derived from red blood cells or released by platelets themselves, stimulates platelets via two G protein-coupled receptors, P2Y1 and P2Y12. In addition, adenosine triphosphate, also contained in the platelet dense granules, activates the P2X1 cation channel. Each of these receptors plays a specific role during platelet activation and aggregation, with relevance to haemostasis, thrombosis and various inflammatory processes where platelets are involved including chronic responses such as atherosclerosis or acute responses such as sepsis, endotoxaemia or allergic asthma. Finally, platelets also express P2Y14, a receptor activated by released uridine diphosphate glucose. Although devoid of any known role in haemostasis, this receptor seems to play a specific role in neutrophil chemotaxis.

Resume Objectif Rechercher des facteurs expliquant les differences de consommation de concentres plaquettaires (CP) entre les regions Lorraine-Champagne (LOCH) et Alsace (ALSA). Materiel et methodes Il s’agit d’une enquete non interventionnelle, prospective, realisee pendant 30 jours en 2015 dans les services de reanimation, chirurgie cardiovasculaire et oncohematologie de 8 etablissements de LOCH et ALSA. Un questionnaire de renseignements clinico-biologiques etait adosse aux ordonnances de produits sanguins labiles (PSL) et rempli a chaque prescription de CP. Resultats Au total, 290 patients, a l’origine de 1093 prescriptions et 1093 delivrances de CP, ont ete analyses. Le contexte pretransfusionnel (poids des patients, transfusion preventive/curative, urgence vitale, recours a une numeration pretransfusionnelle) est homogene. Quatre-vingt-trois pour-cent des prescriptions sont exprimees en quantite de produit actif (QPA) en LOCH vs 22 % en ALSA. L’intervalle moyen entre 2 transfusions est de 2,9 jours en ALSA vs 4,9 jours en LOCH. La QPA moyenne/delivrance est plus elevee en ALSA (5,6.10 11 vs 4,0.10 11 en LOCH). Les QPA delivrees ont ete comparees aux recommandations AFSSaPS de 2003, en vigueur au moment de l’etude. Vingt-sept pour-cent des QPA delivrees sont dans l’intervalle recommande en LOCH, vs 49 % en ALSA. LOCH se demarque par 63 % de QPA, inferieures a 0,5.10 11 plaquettes/7 Kg liees a la delivrance systematique d’un seul CP, y compris pour les poids > 80 Kg. Vingt-deux pour-cent des delivrances en ALSA sont superieures a 0,7.10 11 plaquettes/7 Kg, majoritairement liees a la delivrance simultanee d’un deuxieme CP. Conclusion Cette etude identifie des disparites de pratiques de prescription et de delivrance entre LOCH et ALSA, qui peuvent expliquer les niveaux respectifs de cessions de CP.

Inherited platelet disorders are rare bleeding syndromes due to either platelet function abnormalities or thrombocytopenia which may be associated with functional defects. The haemorrhagic symptoms observed in these patients are mostly muco-cutaneous and of highly variable severity. Although 30 to 50% of the platelet disorders are still of unknown origin, the precise diagnosis of these pathologies by specialized laboratories together with haemorrhagic scores enables an assessment of the risk of bleeding in each patient. Depending on the diagnostic elements collected, an appropriate medical procedure can be proposed for each situation: scheduled or emergency surgical interventions and pregnancy follow-up. The pathologies most at risk correspond to Glanzmann's thrombasthenia, Bernard-Soulier syndrome, severe thrombocytopenia (

Resume Les modifications des recommandations francaises HAS/ANSM de 2015, concernant la dose transfusionnelle plaquettaire optimale pour les patients, nous ont amene a nous interroger sur la pertinence de nos pratiques actuelles de production et de delivrance a l’echelle de l’EFS Grand-Est. Dans un contexte de regroupement des regions transfusionnelles, de generalisation de l’inactivation des pathogenes dans les concentres plaquettaires et de necessite d’harmonisation, notre souci principal est de repondre aux besoins de nos patients, dans le respect de ces recommandations. S’ajoute a cette reflexion la volonte d’optimiser nos couts de production et de contribuer a diminuer le poids financier de la transfusion sur les comptes publics. Nous presentons ici les mesures mises en place dans la region et leur impact.

L’émergence de SARS-CoV-2 a entraîné un afflux massif de patients à l’hôpital et particulièrement en réanimation créant de nombreux problèmes. L’un d’entre eux est la transfusion, tant du côté de l’EFS (sécurité, collecte et stocks) que du transfuseur (soins habituels et demande spécifique inconnue liée au COVID-19). Un des risques était la pénurie avec nécessité de planifier des restrictions transfusionnelles. Nous avons analysé en parallèle l’activité transfusionnelle dans un CHU ainsi que la collecte et la délivrance au niveau de l’EFS entre le 24/02 et le 31/05/2020 et les avons comparées aux données de 2019. L’activité globale a baissé de 33 % sur la période (arrêt des soins réglés hors urgence ou soins ne pouvant être différés et admissions de 2291 patients COVID-19) alors que la transfusion n’a été réduite que de 17 %. Un total de 237 patients COVID-19 (10,3 %) ont nécessité une transfusion, dont 45 pour hémorragie. Parallèlement, la baisse des dons a été contenue à 11 % avec une discrète augmentation des stocks. La diminution de l’activité ne se traduit que par une baisse modérée de l’activité transfusionnelle, celle-ci dépendant principalement de la chirurgie urgente, des syndromes hémorragiques et de la prise en charge des patients en aplasie chimio-induite ou ayant des pathologies hématologiques. Le confinement a entraîné une diminution des dons par suppression des collectes mobiles mais avec un impact limité sur une courte période par mobilisation des donneurs réguliers. Il n’a pas été observé d’inadéquation entre demande et suppléance et il n’a donc pas été nécessaire de mettre en place des restrictions.

The fetal liver is the site of a major expansion of the hematopoietic stem cell (HSC) pool and is also a privileged organ to study megakaryocyte progenitor differentiation. We identified in the mouse fetal liver at day 13.5 a discrete stromal cell population harboring a CD45-TER119-CD31-CD51+VCAM-1+PDGFRα- (V+P-) phenotype that lacked colony-forming unit fibroblast activity and harbored an hepatocyte progenitor signature. This previously undescribed V+P- population efficiently supported megakaryocyte production from mouse bone marrow HSC and human peripheral blood HSC-myeloid progenitors cultured in the presence of limited cytokine concentrations. Megakaryocytes obtained in V+P- cocultures were polyploid, positive for CD41/CD42c, and efficiently produced proplatelets. Megakaryocyte production appeared to be mediated by an expansion of the progenitor compartment through HSC-stromal cell contact. In conclusion, the fetal liver contains a unique cellular microenvironment that could represent a platform for the discovery of regulators of megakaryopoiesis.

Les granules denses plaquettaires sont des organites contenant des molecules non peptidiques : calcium, serotonine, nucleotides et phosphates inorganiques. Lors de l’activation plaquettaire, ils fusionnent avec la membrane plasmique afin de secreter leur contenu dans le milieu exterieur. La maladie du pool vide δ-plaquettaire correspond a une anomalie du nombre et/ou du contenu en granules denses. Les etiologies de cette maladie sont diverses et variees. On distingue (1) les formes constitutionnelles, qui peuvent etre isolees ou s’inscrire dans le cadre de syndromes plus complexes (syndromes d’Hermansky–Pudlak, Chediak–Higashi, ou Griscelli) ; et (2) les formes acquises, qui sont le plus souvent associees a des hemopathies malignes (syndrome myelodysplasique, leucemie aigue, syndrome myeloproliferatif, ou autres). La prevalence de la maladie du pool vide δ-plaquettaire dans sa forme isolee est probablement sous-estimee, principalement parce que cette pathologie est peu connue et que son diagnostic biologique est relativement complexe. Les patients souffrant de deficit en granules denses ont generalement un phenotype hemorragique modere. Neanmoins, certains d’entre eux peuvent avoir des manifestations hemorragiques severes notamment en situation chirurgicale ou traumatique. C’est donc une thrombopathie qui doit etre diagnostiquee et prise en compte, en particulier lorsqu’il est necessaire de realiser des gestes a risque hemorragique. In vitro, les deficits en granules denses sont associes a une reduction de l’agregation dans les conditions ou celle-ci est particulierement sensible, c’est-a-dire aux faibles concentrations d’agonistes. Cependant, le profil d’agregation peut ne pas etre altere ou donner des resultats atypiques et dissocies. C’est pourquoi, la confirmation diagnostique necessite la realisation d’examens tres specialises afin de demontrer la diminution du nombre ou du contenu en granules denses. En l’etat actuel des connaissances, il est recommande, dans la mesure du possible, de realiser des dosages de serotonine, une quantification des nucleotides, ainsi qu’une etude des granules denses par microscopie electronique (whole mount). La maladie du pool vide δ-plaquettaire est donc une pathologie complexe presentant des aspects cliniques et biologiques tres divers, requerant une grande expertise afin de ne pas ignorer son diagnostic.

Blood flow profoundly varies throughout the vascular tree due to its pulsatile nature and to the complex vessel geometry. While thrombus formation has been extensively studied in vitro under constant flow, and in vivo under normal blood flow conditions, increased attention has been paid to the impact of complex hemodynamics such as flow acceleration found in stenosed arteries. We investigated the effect of flow acceleration, characterized by shear rate gradients, on the function of platelets adhering to fibrinogen, a plasma protein which plays a key role in hemostais and thrombosis. While we confirmed that under constant flow, fibrinogen only supports single platelet adhesion, we observed that under shear rate gradients, this surface becomes highly thrombogenic, supporting efficient platelet aggregation leading to occlusive thrombus formation. Interestingly, this phenomenon is general as it occurs on other weak adhesive matrices including laminins and thrombospondin-1. This shear rate gradient-driven thrombosis is biphasic with an initial step of slow platelet recruitment supported by direct plasma von Willebrand factor (VWF) adsorption to immobilized fibrinogen and followed by a second phase of explosive thrombosis initiated by VWF fiber formation on platelet monolayers. In vivo experiments confirmed that shear rate gradients accelerate thrombosis in a VWF-dependent manner. Together, this study characterizes a process of plasma VWF-dependent accelerated thrombosis on weak adhesive proteins such as, fibrinogen in the presence of shear rate gradients.

International audience; Contrary to rat platelets, mouse platelets express both RAB32 and RAB38 that play fully redundant roles for dense granule biogenesis. • Combined RAB32 and RAB38 deficiency mimics severe Hermansky-Pudlak syndrome with albinism and profound defects in hemostasis. The biogenesis of lysosome related organelles is defective in Hermansky-Pudlak syndrome (HPS), a disorder characterized by oculocutaneous albinism and platelet dense granule (DG) defects. The first animal model of HPS was the fawn-hooded rat, harboring a spontaneous mutation inactivating the small guanosine triphosphatase Rab38. This leads to coat color dilution associated with the absence of DGs and lung morphological defects. Another RAB38 mutant, the cht mouse, has normal DGs, which has raised controversy about the role of RAB38 in DG biogenesis. We show here that murine and human, but not rat, platelets also express the closely related RAB32. To elucidate the parts played by RAB32 and RAB38 in the biogenesis of DGs in vivo and their effects on platelet functions, we generated mice inactivated for Rab32, Rab38, and both genes. Single Rab38 inactivation mimicked cht mice, whereas single Rab32 inactivation had no effect in DGs, coat color, or lung morphology. By contrast, Rab32/38 double inactivation mimicked severe HPS, with strong coat and eye pigment dilution, some enlarged lung multilamellar bodies associated with a decrease in the number of DGs. These organelles were morphologically abnormal, decreased in number, and devoid of 5-hydroxytryptamine content. In line with the storage pool defect, platelet activation was affected, resulting in severely impaired thrombus growth and prolongation of the bleeding time. Overall, our study demonstrates the absence of impact of RAB38 or RAB32 single deficiency in platelet biogenesis and function resulting from full redundancy, and characterized a new mouse model mimicking HPS devoid of DG content.

Background The interplay between platelets and fibrinogen is the cornerstone of thrombus formation. Integrin αIIbβ3 is the main platelet adhesion receptor for fibrinogen and mediates an outside‐in signal upon ligand binding that reinforces platelet activation. In addition, FcγRIIA and glycoprotein VI (GPVI) contribute to platelet activation on fibrinogen, thereby participating in thrombus growth and stability. To date, the relative importance of these two immunoreceptor tyrosine‐based activation motif‐bearing receptors in these processes remains unknown. Objective The aim of this study was to evaluate the relative contributions of FcγRIIA and GPVI to platelet activation on fibrinogen and subsequent thrombus growth and stability. Methods We evaluated human and mouse platelet adhesion to fibrinogen in static assays and a flow‐based approach to evaluate the contribution of FcγRIIA and GPVI to thrombus growth and stability. Results We first confirmed that integrin αIIbβ3 is the key receptor supporting platelet adhesion and spreading on fibrinogen. Using human platelets treated with pharmacological blocking agents and transgenic mouse platelets expressing human receptors, data indicate that GPVI, but not FcγRIIA, plays a prominent role in platelet activation on fibrinogen. Moreover, using a flow‐based assay, we observed that blockade of GPVI with 1G5, but not FcγRIIA with IV.3, prevents thrombus growth. Finally, we observed that 1G5, but not IV.3, promotes the disaggregation of thrombi formed on collagen in vitro. Conclusion This study provides evidence that GPVI, but not FcγRIIA, induces platelet activation and spreading on fibrinogen, and promotes thrombus buildup and stability.

Although granule secretion is pivotal in many platelet responses, the fusion routes of α and δ granule release remain uncertain. We used a 3D reconstruction approach based on electron microscopy to visualize the spatial organization of granules in unstimulated and activated platelets. Two modes of exocytosis were identified: a single mode that leads to release of the contents of individual granules and a compound mode that leads to the formation of granule-to-granule fusion, resulting in the formation of large multigranular compartments. Both modes occur during the course of platelet secretion. Single fusion events are more visible at lower levels of stimulation and early time points, whereas large multigranular compartments are present at higher levels of agonist and at later time points. Although α granules released their contents through both modes of exocytosis, δ granules underwent only single exocytosis. To define the underlying molecular mechanisms, we examined platelets from vesicle-associated membrane protein 8 (VAMP8) null mice. After weak stimulation, compound exocytosis was abolished and single exocytosis decreased in VAMP8 null platelets. Higher concentrations of thrombin bypassed the VAMP8 requirement, indicating that this isoform is a key but not a required factor for single and/or compound exocytosis. Concerning the biological relevance of our findings, compound exocytosis was observed in thrombi formed after severe laser injury of the vessel wall with thrombin generation. After superficial injury without thrombin generation, no multigranular compartments were detected. Our studies suggest that platelets use both modes of membrane fusion to control the extent of agonist-induced exocytosis.

Megakaryocyte (MK) differentiation occurs within the bone marrow (BM), a complex 3-dimensional (3D) environment of low stiffness exerting local external constraints. To evaluate the influence of the 3D mechanical constraints that MKs may encounter in vivo, we differentiated mouse BM progenitors in methylcellulose (MC) hydrogels tuned to mimic BM stiffness. We found that MKs grown in a medium of 30- to 60-Pa stiffness more closely resembled those in the BM in terms of demarcation membrane system (DMS) morphological aspect and exhibited higher ploidy levels, as compared with MKs in liquid culture. Following resuspension in a liquid medium, MC-grown MKs displayed twice as much proplatelet formation as cells grown in liquid culture. Thus, the MC gel, by mimicking external constraints, appeared to positively influence MK differentiation. To determine whether MKs adapt to extracellular stiffness through mechanotransduction involving actomyosin-based modulation of the intracellular tension, myosin-deficient (Myh9-/-) progenitors were grown in MC gels. Absence of myosin resulted in abnormal cell deformation and strongly decreased proplatelet formation, similarly to features observed for Myh9-/- MKs differentiated in situ but not in vitro. Moreover, megakaryoblastic leukemia 1 (MKL1), a well-known actor in mechanotransduction, was found to be preferentially relocated within the nucleus of MC-differentiated MKs, whereas its inhibition prevented MC-mediated increased proplatelet formation. Altogether, these data show that a 3D medium mimicking BM stiffness contributes, through the myosin IIA and MKL1 pathways, to a more favorable in vitro environment for MK differentiation, which ultimately translates into increased proplatelet production.

UNLABELLED Essentials Information about the formation of the demarcation membrane system (DMS) is still lacking. We investigated the role of the cytoskeleton in DMS structuration in megakaryocytes. Cdc42/Pak-dependent F-actin remodeling regulates DMS organization for proper megakaryopoiesis. These data highlight the mandatory role of F-actin in platelet biogenesis. SUMMARY Background Blood platelet biogenesis results from the maturation of megakaryocytes (MKs), which involves the development of a complex demarcation membrane system (DMS). Therefore, MK differentiation is an attractive model for studying membrane remodeling. Objectives We sought to investigate the mechanism of DMS structuration in relationship to the cytoskeleton. Results Using three-dimensional (3D) confocal imaging, we have identified consecutive stages of DMS organization that rely on F-actin dynamics to polarize membranes and nuclei territories. Interestingly, microtubules are not involved in this process. We found that the mechanism underlying F-actin-dependent DMS formation required the activation of the guanosine triphosphate hydrolase Cdc42 and its p21-activated kinase effectors (Pak1/2/3). Forster resonance energy transfer demonstrated that active Cdc42 was associated with endomembrane dynamics throughout terminal maturation. Inhibition of Cdc42 or Pak1/2/3 severely destructured the DMS and blocked proplatelet formation. Even though this process does not require containment within the hematopoietic niche, because DMS structuration was observed upon thrombopoietin-treatment in suspension, integrin outside-in signaling was required for Pak activation and probably resulted from secretion of extracellular matrix by MKs. Conclusions These data indicate a functional link, mandatory for MK differentiation, between actin dynamics, regulated by Cdc42/Pak1/2/3, and DMS maturation.

Objective Vascular plug formation by mechanical injury that exposes abundant extracellular matrix is an ideal model to mimic thrombus formation. The objective of this study was to standardize our previously established in vivo mouse model of thrombus formation induced by mechanical injury. Results The mechanical injury was exerted by pinching the abdominal aorta with hemostatic forceps for either 15 (moderate injury) or 60 (severe injury) seconds. Thrombus formation was monitored for 20 min in real time using a fluorescent microscope coupled to a CCD camera. In the moderate injury, thrombus formation peaked at approximately 1 min after injury and resolved within 3 min, with the mean AUC (area under the curve) of 165.2 ± 17.29 mm 2 , whereas a larger thrombus was observed upon the severe injury, with the mean AUC of 600.5 ± 37.77 mm 2 . Using scanning electron microscopy and HE staining, a complete deformation of the endothelium in the moderate injury model and the exposure of the media in the severe injury model were observed. The model was also evaluate for its application on the effects of antithrombotic drugs targeting GP IIb–IIIa (eptifibatide), ADP receptor P2Y 1 (MRS2500) and P2Y 12 (clopidogrel), and thrombin (hirudin) on thrombus formation. Conclusions We have improved a vascular injury model with optimal reproducibility and feasibility that allows evaluating the effect of anti-thrombotic drugs on thrombus formation in vivo.

Background Accurate identification of the proportion of young platelets is important to distinguish peripheral thrombocytopenia from a deficit in platelet production. Young platelets are defined by their higher RNA content and are often assessed as thiazole orange bright (TObright ) by flow cytometry. In clinical practice, their proportion is estimated by automatic blood counter according to their greater RNA content, which identifies a so-called immature platelet fraction (IPF). However, the detected IPFs are not strictly identical to the young TObright platelet population observed by flow cytometry. Objectives The aim of this study was to assess the reliability of HLA I/major histocompatibility I (MHC I) cell surface expression as a marker of young platelets. Methods The HLA I/MHC I expression was evaluated by flow cytometry after costaining blood with TO and antibodies directed against HLA I/MHC I molecules. Results We found that platelets with a higher expression of plasma membrane-localized MHC I molecules displayed an increased TO staining and a higher content in ribosomal P-antigen. Transfusion experiments in mice showed that the number of MHC I molecules expressed on the cell surface of young murine platelets decreased during platelet aging, reaching basal levels within 24 h. Finally, we demonstrated that for patients with thrombocytopenias, the identification of young platelets is better assessed by the flow cytometric determination of the level of HLA I expression than by TO staining or the use of hematological blood counter. Conclusion Overall, our results highlight the relevance of MHC I/HLA I expression as a valuable parameter to identify young platelets.

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.

SummaryThe platelet adenosine 5’-diphosphate (ADP) receptor P2Y12 (P2Y12R) plays a critical role in platelet aggregation. The present report illustrates an update of dysfunctional platelet P2Y12R mutations diagnosed with congenital lifelong bleeding problems. Described patients with heterozygous or homozygous substitution in the P2Y12R gene and qualitative abnormalities of the platelet P2Y12R are summarized. Recently, a further dysfunctional variant of P2Y12R has been identified in two brothers who presented with a lifelong severe bleeding disorder. During in vitro aggregation studies, the patient´s platelets show a markedly reduced and rapid reversible ADP-promoted aggregation. A homozygous c.561T>A substitution that changes the codon for His187 to Gln (p.His187Gln) in the P2Y12R gene has been identified. This mutation causes no change in receptor expression but decreases the affinity of the ligand for the receptor, even at high concentrations. Structure modelling studies indicated that the p.His187Gln mutation, located in the fifth transmembrane spanning domain (TM5), impairs conformational changes of the receptor. Structural integrity of the TM5 region is necessary for agonist and antagonist binding and for correct receptor function.