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Genome-wide platelet transcriptomics is increasingly used to uncover new aspects of platelet biology and as a diagnostic and prognostic tool. Nevertheless, platelet isolation methods for transcriptomic studies are not standardized, introducing challenges for cross-study comparisons, data integration, and replication. In this prospective multicenter study, called "Standardizing Platelet Transcriptomics for Discovery, Diagnostics, and Therapeutics in the Thrombosis and Hemostasis Community (STRIDE)" by the International Society on Thrombosis and Haemostasis Scientific and Standardization Committees, we assessed how 3 of the most commonly used platelet isolation protocols influence metrics from next-generation bulk RNA sequencing and functional assays. Compared with washing alone, more stringent removal of leukocytes by anti-CD45 beads or PALL filters resulted in a sufficient quantity of RNA for next-generation sequencing and similar quality of RNA sequencing metrics. Importantly, stringent removal of leukocytes resulted in the lower relative expression of known leukocyte-specific genes and the higher relative expression of known platelet-specific genes. The results were consistent across enrolling sites, suggesting that the techniques are transferrable and reproducible. Moreover, all 3 isolation techniques did not influence basal platelet reactivity, but agonist-induced integrin αIIbß3 activation is reduced by anti-CD45 bead isolation compared with washing alone. In conclusion, the isolation technique chosen influences genome-wide transcriptional and functional assays in platelets. These results should help the research community make informed choices about platelet isolation techniques in their own platelet studies.
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Background: In hemophilia and von Willebrand disease, the degree of alteration of laboratory assays correlates with bleeding manifestations. Few studies have assessed the predictive value for bleeding of laboratory assays in patients with inherited platelet function disorders (IPFDs). Objectives: To assess whether there is an association between platelet function assay results and bleeding history, as evaluated by the International Society on Thrombosis and Haemostasis (ISTH) bleeding assessment tool (BAT). Methods: Centers participating in the international ISTH-BAT validation study were asked to provide results of the diagnostic assays employed for the patients they enrolled, and the association with the individual patients' bleeding score (BS) was assessed. Results: Sixty-eight patients with 14 different IPFDs were included. Maximal amplitude of platelet aggregation was significantly lower in patients with a pathologic BS and correlated inversely with the BS, a finding largely driven by the subgroup of patients with Glanzmann thrombasthenia and CalDAG-GEFI deficiency; after their exclusion, TRAP-induced aggregation remained significantly lower in patients with a pathologic BS. Bleeding time was significantly more prolonged in patients with a high BS than in those with a normal BS (27.1 ± 6.2 minutes vs 15.1 ± 10.6 minutes; P < .01). Reduced α-granule content was significantly more common among patients with a pathologic BS than among those with a normal BS (80% vs 20%; P < .05). Receiver operating characteristic curve analysis revealed a significant discriminative ability of all the aforementioned tests for pathologic BS (P < .001), also after exclusion of patients with Glanzmann thrombasthenia and CalDAG-GEFI deficiency. Conclusion: This study shows that altered platelet laboratory assay results are associated with an abnormal ISTH-BAT BS in IPFD.
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BACKGROUND: Inherited bleeding, thrombotic, and platelet disorders (BTPDs) are a heterogeneous set of diseases, many of which are very rare globally. Over the past 5 decades, the genetic basis of some of these disorders has been identified, and recently, high-throughput sequencing has become the primary means of identifying disease-causing genetic variants. OBJECTIVES: Knowledge of the clinical validity of a gene-disease relationship is essential to provide an accurate diagnosis based on results of diagnostic gene panel tests and inform the construction of such panels. The Scientific and Standardization Committee for Genetics in Thrombosis and Hemostasis undertook a curation process for selecting 96 TIER1 genes for BTPDs. The purpose of the process was to evaluate the evidence supporting each gene-disease relationship and provide an expert-reviewed classification for the clinical validity of genes associated with BTPDs. METHODS: The Clinical Genome Resource (ClinGen) Hemostasis/Thrombosis Gene Curation Expert Panel assessed the strength of evidence for TIER1 genes using the semiquantitative ClinGen gene-disease clinical validity framework. ClinGen Lumping and Splitting guidelines were used to determine the appropriate disease entity or entities for each gene, and 101 gene-disease relationships were identified for curation. RESULTS: The final outcome included 68 Definitive (67%), 26 Moderate (26%), and 7 Limited (7%) classifications. The summary of each curation is available on the ClinGen website. CONCLUSION: Expert-reviewed assignment of gene-disease relationships by the ClinGen Hemostasis/Thrombosis Gene Curation Expert Panel facilitates accurate molecular diagnoses of BTPDs by clinicians and diagnostic laboratories. These curation efforts can allow genetic testing to focus on genes with a validated role in disease.
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Trastornos de las Plaquetas Sanguíneas , Trombosis , Humanos , Pruebas Genéticas/métodos , Trastornos de las Plaquetas Sanguíneas/genética , Hemostasis/genética , Trombosis/diagnóstico , Trombosis/genética , Variación GenéticaRESUMEN
GATA2 is a transcription factor with key roles in hematopoiesis. Germline GATA2 gene variants have been associated with several inherited and acquired hematologic disorders, including myelodysplastic syndromes. Among the spectrum of GATA2 deficiency- associated manifestations thrombosis has been reported in 25% of patients, but the mechanisms are unknown. GATA2 was shown to be involved in endothelial nitric oxide synthase (eNOS) regulation and vascular development. We assessed eNOS expression and angiogenesis in patients with GATA2 deficiency. Platelets and blood outgrowth endothelial cells (BOEC) from GATA2 variant carriers showed impaired NO production and reduction of eNOS mRNA and protein expression and of eNOS activity. GATA2 binding to the eNOS gene was impaired in BOEC from GATA2-deficient patients, differently from control BOEC. GATA2 deficiency BOEC showed also defective angiogenesis, which was completely restored by treatment with the NO-donor Snitroso- N-acetylpenicillamine (SNAP). Atorvastatin, but not resveratrol, largely restored eNOS expression, NO biosynthesis and neoangiogenesis in GATA2-deficient BOEC by a mechanism involving increased expression of the eNOS transcription factor AP-1/c-JUN, replacing GATA2 when the latter is inactive. Our results unravel a possible thrombogenic mechanism of GATA2 mutations, definitely establish the regulation of eNOS by GATA2 in endothelial cells and show that endothelial angiogenesis is strictly dependent on the eNOS/NO axis. Given the ability of atorvastatin to restore NO production and angiogenesis by GATA2-deficient endothelial cells, the preventive effect of atorvastatin on thrombotic events and possibly on other clinical manifestations of the syndrome related to deranged angiogenesis should be explored in patients with GATA2 deficiency in an ad hoc designed clinical trial.
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Deficiencia GATA2 , Óxido Nítrico Sintasa de Tipo III , Atorvastatina/farmacología , Células Endoteliales/metabolismo , Endotelio Vascular/metabolismo , Factor de Transcripción GATA2/genética , Células Germinativas/metabolismo , Humanos , Óxido Nítrico Sintasa/genética , Óxido Nítrico Sintasa/metabolismo , Óxido Nítrico Sintasa de Tipo III/genética , Óxido Nítrico Sintasa de Tipo III/metabolismo , Óxido Nítrico Sintasa de Tipo III/farmacología , Factor de Transcripción AP-1/genética , Factor de Transcripción AP-1/metabolismo , Regulación hacia ArribaRESUMEN
Platelet-type von Willebrand disease (PT-VWD) is an inherited platelet disorder. It is characterized by macrothrombocytopenia and mucocutaneous bleeding, of variable severity, due to gain-of-function variants of GP1BA conferring to glycoprotein Ibα (GPIbα) enhanced affinity for von Willebrand factor (VWF). The bleeding tendency is conventionally attributed to thrombocytopenia and large VWF-multimer depletion. However, while some indications suggest that platelet dysfunction may contribute to the bleeding phenotype, no information on its characteristics and causes are available. The aim of the present study was to characterize platelet dysfunction in PT-VWD and shed light on its mechanism. Platelets from a PT-VWD patient carrying the p.M239V variant, and from PT-VWD mice carrying the p.G233V variant, showed a remarkable platelet function defect, with impaired aggregation, defective granule secretion and reduced adhesion under static and flow conditions. VWFbinding to GPIbα is known to trigger intracellular signaling involving Src-family kinases (SFK). We found that constitutive phosphorylation of the platelet SFK Lyn induces a negative-feedback loop downregulating platelet activation through phosphorylation of PECAM1 on Tyr686 and that this is triggered by the constitutive binding of VWF to GPIbα. These data show, for the first time, that the abnormal triggering of inhibitory signals mediated by Lyn and PECAM1 may lead to platelet dysfunction. In conclusion, our study unravels the mechanism of platelet dysfunction in PT-VWD caused by deranged inhibitory signaling. This is triggered by the constitutive binding of VWF to GPIbα which may significantly contribute to the bleeding phenotype of these patients.
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Trombocitopenia , Enfermedades de von Willebrand , Animales , Plaquetas/metabolismo , Hemorragia/genética , Ratones , Molécula-1 de Adhesión Celular Endotelial de Plaqueta/metabolismo , Complejo GPIb-IX de Glicoproteína Plaquetaria/genética , Complejo GPIb-IX de Glicoproteína Plaquetaria/metabolismo , Trombocitopenia/genética , Trombocitopenia/metabolismo , Enfermedades de von Willebrand/genética , Enfermedades de von Willebrand/metabolismo , Factor de von Willebrand/genética , Factor de von Willebrand/metabolismoRESUMEN
BACKGROUND: Circulating dysfunctional factor IX (FIX) might modulate distribution of infused FIX in hemophilia B (HB) patients. Recurrent substitutions at FIX activation sites (R191-R226, >300 patients) are associated with variable FIX activity and antigen (FIXag) levels. OBJECTIVES: To investigate the (1) expression of a complete panel of missense mutations at FIX activation sites and (2) contribution of F9 genotypes on the FIX pharmacokinetics (PK). METHODS: We checked FIX activity and antigen and activity assays in plasma and after recombinant expression of FIX variants and performed an analysis of infused FIX PK parameters in patients (n = 30), mostly enrolled in the F9 Genotype and PK HB Italian Study (GePKHIS; EudraCT ID2017-003902-42). RESULTS: The variable FIXag amounts and good relation between biosynthesis and activity of multiple R191 variants results in graded moderate-to-mild severity of the R191C>L>P>H substitutions. Recombinant expression may predict the absence in the HB mutation database of the benign R191Q/W/K and R226K substitutions. Equivalent changes at R191/R226 produced higher FIXag levels for R226Q/W/P substitutions, as also observed in p.R226W female carrier plasma. Pharmacokinetics analysis in patients suggested that infused FIX Alpha distribution and Beta elimination phases positively correlated with endogenous FIXag levels. Mean residence time was particularly prolonged (79.4 h, 95% confidence interval 44.3-114.5) in patients (n = 7) with the R191/R226 substitutions, which in regression analysis were independent predictors (ß coefficient 0.699, P = .004) of Beta half-life, potentially prolonged by the increasing over time ratio between endogenous and infused FIX. CONCLUSIONS: FIX activity and antigen levels and specific features of the dysfunctional R191/R226 variants may exert pleiotropic effects both on HB patients' phenotypes and substitutive treatment.
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Factor IX , Hemofilia B , Pruebas de Coagulación Sanguínea , Factor IX/metabolismo , Femenino , Hemofilia B/diagnóstico , Hemofilia B/tratamiento farmacológico , Hemofilia B/genética , Humanos , Mutación Missense , FenotipoRESUMEN
Gain-of-function (GOF) variants in GP1BA cause platelet-type von Willebrand disease (PT-VWD), a rare inherited autosomal dominant bleeding disorder characterized by enhanced platelet GPIbα to von Willebrand factor (VWF) interaction, and thrombocytopenia. To date, only 6 variants causing PT-VWD have been described, 5 in the C-terminal disulfide loop of the VWF-binding domain of GPIbα and 1 in the macroglycopeptide. GOF GP1BA variants generate a high-affinity conformation of the C-terminal disulfide loop with a consequent allosteric conformational change on another region of GPIbα, the leucine-rich-repeat (LRR) domain. We identified a novel GP1BA variant (p.Arg127Gln) affecting the LRR5 domain of GPIbα in a boy with easy bruising and laboratory test results suggestive of PT-VWD. We thus aimed to investigate the impact of the p.Arg127Gln variant on GPIbα affinity for VWF and GPIbα structure. Chinese hamster ovary cells expressing p.Arg127Gln GPIbα showed increased binding of VWF induced by ristocetin and enhanced tethering on immobilized VWF as compared with cells expressing wild-type GPIbα. Surface plasmon resonance confirmed that p.Arg127Gln enhances the binding affinity of GPIbα for VWF. Hydrogen-deuterium exchange mass spectrometry showed that p.Arg127Gln of LRR, while having little effect on the dynamics of the LRR locally, enhances the conformational dynamics of the GPIbα C-terminal disulfide loop structure. Our data demonstrate for the first time that GOF variants outside the GPIbα C-terminal disulfide loop may be pathogenic and that aminoacidic changes in the LRR may cause allosterically conformational changes in the C-terminal disulfide loop of GPIbα, inducing a conformation with high affinity for VWF.
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Enfermedades de von Willebrand , Factor de von Willebrand , Animales , Células CHO , Cricetinae , Cricetulus , Humanos , Masculino , Complejo GPIb-IX de Glicoproteína Plaquetaria , Unión Proteica , Factor de von Willebrand/metabolismoRESUMEN
ß1-Tubulin plays a major role in proplatelet formation and platelet shape maintenance, and pathogenic variants in TUBB1 lead to thrombocytopenia and platelet anisocytosis (TUBB1-RT). To date, the reported number of pedigrees with TUBB1-RT and of rare TUBB1 variants with experimental demonstration of pathogenicity is limited. Here, we report 9 unrelated families presenting with thrombocytopenia carrying 6 ß1-tubulin variants, p.Cys12LeufsTer12, p.Thr107Pro, p.Gln423*, p.Arg359Trp, p.Gly109Glu, and p.Gly269Asp, the last of which novel. Segregation studies showed incomplete penetrance of these variants for platelet traits. Indeed, most carriers showed macrothrombocytopenia, some only increased platelet size, and a minority had no abnormalities. Moreover, only homozygous carriers of the p.Gly109Glu variant displayed macrothrombocytopenia, highlighting the importance of allele burden in the phenotypic expression of TUBB1-RT. The p.Arg359Trp, p.Gly269Asp, and p.Gly109Glu variants deranged ß1-tubulin incorporation into the microtubular marginal ring in platelets but had a negligible effect on platelet activation, secretion, or spreading, suggesting that ß1-tubulin is dispensable for these processes. Transfection of TUBB1 missense variants in CHO cells altered ß1-tubulin incorporation into the microtubular network. In addition, TUBB1 variants markedly impaired proplatelet formation from peripheral blood CD34+ cell-derived megakaryocytes. Our study, using in vitro modeling, molecular characterization, and clinical investigations provides a deeper insight into the pathogenicity of rare TUBB1 variants. These novel data expand the genetic spectrum of TUBB1-RT and highlight a remarkable heterogeneity in its clinical presentation, indicating that allelic burden or combination with other genetic or environmental factors modulate the phenotypic impact of rare TUBB1 variants.
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Trombocitopenia , Tubulina (Proteína) , Plaquetas , Humanos , Megacariocitos , Trombocitopenia/genética , Tubulina (Proteína)/genéticaRESUMEN
The implementation of high-throughput sequencing (HTS) technologies in research and diagnostic laboratories has linked many new genes to rare bleeding, thrombotic, and platelet disorders (BTPD), and revealed multiple genetic variants linked to those disorders, many of them being of uncertain pathogenicity when considering the accepted evidence (variant consequence, frequency in control datasets, number of reported patients, prediction models, and functional assays). The sequencing effort has also resulted in resources for gathering disease-causing variants associated with specific genes, but for BTPD, such well-curated databases exist only for a few genes. On the other hand, submissions by individuals or diagnostic laboratories to the variant database ClinVar are hampered by the lack of a submission process tailored to capture the specific features of hemostatic diseases. As we move toward the implementation of HTS in the diagnosis of BTPD, the Scientific and Standardization Committee for Genetics in Thrombosis and Haemostasis has developed and tested a REDCap-based interface, aimed at the community, to submit curated genetic variants for diagnostic-grade BTPD genes. Here, we describe the use of the interface and the initial submission of 821 variants from 30 different centers covering 14 countries. This open-access variant resource will be shared with the community to improve variant classification and regular bulk data transfer to ClinVar.
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Trastornos de las Plaquetas Sanguíneas , Trombosis , Trastornos de las Plaquetas Sanguíneas/diagnóstico , Trastornos de las Plaquetas Sanguíneas/genética , Comunicación , Genómica , Hemostasis/genética , Humanos , Trombosis/diagnóstico , Trombosis/genéticaRESUMEN
BACKGROUND: The ISTH Bleeding Assessment Tool (ISTH-BAT) has been validated for clinical screening of suspected von Willebrand disease (VWD) and for bleeding prediction. Recently it has been validated for subjects with inherited platelet disorders (IPD) (BAT-VAL study). OBJECTIVES: To determine whether the ISTH-BAT bleeding score (BS) predicts subsequent bleeding events requiring treatment in IPD patients. METHODS: Patients with IPD, type 1 VWD (VWD-1) and age- and sex-matched healthy controls enrolled in the BAT-VAL study were prospectively followed-up for 2 years and bleeding episodes requiring treatment were recorded. RESULTS: Of the 1098 subjects initially enrolled, 955 were followed-up and 124 suffered hemorrhages during follow-up, 60% of whom had inherited platelet function disorders (IPFD). Total number of events was significantly higher in IPFD (n = 235) than VWD-1 (n = 52) or inherited thrombocytopenia (IT; n = 20). Events requiring transfusions were 66% in IPFD, 5.7% in VWD-1, and 3% in IT. Baseline BS was significantly higher in IPFD patients with a bleeding event at follow-up than in those without (p < .01) and the percentage of subjects suffering a bleeding event increased proportionally to baseline BS quartile. A significant association between the BS and the chance of suffering severe bleeding was found in the overall, IPFD, and VWD-1 populations. Similar results were obtained for the pediatric population. CONCLUSIONS: Inherited platelet function disorder patients with high BS at enrollment are more likely to suffer from bleeding events requiring treatment at follow-up. Moreover, the higher the baseline BS quartile the greater the incidence of subsequent events, suggesting that independently from diagnosis a high BS is associated with a greater risk of subsequent hemorrhage.
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Trastornos de las Plaquetas Sanguíneas , Enfermedades de von Willebrand , Trastornos de las Plaquetas Sanguíneas/diagnóstico , Trastornos de las Plaquetas Sanguíneas/genética , Niño , Comunicación , Hemorragia/diagnóstico , Humanos , Pruebas de Función Plaquetaria , Enfermedades de von Willebrand/diagnóstico , Enfermedades de von Willebrand/genética , Factor de von WillebrandRESUMEN
Inherited thrombocytopenias (IT) are a group of hereditary disorders characterized by a reduced platelet count sometimes associated with abnormal platelet function, which can lead to bleeding but also to syndromic manifestations and predispositions to other disorders. Currently at least 41 disorders caused by mutations in 42 different genes have been described. The pathogenic mechanisms of many forms of IT have been identified as well as the gene variants implicated in megakaryocyte maturation or platelet formation and clearance, while for several of them the pathogenic mechanism is still unknown. A range of therapeutic approaches are now available to improve survival and quality of life of patients with IT; it is thus important to recognize an IT and establish a precise diagnosis. ITs may be difficult to diagnose and an initial accurate clinical evaluation is mandatory. A combination of clinical and traditional laboratory approaches together with advanced sequencing techniques provide the highest rate of diagnostic success. Despite advancement in the diagnosis of IT, around 50% of patients still do not receive a diagnosis, therefore further research in the field of ITs is warranted to further improve patient care.
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Gastrointestinal angiodysplasia (GIA) is the most common cause of occult gastrointestinal bleeding (GIB) requiring often hospitalization and transfusions, especially in patients with hemorrhagic disorders. Thalidomide, impairing neo-angiogenesis, has been successfully used in the management of bleeding in patients with GIA and in particular in patients with inherited bleeding disorders. Only one case of short-term treatment with thalidomide in a patient with Glanzmann thrombasthenia (GT) and recurrent GIB due to GIA has been reported so far.We report the case of a woman with GT developing high frequency recurrent GIB due to GIA requiring repeated blood and platelet transfusions, who was treated with thalidomide obtaining a striking and stable reduction of GIB and of the requirement of platelet and blood transfusions for over 5 years. Moreover, we raise the suspicion that the association between GT and GIA may not be fortuitous.
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Angiodisplasia/complicaciones , Angiodisplasia/tratamiento farmacológico , Talidomida/uso terapéutico , Trombastenia/complicaciones , Trombastenia/tratamiento farmacológico , Anciano , Femenino , Humanos , Talidomida/farmacologíaRESUMEN
The frequent finding of thrombocytopenia in patients with severe SARS-CoV-2 infection (COVID-19) and previous evidence that several viruses enter platelets suggest that SARS-CoV-2 might be internalized by platelets of COVID-19. Aim of our study was to assess the presence of SARS-CoV-2 RNA in platelets from hospitalized patients with aconfirmed diagnosis of COVID-19. RNA was extracted from platelets, leukocytes and serum from 24 COVID-19 patients and 3 healthy controls, real-time PCR and ddPCR for viral genes were carried out. SARS-CoV-2 RNA was not detected in any of the samples analyzed nor in healthy controls, by either RT-PCR or ddPCR, while RNA samples from nasopharyngeal swabs of COVID-19 patients were correctly identified. Viral RNA was not detected independently of viral load, of positive nasopharyngeal swabs, or viremia, the last detected in only one patient (4.1%). SARS-CoV-2 entry in platelets is not acommon phenomenon in COVID-19 patients, differently from other viral infections.
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Plaquetas/virología , COVID-19/sangre , COVID-19/virología , ARN Viral , SARS-CoV-2/fisiología , Anciano , COVID-19/diagnóstico , Prueba de COVID-19 , Femenino , Humanos , Masculino , Persona de Mediana Edad , Reacción en Cadena en Tiempo Real de la Polimerasa , SARS-CoV-2/aislamiento & purificación , Carga ViralRESUMEN
BACKGROUND: Severe acute respiratory syndrome coronavirus 2 infection is associated with hypercoagulability, which predisposes to venous thromboembolism (VTE). We analyzed platelet and neutrophil activation in patients with coronavirus disease 2019 (COVID-19) and their association with VTE. METHODS: Hospitalized patients with COVID-19 and age- and sex-matched healthy controls were studied. Platelet and leukocyte activation, neutrophil extracellular traps (NETs), and matrix metalloproteinase 9, a neutrophil-released enzyme, were measured. Four patients were restudied after recovery. The activating effect of plasma from patients with COVID-19 on control platelets and leukocytes and the inhibiting activity of common antithrombotic agents on it were studied. RESULTS: A total of 36 patients with COVID-19 and 31 healthy controls were studied; VTE developed in 8 of 36 patients with COVID-19 (22.2%). Platelets and neutrophils were activated in patients with COVID-19. NET, but not platelet activation, biomarkers correlated with disease severity and were associated with thrombosis. Plasmatic matrix metalloproteinase 9 was significantly increased in patients with COVID-19. Platelet and neutrophil activation markers, but less so NETs, normalized after recovery. In vitro, plasma from patients with COVID-19 triggered platelet and neutrophil activation and NET formation, the latter blocked by therapeutic-dose low-molecular-weight heparin, but not by aspirin or dypiridamole. CONCLUSIONS: Platelet and neutrophil activation are key features of patients with COVID-19. NET biomarkers may help to predict clinical worsening and VTE and may guide low-molecular-weight heparin treatment.
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COVID-19/sangre , COVID-19/inmunología , Trombosis/sangre , Trombosis/inmunología , Adulto , Anciano , Anciano de 80 o más Años , Biomarcadores/sangre , Plaquetas/inmunología , COVID-19/virología , Trampas Extracelulares , Femenino , Heparina de Bajo-Peso-Molecular/sangre , Humanos , Masculino , Metaloproteinasa 9 de la Matriz/sangre , Persona de Mediana Edad , Activación Neutrófila , Neutrófilos/inmunología , Activación Plaquetaria , SARS-CoV-2/aislamiento & purificación , Trombosis/virología , Tromboembolia Venosa/sangre , Tromboembolia Venosa/inmunología , Tromboembolia Venosa/virologíaRESUMEN
Gray platelet syndrome (GPS) is a rare recessive disorder caused by biallelic variants in NBEAL2 and characterized by bleeding symptoms, the absence of platelet α-granules, splenomegaly, and bone marrow (BM) fibrosis. Due to the rarity of GPS, it has been difficult to fully understand the pathogenic processes that lead to these clinical sequelae. To discern the spectrum of pathologic features, we performed a detailed clinical genotypic and phenotypic study of 47 patients with GPS and identified 32 new etiologic variants in NBEAL2. The GPS patient cohort exhibited known phenotypes, including macrothrombocytopenia, BM fibrosis, megakaryocyte emperipolesis of neutrophils, splenomegaly, and elevated serum vitamin B12 levels. Novel clinical phenotypes were also observed, including reduced leukocyte counts and increased presence of autoimmune disease and positive autoantibodies. There were widespread differences in the transcriptome and proteome of GPS platelets, neutrophils, monocytes, and CD4 lymphocytes. Proteins less abundant in these cells were enriched for constituents of granules, supporting a role for Nbeal2 in the function of these organelles across a wide range of blood cells. Proteomic analysis of GPS plasma showed increased levels of proteins associated with inflammation and immune response. One-quarter of plasma proteins increased in GPS are known to be synthesized outside of hematopoietic cells, predominantly in the liver. In summary, our data show that, in addition to the well-described platelet defects in GPS, there are immune defects. The abnormal immune cells may be the drivers of systemic abnormalities such as autoimmune disease.
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Gránulos Citoplasmáticos/patología , Heterogeneidad Genética , Síndrome de Plaquetas Grises , Sistema Inmunológico/patología , Fenotipo , Biopsia , Proteínas Sanguíneas/genética , Estudios de Casos y Controles , Estudios de Cohortes , Gránulos Citoplasmáticos/metabolismo , Diagnóstico Diferencial , Frecuencia de los Genes , Estudios de Asociación Genética , Síndrome de Plaquetas Grises/clasificación , Síndrome de Plaquetas Grises/genética , Síndrome de Plaquetas Grises/inmunología , Síndrome de Plaquetas Grises/patología , Humanos , Sistema Inmunológico/fisiología , Enfermedades del Sistema Inmune/sangre , Enfermedades del Sistema Inmune/diagnóstico , Enfermedades del Sistema Inmune/genética , Enfermedades del Sistema Inmune/patología , MutaciónRESUMEN
Chemotherapy is the mainstay of treatment for advanced pancreatic cancer however, due to possible myelotoxicity, it is used with caution in patients with thrombocytopenia, especially when severe. TPO-receptor agonists have been employed for chemotherapy-induced thrombocytopenia, however treatment with TPO-receptor agonists to allow chemotherapy in patients with inherited thrombocytopenia has not been reported so far. We report the first successful use of eltrombopag to prevent chemotherapy-induced thrombocytopenia in a patient with MHY9-related disorder and pancreatic cancer. Treatment with eltrombopag allowed to attain a safe and stable platelet count for several months sufficient to permit chemotherapy and to allow the patient to undergo endoscopic placement of a biliary stent with no bleeding complications.
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Benzoatos/uso terapéutico , Hidrazinas/uso terapéutico , Cadenas Pesadas de Miosina , Neoplasias Pancreáticas/tratamiento farmacológico , Pirazoles/uso terapéutico , Trombocitopenia/tratamiento farmacológico , Trombocitopenia/genética , Anciano , Protocolos de Quimioterapia Combinada Antineoplásica/efectos adversos , Benzoatos/farmacología , Pérdida de Sangre Quirúrgica/prevención & control , Endoscopía del Sistema Digestivo , Femenino , Humanos , Hidrazinas/farmacología , Neoplasias Pancreáticas/cirugía , Recuento de Plaquetas , Pirazoles/farmacología , Receptores de Trombopoyetina/agonistas , Trombocitopenia/sangre , Trombocitopenia/congénitoRESUMEN
The heterogeneous manifestations of MYH9-related disorder (MYH9-RD), characterized by macrothrombocytopenia, Döhle-like inclusion bodies in leukocytes, bleeding of variable severity with, in some cases, ear, eye, kidney, and liver involvement, make the diagnosis for these patients still challenging in clinical practice. We collected phenotypic data and analyzed the genetic variants in more than 3,000 patients with a bleeding or platelet disorder. Patients were enrolled in the BRIDGE-BPD and ThromboGenomics Projects and their samples processed by high throughput sequencing (HTS). We identified 50 patients with a rare variant in MYH9. All patients had macrothrombocytes and all except two had thrombocytopenia. Some degree of bleeding diathesis was reported in 41 of the 50 patients. Eleven patients presented hearing impairment, three renal failure and two elevated liver enzymes. Among the 28 rare variants identified in MYH9, 12 were novel. HTS was instrumental in diagnosing 23 patients (46%). Our results confirm the clinical heterogeneity of MYH9-RD and show that, in the presence of an unclassified platelet disorder with macrothrombocytes, MYH9-RD should always be considered. A HTS-based strategy is a reliable method to reach a conclusive diagnosis of MYH9-RD in clinical practice.