RESUMO
BACKGROUND: Aortic stenosis (AS) is driven by progressive inflammatory and fibrocalcific processes regulated by circulating inflammatory and valve resident endothelial and interstitial cells. The impact of platelets, platelet-derived mediators, and platelet-monocyte interactions on the acceleration of local valvular inflammation and mineralization is presently unknown. METHODS: We prospectively enrolled 475 consecutive patients with severe symptomatic AS undergoing aortic valve replacement. Clinical workup included repetitive echocardiography, analysis of platelets, monocytes, chemokine profiling, aortic valve tissue samples for immunohistochemistry, and gene expression analysis. RESULTS: The patients were classified as fast-progressive AS by the median ∆Vmax of 0.45 m/s per year determined by echocardiography. Immunohistological aortic valve analysis revealed enhanced cellularity in fast-progressive AS (slow- versus fast-progressive AS; median [interquartile range], 247 [142.3-504] versus 717.5 [360.5-1234]; P<0.001) with less calcification (calcification area, mm2: 33.74 [27.82-41.86] versus 20.54 [13.52-33.41]; P<0.001). MIF (macrophage migration inhibitory factor)-associated gene expression was significantly enhanced in fast-progressive AS accompanied by significantly elevated MIF plasma levels (mean±SEM; 6877±379.1 versus 9959±749.1; P<0.001), increased platelet activation, and decreased intracellular MIF expression indicating enhanced MIF release upon platelet activation (CD62P, %: median [interquartile range], 16.8 [11.58-23.8] versus 20.55 [12.48-32.28], P=0.005; MIF, %: 4.85 [1.48-9.75] versus 2.3 [0.78-5.9], P<0.001). Regression analysis confirmed that MIF-associated biomarkers are strongly associated with an accelerated course of AS. CONCLUSIONS: Our findings suggest a key role for platelet-derived MIF and its interplay with circulating and valve resident monocytes/macrophages in local and systemic thromboinflammation during accelerated AS. MIF-based biomarkers predict an accelerated course of AS and represent a novel pharmacological target to attenuate progression of AS.
RESUMO
Glycoprotein VI (GPVI), the platelet immunoreceptor tyrosine activating motif (ITAM) receptor for collagen, plays a striking role on vascular integrity in animal models of inflammation and sepsis. Understanding ITAM-receptor signaling defects in humans suffering from sepsis may improve our understanding of the pathophysiology, especially during disease onset. In a pilot study, platelets from 15 patients with sepsis were assessed consecutively at day of admission, day 5 to 7, and the day of intensive care unit (ICU) discharge and subjected to comprehensive analyses by flow cytometry, aggregometry, and immunoblotting. Platelet function was markedly reduced in all patients. The defect was most prominent after GPVI stimulation with collagen-related peptide. In 14 of 15 patients, GPVI dysfunction was already present at time of ICU admission, considerably before the critical drop in platelet counts. Sepsis platelets failed to transduce the GPVI-mediated signal to trigger tyrosine phosphorylation of Syk kinase or LAT. GPVI deficiency was partially inducible in platelets of healthy donors through coincubation in whole blood, but not in plasma from patients with sepsis. Platelet aggregation upon GPVI stimulation increased only in those patients whose condition ameliorated. As blunted GPVI signaling occurred early at sepsis onset, this defect could be exploited as an indicator for early sepsis diagnosis, which needs to be confirmed in prospective studies.
Assuntos
Plaquetas/metabolismo , Agregação Plaquetária , Glicoproteínas da Membrana de Plaquetas/metabolismo , Sepse/metabolismo , Transdução de Sinais , Adulto , Idoso , Idoso de 80 Anos ou mais , Plaquetas/patologia , Estado Terminal , Feminino , Humanos , Masculino , Pessoa de Meia-Idade , Projetos Piloto , Sepse/patologiaRESUMO
The pathophysiology of COVID-19-associated thrombosis seems to be multifactorial. We hypothesized that COVID-19 is accompanied by procoagulant platelets with subsequent alteration of the coagulation system. We investigated depolarization of mitochondrial inner transmembrane potential (ΔΨm), cytosolic calcium (Ca2+) concentration, and phosphatidylserine (PS) externalization. Platelets from COVID-19 patients in the intensive care unit (ICU; n = 21) showed higher ΔΨm depolarization, cytosolic Ca2+, and PS externalization compared with healthy controls (n = 18) and non-ICU COVID-19 patients (n = 4). Moreover, significant higher cytosolic Ca2+ and PS were observed compared with a septic ICU control group (ICU control; n = 5). In the ICU control group, cytosolic Ca2+ and PS externalization were comparable with healthy controls, with an increase in ΔΨm depolarization. Sera from COVID-19 patients in the ICU induced a significant increase in apoptosis markers (ΔΨm depolarization, cytosolic Ca2+, and PS externalization) compared with healthy volunteers and septic ICU controls. Interestingly, immunoglobulin G fractions from COVID-19 patients induced an Fcγ receptor IIA-dependent platelet apoptosis (ΔΨm depolarization, cytosolic Ca2+, and PS externalization). Enhanced PS externalization in platelets from COVID-19 patients in the ICU was associated with increased sequential organ failure assessment score (r = 0.5635) and D-dimer (r = 0.4473). Most importantly, patients with thrombosis had significantly higher PS externalization compared with those without. The strong correlations between markers for apoptosic and procoagulant platelets and D-dimer levels, as well as the incidence of thrombosis, may indicate that antibody-mediated procoagulant platelets potentially contributes to sustained increased thromboembolic risk in ICU COVID-19 patients.
Assuntos
Apoptose , Plaquetas/patologia , COVID-19/patologia , Imunoglobulina G/metabolismo , Adulto , Idoso , Coagulação Sanguínea , Plaquetas/metabolismo , COVID-19/sangue , COVID-19/complicações , COVID-19/metabolismo , Cálcio/metabolismo , Estudos de Coortes , Feminino , Humanos , Masculino , Potencial da Membrana Mitocondrial , Pessoa de Meia-Idade , Fosfatidilserinas/metabolismo , SARS-CoV-2/isolamento & purificação , SARS-CoV-2/fisiologia , Trombose/sangue , Trombose/etiologia , Trombose/metabolismo , Trombose/patologiaRESUMO
Megakaryocytes (MKs), the largest and rarest cells of the hematopoietic system, differentiate by increasing their size, DNA and cytoplasmic contents during maturation in order to release high numbers of blood platelets into the circulation. The gold-standard to study these complex cells is the isolation of primary MKs from the native bone marrow (BM). This is typically achieved by using fluorescence- or magnetic-activated cell sorting. However, both methods are time-consuming and require a trained experimenter who is able to operate highly priced special equipment. Here, we demonstrate a simple and rapid alternative method to enrich mature MKs (≥16 N) from murine adult BM by size exclusion. The purity of the MK fraction reached 70-80% after isolation (100- to 250-fold enrichment). Reanalysis of isolated MKs by confocal microscopy revealed the expected expression of lineage-defining MK- and platelet-specific surface receptors, including CD42a/b/d and CD41/CD61. In addition, we detected a clear enrichment of MK-specific proteins/transcripts like ß1-tubulin, ß3-integrin, GPVI and GPIbα, whereas the neutrophil marker Ly6G was only detectable in the BM sample. Taken together, we demonstrate that the protocol proposed in this Technical Report is a compatible addition to established isolation methods.
Assuntos
Plaquetas , Megacariócitos , Humanos , Adulto , Animais , Camundongos , Megacariócitos/metabolismo , Plaquetas/metabolismo , Complexo Glicoproteico GPIb-IX de Plaquetas/metabolismoRESUMO
Cyclophilin A (CyPA) is widely expressed by all prokaryotic and eukaryotic cells. Upon activation, CyPA can be released into the extracellular space to engage in a variety of functions, such as interaction with the CD147 receptor, that contribute to the pathogenesis of cardiovascular diseases. CyPA was recently found to undergo acetylation at K82 and K125, two lysine residues conserved in most species, and these modifications are required for secretion of CyPA in response to cell activation in vascular smooth muscle cells. Herein we addressed whether acetylation at these sites is also required for the release of CyPA from platelets based on the potential for local delivery of CyPA that may exacerbate cardiovascular disease events. Western blot analyses confirmed the presence of CyPA in human and mouse platelets. Thrombin stimulation resulted in CyPA release from platelets; however, no acetylation was observed-neither in cell lysates nor in supernatants of both untreated and activated platelets, nor after immunoprecipitation of CyPA from platelets. Shotgun proteomics detected two CyPA peptide precursors in the recombinant protein, acetylated at K28, but again, no acetylation was found in CyPA derived from resting or stimulated platelets. Our findings suggest that acetylation of CyPA is not a major protein modification in platelets and that CyPA acetylation is not required for its secretion from platelets.
Assuntos
Plaquetas/metabolismo , Ciclofilina A/metabolismo , Ativação Plaquetária , Acetilação , Animais , Humanos , Lisina , CamundongosRESUMO
During thrombopoiesis, megakaryocytes (MKs) form proplatelets within the bone marrow (BM) and release platelets into BM sinusoids. Phosphoinositide-dependent protein kinase-1 (PDK1) is required for Ca2+-dependent platelet activation, but its role in MK development and regulation of platelet production remained elusive. The present study explored the role of PDK1 in the regulation of MK maturation and polarization during thrombopoiesis using a MK/platelet-specific knockout approach. Pdk1-deficient mice (Pdk1-/-) developed a significant macrothrombocytopenia as compared with wild-type mice (Pdk1fl/fl). Pdk1 deficiency further dramatically increased the number of MKs without sinusoidal contact within the BM hematopoietic compartment, resulting in a pronounced MK hyperplasia and a significantly increased extramedullary thrombopoiesis. Cultured Pdk1-/- BM-MKs showed impaired spreading on collagen, associated with an altered actin cytoskeleton structure with less filamentous actin (F-actin) and diminished podosome formation, whereas the tubulin cytoskeleton remained unaffected. This phenotype was associated with abrogated phosphorylation of p21-activated kinase (PAK) as well as its substrates LIM domain kinase and cofilin, supporting the hypothesis that the defective F-actin assembly results from increased cofilin activity in Pdk1-deficient MKs. Pdk1-/- BM-MKs developed increased ploidy and exhibited an abnormal ultrastructure with disrupted demarcation membrane system (DMS). Strikingly, Pdk1-/- BM-MKs displayed a pronounced defect in DMS polarization and produced significantly less proplatelets, indicating that PDK1 is critically required for proplatelet formation. In human MKs, genetic PDK1 knockdown resulted in increased maturity but reduced platelet-like particles formation. The present observations reveal a pivotal role of PDK1 in the regulation of MK cytoskeletal dynamics and polarization, proplatelet formation, and thrombopoiesis.
Assuntos
Proteínas Quinases Dependentes de 3-Fosfoinositídeo/metabolismo , Plaquetas/metabolismo , Citoesqueleto/metabolismo , Megacariócitos/metabolismo , Trombopoese/fisiologia , Animais , Plaquetas/citologia , Humanos , Megacariócitos/citologia , Camundongos , Camundongos KnockoutRESUMO
BACKGROUND: Coronavirus disease 2019 (COVID-19) associated coagulopathy (CAC) leads to thromboembolic events in a high number of critically ill COVID-19 patients. However, specific diagnostic or therapeutic algorithms for CAC have not been established. In the current study, we analyzed coagulation abnormalities with point-of-care testing (POCT) and their relation to hemostatic complications in patients suffering from COVID-19 induced Acute Respiratory Distress Syndrome (ARDS). Our hypothesis was that specific diagnostic patterns can be identified in patients with COVID-19 induced ARDS at risk of thromboembolic complications utilizing POCT. METHODS: This is a single-center, retrospective observational study. Longitudinal data from 247 rotational thromboelastometries (Rotem®) and 165 impedance aggregometries (Multiplate®) were analysed in 18 patients consecutively admitted to the ICU with a COVID-19 induced ARDS between March 12th to June 30th, 2020. RESULTS: Median age was 61 years (IQR: 51-69). Median PaO2/FiO2 on admission was 122 mmHg (IQR: 87-189), indicating moderate to severe ARDS. Any form of hemostatic complication occurred in 78 % of the patients with deep vein/arm thrombosis in 39 %, pulmonary embolism in 22 %, and major bleeding in 17 %. In Rotem® elevated A10 and maximum clot firmness (MCF) indicated higher clot strength. The delta between EXTEM A10 minus FIBTEM A10 (ΔA10) > 30 mm, depicting the sole platelet-part of clot firmness, was associated with a higher risk of thromboembolic events (OD: 3.7; 95 %CI 1.3-10.3; p = 0.02). Multiplate® aggregometry showed hypoactive platelet function. There was no correlation between single Rotem® and Multiplate® parameters at intensive care unit (ICU) admission and thromboembolic or bleeding complications. CONCLUSIONS: Rotem® and Multiplate® results indicate hypercoagulability and hypoactive platelet dysfunction in COVID-19 induced ARDS but were all in all poorly related to hemostatic complications..
RESUMO
RATIONALE: Regeneration of lost cardiomyocytes is a fundamental unresolved problem leading to heart failure. Despite several strategies developed from intensive studies performed in the past decades, endogenous regeneration of heart tissue is still limited and presents a big challenge that needs to be overcome to serve as a successful therapeutic option for myocardial infarction. OBJECTIVE: One of the essential prerequisites for cardiac regeneration is the identification of endogenous cardiomyocyte progenitors and their niche that can be targeted by new therapeutic approaches. In this context, we hypothesized that the vascular wall, which was shown to harbor different types of stem and progenitor cells, might serve as a source for cardiac progenitors. METHODS AND RESULTS: We describe generation of spontaneously beating mouse aortic wall-derived cardiomyocytes without any genetic manipulation. Using aortic wall-derived cells (AoCs) of WT (wild type), αMHC (α-myosin heavy chain), and Flk1 (fetal liver kinase 1)-reporter mice and magnetic bead-associated cell sorting sorting of Flk1+ AoCs from GFP (green fluorescent protein) mice, we identified Flk1+CD (cluster of differentiation) 34+Sca-1 (stem cell antigen-1)-CD44- AoCs as the population that gives rise to aortic wall-derived cardiomyocytes. This AoC subpopulation delivered also endothelial cells and macrophages with a particular accumulation within the aortic wall-derived cardiomyocyte containing colonies. In vivo, cardiomyocyte differentiation capacity was studied by implantation of fluorescently labeled AoCs into chick embryonic heart. These cells acquired cardiomyocyte-like phenotype as shown by αSRA (α-sarcomeric actinin) expression. Furthermore, coronary adventitial Flk1+ and CD34+ cells proliferated, migrated into the myocardium after mouse myocardial infarction, and expressed Isl-1+ (insulin gene enhancer protein-1) indicative of cardiovascular progenitor potential. CONCLUSIONS: Our data suggest Flk1+CD34+ vascular adventitia-resident stem cells, including those of coronary adventitia, as a novel endogenous source for generating cardiomyocytes. This process is essentially supported by endothelial cells and macrophages. In summary, the therapeutic manipulation of coronary adventitia-resident cardiac stem and their supportive cells may open new avenues for promoting cardiac regeneration and repair after myocardial infarction and for preventing heart failure.
Assuntos
Túnica Adventícia/citologia , Aorta Torácica/citologia , Diferenciação Celular , Proliferação de Células , Miócitos Cardíacos/fisiologia , Células-Tronco/fisiologia , Animais , Antígenos CD34/metabolismo , Antígenos Ly/metabolismo , Células Cultivadas , Embrião de Galinha , Modelos Animais de Doenças , Feminino , Genes Reporter , Separação Imunomagnética , Proteínas Luminescentes/genética , Proteínas Luminescentes/metabolismo , Masculino , Proteínas de Membrana/metabolismo , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Infarto do Miocárdio/metabolismo , Infarto do Miocárdio/patologia , Infarto do Miocárdio/fisiopatologia , Infarto do Miocárdio/cirurgia , Miócitos Cardíacos/metabolismo , Miócitos Cardíacos/transplante , Cadeias Pesadas de Miosina/genética , Fenótipo , Regeneração , Transplante de Células-Tronco , Células-Tronco/metabolismo , Receptor 2 de Fatores de Crescimento do Endotélio Vascular/genética , Receptor 2 de Fatores de Crescimento do Endotélio Vascular/metabolismo , Miosinas Ventriculares/genéticaRESUMO
Defects of platelet intracellular signaling can result in severe platelet dysfunction. Several mutations in each of the linked genes FERMT3 and RASGRP2 on chromosome 11 causing a Glanzmann-like bleeding phenotype have been identified so far. We report on novel variants in two unrelated pediatric patients with severe bleeding diathesis-one with leukocyte adhesion deficiency type III due to a homozygous frameshift in FERMT3 and the other with homozygous variants in both, FERMT3 and RASGRP2. We focus on the challenging genetic and functional variant assessment and aim to accentuate the risk of obtaining misleading results due to the phenomenon of genetic linkage.
Assuntos
Transtornos Plaquetários/patologia , Fatores de Troca do Nucleotídeo Guanina/genética , Transtornos Hemorrágicos/patologia , Proteínas de Membrana/genética , Mutação , Proteínas de Neoplasias/genética , Adolescente , Transtornos Plaquetários/genética , Criança , Feminino , Ligação Genética , Transtornos Hemorrágicos/genética , Homozigoto , Humanos , Masculino , Linhagem , Fenótipo , PrognósticoRESUMO
Platelets, anucleated megakaryocyte (MK)-derived cells, play a major role in hemostasis and arterial thrombosis. Although protein kinase casein kinase 2 (CK2) is readily detected in MKs and platelets, the impact of CK2-dependent signaling on MK/platelet (patho-)physiology has remained elusive. The present study explored the impact of the CK2 regulatory ß-subunit on platelet biogenesis and activation. MK/platelet-specific genetic deletion of CK2ß (ck2ß-/- ) in mice resulted in a significant macrothrombocytopenia and an increased extramedullar megakaryopoiesis with an enhanced proportion of premature platelets. Although platelet life span was only mildly affected, ck2ß-/- MK displayed an abnormal microtubule structure with a drastically increased fragmentation within bone marrow and a significantly reduced proplatelet formation in vivo. In ck2ß-/- platelets, tubulin polymerization was disrupted, resulting in an impaired thrombopoiesis and an abrogated inositol 1,4,5-triphosphate receptor-dependent intracellular calcium (Ca2+) release. Presumably due to a blunted increase in the concentration of cytosolic Ca2+, activation-dependent increases of α and dense-granule secretion and integrin αIIbß3 activation, and aggregation were abrogated in ck2ß-/- platelets. Accordingly, thrombus formation and stabilization under high arterial shear rates were significantly diminished, and thrombotic vascular occlusion in vivo was significantly blunted in ck2ß-/- mice, accompanied by a slight prolongation of bleeding time. Following transient middle cerebral artery occlusion, ck2ß-/- mice displayed significantly reduced cerebral infarct volumes, developed significantly less neurological deficits, and showed significantly better outcomes after ischemic stroke than ck2ßfl/fl mice. The present observations reveal CK2ß as a novel powerful regulator of thrombopoiesis, Ca2+-dependent platelet activation, and arterial thrombosis in vivo.
Assuntos
Caseína Quinase II/fisiologia , Fragmentos de Peptídeos/fisiologia , Ativação Plaquetária , Trombopoese , Trombose/patologia , Animais , Plaquetas , Sinalização do Cálcio , Caseína Quinase II/deficiência , Megacariócitos/metabolismo , Megacariócitos/patologia , Megacariócitos/ultraestrutura , Camundongos , Camundongos Knockout , Fragmentos de Peptídeos/deficiência , Trombose/etiologia , Trombose/metabolismoRESUMO
For several malignant and nonmalignant disorders such as leukemias, lymphomas, or inborn errors of hematopoiesis, stem cell transplantation is the only curative option. Depending on the underlying cause of the disease, the conditioning regimens, source of the stem cells, and graft composition may vary. Possible stem cell donors are selected from databases considering existing major histocompatibility genes of the donor and the recipient. This is currently performed by matching human leukocyte antigen (HLA)-A, -B, and -C for class I, as well as HLA-DRB1 and -DQB1 for class II. Stem cell transplantation for nonmalignant disorders is a specialty of pediatrics. While algorithms for donor selection in these cases are generally similar, the objective of optimizing a possible graft-versus-leukemia effect is less important. In this article, we aim to provide an overview on the current methods for HLA typing and the algorithms for HLA matching. We also address ethical aspects regarding children and minors as stem cell donors.
RESUMO
Platelet collagen interactions at sites of vascular injuries predominantly involve glycoprotein VI (GPVI) and the integrin α2ß1. Both proteins are primarily expressed on platelets and megakaryocytes whereas GPVI expression is also shown on endothelial and integrin α2ß1 expression on epithelial cells. We recently showed that depletion of GPVI improves stroke outcome without increasing the risk of cerebral hemorrhage. Genetic variants associated with higher platelet surface integrin α2 (ITGA2) receptor levels have frequently been found to correlate with an increased risk of ischemic stroke in patients. However until now, no preclinical stroke study has addressed whether platelet integrin α2ß1 contributes to the pathophysiology of ischemia/reperfusion (I/R) injury. Focal cerebral ischemia was induced in C57BL/6 and Itga2-/- mice by a 60 min transient middle cerebral artery occlusion (tMCAO). Additionally, wild-type animals were pretreated with anti-GPVI antibody (JAQ1) or Fab fragments of a function blocking antibody against integrin α2ß1 (LEN/B). In anti-GPVI treated animals, intravenous (IV) recombinant tissue plasminogen activator (rt-PA) treatment was applied immediately prior to reperfusion. Stroke outcome, including infarct size and neurological scoring was determined on day 1 after tMCAO. We demonstrate that targeting the integrin α2ß1 (pharmacologic; genetic) did neither reduce stroke size nor improve functional outcome on day 1 after tMCAO. In contrast, depletion of platelet GPVI prior to stroke was safe and effective, even when combined with rt-PA treatment. Our results underscore that GPVI, but not ITGA2, is a promising and safe target in the setting of ischemic stroke.
Assuntos
Anticorpos/uso terapêutico , Encéfalo/efeitos dos fármacos , Infarto da Artéria Cerebral Média/prevenção & controle , Integrina alfa2beta1/antagonistas & inibidores , Glicoproteínas da Membrana de Plaquetas/antagonistas & inibidores , Substâncias Protetoras/uso terapêutico , Ativador de Plasminogênio Tecidual/uso terapêutico , Animais , Encéfalo/metabolismo , Encéfalo/patologia , Colágeno/metabolismo , Infarto da Artéria Cerebral Média/metabolismo , Infarto da Artéria Cerebral Média/patologia , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Proteínas Recombinantes/uso terapêutico , Acidente Vascular Cerebral/metabolismo , Acidente Vascular Cerebral/patologia , Acidente Vascular Cerebral/prevenção & controleRESUMO
Collagen receptors GPVI (also known as GP6) and integrin α2ß1 are highly expressed on blood platelets and megakaryocytes, their immediate precursors. After vessel injury, subendothelial collagen becomes exposed and induces platelet activation to prevent blood loss. Collagen types I and IV are thought to have opposite effects on platelet biogenesis, directing proplatelet formation (PPF) towards the blood vessels to prevent premature release within the marrow cavity. We used megakaryocytes lacking collagen receptors or treated megakaryocytes with blocking antibodies, and could demonstrate that collagen-I-mediated inhibition of PPF is specifically controlled by GPVI. Other collagen types competed for binding and diminished the inhibitory signal, which was entirely dependent on receptor-proximal Src family kinases, whereas Syk and LAT were dispensable. Adhesion assays indicate that megakaryocyte binding to collagens is mediated by α2ß1, and that collagen IV at the vascular niche might displace collagen I from megakaryocytes and thus contribute to prevention of premature platelet release into the marrow cavity and thereby directionally promote PPF at the vasculature.
Assuntos
Plaquetas/metabolismo , Colágeno Tipo I/metabolismo , Glicoproteínas da Membrana de Plaquetas/metabolismo , Transdução de Sinais , Quinase Syk/metabolismo , Animais , Medula Óssea/metabolismo , Adesão Celular , Diferenciação Celular , Matriz Extracelular/metabolismo , Feminino , Fêmur/metabolismo , Imuno-Histoquímica , Masculino , Megacariócitos/citologia , Camundongos Endogâmicos C57BL , Fenótipo , Receptores de Colágeno/metabolismoRESUMO
A high proportion of patients with mucocutaneous bleeding diathesis and suspected inherited or acquired platelet disorder remain without diagnosis even after comprehensive laboratory testing. Since flow cytometry allows investigation of resting and activated platelets on the single cell level by requiring only minimal amounts of blood, this method has become an important assay within the diagnostic algorithm, especially in pediatrics. We therefore developed a standardized and modular flow cytometric approach that contributes to clarify impaired platelet function in a rational step-by-step manner. Due to simultaneous analysis of four fluorophores in a basic panel design, we are able to readily detect the most common and clinically significant platelet disorders: Glanzmann thrombasthenia or Glanzmann-like diseases (fibrinogen receptor GPIIb-IIIa), Bernard-Soulier syndrome (von Willebrand-factor receptor complex GPIb-IX-V) and less well characterized ß1-integrins that serve as the collagen, laminin or fibronectin receptor (CD29-CD49b, e and f, respectively). Platelet reactivity was investigated in response to the agonists adenosine diphosphate (ADP) and thrombin receptor activator peptide 6 (TRAP6) in suboptimal and optimal concentrations by quantifying surface expression of activation markers CD62P and CD63 as well as binding of PAC-1 antibody to the high affinity conformation of the fibrinogen receptor. For advanced diagnostic questions, several further modules were implemented: (i) calcium mobilization for evaluation of early signal transduction, (ii) a kinetically resolved mepacrine assay for estimation of delta-granule content and release, and (iii) a module to determine platelet reactivity upon additional agonists like the thromboxane A2-analogue U46619 or collagen. Blood withdrawn from a healthy control cohort allowed generating preliminary standard values for all parameters. The modules were validated by analysis of patients with known or suspected platelet defects (leukocyte-adhesion deficiency type III, Wiskott-Aldrich syndrome, acute myeloid leukemia, sickle cell disease and chronic immune thrombocytopenia).
Assuntos
Transtornos Plaquetários/sangue , Plaquetas/metabolismo , Citometria de Fluxo/métodos , Imunofenotipagem/métodos , Adolescente , Adulto , Feminino , Humanos , Masculino , Pessoa de Meia-Idade , Adulto JovemRESUMO
Thrombocytopenia absent radii (TAR) syndrome is clearly defined by the combination of radial aplasia and reduced platelet counts. The genetics of TAR syndrome has recently been resolved and comprises a microdeletion on Chromosome 1 including the RBM8A gene and a single nucleotide polymorphism (SNP) either at the 5' untranslated region (5'UTR) or within the first intron of RBM8A. Although phenotypically readily diagnosed after birth, the genetic determination of particular SNPs in TAR syndrome harbours valuable information to evaluate disease severity and treatment decisions. Here, we present clinical data in a cohort of 38 patients and observed that platelet counts in individuals with 5'UTR SNP are significantly lower compared to patients bearing the SNP in intron 1. Moreover, elevated haemoglobin values could only be assessed in patients with 5'UTR SNP whereas white blood cell count is unaffected, indicating that frequently observed anaemia in TAR patients could also be SNP-dependent whereas leucocytosis does not correlate with genetic background. However, this report on a large cohort provides an overview of important haematological characteristics in TAR patients, facilitating evaluation of the various traits in this disease and indicating the importance of genetic validation for TAR syndrome.
Assuntos
Variação Genética , Hematopoese/genética , Trombocitopenia/genética , Deformidades Congênitas das Extremidades Superiores/genética , Regiões 5' não Traduzidas/genética , Adolescente , Adulto , Criança , Pré-Escolar , Deleção Cromossômica , Cromossomos Humanos Par 1/genética , Estudos de Coortes , Síndrome Congênita de Insuficiência da Medula Óssea , Feminino , Humanos , Lactente , Recém-Nascido , Íntrons/genética , Masculino , Contagem de Plaquetas , Polimorfismo de Nucleotídeo Único/genética , Proteínas de Ligação a RNA/genética , Rádio (Anatomia)/patologia , Trombocitopenia/diagnóstico , Trombocitopenia/patologia , Deformidades Congênitas das Extremidades Superiores/diagnóstico , Deformidades Congênitas das Extremidades Superiores/patologia , Adulto JovemRESUMO
BACKGROUND: Hermansky-Pudlak syndrome (HPS) is a rare autosomal recessive disease characterized by oculocutaneous albinism and platelet dysfunction. We report on a novel HPS6 homozygous frameshift variant (c.1919_1920delTC; p.Val640Glyfs*29) in a nonconsanguineous Caucasian family with two affected siblings (index patients) who presented with oculocutaneous albinism at birth and a mild bleeding phenotype during childhood and adolescence. PROCEDURE: Genetic analysis was conducted by panel-based next-generation sequencing (NGS) and Sanger sequencing. Platelets of the index patients, their parents, and the unaffected sister were then comprehensively evaluated by luminoaggregometry, whole blood flow cytometry, immunoblotting, immunofluorescence, and transmission electron microscopy. RESULTS: The homozygous frameshift variant in HPS6 gene detected by panel-based NGS and its segregation in the family was confirmed by Sanger sequencing. Flow cytometric analysis of the patients' platelets revealed a substantially decreased mepacrine uptake and release upon activation with a thrombin receptor agonist. Electron microscopy of resting platelets confirmed diminished dense granule content and enhanced vacuolization. Reduced release of adenosine triphosphate and CD63 neoexposition upon activation indicated not only a lack of dense granule content, but even an impairment of dense granule release. CONCLUSIONS: Our results demonstrate that the novel loss-of-function variant in the HPS6 subunit of biogenesis of lysosome-related organelles complex 2 is pathologic and leads to a reduced platelet dense granules and their release. The findings are compatible with an impaired platelet function and hence an enhanced bleeding risk. In future, a valid genotype-phenotype correlation may translate into best supportive care, especially regarding elective surgery or trauma management.
Assuntos
Antineoplásicos/metabolismo , Plaquetas/metabolismo , Síndrome de Hermanski-Pudlak/genética , Peptídeos e Proteínas de Sinalização Intracelular/genética , Quinacrina/metabolismo , Trifosfato de Adenosina/metabolismo , Adolescente , Sequência de Bases , Transporte Biológico/genética , Plaquetas/citologia , Criança , Feminino , Citometria de Fluxo , Mutação da Fase de Leitura/genética , Estudos de Associação Genética , Sequenciamento de Nucleotídeos em Larga Escala , Humanos , Masculino , Microscopia Eletrônica , Análise de Sequência de DNA , Deleção de Sequência/genética , Tetraspanina 30/metabolismoRESUMO
Blood platelets are anuclear cell fragments that are essential for blood clotting. Platelets are produced by bone marrow megakaryocytes (MKs), which extend protrusions, or so-called proplatelets, into bone marrow sinusoids. Proplatelet formation requires a profound reorganization of the MK actin and tubulin cytoskeleton. Rho GTPases, such as RhoA, Rac1, and Cdc42, are important regulators of cytoskeletal rearrangements in platelets; however, the specific roles of these proteins during platelet production have not been established. Using conditional knockout mice, we show here that Rac1 and Cdc42 possess redundant functions in platelet production and function. In contrast to a single-deficiency of either protein, a double-deficiency of Rac1 and Cdc42 in MKs resulted in macrothrombocytopenia, abnormal platelet morphology, and impaired platelet function. Double-deficient bone marrow MKs matured normally in vivo but displayed highly abnormal morphology and uncontrolled fragmentation. Consistently, a lack of Rac1/Cdc42 virtually abrogated proplatelet formation in vitro. Strikingly, this phenotype was associated with severely defective tubulin organization, whereas actin assembly and structure were barely affected. Together, these results suggest that the combined action of Rac1 and Cdc42 is crucial for platelet production, particularly by regulating microtubule dynamics.
Assuntos
Células Progenitoras de Megacariócitos/metabolismo , Megacariócitos/metabolismo , Tubulina (Proteína)/metabolismo , Proteína cdc42 de Ligação ao GTP/genética , Proteínas rac1 de Ligação ao GTP/genética , Animais , Western Blotting , Citoesqueleto/metabolismo , Hemostasia/genética , Células Progenitoras de Megacariócitos/citologia , Megacariócitos/citologia , Megacariócitos/ultraestrutura , Camundongos , Camundongos da Linhagem 129 , Camundongos Endogâmicos C57BL , Camundongos Knockout , Microscopia Confocal , Microscopia Eletrônica de Varredura , Microscopia Eletrônica de Transmissão , Microtúbulos/metabolismo , Pseudópodes/genética , Pseudópodes/metabolismo , Trombocitopenia/sangue , Trombocitopenia/genética , Trombocitopenia/metabolismo , Trombose/sangue , Trombose/genética , Trombose/metabolismo , Proteína cdc42 de Ligação ao GTP/deficiência , Proteínas rac1 de Ligação ao GTP/deficiênciaRESUMO
Disabling mutations in integrin-mediated cell signaling have been a major focus of interest over the last decade for patients affected with leukocyte adhesion deficiency-III (LAD-III). In this study, we identified a new C>T point mutation in exon 13 in the FERMT3 gene in an infant diagnosed with LAD-III and showed that KINDLIN-3 expression is required for platelet aggregation and leukocyte function, but also osteoclast-mediated bone resorption. After allogeneic bone marrow transplant, all overt symptoms disappeared. This newly identified mutation along with its novel role in dysregulation of bone homeostasis extends our understanding of KINDLIN-3 in humans.
Assuntos
Plaquetas/fisiologia , Reabsorção Óssea/genética , Códon sem Sentido , Integrinas/fisiologia , Síndrome da Aderência Leucocítica Deficitária/genética , Leucócitos/fisiologia , Proteínas de Membrana/genética , Mutação de Sentido Incorreto , Proteínas de Neoplasias/genética , Osteoclastos/fisiologia , Osteopetrose/genética , Mutação Puntual , Transplante de Medula Óssea , Reabsorção Óssea/patologia , Adesão Celular , Núcleo Celular/ultraestrutura , Éxons/genética , Feminino , Transtornos Hemorrágicos/genética , Homeostase , Humanos , Recém-Nascido , Síndrome da Aderência Leucocítica Deficitária/patologia , Síndrome da Aderência Leucocítica Deficitária/terapia , Proteínas de Membrana/deficiência , Proteínas de Membrana/fisiologia , Proteínas de Neoplasias/deficiência , Proteínas de Neoplasias/fisiologia , Osteoclastos/ultraestrutura , Osteopetrose/patologia , Osteopetrose/terapia , Agregação Plaquetária/genética , Indução de RemissãoRESUMO
The crucial function of blood platelets in hemostasis is to prevent blood loss by stable thrombus formation. This process is driven by orchestrated mechanisms including several signal transduction cascades and morphologic transformations. The cytoplasmic microtubule modulator RanBP10 is a Ran and ß1-tubulin binding protein that is essential for platelet granule release and mice lacking RanBP10 harbor a severe bleeding phenotype. In this study, we demonstrate that RanBP10-nullizygous platelets show normal adhesion on collagen and von Willebrand factor under flow conditions. However, using a ferric chloride-induced arterial thrombosis model, the formation of stable thrombi was significantly impaired, preventing vessel occlusion or leading to recanalization and thromboembolization. Delta-granule secretion was normal in mutant mice, whereas platelet shape change in aggregometry was attenuated. Lack of RanBP10 leads to increased ß1-tubulin protein, which drives α-monomers into polymerized microtubules. In mutant platelets agonists failed to contract the peripheral marginal band or centralize granules. Pretreatment of wild-type platelets with taxol caused microtubule stabilization and phenocopied the attenuated shape change in response to collagen, suggesting that RanBP10 inhibits premature microtubule polymerization of ß1-tubulin and plays a pivotal role in thrombus stabilization.
Assuntos
Plaquetas/metabolismo , Fatores de Troca do Nucleotídeo Guanina/genética , Proteínas Associadas aos Microtúbulos/genética , Microtúbulos/metabolismo , Trombose/metabolismo , Tubulina (Proteína)/metabolismo , Animais , Artérias/metabolismo , Artérias/patologia , Plaquetas/efeitos dos fármacos , Plaquetas/patologia , Cloretos , Colágeno/metabolismo , Grânulos Citoplasmáticos , Compostos Férricos , Expressão Gênica/efeitos dos fármacos , Fatores de Troca do Nucleotídeo Guanina/deficiência , Hemorreologia , Camundongos , Camundongos Knockout , Proteínas Associadas aos Microtúbulos/deficiência , Microtúbulos/efeitos dos fármacos , Microtúbulos/genética , Paclitaxel/farmacologia , Ativação Plaquetária/efeitos dos fármacos , Adesividade Plaquetária/efeitos dos fármacos , Agregação Plaquetária/efeitos dos fármacos , Polimerização , Isoformas de Proteínas/genética , Isoformas de Proteínas/metabolismo , Transdução de Sinais/efeitos dos fármacos , Trombose/induzido quimicamente , Trombose/genética , Tubulina (Proteína)/genética , Fator de von Willebrand/metabolismoRESUMO
BACKGROUND: Extracorporeal membrane oxygenation (ECMO) is a lifesaving therapy in patients with acute respiratory distress syndrome (ARDS). Hemostatic complications are frequently observed in patients on ECMO and limit the success of this therapy. Platelets are key mediators of hemostasis enabling activation, aggregation, and thrombus formation by coming in contact with exposed matrix proteins via their surface receptors such as glycoprotein (GP) VI or GPIb/V/IX. Recent research has elucidated a regulatory role of the GPV subunit. The cleaved soluble GPV (sGPV) ectodomain was identified to spatiotemporally control fibrin formation through complex formation with thrombin. OBJECTIVES: We aimed to decipher the impact of ECMO on platelet phenotype and function, including the role of GPV and plasmatic sGPV. METHODS: We recruited 36 patients with ARDS in the wake of COVID-19 pneumonia and performed a longitudinal comparison of platelet phenotype and function in non-ECMO (n = 23) vs ECMO (n = 13) compared with those of healthy controls. Patients were assessed at up to 3 time points (t1 = days 1-3; t2 = days 4-6; and t3 = days 7-14 after cannulation/study inclusion). RESULTS: Agonist-induced platelet activation was assessed by flow cytometry and revealed decreased GPIIb/IIIa activation and α-granule release in all ARDS patients. During ECMO treatment, agonist-induced δ-granule release continuously decreased, which was independently confirmed by electron microscopy and was associated with a prolonged in vitro bleeding time. GPV expression on the platelet surface markedly decreased in ECMO patients compared with that in non-ECMO patients. Plasma sGPV levels were increased in ECMO patients and were associated with poor outcome. CONCLUSION: Our data demonstrate an ECMO-intrinsic platelet δ-granule deficiency and hemostatic dysfunction beyond the underlying ARDS.