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1.
Int J Mol Sci ; 22(9)2021 Apr 26.
Artigo em Inglês | MEDLINE | ID: mdl-33926054

RESUMO

Platelets play a major role in hemostasis as ppwell as in many other physiological and pathological processes. Accordingly, production of about 1011 platelet per day as well as appropriate survival and functions are life essential events. Inherited platelet disorders (IPDs), affecting either platelet count or platelet functions, comprise a heterogenous group of about sixty rare diseases caused by molecular anomalies in many culprit genes. Their clinical relevance is highly variable according to the specific disease and even within the same type, ranging from almost negligible to life-threatening. Mucocutaneous bleeding diathesis (epistaxis, gum bleeding, purpura, menorrhagia), but also multisystemic disorders and/or malignancy comprise the clinical spectrum of IPDs. The early and accurate diagnosis of IPDs and a close patient medical follow-up is of great importance. A genotype-phenotype relationship in many IPDs makes a molecular diagnosis especially relevant to proper clinical management. Genetic diagnosis of IPDs has been greatly facilitated by the introduction of high throughput sequencing (HTS) techniques into mainstream investigation practice in these diseases. However, there are still unsolved ethical concerns on general genetic investigations. Patients should be informed and comprehend the potential implications of their genetic analysis. Unlike the progress in diagnosis, there have been no major advances in the clinical management of IPDs. Educational and preventive measures, few hemostatic drugs, platelet transfusions, thrombopoietin receptor agonists, and in life-threatening IPDs, allogeneic hematopoietic stem cell transplantation are therapeutic possibilities. Gene therapy may be a future option. Regular follow-up by a specialized hematology service with multidisciplinary support especially for syndromic IPDs is mandatory.


Assuntos
Transtornos Plaquetários/genética , Transtornos Plaquetários/fisiopatologia , Transtornos Plaquetários/terapia , Plaquetas/patologia , Testes Genéticos/métodos , Hemostasia , Sequenciamento de Nucleotídeos em Larga Escala/métodos , Humanos , Testes de Função Plaquetária , Transfusão de Plaquetas , Doenças Raras/genética
2.
Semin Thromb Hemost ; 45(7): 695-707, 2019 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-31041795

RESUMO

Diagnosis of inherited bleeding disorders (IBDs) remains challenging, especially in the case of inherited platelet disorders, due to the heterogeneity of the clinical and laboratory phenotype, the limited specificity of platelet function tests, and the large number of potential culprit genes. Unraveling the underlying molecular defect provides the definitive diagnosis of IBDs, facilitating prognosis and clinical care, which are especially important for severe clinical syndromes and those that may be associated with an increased risk of malignancy. Until recently, Sanger sequencing of candidate genes has been the only method of molecular diagnosis, but this approach is time-consuming and costly and requires phenotype-based identification of any obvious candidate gene(s). Nowadays, high-throughput sequencing (HTS) allows the simultaneous and rapid investigation of multiple genes at a manageable cost. This HTS technology that includes targeted sequencing of prespecified genes, whole-exome sequencing, or whole-genome sequencing, is revolutionizing the genetic diagnosis of human diseases. Through its extensive implementation in research and clinical practice, HTS is rapidly improving the molecular characterization of IBDs. However, despite the availability of this powerful approach, many patients still do not receive a diagnosis. As IBDs are complex and rare diseases, development of more advanced laboratory assays, improvements in bioinformatic pipelines, and the formation of multidisciplinary teams are encouraged to advance our understanding of IBDs.


Assuntos
Transtornos Plaquetários/genética , Transtornos Hemorrágicos/genética , Sequenciamento de Nucleotídeos em Larga Escala/métodos , Humanos
3.
Platelets ; 30(4): 535-539, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-30849270

RESUMO

This manuscript reviews pathogenic variants in RASGRP2, which are the cause of a relatively new autosomal recessive and nonsyndromic inherited platelet function disorder, referred to as platelet-type bleeding disorder-18 (BDPLT18)(OMIM:615888). To date, 18 unrelated BDPLT18 pedigrees have been reported, harboring 19 different homozygous or compound heterozygous RASGRP2 variants. Patients with this disease present with lifelong moderate to severe bleeding, with epistaxis as the most common and relevant bleeding symptom. Biologically, they exhibit normal platelet count and morphology, reduced aggregation responses to ADP, epinephrine and low-dose collagen, and impaired αIIbß3 integrin activation (fibrinogen or PAC-1 binding) in response to most agonists except PMA. Diagnosis is confirmed by genetic analysis of RASGRP2.


Assuntos
Transtornos Plaquetários/genética , Plaquetas/patologia , Variação Genética/genética , Fatores de Troca do Nucleotídeo Guanina/genética , Hemorragia/genética , Transtornos Plaquetários/metabolismo , Hemorragia/metabolismo , Humanos
4.
Haematologica ; 103(1): 148-162, 2018 01.
Artigo em Inglês | MEDLINE | ID: mdl-28983057

RESUMO

Inherited platelet disorders are a heterogeneous group of rare diseases, caused by inherited defects in platelet production and/or function. Their genetic diagnosis would benefit clinical care, prognosis and preventative treatments. Until recently, this diagnosis has usually been performed via Sanger sequencing of a limited number of candidate genes. High-throughput sequencing is revolutionizing the genetic diagnosis of diseases, including bleeding disorders. We have designed a novel high-throughput sequencing platform to investigate the unknown molecular pathology in a cohort of 82 patients with inherited platelet disorders. Thirty-four (41.5%) patients presented with a phenotype strongly indicative of a particular type of platelet disorder. The other patients had clinical bleeding indicative of platelet dysfunction, but with no identifiable features. The high-throughput sequencing test enabled a molecular diagnosis in 70% of these patients. This sensitivity increased to 90% among patients suspected of having a defined platelet disorder. We found 57 different candidate variants in 28 genes, of which 70% had not previously been described. Following consensus guidelines, we qualified 68.4% and 26.3% of the candidate variants as being pathogenic and likely pathogenic, respectively. In addition to establishing definitive diagnoses of well-known inherited platelet disorders, high-throughput sequencing also identified rarer disorders such as sitosterolemia, filamin and actinin deficiencies, and G protein-coupled receptor defects. This included disease-causing variants in DIAPH1 (n=2) and RASGRP2 (n=3). Our study reinforces the feasibility of introducing high-throughput sequencing technology into the mainstream laboratory for the genetic diagnostic practice in inherited platelet disorders.


Assuntos
Transtornos Plaquetários/diagnóstico , Transtornos Plaquetários/genética , Doenças Genéticas Inatas/diagnóstico , Doenças Genéticas Inatas/genética , Testes Genéticos , Sequenciamento de Nucleotídeos em Larga Escala , Adolescente , Adulto , Idoso , Idoso de 80 Anos ou mais , Plaquetas/metabolismo , Criança , Pré-Escolar , Feminino , Estudos de Associação Genética , Predisposição Genética para Doença , Testes Genéticos/métodos , Humanos , Lactente , Masculino , Pessoa de Meia-Idade , Fenótipo , Reprodutibilidade dos Testes , Análise de Sequência de DNA , Adulto Jovem
5.
Platelets ; 29(2): 192-195, 2018 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-28762304

RESUMO

The RASGRP2 gene encodes the Ca2+ and DAG-regulated guanine nucleotide exchange factor I (CalDAG-GEFI), which plays a key role in integrin activation in platelets and neutrophils. We here report two new RASGRP2 variants associated with platelet dysfunction and bleeding in patients. The homozygous patients had normal platelet and neutrophil counts and morphology. Platelet phenotyping showed: prolonged PFA-100 closure times; normal expression of major glycoprotein receptors; severely reduced platelet aggregation response to ADP and collagen (both patients); aggregation response to PAR1 and arachidonic acid markedly impaired in one patient; PMA-induced aggregation unaffected; platelet secretion, clot retraction, and spreading minimally affected. Genetic analysis identified two new homozygous variants in RASGRP2: c.706C>T (p.Q236X) and c.887G>A (p.C296Y). In both patients, CalDAG-GEFI protein was not detectable in platelet lysates, and platelet αIIbß3 activation, as assessed by fibrinogen binding, was greatly impaired in response to all agonists except PMA. Patient neutrophils showed normal integrin expression, but impaired Mn2+-induced fibrinogen binding. In summary, we have identified two new RASGRP2 mutations that can be added to this rapidly growing form of inherited platelet function disorder.


Assuntos
Plaquetas/metabolismo , Fatores de Troca do Nucleotídeo Guanina/sangue , Transtornos Hemorrágicos/sangue , Transtornos Hemorrágicos/genética , Mutação , Complexo Glicoproteico GPIIb-IIIa de Plaquetas/metabolismo , Transtornos Plaquetários/sangue , Transtornos Plaquetários/genética , Plaquetas/patologia , Criança , Pré-Escolar , Feminino , Fatores de Troca do Nucleotídeo Guanina/biossíntese , Fatores de Troca do Nucleotídeo Guanina/genética , Humanos , Masculino , Linhagem
8.
J Thromb Haemost ; 21(5): 1352-1365, 2023 05.
Artigo em Inglês | MEDLINE | ID: mdl-36736831

RESUMO

BACKGROUND: Germline mutations in RUNX1 can cause a familial platelet disorder that may lead to acute myeloid leukemia, an autosomal dominant disorder characterized by moderate thrombocytopenia, platelet dysfunction, and a high risk of developing acute myeloid leukemia or myelodysplastic syndrome. Discerning the pathogenicity of novel RUNX1 variants is critical for patient management. OBJECTIVES: To extend the characterization of RUNX1 variants and evaluate their effects by transcriptome analysis. METHODS: Three unrelated patients with long-standing thrombocytopenia carrying heterozygous RUNX1 variants were included: P1, who is a subject with recent development of myelodysplastic syndrome, with c.802 C>T[p.Gln268∗] de novo; P2 with c.586A>G[p.Thr196Ala], a variant that segregates with thrombocytopenia and myeloid neoplasia in the family; and P3 with c.476A>G[p.Asn159Ser], which did not segregate with thrombocytopenia or neoplasia. Baseline platelet evaluations were performed. Ultrapure platelets were prepared for platelet transcriptome analysis. RESULTS: In P1 and P2, but not in P3, transcriptome analysis confirmed aberrant expression of genes recognized as RUNX1 targets. Data allowed grouping patients by distinct gene expression profiles, which were partitioned with clinical parameters. Functional studies and platelet mRNA expression identified alterations in the actin cytoskeleton, downregulation of GFI1B, defective GPVI downstream signaling, and reduction of alpha granule proteins, such as thrombospondin-1, as features likely implicated in thrombocytopenia and platelet dysfunction. CONCLUSION: Platelet phenotype, familial segregation, and platelet transcriptomics support the pathogenicity of RUNX1 variants p.Gln268∗ and p.Thr196Ala, but not p.Asn159Ser. This study is an additional proof of concept that platelet RNA analysis could be a tool to help classify pathogenic RUNX1 variants and identify novel RUNX1 targets.


Assuntos
Transtornos Plaquetários , Leucemia Mieloide Aguda , Síndromes Mielodisplásicas , Trombocitopenia , Humanos , Subunidade alfa 2 de Fator de Ligação ao Core/genética , Subunidade alfa 2 de Fator de Ligação ao Core/metabolismo , Mutação em Linhagem Germinativa , Transtornos Plaquetários/complicações , Trombocitopenia/genética , Trombocitopenia/complicações , Leucemia Mieloide Aguda/genética , Perfilação da Expressão Gênica , Células Germinativas/metabolismo , Mutação
9.
Cells ; 11(20)2022 10 14.
Artigo em Inglês | MEDLINE | ID: mdl-36291092

RESUMO

The GATA1 transcription factor is essential for normal erythropoiesis and megakaryocytic differentiation. Germline GATA1 pathogenic variants in the N-terminal zinc finger (N-ZF) are typically associated with X-linked thrombocytopenia, platelet dysfunction, and dyserythropoietic anemia. A few variants in the C-terminal ZF (C-ZF) domain are described with normal platelet count but altered platelet function as the main characteristic. Independently performed molecular genetic analysis identified a novel hemizygous variant (c.865C>T, p.H289Y) in the C-ZF region of GATA1 in a German patient and in a Spanish patient. We characterized the bleeding and platelet phenotype of these patients and compared these findings with the parameters of two German siblings carrying the likely pathogenic variant p.D218N in the GATA1 N-ZF domain. The main difference was profound thrombocytopenia in the brothers carrying the p.D218N variant compared to a normal platelet count in patients carrying the p.H289Y variant; only the Spanish patient occasionally developed mild thrombocytopenia. A functional platelet defect affecting αIIbß3 integrin activation and α-granule secretion was present in all patients. Additionally, mild anemia, anisocytosis, and poikilocytosis were observed in the patients with the C-ZF variant. Our data support the concept that GATA1 variants located in the different ZF regions can lead to clinically diverse manifestations.


Assuntos
Anemia Diseritropoética Congênita , Fator de Transcrição GATA1 , Doenças Genéticas Ligadas ao Cromossomo X , Variação Genética , Trombocitopenia , Dedos de Zinco , Humanos , Masculino , Fator de Transcrição GATA1/genética , Fator de Transcrição GATA1/metabolismo , Integrinas/metabolismo , Fenótipo , Trombocitopenia/genética , Dedos de Zinco/genética , Doenças Genéticas Ligadas ao Cromossomo X/genética , Anemia Diseritropoética Congênita/genética , Plaquetas/patologia
10.
J Thromb Haemost ; 20(5): 1248-1255, 2022 05.
Artigo em Inglês | MEDLINE | ID: mdl-35170221

RESUMO

BACKGROUND: Rare inherited thrombocytopenias are caused by alterations in genes involved in megakaryopoiesis, thrombopoiesis and/or platelet release. Diagnosis is challenging due to poor specificity of platelet laboratory assays, large numbers of culprit genes, and difficult assessment of the pathogenicity of novel variants. OBJECTIVES: To characterize the clinical and laboratory phenotype, and identifying the underlying molecular alteration, in a pedigree with thrombocytopenia of uncertain etiology. PATIENTS/METHODS: Index case was enrolled in our Spanish multicentric project of inherited platelet disorders due to lifelong thrombocytopenia and bleeding. Bleeding score was recorded by ISTH-BAT. Laboratory phenotyping consisted of blood cells count, blood film, platelet aggregation and flow cytometric analysis. Genotyping was made by whole-exome sequencing (WES). Cytoskeleton proteins were analyzed in resting/spreading platelets by immunofluorescence and immunoblotting. RESULTS: Five family members displayed lifelong mild thrombocytopenia with a high number of enlarged platelets in blood film, and mild bleeding tendency. Patient's platelets showed normal aggregation and granule secretion response to several agonists. WES revealed a novel nonsense variant (c.322C>T; p.Gln108*) in TPM4 (NM_003290.3), the gene encoding for tropomyosin-4 (TPM4). This variant led to impairment of platelet spreading capacity after stimulation with TRAP-6 and CRP, delocalization of TPM4 in activated platelets, and significantly reduced TPM4 levels in platelet lysates. Moreover, the index case displayed up-regulation of TPM2 and TPM3 mRNA levels. CONCLUSIONS: This study identifies a novel TPM4 nonsense variant segregating with macrothrombocytopenia and impaired platelet cytoskeletal remodeling and spreading. These findings support the relevant role of TPM4 in thrombopoiesis and further expand our knowledge of TPM4-related thrombocytopenia.


Assuntos
Transtornos Plaquetários , Trombocitopenia , Transtornos Plaquetários/genética , Plaquetas/metabolismo , Citoesqueleto/metabolismo , Hemorragia , Humanos , Trombopoese/genética , Tropomiosina/genética , Tropomiosina/metabolismo
11.
Blood Adv ; 6(17): 5244-5255, 2022 09 13.
Artigo em Inglês | MEDLINE | ID: mdl-35349645

RESUMO

Src-related thrombocytopenia (SRC-RT) is a rare autosomal dominant, inherited platelet disorder resulting from the p.E527K heterozygous germline gain-of-function variant of Src. To date, genetic diagnosis of the disease has only been reported in 7 patients from 3 unrelated families. The clinical features ranged from isolated thrombocytopenia to complex syndromic manifestations characterized by thrombocytopenia, bleeding, myelofibrosis, splenomegaly, and bone disease. We report a new 3-generation kindred with the Src p.E527K variant. Patients presented with rather variable platelet counts (38-139 × 109/L), mildly impaired platelet function, >15% immature platelet fraction, and with a significant proportion of large-giant platelets. Four adults from the family were diagnosed with immune thrombocytopenia (ITP) and underwent splenectomy, achieving sustained platelet counts >75 × 109/L for several years; increases in platelet counts were also observed after corticosteroid therapy. Four of 7 Src p.E527K variant carriers showed immune defects and recurrent infections. In addition, a range of neurological symptoms, from specific language impairment to epilepsy, was seen in some family members. Patient platelets exhibited constitutive Src, Bruton tyrosine kinase, and phospholipase Cγ2 activation, and after stimulating CD19 cells by crosslinking surface immunoglobulin M, phosphorylated extracellular signal-regulated kinase (ERK) was significantly increased in B cells from individuals carrying the Src p.E527K substitution. In summary, in addition to causing impaired platelet production, SRC-RT may associate immune dysregulation and increased platelet consumption. In families in whom several members are responsive to ITP-directed therapies, an underlying Src p.E527K variant should be excluded.


Assuntos
Púrpura Trombocitopênica Idiopática , Trombocitopenia , Adulto , Plaquetas , Humanos , Megacariócitos , Púrpura Trombocitopênica Idiopática/diagnóstico , Púrpura Trombocitopênica Idiopática/genética , Trombocitopenia/genética , Trombopoese
12.
Thromb Haemost ; 121(9): 1193-1205, 2021 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-33626581

RESUMO

RUNX1-related disorder (RUNX1-RD) is caused by germline variants affecting the RUNX1 gene. This rare, heterogeneous disorder has no specific clinical or laboratory phenotype, making genetic diagnosis necessary. Although international recommendations have been established to classify the pathogenicity of variants, identifying the causative alteration remains a challenge in RUNX1-RD. Murine models may be useful not only for definitively settling the controversy about the pathogenicity of certain RUNX1 variants, but also for elucidating the mechanisms of molecular pathogenesis. Therefore, we developed a knock-in murine model, using the CRISPR/Cas9 system, carrying the RUNX1 p.Leu43Ser variant (mimicking human p.Leu56Ser) to study its pathogenic potential and mechanisms of platelet dysfunction. A total number of 75 mice were generated; 25 per genotype (RUNX1WT/WT, RUNX1WT/L43S, and RUNX1L43S/L43S). Platelet phenotype was assessed by flow cytometry and confocal microscopy. On average, RUNX1L43S/L43S and RUNX1WT/L43S mice had a significantly longer tail-bleeding time than RUNX1WT/WT mice, indicating the variant's involvement in hemostasis. However, only homozygous mice displayed mild thrombocytopenia. RUNX1L43S/L43S and RUNX1WT/L43S displayed impaired agonist-induced spreading and α-granule release, with no differences in δ-granule secretion. Levels of integrin αIIbß3 activation, fibrinogen binding, and aggregation were significantly lower in platelets from RUNX1L43S/L43S and RUNX1WT/L43S using phorbol 12-myristate 13-acetate (PMA), adenosine diphosphate (ADP), and high thrombin doses. Lower levels of PKC phosphorylation in RUNX1L43S/L43S and RUNX1WT/L43S suggested that the PKC-signaling pathway was impaired. Overall, we demonstrated the deleterious effect of the RUNX1 p.Leu56Ser variant in mice via the impairment of integrin αIIbß3 activation, aggregation, α-granule secretion, and platelet spreading, mimicking the phenotype associated with RUNX1 variants in the clinical setting.


Assuntos
Transtornos Plaquetários/genética , Plaquetas/metabolismo , Proteína 9 Associada à CRISPR/genética , Sistemas CRISPR-Cas , Subunidade alfa 2 de Fator de Ligação ao Core/genética , Mutação , Ativação Plaquetária/genética , Animais , Transtornos Plaquetários/sangue , Proteína 9 Associada à CRISPR/metabolismo , Subunidade alfa 2 de Fator de Ligação ao Core/sangue , Grânulos Citoplasmáticos/genética , Grânulos Citoplasmáticos/metabolismo , Modelos Animais de Doenças , Técnicas de Introdução de Genes , Predisposição Genética para Doença , Hemostasia , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Fenótipo , Complexo Glicoproteico GPIIb-IIIa de Plaquetas/metabolismo , Via Secretória , Trombopoese
13.
Blood Adv ; 5(24): 5453-5467, 2021 12 28.
Artigo em Inglês | MEDLINE | ID: mdl-34516618

RESUMO

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.


Assuntos
Trombocitopenia , Tubulina (Proteína) , Plaquetas , Humanos , Megacariócitos , Trombocitopenia/genética , Tubulina (Proteína)/genética
14.
Neonatology ; 117(1): 15-23, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-31786577

RESUMO

BACKGROUND: The mechanisms underlying neonatal platelets hyporesponsiveness are not fully understood. While previous studies have demonstrated developmental impairment of agonist-induced platelet activation, differences in inhibitory signaling pathways have been scarcely investigated. OBJECTIVE: To compare neonatal and adult platelets with regard to inhibition of platelet reactivity by prostaglandin E1 (PGE1). METHODS: Platelet-rich plasma from umbilical cord (CB) or adult blood was incubated with PGE1 (0-1 µM). We assessed aggregation in response to adenosine diphosphate (ADP), collagen, and thrombin receptor activating peptide as well as cyclic adenosine 3'5'-monophosphate (cAMP) levels (ELISA). Gαs, Gαi2, and total- and phospho-protein kinase A (PKA) were evaluated in adult and CB ultrapure and washed platelets, respectively, by immunoblotting. RESULTS: Neonatal (vs. adult) platelets display hypersensitivity to inhibition by PGE1 of platelet aggregation induced by ADP and collagen (PGE1 IC50: 14 and 117 nM for ADP and collagen, respectively, vs. 149 and 491 nM in adults). They also show increased basal and PGE1-induced cAMP levels. Mechanistically, PGE1 acts by binding to the prostanoid receptor IP (prostacyclin receptor), which couples to the Gαs protein-adenylate cyclase axis and increases intracellular levels of cAMP. cAMP activates PKA, which phosphorylates different target inhibitor proteins. Neonatal platelets showed higher basal and PGE1-induced cAMP levels, higher Gαs protein expression, and a trend to increased PKA-dependent protein phosphorylation compared to adult platelets. CONCLUSION: Neonatal platelets have a functionally increased PGE1-cAMP-PKA axis. This finding supports a downregulation of inhibitory when going from neonate to adult contributing to neonatal platelet hyporesponsiveness.


Assuntos
Fatores Etários , Alprostadil/farmacologia , Agregação Plaquetária/efeitos dos fármacos , Difosfato de Adenosina/fisiologia , Adenilil Ciclases/sangue , Adulto , Plaquetas/efeitos dos fármacos , Plaquetas/enzimologia , AMP Cíclico/sangue , Proteínas Quinases Dependentes de AMP Cíclico/fisiologia , Humanos , Recém-Nascido
15.
Sci Transl Med ; 11(522)2019 12 11.
Artigo em Inglês | MEDLINE | ID: mdl-31826978

RESUMO

Platelet transfusions can fail to prevent bleeding in patients with inherited platelet function disorders (IPDs), such as Glanzmann's thrombasthenia (GT; integrin αIIbß3 dysfunction), Bernard-Soulier syndrome [BSS; glycoprotein (GP) Ib/V/IX dysfunction], and the more recently identified nonsyndromic RASGRP2 variants. Here, we used IPD mouse models and real-time imaging of hemostatic plug formation to investigate whether dysfunctional platelets impair the hemostatic function of healthy donor [wild-type (WT)] platelets. In Rasgrp2-/- mice or mice with platelet-specific deficiency in the integrin adaptor protein TALIN1 ("GT-like"), WT platelet transfusion was ineffective unless the ratio between mutant and WT platelets was ~2:1. In contrast, thrombocytopenic mice or mice lacking the extracellular domain of GPIbα ("BSS-like") required very few transfused WT platelets to normalize hemostasis. Both Rasgrp2-/- and GT-like, but not BSS-like, platelets effectively localized to the injury site. Mechanistic studies identified at least two mechanisms of interference by dysfunctional platelets in IPDs: (i) delayed adhesion of WT donor platelets due to reduced access to GPIbα ligands exposed at sites of vascular injury and (ii) impaired consolidation of the hemostatic plug. We also investigated the hemostatic activity of transfused platelets in the setting of dual antiplatelet therapy (DAPT), an acquired platelet function disorder (APD). "DAPT" platelets did not prolong the time to initial hemostasis, but plugs were unstable and frequent rebleeding was observed. Thus, we propose that the endogenous platelet count and the ratio of transfused versus endogenous platelets should be considered when treating select IPD and APD patients with platelet transfusions.


Assuntos
Transtornos Plaquetários/patologia , Plaquetas/patologia , Hemostasia , Transfusão de Plaquetas , Animais , Sítios de Ligação , Terapia Antiplaquetária Dupla , Fatores de Troca do Nucleotídeo Guanina/deficiência , Fatores de Troca do Nucleotídeo Guanina/metabolismo , Humanos , Integrinas/metabolismo , Ligantes , Camundongos Endogâmicos C57BL , Complexo Glicoproteico GPIb-IX de Plaquetas/metabolismo , Trombastenia/patologia , Doadores de Tecidos
16.
Ann Med ; 51(2): 141-148, 2019 03.
Artigo em Inglês | MEDLINE | ID: mdl-30990103

RESUMO

Background: Hermansky-Pudlak syndrome (HPS) is a rare inherited platelet disorder characterized by bleeding diathesis, oculocutaneous albinism (OCA) and a myriad of often-serious clinical complications. Methods: We established the clinical and laboratory phenotype and genotype of six unrelated pedigrees comprising ten patients with clinical suspicion of HPS; including platelet aggregation, flow cytometry, platelet dense granule content, electron microscopy and high-throughput sequencing (HTS). Results: The clinical presentation showed significant heterogeneity and no clear phenotype-genotype correlations. HTS revealed two known and three novel disease-causing variants. The Spanish patients carried a homozygous p.Pro685Leufs17* deletion (n = 2) in HPS4, or the novel p.Arg822* homozygous variant (n = 1) in HPS3. In the case of two Turkish sisters, a novel missense homozygous HPS4 variant (p.Leu91Pro) was found. In two Portuguese families, genetic studies confirmed a previously reported nonsense variant (p.Gln103*) in DTNBP1 in three patients and a novel duplication (p.Leu22Argfs*33) in HPS6 in two unrelated patients. Conclusions: Our findings expand the mutational spectrum of HPS, which may help in investigating phenotype-genotype relationships and assist genetic counselling for affected individuals. This approach is a proof of principle that HTS can be considered and used in the first-line diagnosis of patients with biological and clinical manifestations suggestive of HPS. Key messages We established the relationships between the clinical and laboratory phenotype and genotype of six unrelated pedigrees comprising ten patients with clinical suspicion of HPS. Molecular analysis is useful in confirming the diagnosis and may offer some prognostic information that will aid in optimizing monitoring and surveillance for early detection of end-organ damage. This approach is a proof of principle that HTS can be considered and used in the first-line diagnosis of patients with biological and clinical manifestations suggestive of HPS.


Assuntos
Síndrome de Hermanski-Pudlak/genética , Sequenciamento de Nucleotídeos em Larga Escala , Adolescente , Adulto , Criança , Feminino , Variação Genética , Síndrome de Hermanski-Pudlak/diagnóstico , Síndrome de Hermanski-Pudlak/fisiopatologia , Humanos , Masculino , Pessoa de Meia-Idade , Linhagem , Fenótipo
17.
Thromb Haemost ; 118(6): 1009-1020, 2018 06.
Artigo em Inglês | MEDLINE | ID: mdl-29695020

RESUMO

Neonatal platelets are hypo-reactive to the tyrosine kinase-linked receptor agonist collagen. Here, we have investigated whether the hypo-responsiveness is related to altered levels of glycoprotein VI (GPVI) and integrin α2ß1, or to defects in downstream signalling events by comparison to platelet activation by C-type lectin-like receptor 2 (CLEC-2). GPVI and CLEC-2 activate a Src- and Syk-dependent signalling pathway upstream of phospholipase C (PLC) γ2. Phosphorylation of a conserved YxxL sequence known as a (hemi) immunotyrosine-based-activation-motif (ITAM) in both receptors is critical for Syk activation. Platelets from human pre-term and full-term neonates display mildly reduced expression of GPVI and CLEC-2, as well as integrin αIIbß3, accounted for at the transcriptional level. They are also hypo-responsive to the two ITAM receptors, as shown by measurement of integrin αIIbß3 activation, P-selectin expression and Syk and PLCγ2 phosphorylation. Mouse platelets are also hypo-responsive to GPVI and CLEC-2 from late gestation to 2 weeks of age, as determined by measurement of integrin αIIbß3 activation. In contrast, the response to G protein-coupled receptor agonists was only mildly reduced and in some cases not altered in neonatal platelets of both species. A reduction in response to GPVI and CLEC-2, but not protease-activated receptor 4 (PAR-4) peptide, was also observed in adult mouse platelets following immune thrombocytopenia, whereas receptor expression was not impaired. Our results demonstrate developmental differences in platelet responsiveness to GPVI and CLEC-2, and also following immune platelet depletion leading to reduced Syk activation. The rapid generation of platelets during development or following platelet depletion is achieved at the expense of signalling by ITAM-coupled receptors.


Assuntos
Plaquetas/fisiologia , Lectinas Tipo C/metabolismo , Glicoproteínas de Membrana/metabolismo , Glicoproteínas da Membrana de Plaquetas/metabolismo , Nascimento Prematuro/metabolismo , Púrpura Trombocitopênica Idiopática/metabolismo , Quinase Syk/metabolismo , Animais , Células Cultivadas , Feminino , Humanos , Recém-Nascido , Integrina alfa2beta1/metabolismo , Camundongos , Selectina-P/metabolismo , Fosfolipase C gama/metabolismo , Ativação Plaquetária , Gravidez , Nascimento Prematuro/patologia , Púrpura Trombocitopênica Idiopática/patologia , Receptores de Trombina/metabolismo , Transdução de Sinais
18.
PLoS One ; 12(8): e0183042, 2017.
Artigo em Inglês | MEDLINE | ID: mdl-28813466

RESUMO

Understanding the underlying mechanisms of the well-substantiated platelet hyporeactivity in neonates is of interest given their implications for the clinical management of newborns, a population at higher bleeding risk than adults (especially sick and preterm infants), as well as for gaining insight into the regulatory mechanisms of platelet biology. Transcriptome analysis is useful in identifying mRNA signatures affecting platelet function. However, human fetal/neonatal platelet transcriptome analysis has never before been reported. We have used mRNA expression array for the first time to compare platelet transcriptome changes during development. Microarray analysis was performed in pure platelet RNA obtained from adult and cord blood, using the same platform in two independent laboratories. A high correlation was obtained between array results for both adult and neonate platelet samples. There was also good agreement between results in our adult samples and outcomes previously reported in three different studies. Gene enrichment analysis showed that immunity- and platelet function-related genes are highly expressed at both developmental stages. Remarkably, 201 genes were found to be differentially expressed throughout development. In particular, neonatal platelets contain higher levels of mRNA that are associated with protein synthesis and processing, while carrying significantly lower levels of genes involved in calcium transport/metabolism and cell signaling (including GNAZ). Overall, our results point to variations in platelet transcriptome as possibly underlining the hypo-functional phenotype of neonatal platelets and provide further support for the role of platelets in cellular immune response. Better characterization of the platelet transcriptome throughout development can contribute to elucidate how transcriptome changes impact different pathological conditions.


Assuntos
Plaquetas/metabolismo , Regulação da Expressão Gênica , Transcriptoma , Adulto , Fatores Etários , Análise por Conglomerados , Biologia Computacional/métodos , Feminino , Perfilação da Expressão Gênica , Humanos , Recém-Nascido , Masculino , Reprodutibilidade dos Testes , Transdução de Sinais
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