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Abstract Zebrafish is widely adopted as a grafting model for studying human development and diseases. Current zebrafish xenotransplantations are performed using embryo recipients, as the adaptive immune system, responsible for host versus graft rejection, only reaches maturity at juvenile stage. However, transplanted primary human hematopoietic stem/progenitor cells (HSC) rapidly disappear even in zebrafish embryos, suggesting that another barrier to transplantation exists before the onset of adaptive immunity. Here, using a labelled macrophage zebrafish line, we demonstrated that engraftment of human HSC induces a massive recruitment of macrophages which rapidly phagocyte transplanted cells. Macrophages depletion, by chemical or pharmacological treatments, significantly improved the uptake and survival of transplanted cells, demonstrating the crucial implication of these innate immune cells for the successful engraftment of human cells in zebrafish. Beyond identifying the reasons for human hematopoietic cell engraftment failure, this work images the fate of human cells in real time over several days in macrophage-depleted zebrafish embryos.

Platelet homeostasis is dependent on a tight regulation of both platelet production and clearance. The small GTPase Rap1 mediates platelet adhesion and hemostatic plug formation. However, Rap1 signaling is also critical for platelet homeostasis as both Rap1 deficiency and uninhibited Rap1 signaling lead to marked thrombocytopenia in mice. Here, we investigated the mechanism by which deficiency in Rasa3, a critical negative regulator of Rap1, causes macrothrombocytopenia in mice. Despite marked morphological and ultrastructural abnormalities, megakaryocytes in hypomorphic Rasa3hlb/hlb (R3hlb/hlb) or Rasa3–/– mice demonstrated robust proplatelet formation in vivo, suggesting that defective thrombopoiesis is not the main cause of thrombocytopenia. Rather, we observed that R3hlb/hlb platelets became trapped in the spleen marginal zone/red pulp interface, with evidence of platelet phagocytosis by macrophages. Clearance of mutant platelets was also observed in the liver, especially in splenectomized mice. Platelet count and platelet life span in Rasa3-mutant mice were restored by genetic or pharmacological approaches to inhibit the Rap1/talin1/αIIbβ3 integrin axis. A similar pattern of splenic clearance was observed in mice injected with anti-αIIbβ3 but not anti–glycoprotein Ibα platelet-depleting antibodies. In summary, we describe a potentially novel, integrin-based mechanism of platelet clearance that could be critical for our understanding of select inherited and acquired thrombocytopenias.

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.

Abstract 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.

Introduction Platelets are increasingly appreciated as key effectors during sepsis, raising the question of the usefulness of antiplatelet drugs to treat patients with sepsis. Objective Evaluate the potential contribution of the platelet P2Y12 receptor in the pathogenesis of polymicrobial-induced sepsis and septic shock in mice. Methods The effects of P2Y12 inhibition using clopidogrel treatment and of platelet-specific deletion of the P2Y12 receptor in mice were examined in two severity grades of cecal ligation and puncture (CLP) leading to mild sepsis or septic shock. Results Twenty hours after induction of the high grade CLP, clopidogrel- and vehicle-treated mice displayed a similar 30% decrease in mean arterial blood pressure (MAP) characteristic of shock. Septic shock-induced thrombocytopenia was not modified by clopidogrel treatment. Plasma concentrations of inflammatory cytokines and myeloperoxidase (MPO) were similarly increased in clopidogrel- and vehicle-treated mice, indicating comparable increase in systemic inflammation. Thrombin-antithrombin (TAT) complexes and the extent of organ damage were also similar. In mild-grade CLP, clopidogrel- and vehicle-treated mice did not display a significant decrease in MAP, while thrombocytopenia and plasma concentrations of TNFα, IL6, IL10, MPO, TAT and organ damage reached similar levels in both groups, although lower than those reached in the high grade CLP. Similarly, mice with platelet-specific deletion of the P2Y12 receptor were not protected from CLP-induced sepsis or septic shock. Conclusion The platelet P2Y12 receptor does not contribute to the pathogenesis of sepsis or septic shock in mice, suggesting that P2Y12 receptor antagonists may not be beneficial in patients with sepsis or septic shock.

Abstract 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.

Abstract: 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.

Abstract Background Podocalyxin (PODXL) is a highly sialylated adhesion glycoprotein that plays an important role in podocyte's physiology. Recently, missense and nonsense dominant variants in the PODXL gene have been associated with focal segmental glomerulosclerosis (FSGS), a leading cause of nephrotic syndrome and kidney failure. Their histologic description, however, was superficial or absent. Methods We performed exome sequencing on a three‐generation family affected by an atypical glomerular nephropathy and characterized the disease by light and electron microscopy. Results The disease was characterized by FSGS features and glomerular basement membrane duplication. Six family members displayed chronic proteinuria, ranging from mild manifestations without renal failure, to severe forms with end‐stage renal disease. Exome sequencing of affected twin sisters, their affected mother, healthy father, and healthy maternal uncle revealed a new nonsense variant cosegregating with the disease (c.1453C>T, NM_001018111) in the PODXL gene, which is known to be expressed in the kidney and to cause nephropathy when mutated. The variant is predicted to lead to a premature stop codon (p.Q485*) that results in the loss of the intracytoplasmic tail of the protein. Conclusion This is the first description of a peculiar association combining a PODXL stop‐gain variant and both FSGS and membranoproliferative glomerulonephritis features, described by light and electron microscopy.

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.

Abstract: 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.

Outcome of patients with coronary artery disease has been significantly improved by percutaneous coronary interventions with stent implantation. However, despite progress made on devices and antithrombotic treatments, stent thrombosis remains an important issue because of serious adverse consequences. Several mechanisms are assumed to favor stent thrombosis as platelet aggregation, fibrin formation, defective healing and local inflammation. The objective of this study was to evaluate in vitro the thrombogenicity, proinflammatory properties and healing capacities of cobalt–chromium (CoCr), an alloy commonly used for cardiovascular implants. Platelet adhesion was quantified in static and flow conditions. Thrombin generation was performed using the calibrated automated thrombogram. Neutrophil adhesion and formation of extracellular traps were visualized by scanning electron microscopy and by immunofluorescence. The phenotype of endothelial cells grown on CoCr was analyzed using specific antibodies, whereas the procoagulant potential was analyzed by measuring thrombin generation and protein C activation. Our results show that human blood platelets adhere to and are activated on CoCr in static and flow conditions. Overall, CoCr significantly induced thrombin generation in the presence or absence of platelets by 1.5- and 4.8-fold, respectively, involving activation of the contact pathway and activation of platelets. CoCr triggered leukocyte adhesion and behaved as a scaffold for the formation of neutrophil extracellular traps in the presence of platelets. Endothelial cells adhered and formed a monolayer covering CoCr. However, they switched from an anticoagulant phenotype to a procoagulant one with a significant 2.2-fold increase in thrombin generation due to a combined 30% reduced capacity to trigger protein C activation and 30% increased expression of tissue factor. Moreover, endothelial cells grown on CoCr acquired an inflammatory phenotype as indicated by the increased expression of ICAM-1 and VCAM-1. These data show that bare CoCr is prothrombotic and proinflammatory due to its capacity to activate platelets and coagulation and to induce leukocyte adhesion and activation. More importantly, even if endothelialization is achievable, the switch in endothelial phenotype prevents effective healing. Furthermore, we propose our methodology for future preclinical in vitro evaluation of the thrombogenicity of stent materials.

Microglia and astrocytes become reactive following traumatic brain injury (TBI). However, the coordination of this reactivity and its relation to pathophysiology are unclear. Here, we show that microglia transform astrocytes into a neuroprotective phenotype via downregulation of the P2Y1 purinergic receptor. TBI initially caused microglial activation in the injury core, followed by reactive astrogliosis in the peri-injured region and formation of a neuroprotective astrocyte scar. Equivalent changes to astrocytes were observed in vitro after injury. This change in astrocyte phenotype resulted from P2Y1 receptor downregulation, mediated by microglia-derived cytokines. In mice, astrocyte-specific P2Y1 receptor overexpression (Astro-P2Y1OE) counteracted scar formation, while astrocyte-specific P2Y1 receptor knockdown (Astro-P2Y1KD) facilitated scar formation, suggesting critical roles of P2Y1 receptors in the transformation. Astro-P2Y1OE and Astro-P2Y1KD mice showed increased and reduced neuronal damage, respectively. Altogether, our findings indicate that microglia-astrocyte interaction, involving a purinergic signal, is essential for the formation of neuroprotective astrocytes.

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 steady flow, and in vivo under normal blood flow conditions, the impact of complex hemodynamics such as flow acceleration found in stenosed arteries has gained increased appreciation. 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 steady 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. This shear rate gradient-driven thrombosis is biphasic with an initial step of slow platelet recruitment supported by direct plasma 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 immobilized fibrinogen in the presence of shear rate gradients.

In vitro, the differentiation of megakaryocytes (MKs) is improved by aryl-hydrocarbon receptor (AHR) antagonists such as StemRegenin 1 (SR1), an effect physiologically recapitulated by the presence of stromal mesenchymal cells (MSC). This inhibition promotes the amplification of a CD34+CD41low population able to mature as MKs with a high capacity for platelet production. In this short report, we showed that the emergence of the thrombocytogenic precursors and the enhancement of platelet production triggered by SR1 involved IKAROS. The downregulation/inhibition of IKAROS (shRNA or lenalidomide) significantly reduced the emergence of SR1-induced thrombocytogenic population, suggesting a crosstalk between AHR and IKAROS. Interestingly, using a proximity ligation assay, we could demonstrate a physical interaction between AHR and IKAROS. This interaction was also observed in the megakaryocytic cells differentiated in the presence of MSCs. In conclusion, our study revealed a previously unknown AHR/ IKAROS -dependent pathway which prompted the expansion of the thrombocytogenic precursors. This AHR- IKAROS dependent checkpoint controlling MK maturation opens new perspectives to platelet production engineering.

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 VI, a major platelet activation receptor for collagen and fibrin, is considered a particularly promising, safe antithrombotic target. In this study, we show that human glycoprotein VI signals upon platelet adhesion to fibrinogen. Full spreading of human platelets on fibrinogen was abolished in platelets from glycoprotein VI- deficient patients suggesting that fibrinogen activates platelets through glycoprotein VI. While mouse platelets failed to spread on fibrinogen, human-glycoprotein VI-transgenic mouse platelets showed full spreading and increased Ca2+ signaling through the tyrosine kinase Syk. Direct binding of fibrinogen to human glycoprotein VI was shown by surface plasmon resonance and by increased adhesion to fibrinogen of human glycoprotein VI-transfected RBL-2H3 cells relative to mock-transfected cells. Blockade of human glycoprotein VI with the Fab of the monoclonal antibody 9O12 impaired platelet aggregation on preformed platelet aggregates in flowing blood independent of collagen and fibrin exposure. These results demonstrate that human glycoprotein VI binds to immobilized fibrinogen and show that this contributes to platelet spreading and platelet aggregation under flow.

Congenital macrothrombocytopenia is a family of rare diseases, of which a significant fraction remains to be genetically characterized. To analyze cases of unexplained thrombocytopenia, 27 individuals from a patient cohort of the Bleeding and Thrombosis Exploration Center of the University Hospital of Marseille were recruited for a high-throughput gene sequencing study. This strategy led to the identification of two novel FLI1 variants (c.1010G>A and c.1033A>G) responsible for macrothrombocytopenia. The FLI1 variant carriers’ platelets exhibited a defect in aggregation induced by low-dose adenosine diphosphate (ADP), collagen and thrombin receptor-activating peptide (TRAP), a defect in adenosine triphosphate (ATP) secretion, a reduced mepacrine uptake and release and a reduced CD63 expression upon TRAP stimulation. Precise ultrastructural analysis of platelet content was performed using transmission electron microscopy and focused ion beam scanning electron microscopy. Remarkably, dense granules were nearly absent in the carriers’ platelets, presumably due to a biogenesis defect. Additionally, 25–29% of the platelets displayed giant α-granules, while a smaller proportion displayed vacuoles (7–9%) and autophagosome-like structures (0–3%). In vitro study of megakaryocytes derived from circulating CD34+ cells of the carriers revealed a maturation defect and reduced proplatelet formation potential. The study of the FLI1 variants revealed a significant reduction in protein nuclear accumulation and transcriptional activity properties. Intraplatelet flow cytometry efficiently detected the biomarker MYH10 in FLI1 variant carriers. Overall, this study provides new insights into the phenotype, pathophysiology and diagnosis of FLI1 variant-associated thrombocytopenia.

Gene 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 bone marrow and in vitro differentiated megakaryocytes. We then 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 bone marrow 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 colony-forming unit megakaryocytes 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 megakaryocyte progenitors and bone marrow megakaryocytes, and the platelet generation, but increased platelet clearance with a consequent reduction in blood cell 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.

Constitutional thrombocytopenias result from platelet production abnormalities of hereditary origin. Long misdiagnosed and poorly studied, knowledge about these rare diseases has increased considerably over the last twenty years due to improved technology for the identification of mutations, as well as an improvement in obtaining megakaryocyte culture from patient hematopoietic stem cells. Simultaneously, the manipulation of mouse genes (transgenesis, total or conditional inactivation, introduction of point mutations, random chemical mutagenesis) have helped to generate disease models that have contributed greatly to deciphering patient clinical and laboratory features. Most of the thrombocytopenias for which the mutated genes have been identified now have a murine model counterpart. This review focuses on the contribution that these mouse models have brought to the understanding of hereditary thrombocytopenias with respect to what was known in humans. Animal models have either i) provided novel information on the molecular and cellular pathways that were missing from the patient studies; ii) improved our understanding of the mechanisms of thrombocytopoiesis; iii) been instrumental in structure-function studies of the mutated gene products; and iv) been an invaluable tool as preclinical models to test new drugs or develop gene therapies. At present, the genetic determinants of thrombocytopenia remain unknown in almost half of all cases. Currently available high-speed sequencing techniques will identify new candidate genes, which will in turn allow the generation of murine models to confirm and further study the abnormal phenotype. In a complementary manner, programs of random mutagenesis in mice should also identify new candidate genes involved in thrombocytopenia.

Previous investigations have indicated that RNAs are mostly present in the minor population of the youngest platelets, whereas translation in platelets could be biologically important. To attempt to solve this paradox, we studied changes in the RNA content of reticulated platelets, i.e., young cells brightly stained by thiazole orange (TObright), a fluorescent probe for RNAs. We provoked in mice strong thrombocytopenia followed by dramatic thrombocytosis characterized by a short period with a vast majority of reticulated platelets. During thrombocytosis, the TObright platelet count rapidly reached a maximum, after which TOdim platelets accumulated, suggesting that most of the former were converted into the latter within 12 h. Experiments on platelets, freshly isolated or incubated ex vivo at 37°C, indicated that their "RNA content", here corresponding to the amounts of extracted RNA, and the percentage of TObright platelets were positively correlated. The "RNA Content" normalized to the number of platelets could be 20 to 40 fold higher when 80-90% of the cells were reticulated (20-40 fg/platelet), than when only 5-10% of control cells were TObright (less than 1fg/platelet). TObright platelets, incubated ex vivo at 37°C or transfused into mice, became TOdim within 24 h. Ex vivo at 37°C, platelets lost about half of their ribosomal and beta actin RNAs within 6 hours, and more than 98% of them after 24 hours. Accordingly, fluorescence in situ hybridization techniques confirmed the presence of beta actin mRNAs in most reticulated-enriched platelets, but detected them in only a minor subset of control platelets. In vitro, constitutive translation decreased considerably within less than 6 hours, questioning how protein synthesis in platelets, especially in non-reticulated ones, could have a biological function in vivo. Nevertheless, constitutive transient translation in young platelets under pathological conditions characterized by a dramatic increase in circulating reticulated platelets could deserve to be investigated.

Growing evidence supports a link between fatty acid metabolism and amyotrophic lateral sclerosis (ALS). Here we determined the fatty acid composition of blood lipids to identify markers of disease progression and survival. We enrolled 117 patients from two clinical centers and 48 of these were age and gender matched with healthy volunteers. We extracted total lipids from serum and blood cells, and separated fatty acid methyl esters by gas chromatography. We measured circulating biochemical parameters indicative of the metabolic status. Association between fatty acid composition and clinical readouts was studied, including ALS functional rating scale-revised (ALSFRS-R), survival, disease duration, site of onset and body mass index. Palmitoleate (16:1) and oleate (18:1) levels, and stearoyl-CoA desaturase indices (16:1/16:0 and 18:1/18:0) significantly increased in blood cells from ALS patients compared to healthy controls. Palmitoleate levels and 16:1/16:0 ratio in blood cells, but not body mass index or leptin concentrations, negatively correlated with ALSFRS-R decline over a six-month period (p

A sensitive procedure is described for the simultaneous determination of vitamin E and coenzyme Q homologues and α-tocopherol oxidation products using two-isocratic step high pressure liquid chromatography (HPLC) and electrochemical detection in the oxidative mode. Zinc-catalyzed reduction in a post-column reactor allows the detection of α-tocopherolquinone, epoxy-tocopherolquinone, and ubiquinones. This technique was used to quantify lipophilic antioxidants in the liver tissue of rats treated or not with α-tocopherolquinone and in a plant oil. α-Tocopherolquinone and its epoxide derivatives, formed from α-tocopherol during iron-catalyzed phospholipid peroxidation, were also determined in a liposome suspension. The high selectivity and sensitivity of the coulometric detection system enabled use of low oxidation potentials giving little baseline noise, while a fast isolation procedure and quantitative recoveries of all oxidized and reduced forms made it possible to measure a high ubiquinol/ubiquinone ratio in liver tissue. Administration of α-tocopherolquinone to rats did not alter the antioxidant status of the liver, despite strong accumulation of both this quinone and its reduced form, α-tocopherolhydroquinone. These results indicate the presence of an efficient reductase and suggest that it could contribute to the protection of cellular membranes from oxidative stress.—Leray, C., M. D. Andriamampandry, M. Freund, C. Gachet, and J-P. Cazenave. Simultaneous determination of homologues of vitamin E and coenzyme Q and products of α-tocopherol oxidation. J. Lipid Res. 1998. 39: 2099–2105.

Methylmercury (MeHg) is a well known environmental pollutant that induces serious neuronal damage. Although MeHg readily crosses the blood-brain barrier, and should affect both neurons and glial cells, how it affects glia or neuron-to-glia interactions has received only limited attention. Here, we report that MeHg triggers ATP/P2Y1 receptor signals in astrocytes, thereby protecting neurons against MeHg via interleukin-6 (IL-6)-mediated pathways. MeHg increased several mRNAs in astrocytes, among which IL-6 was the highest. For this, ATP/P2Y1 receptor-mediated mechanisms were required because the IL-6 production was (i) inhibited by a P2Y1 receptor antagonist, MRS2179, (ii) abolished in astrocytes obtained from P2Y1 receptor-knockout mice, and (iii) mimicked by exogenously applied ATP. In addition, (iv) MeHg released ATP by exocytosis from astrocytes. As for the intracellular mechanisms responsible for IL-6 production, p38 MAP kinase was involved. MeHg-treated astrocyte-conditioned medium (ACM) showed neuro-protective effects against MeHg, which was blocked by anti-IL-6 antibody and was mimicked by the application of recombinant IL-6. As for the mechanism of neuro-protection by IL-6, an adenosine A1 receptor-mediated pathway in neurons seems to be involved. Taken together, when astrocytes sense MeHg, they release ATP that autostimulates P2Y1 receptors to upregulate IL-6, thereby leading to A1 receptor-mediated neuro-protection against MeHg.

The CD1e protein participates in the presentation of lipid antigens in dendritic cells. Its transmembrane precursor is transported to lysosomes where it is cleaved into an active soluble form. In the presence of bafilomycin, which inhibits vacuolar ATPase and consequently the acidification of endosomal compartments, CD1e associates with a 27 kD protein. In this work, we identified this molecular partner as LAPTM5. The latter protein and CD1e colocalize in trans-Golgi and late endosomal compartments. The quantity of LAPTM5/CD1e complexes increases when the cells are treated with bafilomycin, probably due to the protection of LAPTM5 from lysosomal proteases. Moreover, we could demonstrate that LAPTM5/CD1e association occurs under physiological conditions. Although LAPTM5 was previously shown to act as a platform recruiting ubiquitin ligases and facilitating the transport of receptors to lysosomes, we found no evidence that LATPM5 controls either CD1e ubiquitination or the generation of soluble lysosomal CD1e proteins. Notwithstanding these last observations, the interaction of LAPTM5 with CD1e and their colocalization in antigen processing compartments both suggest that LAPTM5 might influence the role of CD1e in the presentation of lipid antigens.

Background Giant platelets and thrombocytopenia are invariable defects in the Bernard-Soulier syndrome caused by deficiency of the GPIb-V-IX complex, a receptor for von Willebrand factor supporting platelet adhesion to the damaged arterial wall. Various properties of this receptor may be considered potential determinants of the macrothrombocytopenia.Design and Methods To explore the underlying mechanisms of the disease, megakaryopoiesis was studied in a mouse model deficient in GPIbβ. Megakaryocytes were initially characterized in situ in the bone marrow of adult mice, after which their capacity to differentiate into proplatelet-bearing cells was evaluated in cultured fetal liver cells.Results The number of megakaryocyte progenitors, their differentiation and progressive maturation into distinct classes and their level of endoreplication were normal in GPIbβ−/− bone marrow. However, the more mature cells exhibited ultrastructural anomalies with a thicker peripheral zone and a less well developed demarcation membrane system. GPIbβ−/− megakaryocytes could be differentiated in culture from Lin− fetal liver cells in normal amounts but the proportion of cells able to extend proplatelets was decreased by 41%. Moreover, the GPIbβ−/− cells extending proplatelets displayed an abnormal morphology characterized by fewer pseudopodial extensions with thicker shaft sections and an increased diameter of the terminal coiled elements. GPIbβ−/− released platelets were larger but retained a typical discoid shape. Proplatelet formation was similarly affected in bone marrow explants from adult mice examined by videomicroscopy. The marginal microtubular ring contained twice as many tubulin fibers in GPIbβ−/− proplatelet buds in cultured and circulating platelets.Conclusions Altogether, these findings point to a role of the GPIb-V-IX complex intrinsic to megakaryocytes at the stage of proplatelet formation and suggest a functional link with the underlying microtubular cytoskeleton in platelet biogenesis.