RESUMO
Immune thrombocytopenia (ITP) is a platelet disorder. Pediatric and adult ITP have been associated with sialic acid alterations, but the pathophysiology of ITP remains elusive, and ITP is often a diagnosis of exclusion. Our analysis of pediatric ITP plasma samples showed increased anti-Thomsen-Friedenreich antigen (TF antigen) antibody representation, suggesting increased exposure of the typically sialylated and cryptic TF antigen in these patients. The O-glycan sialyltransferase St3gal1 adds sialic acid specifically on the TF antigen. To understand if TF antigen exposure associates with thrombocytopenia, we generated a mouse model with targeted deletion of St3gal1 in megakaryocytes (MK) (St3gal1MK-/-). TF antigen exposure was restricted to MKs and resulted in thrombocytopenia. Deletion of Jak3 in St3gal1MK-/- mice normalized platelet counts implicating involvement of immune cells. Interferon-producing Siglec H-positive bone marrow (BM) immune cells engaged with O-glycan sialic acid moieties to regulate type I interferon secretion and platelet release (thrombopoiesis), as evidenced by partially normalized platelet count following inhibition of interferon and Siglec H receptors. Single-cell RNA-sequencing determined that TF antigen exposure by MKs primed St3gal1MK-/- BM immune cells to release type I interferon. Single-cell RNA-sequencing further revealed a new population of immune cells with a plasmacytoid dendritic cell-like signature and concomitant upregulation of the immunoglobulin rearrangement gene transcripts Igkc and Ighm, suggesting additional immune regulatory mechanisms. Thus, aberrant TF antigen moieties, often found in pathological conditions, regulate immune cells and thrombopoiesis in the BM, leading to reduced platelet count.
Assuntos
Megacariócitos/patologia , Contagem de Plaquetas , Polissacarídeos/análise , Púrpura Trombocitopênica Idiopática/patologia , Adolescente , Animais , Antígenos Glicosídicos Associados a Tumores/análise , Criança , Pré-Escolar , Humanos , Lactente , Camundongos Endogâmicos C57BL , Sialiltransferases/análise , beta-Galactosídeo alfa-2,3-SialiltransferaseRESUMO
Cardiovascular (CV) disease prevention with low-dose aspirin can be less effective in patients with a faster recovery of platelet (PLT) cyclooxygenase (COX)-1 activity during the 24-hour dosing interval. We previously showed that incomplete suppression of TXA2 over 24 hours can be rescued by a twice daily aspirin regimen. Here we show that reduced PLT glycoprotein (GP)Ibα shedding characterizes patients with accelerated COX-1 recovery and may contribute to higher thrombopoietin (TPO) production and higher rates of newly formed PLT, escaping aspirin inhibition over 24 hours. Two hundred aspirin-treated patients with high CV risk (100 with type 2 diabetes mellitus) were stratified according to the kinetics of PLT COX-1 activity recovery during the 10- to 24-hour dosing interval. Whole proteome analysis showed that PLT from patients with accelerated COX-1 recovery were enriched in proteins involved in cell survival, inhibition of apoptosis and cellular protrusion formation. In agreement, we documented increased plasma TPO, megakaryocyte maturation and proplatelet formation, and conversely increased PLT galactose and reduced caspase 3, phosphatidylserine exposure and ADAM17 activation, translating into diminished GPIbα cleavage and glycocalicin (GC) release. Treatment of HepG2 cells with recombinant GC led to a dose-dependent reduction of TPO mRNA in the liver, suggesting that reduced GPIbα ectodomain shedding may unleash thrombopoiesis. A cluster of clinical markers, including younger age, non-alcoholic fatty liver disease, visceral obesity and higher TPO/GC ratio, predicted with significant accuracy the likelihood of faster COX-1 recovery and suboptimal aspirin response. Circulating TPO/GC ratio, reflecting a dysregulation of PLT lifespan and production, may provide a simple tool to identify patients amenable to more frequent aspirin daily dosing.
Assuntos
Diabetes Mellitus Tipo 2 , Trombocitopenia , Humanos , Aspirina/farmacologia , Trombopoese , Diabetes Mellitus Tipo 2/metabolismo , Plaquetas/metabolismo , Trombocitopenia/metabolismo , Glicoproteínas da Membrana de Plaquetas/metabolismoRESUMO
BACKGROUND AND AIMS: Thrombocytopenia has been described in most patients with acute and chronic liver failure. Decreased platelet production and decreased half-life of platelets might be a consequence of low levels of thrombopoietin (TPO) in these patients. Platelet production is tightly regulated to avoid bleeding complications after vessel injury and can be enhanced under elevated platelet destruction as observed in liver disease. Thrombopoietin (TPO) is the primary regulator of platelet biogenesis and supports proliferation and differentiation of megakaryocytes. APPROACH AND RESULTS: Recent work provided evidence for the control of TPO mRNA expression in liver and bone marrow (BM) by scanning circulating platelets. The Ashwell-Morell receptor (AMR) was identified to bind desialylated platelets to regulate hepatic thrombopoietin (TPO) production by Janus kinase (JAK2)/signal transducer and activator of transcription (STAT3) activation. Two-thirds partial hepatectomy (PHx) was performed in mice. Platelet activation and clearance by AMR/JAK2/STAT3 signaling and TPO production were analyzed at different time points after PHx. Here, we demonstrate that PHx in mice led to thrombocytopenia and platelet activation defects leading to bleeding complications, but unaltered arterial thrombosis, in these mice. Platelet counts were rapidly restored by up-regulation and crosstalk of the AMR and the IL-6 receptor (IL-6R) to induce JAK2-STAT3-TPO activation in the liver, accompanied by an increased number of megakaryocytes in spleen and BM before liver was completely regenerated. CONCLUSIONS: The AMR/IL-6R-STAT3-TPO signaling pathway is an acute-phase response to liver injury to reconstitute hemostasis. Bleeding complications were attributable to thrombocytopenia and platelet defects induced by elevated PGI2 , NO, and bile acid plasma levels early after PHx that might also be causative for the high mortality in patients with liver disease.
Assuntos
Hepatectomia/efeitos adversos , Trombocitopenia/sangue , Trombopoetina/biossíntese , Animais , Receptor de Asialoglicoproteína/genética , Receptor de Asialoglicoproteína/metabolismo , Modelos Animais de Doenças , Humanos , Janus Quinase 2/metabolismo , Camundongos , Camundongos Knockout , Contagem de Plaquetas , Receptores de Interleucina-6/genética , Receptores de Interleucina-6/metabolismo , Fator de Transcrição STAT3/metabolismo , Organismos Livres de Patógenos Específicos , Trombocitopenia/etiologia , Trombopoetina/sangueRESUMO
G protein-coupled receptors are critical mediators of platelet activation whose signaling can be modulated by members of the regulator of G protein signaling (RGS) family. The 2 most abundant RGS proteins in human and mouse platelets are RGS10 and RGS18. While each has been studied individually, critical questions remain about the overall impact of this mode of regulation in platelets. Here, we report that mice missing both proteins show reduced platelet survival and a 40% decrease in platelet count that can be partially reversed with aspirin and a P2Y12 antagonist. Their platelets have increased basal (TREM)-like transcript-1 expression, a leftward shift in the dose/response for a thrombin receptor-activating peptide, an increased maximum response to adenosine 5'-diphosphate and TxA2, and a greatly exaggerated response to penetrating injuries in vivo. Neither of the individual knockouts displays this constellation of findings. RGS10-/- platelets have an enhanced response to agonists in vitro, but platelet count and survival are normal. RGS18-/- mice have a 15% reduction in platelet count that is not affected by antiplatelet agents, nearly normal responses to platelet agonists, and normal platelet survival. Megakaryocyte number and ploidy are normal in all 3 mouse lines, but platelet recovery from severe acute thrombocytopenia is slower in RGS18-/- and RGS10-/-18-/- mice. Collectively, these results show that RGS10 and RGS18 have complementary roles in platelets. Removing both at the same time discloses the extent to which this regulatory mechanism normally controls platelet reactivity in vivo, modulates the hemostatic response to injury, promotes platelet production, and prolongs platelet survival.
Assuntos
Plaquetas/metabolismo , Ativação Plaquetária/genética , Proteínas RGS/genética , Trombopoese/genética , Animais , Plaquetas/efeitos dos fármacos , Sobrevivência Celular/genética , Camundongos , Camundongos Knockout , Fosforilação , Fator de Ativação de Plaquetas/farmacologia , Ativação Plaquetária/efeitos dos fármacos , Inibidores da Agregação Plaquetária/farmacologia , Contagem de Plaquetas , Proteínas RGS/metabolismo , Trombopoese/efeitos dos fármacosRESUMO
During infection neuraminidase desialylates platelets and induces their rapid clearance from circulation. The underlying molecular basis, particularly the role of platelet glycoprotein (GP)Ibα therein, is not clear. Utilizing genetically altered mice we report that the extracellular domain of GPIbα, but neither von Willebrand factor nor ADAM17 (a disintegrin and metalloprotease 17), is required for platelet clearance induced by intravenous injection of neuraminidase. Lectin binding to platelets following neuraminidase injection over time revealed that the extent of desialylation of O-glycans correlates with the decrease of platelet count in mice. Injection of α2,3-neuraminidase reduces platelet counts in wild-type but not in transgenic mice expressing only a chimeric GPIbα that misses most of its extracellular domain. Neuraminidase treatment induces unfolding of the O-glycosylated mechanosensory domain in GPIbα as monitored by single-molecule force spectroscopy, increases the exposure of the ADAM17 shedding cleavage site in the mechanosensory domain on the platelet surface, and induces ligand-independent GPIb-IX signaling in human and murine platelets. These results suggest that desialylation of O-glycans of GPIbα induces unfolding of the mechanosensory domain, subsequent GPIb-IX signaling including amplified desialylation of N-glycans, and eventually rapid platelet clearance. This new molecular mechanism of GPIbα-facilitated clearance could potentially resolve many puzzling and seemingly contradicting observations associated with clearance of desialylated or hyposialylated platelets.
Assuntos
Plaquetas , Complexo Glicoproteico GPIb-IX de Plaquetas , Animais , Camundongos , Contagem de Plaquetas , Polissacarídeos , Transdução de Sinais , Fator de von WillebrandRESUMO
Glycosyltransferases, usually residing within the intracellular secretory apparatus, also circulate in the blood. Many of these blood-borne glycosyltransferases are associated with pathological states, including malignancies and inflammatory conditions. Despite the potential for dynamic modifications of glycans on distal cell surfaces and in the extracellular milieu, the glycan-modifying activities present in systemic circulation have not been systematically examined. Here, we describe an evaluation of blood-borne sialyl-, galactosyl- and fucosyltransferase activities that act upon the four common terminal glycan precursor motifs, GlcNAc monomer, Gal(ß3)GlcNAc, Gal(ß4)GlcNAc and Gal(ß3)GalNAc, to produce more complex glycan structures. Data from radioisotope assays and detailed product analysis by sequential tandem mass spectrometry show that blood has the capacity to generate many of the well-recognized and important glycan motifs, including the Lewis, sialyl-Lewis, H- and Sialyl-T antigens. While many of these glycosyltransferases are freely circulating in the plasma, human and mouse platelets are important carriers for others, including ST3Gal-1 and ß4GalT. Platelets compartmentalize glycosyltransferases and release them upon activation. Human platelets are also carriers for large amounts of ST6Gal-1 and the α3-sialyl to Gal(ß4)GlcNAc sialyltransferases, both of which are conspicuously absent in mouse platelets. This study highlights the capability of circulatory glycosyltransferases, which are dynamically controlled by platelet activation, to remodel cell surface glycans and alter cell behavior.
Assuntos
Fucosiltransferases/sangue , Galactosiltransferases/sangue , Inflamação/sangue , Sialiltransferases/sangue , Animais , Plaquetas/enzimologia , Glicosilação , Glicosiltransferases , Humanos , Inflamação/enzimologia , Camundongos , Polissacarídeos/biossíntese , Polissacarídeos/químicaRESUMO
Changes in surface glycan determinants, specifically sialic acid loss, determine platelet life span. The gradual loss of stored platelet quality is a complex process that fundamentally involves carbohydrate structures. Here, we applied lipophilic extraction and glycan release protocols to sequentially profile N- and O-linked glycans in freshly isolated and 7-day room temperature-stored platelet concentrates. Analytical methods including matrix assisted laser desorption/ionization time-of-flight mass spectrometry, tandem mass spectrometry, and liquid chromatography were used to obtain structural details of selected glycans and terminal epitopes. The fresh platelet repertoire of surface structures revealed diverse N-glycans, including high mannose structures, complex glycans with polylactosamine repeats, and glycans presenting blood group epitopes. The O-glycan repertoire largely comprised sialylated and fucosylated core-1 and core-2 structures. For both N- and O-linked glycans, we observed a loss in sialylated epitopes with a reciprocal increase in neutral structures as well as increased neuraminidase activity after platelet storage at room temperature. The data indicate that loss of sialylated glycans is associated with diminished platelet quality and untimely removal of platelets after storage.
Assuntos
Plaquetas , Espectrometria de Massas em Tandem , Humanos , Espectrometria de Massas por Ionização e Dessorção a Laser Assistida por Matriz/métodos , Plaquetas/química , Polissacarídeos , EpitoposRESUMO
Platelets are blood components that survive in circulation for 7 to 10 days in humans. Thus, platelet production by bone marrow (BM) megakaryocytes (MKs), and their removal from the blood circulation is precisely orchestrated to maintain an average platelet count. Abnormalities in both processes can result in thrombocytopenia (low platelet count) or thrombocytosis (high platelet count), often associated with the risk of bleeding or overt thrombus formation, respectively. Platelet glycans, particularly sialic acids, are indicators of platelet count. Loss of platelet sialic acids leads to platelet clearance. A State-of-the-Art lecture titled "Platelet and Megakaryocyte Glycobiology" was presented at the ISTH virtual congress 2021 to discuss (i) the loss of O-glycan sialic acid on BM MKs, revealing the Thomsen-Friedenreich (TF) antigen as a new concept of thrombocytopenia; herein, impaired thrombopoiesis is attributed to activation of immune cells with a plasmacytoid dendritic cell signature; and (ii) upregulation of antibodies against the TF antigen in pediatric patients with immune thrombocytopenia (ITP), positing that glycan alterations such as MK asialylation can lead to immune cell responses. Here, we discuss our findings alongside new data presented at the 2020 and 2021 ISTH congresses on the role of sialic acids and glycans in regulating platelet count. Desialylation is a prominent feature in thrombocytopenia, notably in ITP presentation. We compare similarities between ITP mediated with shear-stress and with storage-related asialylation. We also discuss genes involved in sialic acid synthesis leading to thrombocytopenia. Increased awareness in gene-regulating MK and platelet glycans is a giant leap to understanding the underpinning mechanisms of ITP and other forms of thrombocytopenia.
RESUMO
Dnm2fl/fl Pf4-Cre (Dnm2Plt-/- ) mice lacking the endocytic GTPase dynamin 2 (DNM2) in platelets and megakaryocytes (MKs) develop hallmarks of myelofibrosis. At the cellular level, the tyrosine kinase JAK2 is constitutively active but decreased in expression in Dnm2Plt-/- platelets. Additionally, Dnm2Plt-/- platelets cannot endocytose the thrombopoietin (TPO) receptor Mpl, leading to elevated circulating TPO levels. Here, we assessed whether the hyperproliferative phenotype of Dnm2Plt-/- mice was due to JAK2 constitutive activation or to elevated circulating TPO levels. In unstimulated Dnm2Plt-/- platelets, STAT3 and, to a lower extent, STAT5 were phosphorylated, but their phosphorylation was slowed and diminished upon TPO stimulation. We further crossed Dnm2Plt-/- mice in the Mpl-/- background to generate Mpl-/-Dnm2Plt-/- mice lacking Mpl ubiquitously and DNM2 in platelets and MKs. Mpl-/- Dnm2Plt-/- platelets had severely reduced JAK2 and STAT3 but normal STAT5 expression. Mpl-/- Dnm2Plt-/- mice had severely reduced bone marrow MK and hematopoietic stem and progenitor cell numbers. Additionally, Mpl-/- Dnm2Plt-/- mice had severe erythroblast (EB) maturation defects, decreased expression of hemoglobin and heme homeostasis genes and increased expression of ribosome biogenesis and protein translation genes in spleen EBs, and developed anemia with grossly elevated plasma erythropoietin (EPO) levels, leading to early fatality by postnatal day 25. Mpl-/- Dnm2Plt+/+ mice had impaired EB development at three weeks of age, which normalized with adulthood. Together, the data shows that DNM2-dependent Mpl-mediated endocytosis in platelets and MKs is required for steady-state hematopoiesis and provides novel insights into a developmentally controlled role for Mpl in normal erythropoiesis, regulating hemoglobin and heme production.
RESUMO
Lymph nodes (LNs) are the critical sites of immunity, and the stromal cells of LNs are crucial to their function. Our understanding of the stromal compartment of the LN has deepened recently with the characterization of nontraditional stromal cells. CD41 (integrin αIIb) is known to be expressed by platelets and hematolymphoid cells. We identified two distinct populations of CD41+Lyve1+ and CD41+Lyve1- cells in the LNs. CD41+Lyve1- cells appear in the LN mostly at the later stages of the lives of mice. We identified CD41+ cells in human LNs as well. We demonstrated that murine CD41+ cells express mesodermal markers, such as Sca-1, CD105 and CD29, but lack platelet markers. We did not observe the presence of platelets around the HEVs or within proximity to fibroblastic reticular cells of the LN. Examination of thoracic duct lymph fluid showed the presence of CD41+Lyve1- cells, suggesting that these cells recirculate throughout the body. FTY720 reduced their trafficking to lymph fluid, suggesting that their egress is controlled by the S1P1 pathway. CD41+Lyve1- cells of the LNs were sensitive to radiation, suggestive of their replicative nature. Single cell RNA sequencing data showed that the CD41+ cell population in naïve mouse LNs expressed largely stromal cell markers. Further studies are required to examine more deeply the role of CD41+ cells in the function of LNs.
Assuntos
Linfonodos , Células Estromais , Animais , Fibroblastos , Humanos , CamundongosRESUMO
Glycosylation is highly prevalent, and also one of the most complex and varied posttranslational modifications. This large glycan diversity results in a wide range of biological functions. Functional diversity includes protein degradation, protein clearance, cell trafficking, cell signaling, host-pathogen interactions, and immune defense, including both innate and acquired immunity. Glycan-based ABO(H) antigens are critical in providing compatible products in the setting of transfusion and organ transplantation. However, evidence also suggests that ABO expression may influence cardiovascular disease, thrombosis, and hemostasis disorders, including alterations in platelet function and von Willebrand factor blood levels. Glycans also regulate immune and hemostasis function beyond ABO(H) antigens. Mutations in glycogenes (PIGA, COSMC) lead to serious blood disorders, including Tn syndrome associated with hyperagglutination, hemolysis, and thrombocytopenia. Alterations in genes responsible for sialic acids (Sia) synthesis (GNE) and UDP-galactose (GALE) and lactosamine (LacNAc) (B4GALT1) profoundly affect circulating platelet counts. Desialylation (removal of Sia) is affected by human and pathogenic neuraminidases. This review addresses the role of glycans in transfusion medicine, hemostasis and thrombosis, and red blood cell and platelet survival.
Assuntos
Plaquetas , Medicina Transfusional , Plaquetas/metabolismo , Eritrócitos/metabolismo , Glicosilação , Humanos , Fator de von Willebrand/metabolismoRESUMO
Glycosylation is critical to megakaryocyte (MK) and thrombopoiesis in the context of gene mutations that affect sialylation and galactosylation. Here, we identify the conserved B4galt1 gene as a critical regulator of thrombopoiesis in MKs. ß4GalT1 deficiency increases the number of fully differentiated MKs. However, the resulting lack of glycosylation enhances ß1 integrin signaling leading to dysplastic MKs with severely impaired demarcation system formation and thrombopoiesis. Platelets lacking ß4GalT1 adhere avidly to ß1 integrin ligands laminin, fibronectin, and collagen, while other platelet functions are normal. Impaired thrombopoiesis leads to increased plasma thrombopoietin (TPO) levels and perturbed hematopoietic stem cells (HSCs). Remarkably, ß1 integrin deletion, specifically in MKs, restores thrombopoiesis. TPO and CXCL12 regulate ß4GalT1 in the MK lineage. Thus, our findings establish a non-redundant role for ß4GalT1 in the regulation of ß1 integrin function and signaling during thrombopoiesis. Defective thrombopoiesis and lack of ß4GalT1 further affect HSC homeostasis.
Assuntos
Galactosiltransferases/metabolismo , Células-Tronco Hematopoéticas/metabolismo , Homeostase , Integrina beta1/metabolismo , Trombopoese/fisiologia , Animais , Plaquetas/metabolismo , Plaquetas/patologia , Adesão Celular , Diferenciação Celular , Quimiocina CXCL12/metabolismo , Colágeno , Modelos Animais de Doenças , Fibronectinas , Galactosiltransferases/genética , Predisposição Genética para Doença , Hemorragia/genética , Hemorragia/metabolismo , Hemorragia/patologia , Integrina beta1/genética , Laminina , Ligantes , Megacariócitos , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Camundongos Transgênicos , Transdução de Sinais , Trombocitopenia/genética , Trombocitopenia/metabolismo , Trombocitopenia/patologia , Trombopoese/genética , Trombopoetina/sangueRESUMO
The immune response relies on the integration of cell-intrinsic processes with cell-extrinsic cues. During infection, B cells vacate the marrow during emergency granulopoiesis but return upon restoration of homeostasis. Here we report a novel glycosylation-mediated crosstalk between marrow B cells and hematopoietic progenitors. Human B cells secrete active ST6GAL1 sialyltransferase that remodels progenitor cell surface glycans to suppress granulopoiesis. In mouse models, ST6GAL1 from B cells alters the sialylation profile of bone marrow populations, and mature IgD+ B cells were enriched in sialylated bone marrow niches. In clinical multiple myeloma, ST6GAL1 abundance in the multiple myeloma cells negatively correlated with neutrophil abundance. These observations highlight not only the ability of medullary B cells to influence blood cell production, but also the disruption to normal granulopoiesis by excessive ST6GAL1 in malignancy.