RESUMEN
Most patients with rare diseases do not receive a molecular diagnosis and the aetiological variants and causative genes for more than half such disorders remain to be discovered1. Here we used whole-genome sequencing (WGS) in a national health system to streamline diagnosis and to discover unknown aetiological variants in the coding and non-coding regions of the genome. We generated WGS data for 13,037 participants, of whom 9,802 had a rare disease, and provided a genetic diagnosis to 1,138 of the 7,065 extensively phenotyped participants. We identified 95 Mendelian associations between genes and rare diseases, of which 11 have been discovered since 2015 and at least 79 are confirmed to be aetiological. By generating WGS data of UK Biobank participants2, we found that rare alleles can explain the presence of some individuals in the tails of a quantitative trait for red blood cells. Finally, we identified four novel non-coding variants that cause disease through the disruption of transcription of ARPC1B, GATA1, LRBA and MPL. Our study demonstrates a synergy by using WGS for diagnosis and aetiological discovery in routine healthcare.
Asunto(s)
Internacionalidad , Programas Nacionales de Salud , Enfermedades Raras/diagnóstico , Enfermedades Raras/genética , Secuenciación Completa del Genoma , Complejo 2-3 Proteico Relacionado con la Actina/genética , Proteínas Adaptadoras Transductoras de Señales/genética , Alelos , Bases de Datos Factuales , Eritrocitos/metabolismo , Factor de Transcripción GATA1/genética , Humanos , Fenotipo , Sitios de Carácter Cuantitativo , Receptores de Trombopoyetina/genética , Medicina Estatal , Reino UnidoRESUMEN
Fibrinogen is a key component of the coagulation cascade, and variation in its circulating levels may contribute to thrombotic diseases, such as venous thromboembolism (VTE) and ischemic stroke. Gamma prime (γ') fibrinogen is an isoform of fibrinogen that has anticoagulant properties. We applied 2-sample Mendelian randomization (MR) to estimate the causal effect of total circulating fibrinogen and its isoform, γ' fibrinogen, on risk of VTE and ischemic stroke subtypes using summary statistics from genome-wide association studies. Genetic instruments for γ' fibrinogen and total fibrinogen were selected, and the inverse-variance weighted MR approach was used to estimate causal effects in the main analysis, complemented by sensitivity analyses that are more robust to the inclusion of pleiotropic variants, including MR-Egger, weighted median MR, and weighted mode MR. The main inverse-variance weighted MR estimates based on a combination of 16 genetic instruments for γ' fibrinogen and 75 genetic instruments for total fibrinogen indicated a protective effect of higher γ' fibrinogen and higher total fibrinogen on VTE risk. There was also a protective effect of higher γ' fibrinogen levels on cardioembolic and large artery stroke risk. Effect estimates were consistent across sensitivity analyses. Our results provide evidence to support effects of genetically determined γ' fibrinogen on VTE and ischemic stroke risk. Further research is needed to explore mechanisms underlying these effects and their clinical applications.
Asunto(s)
Fibrinógeno , Variación Genética , Accidente Cerebrovascular Isquémico , Análisis de la Aleatorización Mendeliana , Tromboembolia Venosa , Femenino , Fibrinógeno/genética , Fibrinógeno/metabolismo , Estudio de Asociación del Genoma Completo , Humanos , Accidente Cerebrovascular Isquémico/sangre , Accidente Cerebrovascular Isquémico/epidemiología , Accidente Cerebrovascular Isquémico/genética , Masculino , Factores de Riesgo , Tromboembolia Venosa/sangre , Tromboembolia Venosa/epidemiología , Tromboembolia Venosa/genéticaRESUMEN
To identify novel causes of hereditary thrombocytopenia, we performed a genetic association analysis of whole-genome sequencing data from 13 037 individuals enrolled in the National Institute for Health Research (NIHR) BioResource, including 233 cases with isolated thrombocytopenia. We found an association between rare variants in the transcription factor-encoding gene IKZF5 and thrombocytopenia. We report 5 causal missense variants in or near IKZF5 zinc fingers, of which 2 occurred de novo and 3 co-segregated in 3 pedigrees. A canonical DNA-zinc finger binding model predicts that 3 of the variants alter DNA recognition. Expression studies showed that chromatin binding was disrupted in mutant compared with wild-type IKZF5, and electron microscopy revealed a reduced quantity of α granules in normally sized platelets. Proplatelet formation was reduced in megakaryocytes from 7 cases relative to 6 controls. Comparison of RNA-sequencing data from platelets, monocytes, neutrophils, and CD4+ T cells from 3 cases and 14 healthy controls showed 1194 differentially expressed genes in platelets but only 4 differentially expressed genes in each of the other blood cell types. In conclusion, IKZF5 is a novel transcriptional regulator of megakaryopoiesis and the eighth transcription factor associated with dominant thrombocytopenia in humans.
Asunto(s)
Plaquetas , Enfermedades Genéticas Congénitas , Mutación de Línea Germinal , Factor de Transcripción Ikaros , Mutación Missense , Trombocitopenia , Trombopoyesis/genética , Plaquetas/metabolismo , Plaquetas/ultraestructura , Cromatina/genética , Cromatina/metabolismo , Cromatina/ultraestructura , Gránulos Citoplasmáticos/genética , Gránulos Citoplasmáticos/metabolismo , Gránulos Citoplasmáticos/ultraestructura , Femenino , Regulación de la Expresión Génica , Enfermedades Genéticas Congénitas/genética , Enfermedades Genéticas Congénitas/metabolismo , Enfermedades Genéticas Congénitas/patología , Células HEK293 , Humanos , Factor de Transcripción Ikaros/genética , Factor de Transcripción Ikaros/metabolismo , Masculino , Trombocitopenia/genética , Trombocitopenia/metabolismo , Trombocitopenia/patologíaRESUMEN
A targeted high-throughput sequencing (HTS) panel test for clinical diagnostics requires careful consideration of the inclusion of appropriate diagnostic-grade genes, the ability to detect multiple types of genomic variation with high levels of analytic sensitivity and reproducibility, and variant interpretation by a multidisciplinary team (MDT) in the context of the clinical phenotype. We have sequenced 2396 index patients using the ThromboGenomics HTS panel test of diagnostic-grade genes known to harbor variants associated with rare bleeding, thrombotic, or platelet disorders (BTPDs). The molecular diagnostic rate was determined by the clinical phenotype, with an overall rate of 49.2% for all thrombotic, coagulation, platelet count, and function disorder patients and a rate of 3.2% for patients with unexplained bleeding disorders characterized by normal hemostasis test results. The MDT classified 745 unique variants, including copy number variants (CNVs) and intronic variants, as pathogenic, likely pathogenic, or variants of uncertain significance. Half of these variants (50.9%) are novel and 41 unique variants were identified in 7 genes recently found to be implicated in BTPDs. Inspection of canonical hemostasis pathways identified 29 patients with evidence of oligogenic inheritance. A molecular diagnosis has been reported for 894 index patients providing evidence that introducing an HTS genetic test is a valuable addition to laboratory diagnostics in patients with a high likelihood of having an inherited BTPD.
Asunto(s)
Trastornos de las Plaquetas Sanguíneas , Hemorragia , Secuenciación de Nucleótidos de Alto Rendimiento , Trombosis , Trastornos de las Plaquetas Sanguíneas/diagnóstico , Trastornos de las Plaquetas Sanguíneas/genética , Femenino , Dosificación de Gen , Hemorragia/diagnóstico , Hemorragia/genética , Hemostasis/genética , Humanos , Masculino , Trombosis/diagnóstico , Trombosis/genéticaRESUMEN
We have identified a rare missense variant on chromosome 9, position 125145990 (GRCh37), in exon 8 in PTGS1 (the gene encoding cyclo-oxygenase 1, COX-1, the target of anti-thrombotic aspirin therapy). We report that in the homozygous state within a large consanguineous family this variant is associated with a bleeding phenotype and alterations in platelet reactivity and eicosanoid production. Western blotting and confocal imaging demonstrated that COX-1 was absent in the platelets of three family members homozygous for the PTGS1 variant but present in their leukocytes. Platelet reactivity, as assessed by aggregometry, lumi-aggregometry and flow cytometry, was impaired in homozygous family members, as were platelet adhesion and spreading. The productions of COX-derived eicosanoids by stimulated platelets were greatly reduced but there were no changes in the levels of urinary metabolites of COX-derived eicosanoids. The proband exhibited additional defects in platelet aggregation and spreading which may explain why her bleeding phenotype was slightly more severe than those of other homozygous affected relatives. This is the first demonstration in humans of the specific loss of platelet COX-1 activity and provides insight into its consequences for platelet function and eicosanoid metabolism. Notably despite the absence of thromboxane A2 (TXA2) formation by platelets, urinary TXA2 metabolites were in the normal range indicating these cannot be assumed as markers of in vivo platelet function. Results from this study are important benchmarks for the effects of aspirin upon platelet COX-1, platelet function and eicosanoid production as they define selective platelet COX-1 ablation within humans.
Asunto(s)
Aspirina , Pruebas de Función Plaquetaria , Plaquetas , Ciclooxigenasa 1/genética , Femenino , Humanos , Agregación Plaquetaria/genética , Tromboxano A2RESUMEN
Heritable platelet function disorders (PFDs) are genetically heterogeneous and poorly characterized. Pathogenic variants in RASGRP2, which encodes calcium and diacylglycerol-regulated guanine exchange factor I (CalDAG-GEFI), have been reported previously in 3 pedigrees with bleeding and reduced platelet aggregation responses. To better define the phenotype associated with pathogenic RASGRP2 variants, we compared high-throughput sequencing and phenotype data from 2042 cases in pedigrees with unexplained bleeding or platelet disorders to data from 5422 controls. Eleven cases harbored 11 different, previously unreported RASGRP2 variants that were biallelic and likely pathogenic. The variants included 5 high-impact variants predicted to prevent CalDAG-GEFI expression and 6 missense variants affecting the CalDAG-GEFI CDC25 domain, which mediates Rap1 activation during platelet inside-out αIIbß3 signaling. Cases with biallelic RASGRP2 variants had abnormal mucocutaneous, surgical, and dental bleeding from childhood, requiring ≥1 blood or platelet transfusion in 78% of cases. Platelets displayed reduced aggregation in response to adenosine 5'-diphosphate and epinephrine, but variable aggregation defects with other agonists. There were no other consistent clinical or laboratory features. These data enable definition of human CalDAG-GEFI deficiency as a nonsyndromic, recessive PFD associated with a moderate or severe bleeding phenotype and complex defects in platelet aggregation.
Asunto(s)
Plaquetas/patología , Factores de Intercambio de Guanina Nucleótido/genética , Hemorragia/genética , Mutación/genética , Alelos , Secuencia de Bases , Femenino , Humanos , Masculino , LinajeRESUMEN
The von Willebrand receptor complex, which is composed of the glycoproteins Ibα, Ibß, GPV, and GPIX, plays an essential role in the earliest steps in hemostasis. During the last 4 decades, it has become apparent that loss of function of any 1 of 3 of the genes encoding these glycoproteins (namely, GP1BA, GP1BB, and GP9) leads to autosomal recessive macrothrombocytopenia complicated by bleeding. A small number of variants in GP1BA have been reported to cause a milder and dominant form of macrothrombocytopenia, but only 2 tentative reports exist of such a variant in GP1BB By analyzing data from a collection of more than 1000 genome-sequenced patients with a rare bleeding and/or platelet disorder, we have identified a significant association between rare monoallelic variants in GP1BB and macrothrombocytopenia. To strengthen our findings, we sought further cases in 2 additional collections in the United Kingdom and Japan. Across 18 families exhibiting phenotypes consistent with autosomal dominant inheritance of macrothrombocytopenia, we report on 27 affected cases carrying 1 of 9 rare variants in GP1BB.
Asunto(s)
Plaquetas/metabolismo , Hemorragia/genética , Mutación , Complejo GPIb-IX de Glicoproteína Plaquetaria/genética , Trombocitopenia/genética , Alelos , Plaquetas/patología , Estudios de Casos y Controles , Femenino , Expresión Génica , Genes Dominantes , Genoma Humano , Hemorragia/diagnóstico , Hemorragia/metabolismo , Hemorragia/patología , Secuenciación de Nucleótidos de Alto Rendimiento , Humanos , Masculino , Linaje , Recuento de Plaquetas , Trombocitopenia/diagnóstico , Trombocitopenia/metabolismo , Trombocitopenia/patologíaRESUMEN
Variations in platelet number, volume, and function are largely genetically controlled, and many loci associated with platelet traits have been identified by genome-wide association studies (GWASs).(1) The genome also contains a large number of rare variants, of which a tiny fraction underlies the inherited diseases of humans. Research over the last 3 decades has led to the discovery of 51 genes harboring variants responsible for inherited platelet disorders (IPDs). However, the majority of patients with an IPD still do not receive a molecular diagnosis. Alongside the scientific interest, molecular or genetic diagnosis is important for patients. There is increasing recognition that a number of IPDs are associated with severe pathologies, including an increased risk of malignancy, and a definitive diagnosis can inform prognosis and care. In this review, we give an overview of these disorders grouped according to their effect on platelet biology and their clinical characteristics. We also discuss the challenge of identifying candidate genes and causal variants therein, how IPDs have been historically diagnosed, and how this is changing with the introduction of high-throughput sequencing. Finally, we describe how integration of large genomic, epigenomic, and phenotypic datasets, including whole genome sequencing data, GWASs, epigenomic profiling, protein-protein interaction networks, and standardized clinical phenotype coding, will drive the discovery of novel mechanisms of disease in the near future to improve patient diagnosis and management.
Asunto(s)
Trastornos de las Plaquetas Sanguíneas/diagnóstico , Trastornos de las Plaquetas Sanguíneas/genética , Técnicas de Diagnóstico Molecular/tendencias , Plaquetas/fisiología , ADN/análisis , ADN/sangre , Estudio de Asociación del Genoma Completo , Secuenciación de Nucleótidos de Alto Rendimiento , Humanos , Técnicas de Diagnóstico Molecular/métodos , Enfermedades Raras/diagnóstico , Enfermedades Raras/genética , Trombopoyesis/genéticaRESUMEN
Macrothrombocytopenia (MTP) is a heterogeneous group of disorders characterized by enlarged and reduced numbers of circulating platelets, sometimes resulting in abnormal bleeding. In most MTP, this phenotype arises because of altered regulation of platelet formation from megakaryocytes (MKs). We report the identification of DIAPH1, which encodes the Rho-effector diaphanous-related formin 1 (DIAPH1), as a candidate gene for MTP using exome sequencing, ontological phenotyping, and similarity regression. We describe 2 unrelated pedigrees with MTP and sensorineural hearing loss that segregate with a DIAPH1 R1213* variant predicting partial truncation of the DIAPH1 diaphanous autoregulatory domain. The R1213* variant was linked to reduced proplatelet formation from cultured MKs, cell clustering, and abnormal cortical filamentous actin. Similarly, in platelets, there was increased filamentous actin and stable microtubules, indicating constitutive activation of DIAPH1. Overexpression of DIAPH1 R1213* in cells reproduced the cytoskeletal alterations found in platelets. Our description of a novel disorder of platelet formation and hearing loss extends the repertoire of DIAPH1-related disease and provides new insight into the autoregulation of DIAPH1 activity.
Asunto(s)
Proteínas Adaptadoras Transductoras de Señales/genética , Pérdida Auditiva/genética , Mutación , Trombocitopenia/genética , Células A549 , Adolescente , Adulto , Anciano , Estudios de Casos y Controles , Células Cultivadas , Niño , Femenino , Forminas , Estudios de Asociación Genética , Predisposición Genética a la Enfermedad , Células HEK293 , Pérdida Auditiva/complicaciones , Humanos , Masculino , Persona de Mediana Edad , Linaje , Polimorfismo de Nucleótido Simple , Síndrome , Trombocitopenia/complicaciones , Adulto JovenRESUMEN
OBJECTIVE: Iron status is a modifiable trait that has been implicated in cardiovascular disease. This study uses the Mendelian randomization technique to investigate whether there is any causal effect of iron status on risk of coronary artery disease (CAD). APPROACH AND RESULTS: A 2-sample Mendelian randomization approach is used to estimate the effect of iron status on CAD risk. Three loci (rs1800562 and rs1799945 in the HFE gene and rs855791 in TMPRSS6) that are each associated with serum iron, transferrin saturation, ferritin, and transferrin in a pattern suggestive of an association with systemic iron status are used as instruments. SNP (single-nucleotide polymorphism)-iron status association estimates are based on a genome-wide association study meta-analysis of 48 972 individuals. SNP-CAD estimates are derived by combining the results of a genome-wide association study meta-analysis of 60 801 CAD cases and 123 504 controls with those of a meta-analysis of 63 746 CAD cases and 130 681 controls obtained from Metabochip and genome-wide association studies. Combined Mendelian randomization estimates are obtained for each marker by pooling results across the 3 instruments. We find evidence of a protective effect of higher iron status on CAD risk (iron odds ratio, 0.94 per SD unit increase; 95% confidence interval, 0.88-1.00; P=0.039; transferrin saturation odds ratio, 0.95 per SD unit increase; 95% confidence interval, 0.91-0.99; P=0.027; log-transformed ferritin odds ratio, 0.85 per SD unit increase; 95% confidence interval, 0.73-0.98; P=0.024; and transferrin odds ratio, 1.08 per SD unit increase; 95% confidence interval, 1.01-1.16; P=0.034). CONCLUSIONS: This Mendelian randomization study supports the hypothesis that higher iron status reduces CAD risk. These findings may highlight a therapeutic target.
Asunto(s)
Enfermedad de la Arteria Coronaria/genética , Proteína de la Hemocromatosis/genética , Trastornos del Metabolismo del Hierro/genética , Hierro/sangre , Proteínas de la Membrana/genética , Polimorfismo de Nucleótido Simple , Serina Endopeptidasas/genética , Estudios de Casos y Controles , Enfermedad de la Arteria Coronaria/diagnóstico , Enfermedad de la Arteria Coronaria/prevención & control , Bases de Datos Genéticas , Ferritinas/sangre , Estudios de Asociación Genética , Predisposición Genética a la Enfermedad , Humanos , Trastornos del Metabolismo del Hierro/sangre , Trastornos del Metabolismo del Hierro/diagnóstico , Análisis de la Aleatorización Mendeliana , Oportunidad Relativa , Fenotipo , Factores Protectores , Medición de Riesgo , Factores de Riesgo , Transferrina/análisisRESUMEN
Tissue factor (TF) (CD142) is a 47 kDa transmembrane cell surface glycoprotein that triggers the extrinsic coagulation cascade and links thrombosis with inflammation. Although macrophage TF expression is known to be regulated at the RNA level, very little is known about the mechanisms involved. Poly(adenosine 5'-diphosphate [ADP]-ribose)-polymerase (PARP)-14 belongs to a family of intracellular proteins that generate ADP-ribose posttranslational adducts. Functional screening of PARP-14-deficient macrophages mice revealed that PARP-14 deficiency leads to increased TF expression and functional activity in macrophages after challenge with bacterial lipopolysaccharide. This was related to an increase in TF messenger RNA (mRNA) stability. Ribonucleoprotein complex immunoprecipitation and biotinylated RNA pull-down assays demonstrated that PARP-14 forms a complex with the mRNA-destabilizing protein tristetraprolin (TTP) and a conserved adenylate-uridylate-rich element in the TF mRNA 3' untranslated region. TF mRNA regulation by PARP-14 was selective, as tumor necrosis factor (TNF)α mRNA, which is also regulated by TTP, was not altered in PARP-14 deficient macrophages. Consistent with the in vitro data, TF expression and TF activity, but not TNFα expression, were increased in Parp14(-/-) mice in vivo. Our study provides a novel mechanism for the posttranscriptional regulation of TF expression, indicating that this is selectively regulated by PARP-14.
Asunto(s)
Regulación de la Expresión Génica , Macrófagos/metabolismo , Poli(ADP-Ribosa) Polimerasas/metabolismo , Tromboplastina/biosíntesis , Tristetraprolina/metabolismo , Regiones no Traducidas 3'/fisiología , Animales , Lipopolisacáridos/farmacología , Ratones , Ratones Noqueados , Poli(ADP-Ribosa) Polimerasas/genética , Estabilidad del ARN/efectos de los fármacos , Estabilidad del ARN/fisiología , Tromboplastina/genética , Tristetraprolina/genética , Factor de Necrosis Tumoral alfa/biosíntesis , Factor de Necrosis Tumoral alfa/genéticaRESUMEN
We have examined the effect of the O-linked glycan (OLG) structures of VWF on its interaction with the platelet receptor glycoprotein Ibα. The 10 OLGs were mutated individually and as clusters (Clus) on either and both sides of the A1 domain: Clus1 (N-terminal side), Clus2 (C-terminal side), and double cluster (DC), in both full-length-VWF and in a VWF construct spanning D' to A3 domains. Mutations did not alter VWF secretion by HEK293T cells, multimeric structure, or static collagen binding. The T1255A, Clus1, and DC variants caused increased ristocetin-mediated GPIbα binding to VWF. Platelet translocation rate on OLG mutants was increased because of reduced numbers of GPIbα binding sites but without effect on bond lifetime. In contrast, OLG mutants mediated increased platelet capture on collagen under high shear stress that was associated with increased adhesion of these variants to the collagen under flow. These findings suggest that removal of OLGs increases the flexibility of the hinge linker region between the D3 and A1 domain, facilitating VWF unfolding by shear stress, thereby enhancing its ability to bind collagen and capture platelets. These data demonstrate an important functional role of VWF OLGs under shear stress conditions.
Asunto(s)
Plaquetas/fisiología , Glicoproteínas de Membrana/metabolismo , Polisacáridos/metabolismo , Factor de von Willebrand/genética , Factor de von Willebrand/metabolismo , Antibacterianos/farmacología , Sitios de Unión/fisiología , Colágeno/metabolismo , Variación Genética , Glicosilación , Células HEK293 , Humanos , Glicoproteínas de Membrana/química , Mutagénesis/fisiología , Complejo GPIb-IX de Glicoproteína Plaquetaria , Unión Proteica/efectos de los fármacos , Unión Proteica/fisiología , Estructura Terciaria de Proteína/fisiología , Proteínas Recombinantes/metabolismo , Flujo Sanguíneo Regional/fisiología , Ristocetina/farmacología , Estrés Mecánico , Factor de von Willebrand/químicaRESUMEN
Heritable dysfibrinogenaemia (HD) is a rare qualitative disorder of fibrinogen (FGN). To better describe the clinical, laboratory and genotypic spectrum of HD, we evaluated 35 subjects identified at two UK centres using laboratory criteria. 12/35(34%) subjects with HD experienced bleeding (bleeding score >1 at any site), 3/35(9%) thrombosis and 20/35(57%) were asymptomatic. Amongst subjects with bleeding, symptoms were typically mild, at one anatomical site and seldom occurred after invasive procedures. All subject showed dry clot weight within or above laboratory reference interval (median 3·2 g/l; range 1·9-5·1), reduced Clauss fibrinogen (median 0·52 g/l; range 0·21-1·3), and prolonged thrombin (median 30·7 s; range 21·3-45·7) and reptilase (median 42·0 s; range 20·0-68·0) times. In all subjects, the prothrombin time ratio (PTR), determined by Sysmex CA-1500 coagulometer and Innovin activator, was abnormal (median 1·42; range 1·22-1·61). The activated partial thromboplastin time ratio and PTR with other coagulometers and activators were comparatively insensitive to HD. All subjects with HD harboured heterozygous candidate nucleotide variations within known hotspots in the FGN genes. The HD variants identified in this cross-sectional study seldom have significant clinical manifestations and show similar laboratory features irrespective of genotype. Selection of coagulometer and PT activator may markedly affect the detection of new HD cases using coagulation screening tests.
Asunto(s)
Afibrinogenemia/epidemiología , Fibrinógenos Anormales/genética , Adolescente , Adulto , Afibrinogenemia/sangre , Afibrinogenemia/genética , Anciano , Alelos , Pruebas de Coagulación Sanguínea/instrumentación , Pruebas de Coagulación Sanguínea/métodos , Niño , Estudios Transversales , Análisis Mutacional de ADN , Inglaterra/epidemiología , Femenino , Frecuencia de los Genes , Genotipo , Hemorragia/epidemiología , Hemorragia/etiología , Humanos , Masculino , Tamizaje Masivo , Persona de Mediana Edad , Fenotipo , Mutación Puntual , Polimorfismo de Nucleótido Simple , Reproducibilidad de los Resultados , Adulto JovenRESUMEN
BACKGROUND: The von Willebrand factor (VWF) is a multimeric plasma glycoprotein essential for hemostasis, inflammation, and angiogenesis. The majority of VWF is synthesized by endothelial cells (ECs) and stored in Weibel-Palade bodies (WPB). Among the range of proteins shown to co-localize to WPB is angiopoietin-2 (Angpt-2), a ligand of the receptor tyrosine kinase Tie-2. We have previously shown that VWF itself regulates angiogenesis, raising the hypothesis that some of the angiogenic activity of VWF may be mediated by its interaction with Angpt-2. METHODS: Static-binding assays were used to probe the interaction between Angpt-2 and VWF. Binding in media from cultured human umbilical vein ECs s and in plasma was determined by immunoprecipitation experiments. Immunofluorescence was used to detect the presence of Angpt-2 on VWF strings, and flow assays were used to investigate the effect on VWF function. RESULTS: Static-binding assays revealed that Angpt-2 bound to VWF with high affinity (KD,app â¼3 nM) in a pH and calcium-dependent manner. The interaction was localized to the VWF A1 domain. Co-immunoprecipitation experiments demonstrated that the complex persisted following stimulated secretion from ECs and was present in plasma. Angpt-2 was also visible on VWF strings on stimulated ECs. The VWF-Angpt-2 complex did not inhibit the binding of Angpt-2 to Tie-2 and did not significantly interfere with VWF-platelet capture. CONCLUSIONS: Together, these data demonstrate a direct binding interaction between Angpt-2 and VWF that persists after secretion. VWF may act to localize Angpt-2; further work is required to establish the functional consequences of this interaction.
Asunto(s)
Cuerpos de Weibel-Palade , Factor de von Willebrand , Humanos , Factor de von Willebrand/metabolismo , Cuerpos de Weibel-Palade/metabolismo , Angiopoyetina 2/metabolismo , Exocitosis , Células Endoteliales de la Vena Umbilical Humana/metabolismo , Células CultivadasRESUMEN
BACKGROUND: Elevated plasma levels of coagulation factor VIII are a strong risk factor for pulmonary emboli and deep venous thromboses. OBJECTIVES: To identify reversible biomarkers associated with high factor VIII and assess potential significance in a specific at-risk population. PATIENTS/METHODS: 609 patients with hereditary haemorrhagic telangiectasia were recruited prospectively in two separate series at a single centre. Associations between log-transformed factor VIII measured 6 months from any known thrombosis/illness, and patient-specific variables including markers of inflammation and iron deficiency, were assessed in stepwise multiple regression analyses. Age-specific incidence rates of radiologically proven pulmonary emboli/deep venous thromboses were calculated, and logistic regression analyses performed. RESULTS: In each series, there was an inverse association between factor VIII and serum iron that persisted after adjustment for age, inflammation and/or von Willebrand factor. Iron response elements within untranslated regions of factor VIII transcripts provide potential mechanisms for the association. Low serum iron levels were also associated with venous thromboemboli (VTE): the age-adjusted OR of 0.91 (95% CI 0.86 to 0.97) per 1 µmol/litre increase in serum iron implied a 2.5-fold increase in VTE risk for a serum iron of 6 µmol/litre compared with the mid-normal range (17 µmol/litre). The association appeared to depend on factor VIII, as once adjusted for factor VIII, the association between VTE and iron was no longer evident. CONCLUSIONS: In this population, low serum iron levels attributed to inadequate replacement of haemorrhagic iron losses are associated with elevated plasma levels of coagulation factor VIII and venous thromboembolic risk. Potential implications for other clinical populations are discussed.
Asunto(s)
Factor VIII/análisis , Hierro/sangre , Embolia Pulmonar/sangre , Telangiectasia Hemorrágica Hereditaria/sangre , Trombosis de la Vena/sangre , Biomarcadores/sangre , Estudios de Cohortes , Diagnóstico por Imagen , Femenino , Humanos , Masculino , Plasma/química , Estudios Prospectivos , Embolia Pulmonar/diagnóstico , Análisis de Regresión , Factores de Riesgo , Suero/química , Trombosis de la Vena/diagnóstico , Factor de von Willebrand/análisisRESUMEN
We examined the role that N-linked glycans play in the synthesis and expression of von Willebrand Factor (VWF). Blocking the addition of N-linked glycans (NLGs) or inhibiting initial glycan processing prevented secretion of VWF. To determine whether specific glycosylation sites were important, the 16 VWF N-linked glycosylation sites were mutated followed by expression in HEK293T cells. Four NLG mutants affected VWF expression: N99Q (D1 domain), N857Q (D' domain), N2400Q (B1 domain), and N2790Q (CK domain) either abolished or reduced secretion of VWF and this was confirmed by metabolic labeling. Multimer analysis of mutant N2790Q cell lysate revealed an increase in VWF monomers, which was also observed when the isolated CK domain was expressed with N2790 mutated. Immunofluorescence microscopy showed that mutants N99Q, N857Q, and N2790Q were primarily retained within the ER, producing only few pseudo Weibel-Palade bodies over longer time periods compared with wtVWF. All the variants also showed an increase in free thiol reactivity. This was greatest with N857Q and D4-C2 NLG mutants, which had approximately 6-fold and 3- to 4-fold more free thiol reactivity than wtVWF. These data provide further evidence of the critical role that individual N-linked glycans play in determining VWF synthesis and expression.
Asunto(s)
Factor de von Willebrand/biosíntesis , Factor de von Willebrand/metabolismo , Sustitución de Aminoácidos/fisiología , Antígenos Bacterianos/química , Antígenos Bacterianos/metabolismo , Dominio Catalítico/genética , Células Cultivadas , Expresión Génica , Glicosilación , Humanos , Modelos Biológicos , Proteínas Mutantes/metabolismo , Mutación/fisiología , Polisacáridos/metabolismo , Procesamiento Proteico-Postraduccional/fisiología , Transporte de Proteínas , Especificidad por Sustrato , Distribución Tisular , Factor de von Willebrand/química , Factor de von Willebrand/genéticaRESUMEN
BACKGROUND: Post-marketing surveillance for COVID-19 vaccines during the pandemic identified an extremely rare thrombosis with thrombocytopenia syndrome (TTS) reported post-vaccination, requiring further characterisation to improve diagnosis and management. METHODS: We searched the AstraZeneca Global Safety Database (through April 26, 2021) for cases with co-reported thrombocytopenia and thrombosis (using standardised MedDRA queries/high-level terms) following AZD1222 (ChAdOx1 nCoV-19). Cases were adjudicated by experts as 'typical','possible', 'no' or 'unknown' according to available TTS criteria. Additional confirmatory datasets (May 20-June 20, October 1-December 28) were evaluated. FINDINGS: We identified 573 reports, including 273 (47.6 %) 'typical' and 171 (29.8 %) 'possible' TTS cases. Of these 444 cases, 275 (61.9 %) were female, median age was 50.0 years (IQR: 38.0-60.0). Cerebral venous sinus thrombosis was reported in 196 (44.1 %) cases, splanchnic venous thrombosis in 65 (14.6 %) and thromboses at multiple sites in 119 (26.8 %). Median time to onset was 12.0 days (IQR: 9.0-15.0). Comparison with a pre-pandemic reference population indicated higher rates of autoimmune disorders (13.8 %, 4.4 %), previous heparin therapy (7.4 %, 1.2 %), history of thrombosis (5.5 %, 1.4 %), and immune thrombocytopenia (6.1 %, 0.2 %). Fatality rate was 22.2 % (127/573) overall and 23.6 % (105/444) in 'typical'/'possible' TTS, which decreased from 39.0 % (60/154) in February/March to 15.5 % (45/290) in April. Overall patterns were similar in confirmatory datasets. CONCLUSIONS: The reporting rate of 'typical'/'possible' TTS post first-dose vaccination in this dataset is 7.5 per million vaccinated persons; few cases were reported after subsequent doses, including booster doses. Peak reporting coincided with media-driven attention. Medical history differences versus a reference population indicate potentially unidentified risk factors. The decreasing fatality rate correlates with increasing awareness and publication of diagnostic/treatment guidelines. Adjudication was hindered by unreported parameters, and an algorithm was developed to classify potential TTS cases; comprehensive reporting could help further improve definition and management of this extremely rare syndrome.
Asunto(s)
Vacunas contra la COVID-19 , COVID-19 , Trombocitopenia , Trombosis , COVID-19/epidemiología , COVID-19/prevención & control , Vacunas contra la COVID-19/efectos adversos , ChAdOx1 nCoV-19 , Femenino , Humanos , Masculino , Persona de Mediana Edad , Trombocitopenia/inducido químicamente , Trombocitopenia/epidemiología , Trombosis/inducido químicamente , Trombosis/epidemiología , Vacunación/efectos adversosRESUMEN
BACKGROUND: Von Willebrand factor (VWF) contains a number of free thiols, the majority of which are located in its C-domains, and these have been shown to alter VWF function, However, the impact of free thiols on function following acute exposure of VWF to collagen under high and pathological shear stress has not been determined. METHODS: VWF free thiols were blocked with N-ethylmaleimide and flow assays performed under high and pathological shear rates to determine the impact on platelet capture and collagen binding function. Atomic force microscopy (AFM) was used to probe the interaction of VWF with collagen and molecular simulations conducted to determine the effect of free thiols on the flexibility of the VWF-C4 domain. RESULTS: Blockade of VWF free thiols reduced VWF-mediated platelet capture to collagen in a shear-dependent manner, with platelet capture virtually abolished above 5000 s-1 and in regions of stenosis in microfluidic channels. Direct visualization of VWF fibers formed under extreme pathological shear rates and analysis of collagen-bound VWF attributed the effect to altered binding of VWF to collagen. AFM measurements showed that thiol-blockade reduced the lifetime and strength of the VWF-collagen bond. Pulling simulations of the VWF-C4 domain demonstrated that with one or two reduced disulphide bonds the C4 domain has increased flexibility and the propensity to undergo free-thiol exchange. CONCLUSIONS: We conclude that free thiols in the C-domains of VWF enhance the flexibility of the molecule and enable it to withstand high shear forces following collagen binding, demonstrating a previously unrecognized role for VWF free thiols.
Asunto(s)
Compuestos de Sulfhidrilo , Factor de von Willebrand , Plaquetas/metabolismo , Colágeno/metabolismo , Humanos , Adhesividad Plaquetaria , Unión Proteica , Estrés Mecánico , Factor de von Willebrand/metabolismoRESUMEN
Thrombosis affecting the pulmonary and systemic vasculature is common during severe COVID-19 and causes adverse outcomes. Although thrombosis likely results from inflammatory activation of vascular cells, the mediators of thrombosis remain unconfirmed. In a cross-sectional cohort of 36 severe COVID-19 patients, we show that markedly increased plasma von Willebrand factor (VWF) levels were accompanied by a partial reduction in the VWF regulatory protease ADAMTS13. In all patients we find this VWF/ADAMTS13 imbalance to be associated with persistence of ultra-high-molecular-weight (UHMW) VWF multimers that are highly thrombogenic in some disease settings. Incubation of plasma samples from patients with severe COVID-19 with recombinant ADAMTS13 (rADAMTS13) substantially reduced the abnormally high VWF activity, reduced overall multimer size and depleted UHMW VWF multimers in a time and concentration dependent manner. Our data implicate disruption of normal VWF/ADAMTS13 homeostasis in the pathogenesis of severe COVID-19 and indicate that this can be reversed ex vivo by correction of low plasma ADAMTS13 levels. These findings suggest a potential therapeutic role for rADAMTS13 in helping restore haemostatic balance in COVID-19 patients.