RESUMO
Methicillin-resistant Staphylococcus aureus (MRSA) is a global health concern, but there are few data from Central Africa. The objective of our study was to characterise S. aureus colonisation isolates from healthcare-exposed professionals in the Democratic Republic of the Congo (DRC). Healthcare workers and medical students (n = 380) in Kisangani, DRC were screened for S. aureus nasal carriage in a single-centre cross-sectional study in the University Hospital of Kisangani. The isolates were identified and characterised using phenotypic and genotypic methods. The nasal carriage rate of S. aureus was 16.6 % and 10 out of 63 isolates (15.9 %) were MRSA. We found 28 different spa types. Most MRSA isolates belonged to ST8-spa t1476-SCCmec V. The majority of MRSA were multidrug-resistant to non-beta-lactam antibiotics. Overall, 28.5 % of S. aureus carried Panton-Valentine leucocidin (PVL)-encoding genes (all methicillin-sensitive) and 17.5 % carried toxic shock syndrome toxin-1 (TSST-1)-encoding genes. The finding of MRSA carriage among healthcare workers in a setting with limited access to diagnostic microbiology and appropriate therapy calls for improved education on infection control practices and supports the introduction of surveillance programmes.
Assuntos
Portador Sadio/epidemiologia , Pessoal de Saúde , Mucosa Nasal/microbiologia , Infecções Estafilocócicas/epidemiologia , Staphylococcus aureus/isolamento & purificação , Estudantes de Medicina , Adulto , Toxinas Bacterianas/genética , Estudos Transversais , República Democrática do Congo/epidemiologia , Farmacorresistência Bacteriana , Enterotoxinas/genética , Exotoxinas/genética , Feminino , Genótipo , Hospitais Universitários , Humanos , Leucocidinas/genética , Masculino , Pessoa de Meia-Idade , Tipagem Molecular , Superantígenos/genética , Adulto JovemRESUMO
Salmonella enterica is the leading cause of bloodstream infection in children in sub-Saharan Africa, but few data are available from Central-Africa. We documented during the period November 2011 to May 2012 an epidemic increase in invasive Salmonella bloodstream infections in HGR Bwamanda, a referral hospital in Equateur Province, DR Congo. Salmonella spp. represented 90.4 % (103 out of 114) of clinically significant blood culture isolates and comprised Salmonella Typhimurium (54.4 %, 56 out of 103), Salmonella Enteritidis (28.2 %, 29 out of 103) and Salmonella Typhi (17.5 %, 18 out of 103), with Salmonella Enteritidis accounting for most of the increase. Most (82 out of 103, 79.6 %) isolates were obtained from children < 5 years old. Median ages of patients infected with Salmonella Typhimurium and Salmonella Enteritidis were 14 months (14 days to 64 years) and 19 months (3 months to 8 years) respectively. Clinical presentation was non-specific; the in-hospital case fatality rate was 11.1 %. More than two thirds (69.7 %, 53 out of 76) of children < 5 years for whom laboratory data were available had Plasmodium falciparum infection. Most (83/85, 97.6 %) non-typhoid Salmonella isolates as well as 6/18 (33.3 %) Salmonella Typhi isolates were multidrug resistant (i.e. resistant to the first-line oral antibiotics amoxicillin, trimethoprim-sulfamethoxazole and chloramphenicol), one (1.0 %) Salmonella Typhimurium had decreased ciprofloxacin susceptibility owing to a point mutation in the gyrA gene (Gly81Cys). Multilocus variable-number tandem-repeat (MLVA) analysis of the Salmonella Enteritidis isolates revealed closely related patterns comprising three major and four minor profiles, with differences limited to one out of five loci. These data show an epidemic increase in clonally related multidrug-resistant Salmonella bloodstream infection in children in DR Congo.
Assuntos
Bacteriemia/epidemiologia , Infecções por Salmonella/epidemiologia , Salmonella enteritidis/isolamento & purificação , Salmonella typhi/isolamento & purificação , Salmonella typhimurium/isolamento & purificação , Adolescente , Distribuição por Idade , Bacteriemia/microbiologia , Criança , Pré-Escolar , Análise por Conglomerados , República Democrática do Congo/epidemiologia , Farmacorresistência Bacteriana , Feminino , Humanos , Incidência , Lactente , Masculino , Tipagem Molecular , Infecções por Salmonella/microbiologia , Salmonella enteritidis/classificação , Salmonella enteritidis/genética , Análise de SobrevidaRESUMO
Male subjects with iron deficiency from the general population were examined for polymorphisms or sporadic mutations in TMPRSS6 to identify genetic risk factors for iron deficiency anemia. Three uncommon non-synonymous polymorphisms were identified, G228D, R446W, and V795I (allele frequencies 0.0074, 0.023 and 0.0074 respectively), of which the R446W polymorphism appeared to be overrepresented in the anemic population. In addition, three children with iron refractory iron deficiency anemia, and one sibling with iron responsive iron deficiency anemia were also examined for polymorphisms or sporadic mutations in TMPRSS6. Two children (family 1) were compound heterozygotes for a L674F mutation and a previously described splicing defect predicted to cause skipping of exon 13 (IVS13+1 G>A). One child from the second family was homozygous for a deletion (497T) causing a frameshift (L166X+36) and premature termination. The sibling and mother from the second family were compound heterozygotes for the L166X mutation and the uncommon R446W polymorphism. Although in vitro expression studies demonstrated that the R446W isoform was biologically similar to wildtype Tmprss6, clinical data indicate that the R446W produces a milder disease when carried in trans with severe mutation in Tmprss6. The four children carrying mutations in TMPRSS6 all exhibited inappropriately high urinary hepcidin levels for the degree of iron deficiency.
Assuntos
Anemia Ferropriva/genética , Anemia Refratária/genética , Proteínas de Membrana/genética , Mutação , Polimorfismo Genético , Serina Endopeptidases/genética , Idoso , Substituição de Aminoácidos/genética , Peptídeos Catiônicos Antimicrobianos/sangue , Peptídeos Catiônicos Antimicrobianos/urina , Criança , Estudos de Coortes , Análise Mutacional de DNA , Feminino , Frequência do Gene , Células Hep G2 , Hepcidinas , Humanos , Lactente , Masculino , Proteínas de Membrana/metabolismo , Pessoa de Meia-Idade , Linhagem , Serina Endopeptidases/metabolismo , População BrancaRESUMO
BACKGROUND: Pituitary adenylate cyclase-activating polypeptide (PACAP) was recently identified as an inhibitor of megakaryopoiesis and platelet aggregability. OBJECTIVE: We studied PACAP levels in children with nephrotic syndrome (NS), which is associated with thrombocytosis, platelet hyperaggregability, and an increased risk of thrombosis. PATIENTS/METHODS: In four children with congenital NS (CNS) and 24 children with idiopathic NS (INS), plasma and urine levels of PACAP and ceruloplasmin were measured, as were platelet counts and platelet aggregation responses to collagen. In CNS patients, in vitro megakaryopoiesis and nuclear factor-κB expression in platelet lysates were also measured. All tests were performed during the nephrotic state and the non-nephrotic state. RESULTS: Urinary losses of PACAP and ceruloplasmin were observed during the nephrotic state, and disappeared during the non-nephrotic state. Plasma PACAP deficiency was more pronounced in CNS patients than in INS patients. Thrombocytosis was observed in all CNS patients and in 11 of 29 INS patients during the nephrotic state. During the PACAP-deficient state, in vitro megakaryopoiesis was increased for CNS patients, and this effect could be reversed by the addition of recombinant PACAP. Platelet hyperaggregability was observed during the nephrotic state in both CNS and INS patients. In INS patients, the addition of recombinant PACAP to patients' platelets was studied, and resulted in decreased aggregation during the nephrotic state. Platelet aggregation correlated inversely with plasma PACAP levels, but not with serum albumin levels. CONCLUSIONS: We demonstrate urinary losses of PACAP and plasma PACAP deficiency in children with NS, associated with thrombocytosis and platelet hyperaggregability.
Assuntos
Síndrome Nefrótica/sangue , Polipeptídeo Hipofisário Ativador de Adenilato Ciclase/metabolismo , Agregação Plaquetária , Contagem de Plaquetas , Adolescente , Criança , Pré-Escolar , Humanos , LactenteRESUMO
Recombinant factor VIIa (rFVIIa; NovoSeven, Novo Nordisk, Bagsvaerd, Denmark) appears effective and relatively safe for the treatment of bleeding and for surgical prophylaxis in patients with Glanzmann thrombasthenia as reported to the International Registry on rFVIIa and Congenital Platelet Disorders. One of the shortcomings of the Registry data is the heterogeneity of treatment protocol, including dosage, number of doses used, duration of treatment before declaration of failure, and mode of rFVIIa administration (bolus v continuous infusion). The data are not yet sufficient to define optimal regimens for various indications such as the type of bleeding or the type of procedures. The place of this drug compared to platelet transfusion in the overall management of patients with Glanzmann thrombasthenia will need to be determined in relationship to a number of challenges and unresolved issues in the clinical care of these patients. These issues include: how to improve local measures for patients with mucosal bleeds, optimal management of young women during menarche, optimal platelet transfusion regimens for various indications, the relationship between antiplatelet antibodies detected by monoclonal antibody-specific immobilization of platelet antigens (MAIPA) and effectiveness of platelet transfusion, whether there are other biological tests that may correlate with effectiveness of platelet transfusion, and management of pregnancy and delivery regarding antiplatelet immunization.
Assuntos
Fator VII/uso terapêutico , Proteínas Recombinantes/uso terapêutico , Trombastenia/tratamento farmacológico , Coagulantes/uso terapêutico , Fator VIIa , Feminino , Humanos , Masculino , Transfusão de Plaquetas/efeitos adversos , Gravidez , Complicações Hematológicas na Gravidez/tratamento farmacológico , Complicações Hematológicas na Gravidez/terapia , Trombastenia/diagnóstico , Trombastenia/terapiaRESUMO
Recombinant human erythropoietin was administered for up to 40 weeks to nine patients on chronic haemodialysis. From the third week of administration onwards, not only haemoglobin and haematocrit but also the platelet count rose, the latter, however, transiently. Subnormal platelet aggregation before therapy also improved transiently and in parallel with the erythropoietin dosage. The bleeding time normalized in almost all patients. There were no major side-effects. We conclude that recombinant erythropoietin improves haemostasis in chronic haemodialysis patients by increasing the haematocrit and in addition transiently enhances platelet number and function.
Assuntos
Eritropoetina/uso terapêutico , Hemostasia/efeitos dos fármacos , Diálise Renal , Adulto , Tempo de Sangramento , Feminino , Hemoglobinas/efeitos dos fármacos , Humanos , Masculino , Agregação Plaquetária/efeitos dos fármacos , Contagem de Plaquetas/efeitos dos fármacos , Proteínas Recombinantes/uso terapêuticoRESUMO
The combination of thromboxane synthase inhibition with thromboxane receptor antagonism has been shown to result in a strong inhibition of platelet aggregation and a prolongation of the bleeding time (Gresele et al., J. Clin Invest 1987; 80: 1435-45). Ridogrel is a single molecule that efficiently achieves both inhibitions in human volunteers. The present study was performed in patients with obstructive peripheral arterial disease and elevated plasma beta-thromboglobulin levels. Patients were treated with either 2 x 300 mg ridogrel or 2 x 300 mg placebo per day for 2 1/2 days, according to a double blind randomised parallel design. Plasma beta-thromboglobulin decreased significantly throughout active treatment starting within 2 h after administration; serum and urinary immunoreactive TxB2 levels and urinary 11-dehydro-TxB2 excretion were significantly lower and serum PGE2 and 6-keto-PGF1 alpha levels significantly higher with ridogrel; no changes were observed in the placebo-treated group. In conclusion this study demonstrates a reduction of platelet activation in vivo by ridogrel.
Assuntos
Arteriopatias Oclusivas/tratamento farmacológico , Ácidos Pentanoicos/uso terapêutico , Piridinas/uso terapêutico , Receptores de Prostaglandina/antagonistas & inibidores , Tromboxano-A Sintase/antagonistas & inibidores , beta-Tromboglobulina/efeitos dos fármacos , Idoso , Idoso de 80 Anos ou mais , Arteriopatias Oclusivas/sangue , Método Duplo-Cego , Humanos , Ativação Plaquetária/efeitos dos fármacos , Fator Plaquetário 4/metabolismo , Prostaglandinas/sangue , Receptores de Tromboxanos , Tromboxano B2/sangueRESUMO
Recombinant human erythropoietin was administered to 10 uraemic children on chronic haemodialysis, all of whom responded by correcting their haemoglobin. In addition, they showed an increase in blood pressure; platelet aggregations, subnormal before therapy, improved during treatment. The intracellular free calcium concentration in platelets after thrombin stimulation also increased significantly during erythropoietin administration. We hypothesize that the effect of erythropoietin on platelet aggregability and on blood pressure may be due to an increase in the intracellular free calcium mobilisation in platelets and possibly in smooth muscle cells respectively.
Assuntos
Plaquetas/efeitos dos fármacos , Pressão Sanguínea/efeitos dos fármacos , Cálcio/sangue , Eritropoetina/farmacologia , Uremia/tratamento farmacológico , Adolescente , Plaquetas/metabolismo , Criança , Pré-Escolar , Feminino , Humanos , Masculino , Agregação Plaquetária/efeitos dos fármacos , Proteínas Recombinantes/farmacologia , Uremia/sangue , Uremia/fisiopatologiaRESUMO
Alternatively spliced GNAS1 and XL-GNAS1, encoding respectively the stimulatory G-protein alpha-subunit (Gsalpha) and the extra-large stimulatory G-protein alpha-subunit (XLsalpha), are located on the imprinted chromosomal region 20q13.12-13. We presently report a functional polymorphism in the imprinted XL-GNAS1 gene. In three patients, a 36 bp insertion and two basepair substitutions flanking this insertion were found in the paternally inherited XL-GNAS1 exon 1. They clinically manifest an enhanced trauma-related bleeding tendency and a variable degree of mental retardation. A platelet aggregation inhibition test to evaluate Gs function was developed. Their platelets display Gs hyperfunction and an enhanced cAMP generation upon stimulation of Gs-coupled receptors. The prevalence of the XLsalpha insertion in a normal control group was 2.2%. Normal controls, inheriting the insertion maternally, had a normal platelet Gs activity, whereas controls inheriting the insertion paternally had increased inducible platelet Gs activity, defining the insertion as a functional polymorphism. This paternally inherited XLsalpha insertion represents a new genetic cause of an inherited bleeding tendency, although to a variable degree.
Assuntos
Anormalidades Múltiplas/genética , Plaquetas/fisiologia , Subunidades alfa Gs de Proteínas de Ligação ao GTP/genética , Transtornos Hemorrágicos/genética , Proteínas Heterotriméricas de Ligação ao GTP , Hipoparatireoidismo/genética , Deficiência Intelectual/genética , Proteínas do Tecido Nervoso , Adenosina/farmacologia , Adenilil Ciclases/metabolismo , Adolescente , Alprostadil/farmacologia , Processamento Alternativo , Sequência de Aminoácidos , Substituição de Aminoácidos , Tempo de Sangramento , Criança , Cromograninas , AMP Cíclico/biossíntese , Feminino , Dedos/anormalidades , Frequência do Gene , Impressão Genômica , Transtornos Hemorrágicos/epidemiologia , Humanos , Hipercinese/genética , Iloprosta/farmacologia , Masculino , Dados de Sequência Molecular , Hipotonia Muscular/genética , Mutagênese Insercional , Fenótipo , Agregação Plaquetária/efeitos dos fármacos , Testes de Função Plaquetária , Polimorfismo Genético , Subunidades Proteicas , Sequências Repetitivas de Aminoácidos , Fatores de Risco , Sistemas do Segundo Mensageiro , Relação Estrutura-Atividade , Ferimentos e Lesões/complicaçõesRESUMO
AIM: To evaluate the clinical features, angiographic findings and evolution of Takayasu's arteritis in a Belgian tertiary center, and to compare the findings with published series of Western patients. METHODS: Retrospective analysis of 15 patients with Takayasu's arteritis, satisfying the American College of Rheumathology criteria, in the period 1986 to 2002. Published series of Western patients were identified by means of a Medline search and citation-tracking. RESULTS: Diagnosis was often delayed, with a median period of 9 months. Patients presented with a variety of symptoms and clinical signs and had on average 4.5 arterial segments involved at angiography. Twelve patients received corticosteroid treatment and 4 of them additional immunosuppressive drugs. Five patients underwent angioplasty and/or stenting and 8 patients had open surgical procedures. During follow-up, there were 2 cardiac deaths and 2 other patients died from intracranial hemorrhage. Comparison with published series of other Western patients did not reveal major differences of anatomical distributions of the lesions, but left the impression that more aggressive use of immunosuppressive drugs might have improved the outcome. CONCLUSION: Takayasu's arteritis results in an important morbidity and mortality. More aggressive medical therapy may be advantageous, but this would require adequate investigation in a controlled trial for which a multicenter effort is needed because of the rarity of the disease.
Assuntos
Arterite de Takayasu , Adolescente , Adulto , Bélgica , Criança , Pré-Escolar , Feminino , Seguimentos , Humanos , Masculino , Pessoa de Meia-Idade , Estudos Retrospectivos , Arterite de Takayasu/diagnóstico , Arterite de Takayasu/terapia , Fatores de TempoRESUMO
Although platelets were already discovered light-microscopically in the 19th century, it became only clear in 1906 that they originate from megakaryocytes. The discovery of thrombopoietin in 1994 facilitated research on the origin of platelets, because this growth factor enabled expansion of megakaryocytes in culture. Thus, cell biological and molecular studies could be undertaken. Knowledge accumulated further by studying mouse models and by unravelling the defect in patients with hereditary thrombocytopenia. How a megakaryocyte originates from a hematopoietic stem cell and how this cell undergoes further maturation and differentiation is a complex process, controlled at different stages by several transcription factor. These transcription factors influence the expression of their own target genes, leading to megakaryocyte maturation and platelet release. One of the transcription factors that is most studied in this regard is GATA1, that forms a transcriptional complex with its cofactor FOG1. Families with hereditary thrombocytopenia have been described due to mutations in the GATA1 gene, and target genes were studied. This broadens our insight in normal megakaryocyte differentiation. The final release of platelets from the megakaryocyte is strongly dependent on the formation of so-called proplatelets. The formation of pseudopodia requires the presence of microtubuli. Beta 1-tubulin is a major part of these microtubuli and plays an important role not only in the genesis of platelets but also in the final discoid form of the platelet. Despite a renewed interest and expanding knowledge in this area, there are more questions than answers at this day.
Assuntos
Plaquetas/citologia , Megacariócitos/citologia , Trombopoese/fisiologia , Trombopoetina/fisiologia , Fatores de Transcrição/fisiologia , Plaquetas/metabolismo , Diferenciação Celular , Humanos , Megacariócitos/metabolismo , Biologia Molecular , Pesquisa , Trombopoese/genética , Fatores de Transcrição/genéticaRESUMO
In four patients, all girls, aged 2, 3.5, 4 and 5 years, transient erythroblastopenia was diagnosed. The children were presented because of acute pallor. The haemoglobin levels were 2.8 to 5.0 mmol/l. After 3 weeks all patients had recovered or were recovering with increasing haemoglobin values. Three of the four patients needed one blood transfusion. In two patients there was evidence of a parvovirus B19 infection. Transient erythroblastopenia is mostly seen in patients aged 1-4 years. Most cases are postinfectious and there is evidence that human parvovirus B19 is responsible for many cases. In the very young child the differential diagnosis from Blackfan-Diamond anaemia may be very difficult.
Assuntos
Eritema Infeccioso/complicações , Aplasia Pura de Série Vermelha/sangue , Transfusão de Sangue , Pré-Escolar , Diagnóstico Diferencial , Feminino , Hemoglobinas/análise , Humanos , Lactente , Aplasia Pura de Série Vermelha/terapiaRESUMO
Platelets are derived from megakaryocytes in the bone marrow that create the cellular machinery the platelet needs to participate in the different processes of primary hemostasis including adhesion, activation and clot formation at the site of injury. Defects related to megakaryocyte differentiation, platelet formation, and/or platelet function can result in bleeding. Patients with thrombopathies can present with mucous membrane bleeding but may also present with bleeding following trauma or surgery. In this review, we have classified inherited platelet bleeding disorders (IPD) according to their underlying defective pathway: transcription regulation, TPO signaling, cytoskeletal organization, apoptosis, granule trafficking, and receptor signaling. Platelet function testing has provided insights into the underlying molecular defects that can result in bleeding. A major step forward was made during the last 3 years using new-generation genetic approaches that resulted in the discovery of novel genes such as NBEAL2, RBM8A, ACTN1, and GFI1B for the well-known IPD that cause gray platelet syndrome, thrombocytopenia-absent radius syndrome, and autosomal dominant thrombocytopenias, respectively. In the near future, it is expected that a similar approach will identify many novel genes that cause IPD of unknown etiology, which are common. The future challenge will be to use a functional, systems biology approach to study the genes mutated in IPD and determine their roles in megakaryocyte and platelet biology and pathology.
Assuntos
Transtornos Plaquetários/etiologia , Transtornos Plaquetários/fisiopatologia , Plaquetas/metabolismo , HumanosRESUMO
Endocrinopathies in patients with hypocalcemia and hyperphosphatemia that share resistance to parathyroid hormone (PTH) are grouped under the term pseudohypoparathyroidism (PHP). Patients with PHP type Ia (PHP-Ia) often present with additional hormonal resistance and show characteristic physical features that are jointly termed as having an Albright's hereditary osteodystrophy (AHO) phenotype. Alternatively, PHPIb patients predominantly have PTH and sometimes TSH resistance but do not present with AHO features. Most of these PHP forms are caused by defects in GNAS, an imprinted gene locus consisting of maternal, paternal and biallelic transcripts. PHP-Ia is caused by heterozygous inactivating mutations in those exons of GNAS encoding the alpha subunit of the stimulatory guanine nucleotide-binding protein (Gsalpha) while PHPIb results from epigenetic GNAS defects. Familial and sporadic forms of PHP-Ib have distinct GNAS imprinting patterns: familial PHP-Ib patients have an exon A/B-only imprinting defect whereas sporadic PHP-Ib cases have abnormal imprinting of the three differentially methylated regions (DMRs) in GNAS. This classification of PHP was made years ago but was recently questioned since different studies showed GNAS epigenetic defects in PHP-Ia patients. In this review, we focus on the epigenetic description and screening methods of GNAS, the associated pathology and the recent need for a PHP reclassification.
Assuntos
Epigenômica/métodos , Subunidades alfa Gs de Proteínas de Ligação ao GTP/genética , Pseudo-Hipoparatireoidismo/genética , Animais , Metilação de DNA/genética , Impressão Genômica/genética , HumanosRESUMO
Regulators of G protein signaling (RGS) are intracellular signaling regulators that bind activated G protein α subunits (Gα) and increase their intrinsic GTPase activity via their common RGS homology domain. In addition to their GTPase accelerating activity (GAP), RGS proteins also contain other domains that regulate their receptor selectivity, their interaction with other proteins such as adenylyl cyclase or their subcellular localization via interaction with scaffold proteins such as tubulin, 14-3-3 or spinophilin. There are at least 37 different RGS family members in humans and numerous physiological functions have been assigned to these proteins, which have rather a tissue-specific expression pattern. The role of some RGS proteins was shown to be important for hematopoiesis. More recent studies also focused on their expression in platelets, and for R4 RGS subfamily members RGS2, RGS16 and RGS18, it could be demonstrated that they regulate megakaryopoiesis and/or platelet function. These functional studies mostly comprised in vitro experiments and in vivo studies using small animal models. Their role in human pathology related to platelet dysfunction remains still largely unknown, except for a case report with a RGS2 gain of function mutation. In addition to an introduction on RGS signaling and different effectors with a special focus on the R4 subfamily members, we here will give an overview of the studies related to the role of RGS proteins in hematopoiesis, megakaryopoiesis and platelet function.
Assuntos
Plaquetas/fisiologia , Subunidades alfa de Proteínas de Ligação ao GTP/metabolismo , Hematopoese/fisiologia , Megacariócitos/fisiologia , Proteínas RGS/fisiologia , Transdução de Sinais , Proteínas 14-3-3/metabolismo , Animais , Regulação da Expressão Gênica , Hemostasia , Humanos , Proteínas dos Microfilamentos/metabolismo , Proteínas do Tecido Nervoso/metabolismo , Fosforilação , Ligação Proteica , Tirosina/químicaRESUMO
Blood platelets play a fundamental role in primary haemostasis and wound repair, but are also involved in several thrombotic and bleeding disorders for which the underlying mechanisms are still largely unknown. Elucidating platelet biology would help in finding novel disease biomarkers and drug targets in complex and/or genetically unknown platelet-related disorders. Proteomics, which allows studying thousands of gene products at once, represents an efficient tool to quali-quantitatively analyze and compare the platelet protein patterns of different samples (i.e. control/patient, treated/untreated, drug sensitive/resistant), to investigate post-translation modifications, protein-protein interactions and the underlying molecular pathways. This review gives an overview of the applications of proteomic strategies to study platelet biology and function, as well as to unravel differences in protein expression according to specific platelet conditions (i.e. basic versus activated), compartments (i.e. membrane or granules) and fractions (i.e. phosphoproteins and glycoproteins). The use of innovative powerful proteomic technologies can lead to the identification of proteins whose expression is altered in pathological conditions, allowing the identification of candidate biomarkers for: i) understanding the molecular defects underlying platelet disorders, ii) obtaining novel insights in more complex diseases that involve platelets, iii) unraveling the drug mode of action or identifying the mechanisms of drug resistance and iv) detecting novel therapeutic antiplatelet targets based on fundamental platelet research studies. Several studies on how proteomics proved to be useful in our understanding of platelet function and its diseases are discussed. Eventually, this could result in the discovery of novel drug targets for antiplatelet therapy.
Assuntos
Transtornos Plaquetários/metabolismo , Plaquetas/metabolismo , Inibidores da Agregação Plaquetária/uso terapêutico , Proteômica , Biomarcadores/metabolismo , Transtornos Plaquetários/tratamento farmacológico , Transtornos Plaquetários/patologia , Humanos , Espectrometria de Massas , Inibidores da Agregação Plaquetária/química , Proteoma/metabolismoRESUMO
Platelets are indispensable for primary haemostasis, but their function needs to be tightly regulated to prevent excessive platelet activity, possibly leading to atherothrombotic events. An important mediator of the platelet activity is cyclic AMP (cAMP), which inhibits platelet aggregation. Intracellular cAMP levels are regulated via the Gs and Gi alpha subunits of heterotrimeric G proteins, which couple to adenylyl cyclase to respectively stimulate or inhibit cAMP production. Binding of a ligand to its G protein-coupled seven-transmembrane receptor activates these G proteins. In this review, we discuss a Gs-coupled receptor on platelets, VPAC1, and 2 important Gi-coupled receptors, the ADP receptor P2Y(12) and the prostaglandin E(2) receptor EP3. The regulation of platelet cAMP levels at the level of the receptors themselves or the G proteins coupled to them is analyzed. Alterations in Gsα and Giα function are associated with altered platelet reactivity. An increase in Gs function, or alternatively a defective Gi signaling, can be a risk factor for bleeding, while a loss of Gs function can result in a prothrombotic state. Regulator of G protein signaling (RGS) proteins accelerate the rate of inactivation of G protein-mediated signaling. One of the RGS proteins, RGS2, inhibits Gs signaling by interacting directly with adenylyl cyclase. The thienopyridine class of antiplatelet agents is based on cAMP-mediated regulation of platelet function through modification of the P2Y(12) receptor. Clopidogrel and some other novel cAMP regulators are discussed. Secondly, we review the use of prostacyclin derivatives to treat pulmonary arterial hypertension.
Assuntos
Plaquetas/efeitos dos fármacos , Plaquetas/metabolismo , AMP Cíclico/metabolismo , Inibidores da Agregação Plaquetária/farmacologia , Inibidores da Agregação Plaquetária/uso terapêutico , Antagonistas do Receptor Purinérgico P2Y/farmacologia , Antagonistas do Receptor Purinérgico P2Y/uso terapêutico , Clopidogrel , AMP Cíclico/sangue , Hipertensão Pulmonar Primária Familiar , Humanos , Hipertensão Pulmonar/tratamento farmacológico , Antagonistas do Receptor Purinérgico P2Y/química , Receptores Purinérgicos P2Y12/metabolismo , Receptores Tipo I de Polipeptídeo Intestinal Vasoativo/metabolismo , Transdução de Sinais/efeitos dos fármacos , Ticlopidina/análogos & derivados , Ticlopidina/química , Ticlopidina/farmacologiaRESUMO
BACKGROUND: The effect of cholestatic liver disease on primary hemostasis function remains ill-defined. OBJECTIVES: To determine platelet function and identify the mechanisms involved in the observed platelet function in cholestatic rats. METHODS: Platelet function was studied in a model of 2-week bile duct ligation and compared to that in sham-operated rats with and without a storage pool defect. RESULTS: ADP-induced and collagen-induced platelet aggregation were clearly impaired following bile duct ligation (P<0.01 for areas under the curve). Crossover experiments, with sham platelets in bile duct-ligated plasma and vice versa, demonstrated that this is due to inhibition by a plasmatic factor, as sham platelets aggregated less in cholestatic plasma (P<0.03) and to an equal extent as platelets from bile duct-ligated rats when they were in the same sham or cholestatic plasma. Moreover, in bile duct-ligated rats, platelet ultrastructure was unaffected and platelet aggregation was similar to that of sham platelets when resuspended in the same plasma (P-value not significant). Additionally, studies in storage pool-deficient rats showed no role of platelet exhaustion. The plasmatic factor causing impaired aggregation was shown to be increased total activity of ADP-degrading enzymes upon bile duct ligation (P<0.01), as there was no decreased aggregation with a stable ADP analog in bile duct-ligated rats (P-value not significant vs. sham-operated rats). Furthermore, preincubation of plasma from bile duct-ligated rats with ADP decreased aggregation more than was seen with sham plasma (P<0.01). CONCLUSIONS: Bile duct ligation does not affect intrinsic platelet function, but impairs platelet activation via release of ADP-degrading enzymes in the circulation.
Assuntos
5'-Nucleotidase/sangue , Difosfato de Adenosina/sangue , Fosfatase Alcalina/sangue , Plaquetas/enzimologia , Colestase/sangue , Agregação Plaquetária , Animais , Ductos Biliares/cirurgia , Plaquetas/ultraestrutura , Colestase/enzimologia , Colestase/patologia , Colágeno , Modelos Animais de Doenças , Humanos , Ligadura , Contagem de Plaquetas , Ratos , Ratos Wistar , Fatores de TempoRESUMO
SUMMARY BACKGROUND: Regulator of G-protein signaling (RGS) 2 negatively regulates Gs signaling by inhibiting the activation of adenylyl cyclase (AC). RGS2 mRNA contains four translation initiation sites, leading to four isoforms with different abilities to inhibit AC activity; the largest isoform is the most pronounced inhibitor. A role for RGS2 in platelets is not known. OBJECTIVE: To describe a heterozygous RGS2 mutation (G23D) in three related patients, leading to Gs hypofunction in their platelets, and to study the mechanism behind the effect of the RGS2 mutation on platelet function and morphology. METHODS: Gs signaling was studied ex vivo in platelets and in vitro in transfected cells. Translation initiation was evaluated in vitro, and the interaction of wild-type and G23D RGS2 with AC was unraveled via immunoprecipitation. Platelet granule content was analyzed with proteomics. RESULTS: The mutation leads to reduced cAMP production after stimulation of Gs-coupled receptors. The largest RGS2 isoforms, with strong AC inhibitor activity, are enriched when the mutation is present, as compared with wild-type RGS2. Moreover, the mutation results in a stronger interaction of RGS2 with AC. G23D RGS2 carriers have enlarged, round platelets with abnormal alpha-granules. Proteomics of the platelet releasate revealed altered expression of some proteins involved in actin assembly, and carriers seemed to have a reduced platelet shape change. CONCLUSIONS: We present the first platelet Gs signaling defect caused by a heterozygous RGS2 variant that results in a unique mutational mechanism, such as the differential use of translation initiation sites resulting in different functional RGS2 isoforms.
Assuntos
Plaquetas/metabolismo , Proteínas de Ligação ao GTP/metabolismo , Mutação de Sentido Incorreto , Proteínas RGS/fisiologia , Inibidores de Adenilil Ciclases , Plaquetas/patologia , Plaquetas/fisiologia , Forma Celular/genética , Heterozigoto , Humanos , Biossíntese de Proteínas , Isoformas de Proteínas , Proteínas RGS/genética , Transdução de SinaisRESUMO
Platelets are highly responsive to signals from their environment. The sensing and processing of some of these stimuli are mediated by G-protein signal transduction cascades. It is well established that proteins involved in signal transduction may be targets for naturally occurring mutations resulting in human diseases. The best-studied molecules in platelets in relation to disease are the G-protein coupled receptors being the most platelet-specific. Many of the other signal transduction genes are often not only present in platelets but also in other tissues. Therefore, the clinical phenotype of signaling defects in platelets, apart from the membrane receptor defects, is seldom isolated to a hemostatic phenotype. Moreover, as platelets are easily accessible cells, and one of the best-studied models regarding signaling, platelets are easily applicable to investigate defects in ubiquitously expressed genes. Apart from a discussion on classical thrombopathies, this review will also deal with the less commonly known relation between platelet signaling defects and disorders with a broader clinical phenotype.