RESUMO
Increasing evidence suggests that platelets play a predominant role in colon and breast cancer metastasis, but the underlying molecular mechanisms remain elusive. Glycoprotein VI (GPVI) is a platelet-specific receptor for collagen and fibrin that triggers platelet activation through immunoreceptor tyrosine-based activation motif (ITAM) signaling and thereby regulates diverse functions, including platelet adhesion, aggregation, and procoagulant activity. GPVI has been proposed as a safe antithrombotic target, because its inhibition is protective in models of arterial thrombosis, with only minor effects on hemostasis. In this study, the genetic deficiency of platelet GPVI in mice decreased experimental and spontaneous metastasis of colon and breast cancer cells. Similar results were obtained with mice lacking the spleen-tyrosine kinase Syk in platelets, an essential component of the ITAM-signaling cascade. In vitro and in vivo analyses supported that mouse, as well as human GPVI, had platelet adhesion to colon and breast cancer cells. Using a CRISPR/Cas9-based gene knockout approach, we identified galectin-3 as the major counterreceptor of GPVI on tumor cells. In vivo studies demonstrated that the interplay between platelet GPVI and tumor cell-expressed galectin-3 uses ITAM-signaling components in platelets and favors the extravasation of tumor cells. Finally, we showed that JAQ1 F(ab')2-mediated inhibition of GPVI efficiently impairs platelet-tumor cell interaction and tumor metastasis. Our study revealed a new mechanism by which platelets promote the metastasis of colon and breast cancer cells and suggests that GPVI represents a promising target for antimetastatic therapies.
Assuntos
Plaquetas/patologia , Neoplasias da Mama/patologia , Neoplasias do Colo/patologia , Galectina 3/metabolismo , Glicoproteínas da Membrana de Plaquetas/metabolismo , Animais , Plaquetas/metabolismo , Neoplasias da Mama/metabolismo , Linhagem Celular Tumoral , Movimento Celular , Neoplasias do Colo/metabolismo , Feminino , Humanos , Neoplasias Pulmonares/patologia , Neoplasias Pulmonares/secundário , Masculino , Camundongos Endogâmicos BALB C , Camundongos Endogâmicos C57BL , Metástase Neoplásica/patologia , Ativação Plaquetária , Glicoproteínas da Membrana de Plaquetas/genética , Mapas de Interação de ProteínasRESUMO
Charge interactions play a critical role in the activation of the innate immune system by damage- and pathogen-associated molecular pattern receptors. The ability of these receptors to recognize a wide spectrum of ligands through a common mechanism is critical in host defense. In this article, we argue that platelet glycoprotein receptors that signal through conserved tyrosine-based motifs function as pattern recognition receptors (PRRs) for charged endogenous and exogenous ligands, including sulfated polysaccharides, charged proteins and nanoparticles. This is exemplified by GPVI, CLEC-2 and PEAR1 which are activated by a wide spectrum of endogenous and exogenous ligands, including diesel exhaust particles, sulfated polysaccharides and charged surfaces. We propose that this mechanism has evolved to drive rapid activation of platelets at sites of injury, but that under some conditions it can drive occlusive thrombosis, for example, when blood comes into contact with infectious agents or toxins. In this Opinion Article, we discuss mechanisms behind charge-mediated platelet activation and opportunities for designing nanoparticles and related agents such as dendrimers as novel antithrombotics.
Assuntos
Plaquetas/metabolismo , Nanopartículas/metabolismo , Ativação Plaquetária/imunologia , Glicoproteínas da Membrana de Plaquetas/metabolismo , Receptores de Reconhecimento de Padrão/metabolismo , Humanos , Ligantes , Transdução de SinaisRESUMO
Platelet membrane glycoprotein VI (GPVI) is increasingly recognized as an important receptor for thrombus formation and growth. Numerous arguments have been published indicating that GPVI plays a major role in thrombosis without being essential for physiological hemostasis. In humans, GPVI deficiencies are rarely reported. These are most often deficiencies occurring in the context of autoimmunity and, more rarely, genetic deficits. The purpose of this review is to compile data on the quantitative and qualitative genetic abnormalities of GPVI.
Assuntos
Plaquetas/metabolismo , Glicoproteínas da Membrana de Plaquetas/genética , Humanos , Glicoproteínas da Membrana de Plaquetas/deficiênciaRESUMO
Glycoprotein VI, a major platelet activation receptor for collagen and fibrin, is considered a particularly promising, safe antithrombotic target. In this study, we show that human glycoprotein VI signals upon platelet adhesion to fibrinogen. Full spreading of human platelets on fibrinogen was abolished in platelets from glycoprotein VI- deficient patients suggesting that fibrinogen activates platelets through glycoprotein VI. While mouse platelets failed to spread on fibrinogen, human-glycoprotein VI-transgenic mouse platelets showed full spreading and increased Ca2+ signaling through the tyrosine kinase Syk. Direct binding of fibrinogen to human glycoprotein VI was shown by surface plasmon resonance and by increased adhesion to fibrinogen of human glycoprotein VI-transfected RBL-2H3 cells relative to mock-transfected cells. Blockade of human glycoprotein VI with the Fab of the monoclonal antibody 9O12 impaired platelet aggregation on preformed platelet aggregates in flowing blood independent of collagen and fibrin exposure. These results demonstrate that human glycoprotein VI binds to immobilized fibrinogen and show that this contributes to platelet spreading and platelet aggregation under flow.
Assuntos
Plaquetas/fisiologia , Fibrinogênio/metabolismo , Leucemia Basofílica Aguda/patologia , Ativação Plaquetária , Glicoproteínas da Membrana de Plaquetas/metabolismo , Animais , Humanos , Leucemia Basofílica Aguda/genética , Leucemia Basofílica Aguda/metabolismo , Camundongos , Adesividade Plaquetária , Glicoproteínas da Membrana de Plaquetas/genética , Ratos , Quinase Syk/genética , Quinase Syk/metabolismo , Trombose , Células Tumorais CultivadasRESUMO
BACKGROUND: An increase in circulating platelets, or thrombocytosis, is recognized as an independent risk factor of bad prognosis and metastasis in patients with ovarian cancer; however the complex role of platelets in tumor progression has not been fully elucidated. Platelet activation has been associated with an epithelial to mesenchymal transition (EMT), while Tissue Factor (TF) protein expression by cancer cells has been shown to correlate with hypercoagulable state and metastasis. The aim of this work was to determine the effect of platelet-cancer cell interaction on TF and "Metastasis Initiating Cell (MIC)" marker levels and migration in ovarian cancer cell lines and cancer cells isolated from the ascetic fluid of ovarian cancer patients. METHODS: With informed patient consent, ascitic fluid isolated ovarian cancer cells, cell lines and ovarian cancer spheres were co-cultivated with human platelets. TF, EMT and stem cell marker levels were determined by Western blotting, flow cytometry and RT-PCR. Cancer cell migration was determined by Boyden chambers and the scratch assay. RESULTS: The co-culture of patient-derived ovarian cancer cells with platelets causes: 1) a phenotypic change in cancer cells, 2) chemoattraction and cancer cell migration, 3) induced MIC markers (EMT/stemness), 3) increased sphere formation and 4) increased TF protein levels and activity. CONCLUSIONS: We present the first evidence that platelets act as chemoattractants to cancer cells. Furthermore, platelets promote the formation of ovarian cancer spheres that express MIC markers and the metastatic protein TF. Our results suggest that platelet-cancer cell interaction plays a role in the formation of metastatic foci.
Assuntos
Plaquetas/metabolismo , Neoplasias Ovarianas/metabolismo , Neoplasias Ovarianas/patologia , Tromboplastina/metabolismo , Biomarcadores , Comunicação Celular , Movimento Celular , Fatores Quimiotáticos/metabolismo , Feminino , Humanos , Estadiamento de Neoplasias , Neoplasias Ovarianas/sangue , Neoplasias Ovarianas/cirurgia , Fenótipo , Complexo Glicoproteico GPIb-IX de Plaquetas/metabolismo , Tromboplastina/genética , Células Tumorais CultivadasRESUMO
OBJECTIVE: Cocaine consumption is a risk factor for vascular ischemic complications. Although endothelial dysfunction and accelerated atherosclerosis have been observed in cocaine consumers, the mechanisms underlying their pathogenesis are not fully understood. This study aimed at identifying the effects of atorvastatin in relation to a proadhesive and prothrombotic phenotype induced by cocaine and plasma from chronic cocaine users on endothelial cells. APPROACH AND RESULTS: Human umbilical vein endothelial cells were exposed to either cocaine or platelet-free plasma (PFP) from chronic cocaine consumers in the presence or absence of 10 µmol/L of atorvastatin. Atorvastatin significantly reduced the enhanced platelet adhesion that was induced by cocaine and PFP from chronic cocaine consumers, as well as the release of the von Willebrand factor. Atorvastatin also avoided striking alterations on cell monolayer structure triggered by both stimuli and enhanced NO reduction because of cocaine stimulation through disrupting interactions between endothelial nitric oxide synthase (eNOS) and caveolin-1, thus increasing eNOS bioavailability. Cocaine-increased tissue factor-dependent procoagulant activity and reactive oxygen species generation were not counteracted by atorvastatin. Although monocyte chemoattractant protein-1 levels were not significantly higher than controls either under cocaine or PFP stimulation, atorvastatin completely avoided monocyte chemoattractant protein-1 release in both conditions. Platelets stimulated with cocaine or PFP did not express P-selectin, glycoprotein IIb/IIIa, or CD40L and failed to adhere to resting human umbilical vein endothelial cell. CONCLUSIONS: Cocaine and patient plasma equally induced a proadhesive and prothrombotic phenotype in endothelial cells, except for von Willebrand Factor release, which was only induced by PFP from chronic cocaine consumers. Atorvastatin improved endothelial cell function by reducing cocaine-induced and PFP from chronic cocaine consumer-induced effects on platelet adhesion, cell architecture, and NO production.
Assuntos
Adesão Celular/efeitos dos fármacos , Cocaína/farmacologia , Endotélio Vascular/patologia , Ácidos Heptanoicos/farmacologia , Fenótipo , Plasma , Pirróis/farmacologia , Trombose/patologia , Anticolesterolemiantes/farmacologia , Atorvastatina , Caveolina 1/metabolismo , Células Cultivadas , Transtornos Relacionados ao Uso de Cocaína/sangue , Endotélio Vascular/efeitos dos fármacos , Endotélio Vascular/metabolismo , Humanos , Técnicas In Vitro , Óxido Nítrico/metabolismo , Óxido Nítrico Sintase Tipo III/metabolismo , Espécies Reativas de Oxigênio/metabolismo , Trombose/metabolismo , Fator de von Willebrand/metabolismoRESUMO
Background: Mild secretion defects are the most frequent and challenging blood platelet disorders to diagnose. Most δ-granule secretion tests lack validation, are not quantitative, or have unreliable response to weak platelet agonists. Objectives: To compare platelet serotonin secretion by HPLC-electrochemical detection technique (HPLC-ECD) with the reference isotopic test (3H-5-HT), evaluating its performance in clinical laboratories. Methods: The assay validation followed STARD-2015 recommendations. HPLC-ECD measured the nonsecreted serotonin remaining in platelet pellets after aggregation, comparing it with the reference 3H-5-HT assay. We studied subjects with inherited and aspirin-induced blood platelet disorders and assessed the HPLC-ECD operation for routine clinical diagnosis. Results: Calibration curves were linear (R2 = 0.997), with SD for residuals of 3.91% and analytical sensitivity of 5ng/mL. Intra- and interassay imprecision bias ranged between -8.5% and 2.1% and -9% and 3.1%, respectively. Serotonin recovery and stability were >95%, and the variability range of measurements was -5.5% to 4.6%. Statistical differences detected between tests were biologically irrelevant, with bias of 1.48% (SD, 8.43) and CI agreement of -18% to 15%. Both assays distinctly detected platelet secretion induced by 10 µM epinephrine and 4 µmM adenosine diphosphate. However, HPLC-ECD is quantitative and more sensitive to low serotonin content in blood platelets. Reference cutoffs for each agonist were determined in 87 subjects. Initially, the HPLC-ECD requires relatively expensive equipment and trained operators but has remarkably cheap running costs and a turn-around time of 24-36 hours. We have used this diagnostic tool routinely for >8 years. Conclusion: HPLC-ECD assay for platelet serotonin secretion is highly accurate, has advantages over the reference 3H-5-HT test, and is suitable as a clinical laboratory technique.
RESUMO
Fibrinolysis dysfunctions cause bleeding or predisposition to thrombosis. Platelets contain several factors of the fibrinolytic system, which could up or down regulate this process. However, the temporal relationship and relative contributions of plasma and platelet components in clot lysis are mostly unknown. We developed a clot lysis time (CLT) assay in platelet-rich plasma (PRP-CLT, with and without stimulation) and compared it to a similar one in platelet-free plasma (PFP) and to another previously reported test in platelet-poor plasma (PPP). We also studied the differential effects of a single dose of tranexamic acid (TXA) on these tests in healthy subjects. PFP- and PPP-CLT were significantly shorter than PRP-CLT, and the three assays were highly correlated (p < 0.0001). PFP- and PPP-, but more significantly PRP-CLT, were positively correlated with age and plasma PAI-1, von Willebrand factor, fibrinogen, LDL-cholesterol, and triglycerides (p < 0.001). All these CLT assays had no significant correlations with platelet aggregation/secretion, platelet counts, and pro-coagulant tests to explore factor X activation by platelets, PRP clotting time, and thrombin generation in PRP. Among all the studied variables, PFP-CLT was independently associated with plasma PAI-1, LDL-cholesterol, and triglycerides and, additionally, stimulated PRP-CLT was also independently associated with plasma fibrinogen. A single 1 g dose of TXA strikingly prolonged all three CLTs, but in contrast to the results without the drug, the lysis times were substantially shorter in non-stimulated or stimulated PRP than in PFP and PPP. This standardized PRP-CLT may become a useful tool to study the role of platelets in clot resistance and lysis. Our results suggest that initially, the platelets enmeshed in the clot slow down the fibrinolysis process. However, the increased clot resistance to lysis induced by TXA is overcome earlier in platelet-rich clots than in PFP or PPP clots. This is likely explained by the display of platelet pro-fibrinolytic effects. Focused research is needed to disclose the mechanisms for the relationship between CLT and plasma cholesterol and its potential pathophysiologic and clinical relevance.
Assuntos
Antifibrinolíticos/farmacologia , Plaquetas/metabolismo , Fibrinólise/efeitos dos fármacos , Plasma , Ácido Tranexâmico/farmacologia , Adolescente , Adulto , Fatores Etários , Idoso , Proteínas Sanguíneas/metabolismo , Feminino , Humanos , Masculino , Pessoa de Meia-Idade , Testes de Função Plaquetária/métodos , Fatores de Tempo , Triglicerídeos/metabolismoRESUMO
Assessment of platelet secretion is crucial for diagnosing suspected inherited platelet function disorders (IPFD). A previous survey of the SSC on Platelet Physiology of the ISTH and a comprehensive review highlighted that most of the platelet secretion assays (PSAs) lack standardization and validation. The aim of this study was to provide expert consensus guidance on the use of PSAs for IPFD diagnosis. We surveyed 26 experts from 10 different countries using the RAND/UCLA methodology, to attain a consensus on sensitivity, specificity, feasibility, time to readout, and cost of most PSAs. Answers were then graded in three categories: appropriate, uncertain, and inappropriate. Equivocal or misinterpretable statements required a second and third round survey involving 14 of the original 26 experts. We report here the consolidated results of the entire procedure. There was uniform agreement on several general statements, including that PSAs should be performed in hemostasis laboratories as first line diagnostic tests even in patients with normal platelet aggregation, and should include a δ-granule secretion marker. Among the specific assays examined, lumiaggregometry, other luciferin/luciferase-based assays, high-performance liquid chromatography methods, radiolabeled-serotonin based assays, and whole-mount transmission electron microscopy were rated as appropriate for the measurement of δ-granule release, and platelet P-selectin expression by flow cytometry and released proteins by ELISA for α-granule release. For most of the other PSAs, the expert opinions were widely dispersed. Lack of expert consensus on many PSAs clearly indicates an unmet need for rigorous standardization, multicenter comparison of results, and validation of PSAs for clinical laboratory practice.
Assuntos
Transtornos Plaquetários , Trombastenia , Transtornos Plaquetários/diagnóstico , Transtornos Plaquetários/metabolismo , Plaquetas/metabolismo , Comunicação , Prova Pericial , Hemostasia , Humanos , Estudos Multicêntricos como Assunto , Testes de Função Plaquetária/métodosRESUMO
BACKGROUND: Genome-wide association studies are currently identifying new loci with potential roles in thrombosis and hemostasis: these loci include novel polymorphisms associated with platelet function traits and count. However, no genome-wide study performed on children has been reported to date, in spite of the potential that these subjects have in genetic studies, when compared to adults, given the minimal degree of confounders, i.e., acquired and environmental factors, such as smoking, physical activity, diet, and drug or hormone intake, which are particularly important in platelet function. DESIGN AND METHODS: To identify new genetic variants involved in platelet reactivity and count, we performed a genome-wide association study on 75 children (8.5±1.8 years) using the Illumina Sentrix Human CNV370-Quad BeadChip containing 320,610 single nucleotide polymorphisms. Functional analyses included assessment of platelet aggregation and granule secretion triggered by different agonists (arachidonic acid, collagen, epinephrine, ADP), as well as platelet count. Associations were selected based on statistical significance and physiological relevance for a subsequent replication study in a similar sample of 286 children. RESULTS: We confirmed previously established associations with plasma levels of factors XII, VII and VIII as well as associations with platelet responses to ADP. Additionally, we identified 82 associations with platelet reactivity and count with a P value less than 10(-5). From the associations selected for further replication, we validated two single nucleotide polymorphisms with mildly increased platelet reactivity (rs4366150 and rs1787566) on the LPAR1 and MYO5B genes, encoding lisophosphatidic acid receptor-1 and myosin VB, respectively; and rs1937970, located on the NRG3 gene coding neuroregulin-3, associated with platelet count. CONCLUSIONS: Our genome-wide association study performed in children, followed by a validation analysis, led us to the identification of new genes potentially relevant in platelet function and biogenesis.
Assuntos
Plaquetas/metabolismo , Loci Gênicos , Estudo de Associação Genômica Ampla , Contagem de Plaquetas , Alelos , Criança , Pré-Escolar , Feminino , Frequência do Gene , Predisposição Genética para Doença , Humanos , Masculino , Polimorfismo de Nucleotídeo Único , Reprodutibilidade dos TestesRESUMO
Cocaine abuse increases the risk of cardiac and cerebrovascular events, such as myocardial infarction and ischemic stroke. The underlying mechanisms leading to these complications are not fully understood although intravascular thrombus formation has been observed. The aim of this study was to investigate the existence of platelet activation and the effect of short-term abstinence in chronic cocaine consumers. We studied 23 cocaine dependent individuals (aged 20-54 years) who met DSM-IV criteria for cocaine dependence and 20 controls. Samples were obtained at baseline, within 72 h of last drug exposure and after 4 weeks of controlled abstinence. Monocyte-platelet aggregates (MPA) were measured by flow cytometry. Plasma levels of soluble CD40L (sCD40L), Neutrophil-Activating Peptide-2 (NAP-2) and regulated on activation normal T cells expressed and secreted (RANTES) were determined by ELISA. Levels of MPA, sCD40L, NAP-2 and RANTES were significantly higher (all p < 0.05) in cocaine addicts compared to controls at baseline. All the parameters returned to values similar to the control group after 4-weeks' abstinence. Levels of sCD40L and RANTES were associated with an index of intensity of drug consumption (p < 0.02). Our results demonstrate that cocaine use induces platelet activation which is a prominent finding after recent consumption. The persistence over time of this condition may contribute not only to acute thrombotic complications but also to the development of early-onset atherosclerotic process observed in cocaine abusers.
Assuntos
Transtornos Relacionados ao Uso de Cocaína/metabolismo , Cocaína/farmacologia , Ativação Plaquetária/efeitos dos fármacos , Adulto , Biomarcadores/sangue , Ligante de CD40/sangue , Agregação Celular/efeitos dos fármacos , Transtornos Relacionados ao Uso de Cocaína/sangue , Feminino , Humanos , Masculino , Pessoa de Meia-Idade , Monócitos/efeitos dos fármacos , Monócitos/metabolismo , Agregação Plaquetária/efeitos dos fármacos , Adulto Jovem , beta-Tromboglobulina/metabolismoRESUMO
Thromboembolic disease (TED) is the leading cause of morbidity and mortality worldwide. The hallmark of oral long-term anticoagulant therapy has been the use of vitamin K antagonists, whose anticoagulant effect is exerted inhibiting vitamin K epoxide reductase. Warfarin and acenocoumarol are the most commonly used. In the last five years several new drugs for long term anticoagulation have been developed, which can inhibit single clotting factors with the purpose of improving drug therapeutic range and, ideally, minimizing bleeding risks. This review addresses the state of the art on the clinical use of inhibitors of activated factor X and thrombin.
Assuntos
Anticoagulantes/classificação , Inibidores do Fator Xa , Trombina/antagonistas & inibidores , Vitamina K/antagonistas & inibidores , Administração Oral , HumanosRESUMO
Hereditary disorders of primary hemostasis, characterized by mucocutaneous bleeding (MCB), are highly prevalent in children. Few cases are clearly monogenic, but the overwhelming majority are classified as mild bleeding disorders, with wide clinical and laboratory heterogeneity suggestive of complex polygenic diseases. In this framework, and by homology with venous thrombosis, some functional polymorphisms affecting the hemostatic system should be considered. We evaluated the role of 18 common hemostatic polymorphisms on the occurrence and severity of MCB in a case-control study including 269 patients and 286 matched controls consecutively recruited. FV Leiden was associated with milder bleeding severity, assessed by a standardized bleeding score (p = 0.013). Multivariate analysis revealed that three additional polymorphisms protected against MCB (F13 Leu34, OR = 0.66; 95% CI, 0.47-0.94; p = 0.024; VKORC1 1173T, OR = 0.59; 95% CI, 0.40-0.87; p = 0.009; and non-O blood group alleles, OR = 0.59; 95% CI, 0.41-0.86; p = 0.006). When combined, these polymorphisms showed an additive protection (OR = 0.24; 95% CI, 0.11-0.52), supporting the polygenic nature of MCB. Our data suggest that some common polymorphisms affecting hemostasis-related genes could protect from bleeding.
Assuntos
Transtornos Hemorrágicos/genética , Hemostasia/genética , Mucosa/fisiopatologia , Polimorfismo Genético , Adolescente , Adulto , Animais , Estudos de Casos e Controles , Criança , Pré-Escolar , Fator V/genética , Feminino , Predisposição Genética para Doença , Genótipo , Humanos , Pessoa de Meia-Idade , Dados de Sequência Molecular , Adulto JovemRESUMO
Cerebral amyloid angiopathy (CAA) and ß-amyloid (Aß) deposition in the brain parenchyma are hallmarks of Alzheimer's disease (AD). We previously reported that platelets contribute to Aß aggregation in cerebral vessels by secreting the factor clusterin upon binding of Aß40 to the fibrinogen receptor integrin αIIbß3 Here, we investigated the contribution of the collagen receptor GPVI (glycoprotein VI) in platelet-induced amyloid aggregation. Using platelets isolated from GPVI-wild type and GPVI-deficient human donors and mice, we found that Aß40 bound to GPVI, which induced the release of ATP and fibrinogen, resulting in platelet aggregation. Binding of Aß40 to integrin αIIbß3, fibrinogen, and GPVI collectively contributed to the formation of amyloid clusters at the platelet surface. Consequently, blockade of αIIbß3 or genetic loss of GPVI reduced amyloid fibril formation in cultured platelets and decreased the adhesion of Aß-activated platelets to injured carotid arteries in mice. Application of losartan to inhibit collagen binding to GPVI resulted in decreased Aß40-stimulated platelet activation, factor secretion, and platelet aggregation. Furthermore, the application of GPVI- or integrin-blocking antibodies reduced the formation of platelet-associated amyloid aggregates. Our findings indicate that Aß40 promotes platelet-mediated amyloid aggregation by binding to both GPVI and integrin αIIbß3 Blocking these pathways may therapeutically reduce amyloid plaque formation in cerebral vessels and the brain parenchyma of patients.
Assuntos
Peptídeos beta-Amiloides/metabolismo , Plaquetas/metabolismo , Fragmentos de Peptídeos/metabolismo , Glicoproteínas da Membrana de Plaquetas/metabolismo , Agregação Patológica de Proteínas/metabolismo , Receptores de Colágeno/metabolismo , Adulto , Doença de Alzheimer/metabolismo , Animais , Plaquetas/citologia , Células Cultivadas , Fibrinogênio/metabolismo , Humanos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Pessoa de Meia-Idade , Agregação Plaquetária , Complexo Glicoproteico GPIIb-IIIa de Plaquetas/metabolismo , Glicoproteínas da Membrana de Plaquetas/genética , Ligação Proteica , Receptores de Colágeno/genética , Transdução de SinaisRESUMO
The role of glycoprotein VI (GPVI) in platelets was investigated in 3 families bearing an insertion within the GP6 gene that introduces a premature stop codon prior to the transmembrane domain, leading to expression of a truncated protein in the cytoplasm devoid of the transmembrane region. Western blotting and flow cytometry of GP6hom (homozygous) platelets confirmed loss of the full protein. The level of the Fc receptor γ-chain, which associates with GPVI in the membrane, was partially reduced, but expression of other receptors and signaling proteins was not altered. Spreading of platelets on collagen and von Willebrand factor (which supports partial spreading) was abolished in GP6hom platelets, and spreading on uncoated glass was reduced. Anticoagulated whole blood flowed over immobilized collagen or a mixture of von Willebrand factor, laminin, and rhodocytin (noncollagen surface) generated stable platelet aggregates that express phosphatidylserine (PS). Both responses were blocked on the 2 surfaces in GP6hom individuals, but adhesion was not altered. Thrombin generation was partially reduced in GP6hom blood. The frequency of the GP6het (heterozygous) variant in a representative sample of the Chilean population (1212 donors) is 2.9%, indicating that there are â¼4000 GP6hom individuals in Chile. These results demonstrate that GPVI supports aggregation and PS exposure under flow on collagen and noncollagen surfaces, but not adhesion. The retention of adhesion may contribute to the mild bleeding diathesis of GP6hom patients and account for why so few of the estimated 4000 GP6hom individuals in Chile have been identified.
Assuntos
Transtornos da Coagulação Sanguínea , Glicoproteínas da Membrana de Plaquetas , Plaquetas , Colágeno , Humanos , Glicoproteínas da Membrana de Plaquetas/genética , Fator de von WillebrandRESUMO
Light transmission platelet aggregation (PA), adapted to measure platelet secretion (PS), is the reference test for diagnosing platelet functional disorders (PFD). Problems with these assays include lack of standardisation, unknown reproducibility and lack of universally accepted diagnostic criteria. We addressed these issues in patients with inherited mucocutaneous bleeding (MCB). Normal and abnormal PA tests in 213 patients were reproducible in 93.3% and 90.4% of the cases, respectively. Mean intra-subject coefficient of variation for PA with strong agonists were <9% and mean intra-class correlation coefficient for weak agonists were >0.86 (P < 0.0001). Concomitant impaired PA with 10 micromol/l-adrenaline and 4 micromol/l-ADP was observed in 13.7% of the controls. This combination was not considered per se a criterion for PFD. PA with adrenaline > or = 42% or irreversible aggregation with 4 micromol/l ADP had 93% and 95% Negative Predictive Value for diagnosing PFD, respectively. PA defects were consistently associated with abnormal PS. In contrast, 14.3% of patients with MCB had isolated PS. Thus, standardized PA/PS assays are highly reproducible and concordant in normal and patient populations. Normal PA with adrenaline and low ADP concentration robustly predict a normal PA. Simultaneous PA/PS assays enable the diagnosis of isolated PS defects. This study confirmed that hereditary PA-PS defects are highly prevalent.
Assuntos
Transtornos Plaquetários/diagnóstico , Agregação Plaquetária/fisiologia , Serotonina/metabolismo , Adolescente , Adulto , Transtornos Plaquetários/sangue , Plaquetas/metabolismo , Criança , Pré-Escolar , Feminino , Humanos , Lactente , Masculino , Pessoa de Meia-Idade , Testes de Função Plaquetária/métodos , Estudos Prospectivos , Reprodutibilidade dos Testes , Adulto JovemRESUMO
Polymorphisms affecting platelet receptors and intracellular proteins have been extensively studied in relation to their potential influence in thrombosis and haemorrhages. However, few reports have addressed their impact on platelet function, with contradictory results. Limitations of these studies include, among others, small number of patients, the platelet functional parameters analyzed and their known variability in the healthy population. We studied the effect of six polymorphisms [ITGB3 1565T > C (HPA-1), GPIBA variable number tandem repeat and 524C > T (HPA-2), ITGA2 807C > T, ADRA2A 1780A > G, and TUBB1 Q43P] on platelet function in 286 healthy subjects and their potential pathogenetic role in 160 patients with hereditary mucocutaneous bleeding of unknown cause. We found no effect of any of these polymorphisms on platelet aggregation, secretion, PFA-100, and thrombin generation in platelet rich plasma. Furthermore, patients and controls showed no significant differences in the frequency of any of these polymorphisms. Thus, our study demonstrated that polymorphisms in genes affecting platelet function do not influence significantly major platelet functions and appear irrelevant in the pathogenesis of bleeding disorders.
Assuntos
Antígenos de Plaquetas Humanas/genética , Plaquetas/fisiologia , Transtornos Hemorrágicos/genética , Polimorfismo Genético , Adolescente , Adulto , Estudos de Casos e Controles , Criança , Pré-Escolar , Feminino , Predisposição Genética para Doença , Genótipo , Humanos , Masculino , Pessoa de Meia-Idade , Fenótipo , Agregação Plaquetária , Testes de Função Plaquetária , Serotonina/metabolismo , Estatísticas não Paramétricas , Trombina/biossíntese , Adulto JovemRESUMO
The major advances from research on platelet molecular and cell biology, physiology, and pathophysiology over the past decades have not been adequately translated to clinical laboratory diagnosis. Hereditary platelet function disorders (PFDs) are at least as prevalent in the general population as von Willebrand disease (VWD) although PFDs tend not be as well recognized or evaluated. Clinical mucous and skin bleeding in patients with PFDs is prototypic of primary hemostasis disorders, and the bleeding pattern is not distinguishable from that of other primary hemostasis disorders such as VWD. However, different treatment needs, between these discrete disorders, make a precise diagnosis mandatory. Currently, clinicians receive reliable laboratory reports when testing patients with severe PFDs, such as Glanzmann thrombasthenia and Bernard-Soulier syndrome, due to the distinctive laboratory defects that these disorders present, together with the availability of differential diagnostic tests. This is not the case for the majority of PFDs generically classified as "platelet secretion disorders," which are a heterogeneous group of "mild bleeding disorders," for which there are not universally accepted diagnostic criteria. An important reason for robust diagnostic tests is the high proportion (more than 50% in some reports) of patients with unequivocal bleeding who have no precise diagnosis established after a complete laboratory workup. It is paradoxical that the current "gold standard" test for PFD diagnosis, light transmission aggregometry (LTA), has not been standardized after more than four decades of worldwide clinical use. This review describes current diagnostic assays for PFD in a clinical hemostasis laboratory, relating these with current knowledge on platelet function and pathophysiology. Special emphasis will be given to LTA and platelet secretion tests, as well as to the reasons why sensitive tests are needed to explore the lesser known participation of platelets in blood clotting and fibrinolytic processes.
Assuntos
Transtornos Plaquetários/diagnóstico , Plaquetas/fisiologia , Agregação Plaquetária/fisiologia , Transtornos Plaquetários/sangue , Técnicas de Laboratório Clínico , HumanosRESUMO
OBJECTIVE: To evaluate the feasibility of diet mediterranisation, in a food-at-work context, and its consequence on metabolic syndrome in a mid-age unselected healthy male population group. DESIGN: One-year longitudinal intervention study. Physical exercise was not modified. SETTING: All workers of the Santiago division of 'Maestranza Diesel', a metal-mechanic company servicing the mining industry, were invited to participate. SUBJECTS: Initially, 145 workers of a total of 171, of average age 39 years, accepted to participate (sixteen women and 129 men). A subgroup of ninety-six men fully completed the controls programmed for the intervention study. Losses from the original group correspond to missing one control (sixteen), leaving the company (eleven) or blood sampling discomfort (six). The women and sixteen male workers, hired post study initiation, did participate but were excluded from this 12-month analysis. RESULTS: Diet mediterranisation was successful, reflected in the daily food consumption at the canteen and the evolution of the Mediterranean diet score (MDS) from 4.8 +/- 1.4 to 7.4 +/- 1.5 (limits 0-14). Some metabolic syndrome components showed statistically significant improvement and also statistically significant correlation with the MDS: waist circumference, HDL-cholesterol, systolic and diastolic blood pressure. After 12 months, the reversion rate for metabolic syndrome was 48 % (12/23) with an incidence rate of 4.1 % for new cases (3/73). In total, metabolic syndrome decreased from 24.0 % to 15.6 % (23/96 to 15/96) (P = 0.029). CONCLUSIONS: Diet mediterranisation is feasible in a food-at-work intervention, affecting lunch consumption at the workers canteen and overall consumption evaluated with MDS, together with a significant reduction in metabolic syndrome.
Assuntos
Dieta Mediterrânea , Serviços de Alimentação , Síndrome Metabólica/dietoterapia , Adulto , Comportamentos Relacionados com a Saúde , Humanos , Incidência , Estudos Longitudinais , Masculino , Síndrome Metabólica/epidemiologia , Síndrome Metabólica/prevenção & controle , Local de TrabalhoRESUMO
Healthy subjects frequently report minor bleedings that are frequently 'background noise' of normality rather than a true disorder. Nevertheless, unexpected or unusual bleeding may be alarming. Thus, the distinction between normal and pathologic bleeding is critical. Understanding the underlying pathologic mechanism in patients with an excessive bleeding is essential for their counseling and treatment. Most of these patients with significant bleeding will result affected by non-severe inherited bleeding disorders (BD), collectively denominated mild or moderate BD for their relatively benign course. Unfortunately, practical recommendations for the management of these disorders are still lacking due to the current state of fragmented knowledge of pathophysiology and lack of a systematic diagnostic approach. To address this gap, an International Working Group (IWG) was established by the European Hematology Association (EHA) to develop consensus-based guidelines on these disorders. The IWG agreed that grouping these disorders by their clinical phenotype under the single category of mild-to-moderate bleeding disorders (MBD) reflects current clinical practice and will facilitate a systematic diagnostic approach. Based on standardized and harmonized definitions a conceptual unified framework is proposed to distinguish normal subjects from affected patients. The IWG proposes a provisional comprehensive patient-centered initial diagnostic approach that will result in classification of MBD into distinct clinical-pathological entities under the overarching principle of clinical utility for the individual patient. While we will present here a general overview of the global management of patients with MBD, this conceptual framework will be adopted and validated in the evidence-based, disease-specific guidelines under development by the IWG.