RESUMO
Platelet inhibition is a fundamental objective to prevent and treat thrombus formation. Platelet activation depends on mitochondrial function. This study aims to identify a new mitochondria-targeting compound with antiplatelet activity at safe concentrations in vitro. Cytotoxicity and viability tests were performed on human platelets from volunteer donors, together with experiments on aggregation, platelet activation, mitochondrial function, mitochondrial respiration, and thioredoxin reductase 2 (TrxR2) enzymatic activity in isolated platelet mitochondria. The compound MitoCDNB, corresponding to the molecule 5-chloro-2,4-dinitrophenylamino linked with triphenylphosphonium cation (TPP+) by a butyl chain and methanesulfonate as the counterion, was evaluated. MitoCDNB demonstrates potent, high mitochondria-selective antiplatelet effects that provide a novel approach to platelet inhibition with potentially minimized systemic risks. Here, we describe the first compound that inhibits platelet activation by decreasing TrxR2 enzymatic activity and collagen-stimulated maximal mitochondrial respiration, preventing aggregation and platelet activation. These results can be used to develop new antiplatelet drugs targeting mitochondria.
Assuntos
Plaquetas , Mitocôndrias , Ativação Plaquetária , Inibidores da Agregação Plaquetária , Agregação Plaquetária , Humanos , Mitocôndrias/efeitos dos fármacos , Mitocôndrias/metabolismo , Ativação Plaquetária/efeitos dos fármacos , Inibidores da Agregação Plaquetária/farmacologia , Plaquetas/efeitos dos fármacos , Plaquetas/enzimologia , Plaquetas/metabolismo , Agregação Plaquetária/efeitos dos fármacos , Tiorredoxina Dissulfeto Redutase/metabolismo , Tiorredoxina Dissulfeto Redutase/antagonistas & inibidores , Compostos Organofosforados/farmacologia , Tiorredoxina Redutase 2/metabolismoRESUMO
BACKGROUND/AIM: SARS-CoV-2 infection presents different severity levels that suggest the influence of genetic factors on the clinical outcome of the disease. In cases of severe COVID-19, the presence of elevated coagulation markers, increased platelet activation and aggregation and the risk of thrombotic complications are described. Given the participation of these cells in several serious viral infections and their negative role when associated with a prothrombotic response, it is important to understand the mechanistic role of SARS-CoV-2 in platelet physiology. This study evaluated the hyperexpression of platelet-activating factor receptor (PTAFR) and platelet factor 4 (PF4) in unvaccinated and hospitalized patients with COVID-19. PATIENTS AND METHODS: The study included 43 COVID-19 patients stratified according to WHO guidelines. Subsequently, the expression of the PTAFR and PF4 genes were evaluated using the real-time quantitative PCR and their possible correlation with the severity of the disease and clinical variables including hospitalization, outcome, sex, age and laboratory parameters (platelet count, INR and D-dimer). RESULTS: The analysis demonstrated a significant (p<0.05) hyperexpression of these genes COVID-19 patients (n=43) compared to healthy controls. Expression of these genes in patients was not statistically significant (p>0.05) different between patients stratified according to clinical variables. CONCLUSION: The expression of PTAFR and PF4 suggests an important molecular pathway in the pathophysiology of the disease and may be valuable platelet biomarkers to indicate increased risk in patients with COVID-19 who require hospital care, contributing to personalized intervention strategies and improving their clinical management.
Assuntos
Biomarcadores , Plaquetas , COVID-19 , Fator Plaquetário 4 , SARS-CoV-2 , Humanos , COVID-19/sangue , Masculino , Feminino , Fator Plaquetário 4/sangue , Fator Plaquetário 4/genética , Pessoa de Meia-Idade , Biomarcadores/sangue , Plaquetas/metabolismo , Plaquetas/patologia , Idoso , Receptores Acoplados a Proteínas G/genética , Receptores Acoplados a Proteínas G/metabolismo , Adulto , Glicoproteínas da Membrana de Plaquetas/metabolismo , Glicoproteínas da Membrana de Plaquetas/genética , Ativação Plaquetária/genética , Índice de Gravidade de Doença , Contagem de Plaquetas , Fatores de RiscoRESUMO
Platelets are small cell fragments that play a crucial role in hemostasis, requiring fast response times and fine signaling pathway regulation. For this regulation, platelets require a balance between two pathway types: the activatory and negative signaling pathways. Activatory signaling mediators are positive responses that enhance stimuli initiated by a receptor in the platelet membrane. Negative signaling regulates and controls the responses downstream of the same receptors to roll back or even avoid spontaneous thrombotic events. Several blood-related pathologies can be observed when these processes are unregulated, such as massive bleeding in activatory signaling inhibition or thrombotic events for negative signaling inhibition. The study of each protein and metabolite in isolation does not help to understand the role of the protein or how it can be contrasted; however, understanding the balance between active and negative signaling could help develop effective therapies to prevent thrombotic events and bleeding disorders.
Assuntos
Plaquetas , Hemorragia , Ativação Plaquetária , Transdução de Sinais , Trombose , Humanos , Trombose/metabolismo , Trombose/etiologia , Plaquetas/metabolismo , Hemorragia/metabolismo , Hemorragia/etiologia , Animais , HemostasiaRESUMO
BACKGROUND: Cutaneous melanoma (CM) is a malignancy with a variable incidence worldwide and a poor advanced-stage prognosis. Melanoma growth is closely associated with the immune system. METHODS: A cross-sectional study was performed on CM patients admitted at the Hospital de Cancer de Pernambuco (HCP) between 2015 and 2018. Fifty-one CM patients were included, and 30 healthy individuals. The study aimed to evaluate the association of platelet activation mechanisms and inflammatory response in patients with cutaneous melanoma. RESULTS: Elevated serum IL10 and low serum TNF levels in CM patients compared to controls (p < 0.05). High IL6 levels in patients with negative lymph nodes LN (-) compared to positive lymph nodes group (LN +, p = 0.0005). Low RANTES levels in patients compared to controls (p < 0.05). Elevated levels of platelet-lymphocyte (PLA), platelet-monocytes (PMA), and platelet-neutrophils (PNA) aggregates were observed in patients compared to controls (p < 0.05). CM patients with stage II had lower PMA levels than stages I and III (p < 0.05). High PMA levels were observed in patients with LN (+) compared to the LN (-) group (p < 0.0001). Patients with SSM had high levels of sCD40L and sCD62P compared to controls (p < 0.05)). High sCD40L levels in stage II compared to the stage III group, and sCD62P in stages I and II compared to the stage III group (p < 0.05). High sCD62P levels in patients with LN (-) compared to the group LN (+) (p < 0.05). CONCLUSION: It was observed the immunosuppressive profile in CM may favor tumor progression. High levels of platelet-leukocyte aggregates, sCD40L, and sCD62P may be associated with the worst prognosis.
Assuntos
Plaquetas , Ligante de CD40 , Melanoma , Neoplasias Cutâneas , Humanos , Melanoma/patologia , Melanoma/sangue , Melanoma/metabolismo , Masculino , Feminino , Estudos Transversais , Neoplasias Cutâneas/patologia , Neoplasias Cutâneas/sangue , Pessoa de Meia-Idade , Ligante de CD40/sangue , Ligante de CD40/metabolismo , Plaquetas/patologia , Plaquetas/metabolismo , Estudos de Casos e Controles , Prognóstico , Citocinas/sangue , Idoso , Melanoma Maligno Cutâneo , Adulto , Seguimentos , Leucócitos/metabolismo , Leucócitos/patologia , Quimiocinas/sangue , Biomarcadores Tumorais/sangue , Biomarcadores Tumorais/metabolismo , Selectina-P/sangueRESUMO
Platelets are enucleated fragments of cells with a diversity of internal granules. They are responsible for functions related to hemostasis, coagulation, and inflammation. The activation of these processes depends on a cascade coordinated by cytokines, chemokines, and components of purinergic signaling, such as ATP, ADP, and adenosine. Platelets express distinct components of the purinergic system: P2X1, P2Y1, PY12, and P2Y14 receptors; and the ectonucleotidases NTPDase, NPP, and 5NTE (ecto-5'-nucleotidase). Except for P2Y14, which has not yet exhibited a known function, all other components relate to the biological processes mentioned before. Platelets are known to display specific responses to microorganisms, being capable of recognizing pathogen-associated molecular patterns (PAMPs), engulfing certain classes of viruses, and participating in NETosis. Platelet function dysregulation implicates various pathophysiological processes, including cardiovascular diseases (CVDs) and infections. In COVID-19 patients, platelets exhibit altered purinergic signaling and increased activation, contributing to inflammation. Excessive platelet activation can lead to complications from thrombosis, which can affect the circulation of vital organs. Therefore, controlling the activation is necessary to end the inflammatory process and restore homeostasis. Ectonucleotidases, capable of hydrolyzing ATP, ADP, and AMP, are of fundamental importance in activating platelets, promising pharmacological targets for clinical use as cardiovascular protective drugs. In this review, we revisit platelet biology, the purinergic receptors and ectonucleotidases on their surface, and their importance in platelet activity. Additionally, we describe methods for isolating platelets in humans and murine, as well as the main techniques for detecting the activity of ectonucleotidases in platelets. Considering the multitude of functions revealed by platelets and their potential use as potent bioreactors able to secrete and present molecules involved in the communication of the vasculature with the immune system, it is crucial to deeply understand platelet biology and purinergic signaling participation to contribute to the developing of therapeutic strategies in diseases of the cardiovascular, inflammatory, and immune systems.
Assuntos
Plaquetas , COVID-19 , Ativação Plaquetária , Animais , Humanos , 5'-Nucleotidase/metabolismo , Adenosina Trifosfatases/metabolismo , Plaquetas/metabolismo , Plaquetas/imunologia , Separação Celular/métodos , COVID-19/imunologia , COVID-19/sangue , Ativação Plaquetária/imunologia , SARS-CoV-2/imunologia , Transdução de SinaisRESUMO
Clopidogrel is widely used worldwide as an antiplatelet therapy in patients with acute coronary disease. Genetic factors influence interindividual variability in response. Some studies have explored the polygenic contributions in the drug response, generating pharmacogenomic risk scores (PgxPRS). Importantly, these factors are less explored in underrepresented populations, such as Latin-American countries. Identifying patients at risk of high-on-treatment platelet reactivity (HTPR) is highly valuable in translational medicine. In this study we used a custom next-generation sequencing (NGS) panel composed of 91 single nucleotide polymorphisms (SNPs) and 28 genes related to clopidogrel metabolism, to analyze 70 patients with platelet reactivity values, assessed through closure time (CT). Our results demonstrated the association of SNPs with HTPR and non-HTPR, revealing the strongest associations with rs2286823 (OR: 5,0; 95% CI: 1,02-24,48; p: 0,03), rs2032582 (OR: 4,41; 95% CI: 1,20-16,12; p: 0,019), and rs1045642 (OR: 3,38; 95% CI: 0,96-11,9; p: 0,05). Bivariate regression analysis demonstrated the significant association of several SNPs with the CT value, a "surrogate" biomarker of clopidogrel response. Exploratory results from the LASSO regression model showed a high discriminatory capacity between HTPR and non-HTPR patients (AUC: 0,955), and the generated PgxPRS demonstrated a significant negative association between the risk score, CT value, and the condition of HTPR and non-HTPR. To our knowledge, our study addresses for the first time the analysis of the polygenic contribution in platelet reactivity using NGS and establishes PgxPRS derived from the LASSO model. Our results demonstrate the polygenic implication of clopidogrel response and offer insights applicable to the translational medicine of antiplatelet therapy in an understudied population.
Assuntos
Plaquetas , Clopidogrel , Sequenciamento de Nucleotídeos em Larga Escala , Inibidores da Agregação Plaquetária , Polimorfismo de Nucleotídeo Único , Humanos , Clopidogrel/uso terapêutico , Clopidogrel/farmacologia , Masculino , Sequenciamento de Nucleotídeos em Larga Escala/métodos , Feminino , Inibidores da Agregação Plaquetária/uso terapêutico , Inibidores da Agregação Plaquetária/farmacologia , Pessoa de Meia-Idade , Plaquetas/efeitos dos fármacos , Plaquetas/metabolismo , Idoso , Herança Multifatorial/genética , Ticlopidina/análogos & derivados , Ticlopidina/uso terapêutico , Ticlopidina/farmacologiaRESUMO
The study aimed to evaluate the antithrombotic action of Acrocomia aculeata pulp oil (AAPO) in natura, in an in vitro experimental model. AAPO was obtained by solvent extraction, and its chemical characterization was performed by gas chromatography coupled to a mass spectrometer (GC-MS). In vitro toxicity was evaluated with the Trypan Blue exclusion test and in vivo by the Galleria mellonella model. ADP/epinephrine-induced platelet aggregation after treatment with AAPO (50, 100, 200, 400, and 800 µg/mL) was evaluated by turbidimetry, and coagulation was determined by prothrombin activity time (PT) and activated partial thromboplastin time (aPTT). Platelet activation was measured by expression of P-selectin on the platelet surface by flow cytometry and intraplatelet content of reactive oxygen species (ROS) by fluorimetry. The results showed that AAPO has as major components such as oleic acid, palmitic acid, lauric acid, caprylic acid, and squalene. AAPO showed no toxicity in vitro or in vivo. Platelet aggregation decreased against agonists using treatment with different concentrations of AAPO. Oil did not interfere in PT and aPTT. Moreover, it expressively decreased ROS-induced platelet activation and P-selectin expression. Therefore, AAPO showed antiplatelet action since it decreased platelet activation verified by the decrease in P-selectin expression as well as in ROS production.
Assuntos
Fibrinolíticos , Selectina-P , Óleos de Plantas , Agregação Plaquetária , Espécies Reativas de Oxigênio , Animais , Agregação Plaquetária/efeitos dos fármacos , Selectina-P/metabolismo , Humanos , Óleos de Plantas/farmacologia , Óleos de Plantas/química , Espécies Reativas de Oxigênio/metabolismo , Fibrinolíticos/farmacologia , Plaquetas/efeitos dos fármacos , Plaquetas/metabolismo , Coagulação Sanguínea/efeitos dos fármacos , Ativação Plaquetária/efeitos dos fármacosRESUMO
Understanding the mechanisms controlling platelet function is crucial for exploring potential therapeutic targets related to atherothrombotic pathologies and primary hemostasis disorders. Our research, which focuses on the role of platelet mitochondria and Ca2+ fluxes in platelet activation, the formation of the procoagulant phenotype, and thrombosis, has significant implications for the development of new therapeutic strategies. Traditionally, Ca2+-dependent cellular signaling has been recognized as a determinant process throughout the platelet activation, controlled primarily by store-operated Ca2+ entry and the PLC-PKC signaling pathway. However, despite the accumulated knowledge of these regulatory mechanisms, the effectiveness of therapy based on various commonly used antiplatelet drugs (such as acetylsalicylic acid and clopidogrel, among others) has faced challenges due to bleeding risks and reduced efficacy associated with the phenomenon of high platelet reactivity. Recent evidence suggests that platelet mitochondria could play a fundamental role in these aspects through Ca2+-dependent mechanisms linked to apoptosis and forming a procoagulant phenotype. In this context, the present review describes the latest advances regarding the role of platelet mitochondria and Ca2+ fluxes in platelet activation, the formation of the procoagulant phenotype, and thrombosis.
Assuntos
Envelhecimento , Plaquetas , Cálcio , Mitocôndrias , Ativação Plaquetária , Humanos , Mitocôndrias/metabolismo , Ativação Plaquetária/fisiologia , Cálcio/metabolismo , Plaquetas/metabolismo , Envelhecimento/metabolismo , Animais , Trombose/metabolismo , Sinalização do Cálcio/fisiologiaRESUMO
Bioproducts derived from platelets have been extensively used across various medical fields, with a recent notable surge in their application in dermatology and aesthetic procedures. These products, such as platelet-rich plasma (PRP) and platelet-rich fibrin (PRF), play crucial roles in inducing blood vessel proliferation through growth factors derived from peripheral blood. PRP and PRF, in particular, facilitate fibrin polymerization, creating a robust structure that serves as a reservoir for numerous growth factors. These factors contribute to tissue regeneration by promoting cell proliferation, differentiation, and migration and collagen/elastin production. Aesthetic medicine harnesses these effects for diverse purposes, including hair restoration, scar treatment, striae management, and wound healing. Furthermore, these biological products can act as adjuvants with other treatment modalities, such as laser therapy, radiofrequency, and microneedling. This review synthesizes the existing evidence, offering insights into the applications and benefits of biological products in aesthetic medicine.
Assuntos
Fibrina Rica em Plaquetas , Plasma Rico em Plaquetas , Medicina Regenerativa , Humanos , Plasma Rico em Plaquetas/metabolismo , Plasma Rico em Plaquetas/química , Medicina Regenerativa/métodos , Fibrina Rica em Plaquetas/metabolismo , Cicatrização , Plaquetas/metabolismo , Animais , Regeneração , Proliferação de CélulasRESUMO
Uropathogenic strains of E. coli (UPEC) is a leading cause of sepsis, deploying multiple virulence factors to evade host immune responses. Notably, alpha-hemolysin (HlyA) produced by UPEC is implicated in septic symptoms associated with bacteremia, correlating with thrombocytopenia, a critical indicator of organ dysfunction and a predictor of poorer patient prognosis. This study meticulously explores the impact of sublytic concentrations of HlyA on platelets. Findings reveal that HlyA triggers an increase in intracellular calcium, activating calpain and exposing phosphatidylserine to the cell surface, as validated by flow cytometric experiments. Electron microscopy reveals a distinctive balloon-like shape in HlyA-treated platelets, indicative of a procoagulant state. The toxin induces the release of procoagulant extracellular vesicles and the secretion of alpha and dense granules. Overall, the results point to HlyA inducing a necrotic-like procoagulant state in platelets. The effects of sublytic concentrations of HlyA on both erythrocytes and platelets could have a potential impact on capillary microcirculation. Targeting HlyA emerges as a viable therapeutic strategy to mitigate the adverse effects of UPEC infections, especially in South American countries where these infections are endemic, underscoring its significance as a potential therapeutic target.
Assuntos
Plaquetas , Proteínas de Escherichia coli , Proteínas Hemolisinas , Proteínas Hemolisinas/metabolismo , Humanos , Plaquetas/metabolismo , Proteínas de Escherichia coli/metabolismo , Necrose , Coagulação Sanguínea/efeitos dos fármacos , Escherichia coli , Escherichia coli Uropatogênica/efeitos dos fármacos , Escherichia coli Uropatogênica/patogenicidade , Cálcio/metabolismoRESUMO
Cervical cancer ranks as the fourth most common and fatal cancer among women worldwide. Studies have demonstrated a strong association between purinergic platelet signaling and tumor progression in this type of cancer. The literature shows that neoplastic cells, when in the bloodstream, secrete adenosine triphosphate (ATP) and adenosine nucleotide diphosphate (ADP) that act on their corresponding platelet P2Y and P2X receptors. The interaction of these nucleotides with their receptors results in platelet activation and degranulation, ensuing several consequences, such as vascular endothelial growth factor (VEGF), platelet-derived growth factor, matrix metalloproteinases, ADP, and ATP. These molecules play essential roles in angiogenesis and tumor metastasis in cervical cancer. Several purinergic receptors are found in endothelial cells. Their activation, especially P2Y2, by the nucleotides released by platelets can induce relaxation of the endothelial barrier and consequent extravasation of tumor cells, promoting the development of metastases. Cancer cells that enter the bloodstream during the metastatic process are also subject to high shear stress and immune surveillance. In this context, activated platelets bind to circulating tumor cells and protect them against shear stress and the host's immune system, especially against natural killer cells, facilitating their spread throughout the body. Furthermore, activation of the P2Y12 receptor present on the platelet surface promotes the release of VEGF, the main inducer of angiogenesis in cervical cancer, in addition to increasing the concentration of several other pro-angiogenic molecules. Therefore, this review will address the role of platelet purinergic signaling in tumor progression of cervical cancer and propose possible therapeutic targets.
Assuntos
Plaquetas , Metástase Neoplásica , Neovascularização Patológica , Receptores Purinérgicos , Transdução de Sinais , Neoplasias do Colo do Útero , Humanos , Neoplasias do Colo do Útero/metabolismo , Neoplasias do Colo do Útero/patologia , Neovascularização Patológica/metabolismo , Neovascularização Patológica/patologia , Plaquetas/metabolismo , Feminino , Transdução de Sinais/fisiologia , Receptores Purinérgicos/metabolismo , Animais , AngiogêneseRESUMO
Gamma-terpinene (γ-TPN) is a cyclohexane monoterpene isolated from plant essential oils, such as tea tree (Melaleuca alternifolia), oregano (Origanum vulgare), rosemary (Rosmarinus officinalis L.), thyme (Thymus vulgaris Marchand), and eucalyptus (Eucalyptus sp.). Terpenes are widely studied molecules pharmacologically active on the cardiovascular system, hemostasis, and antioxidant actions. Herein, it was investigated the cytotoxic and antiplatelet activity of γ-TPN using different non-clinical laboratory models. For in silico evaluation, the PreADMET, SwissADME, and SwissTargetPrediction softwares were used. Molecular docking was performed using the AutoDockVina and BIOVIA Discovery Studio databases. The cytotoxicity of γ-TPN was analyzed by the MTT assay upon normal murine endothelial SVEC4-10 and fibroblast L-929 cells. Platelet aggregation was evaluated with platelet-rich (PRP) and platelet-poor (PPP) plasma from spontaneously hypertensive rats (SHR), in addition to SVEC4-10 cells pre-incubated with γ-TPN (50, 100, and 200 µM) for 24 h. SHR animals were pre-treated by gavage with γ-TPN for 7 days and divided into four groups (negative control, 25, 50, and 100 mg/kg). Blood samples were collected to measure nitrite using the Griess reagent. Gamma-TPN proved to be quite lipid-soluble (Log P = +4.50), with a qualified profile of similarity to the drug, good bioavailability, and adequate pharmacokinetics. It exhibited affinity mainly for the P2Y12 receptor (6.450 ± 0.232 Kcal/mol), moderate cytotoxicity for L-929 (CC50 = 333.3 µM) and SVEC 4-10 (CC50 = 366.7 µM) cells. The presence of γ-TPN in SVEC 4-10 cells was also able to reduce platelet aggregation by 51.57 and 44.20% at lower concentrations (50 and 100 µM, respectively). Then, γ-TPN has good affinity with purinergic receptors and an effect on the reversal of platelet aggregation and oxidative stress, being promising and safe for therapeutic targets and subsequent studies on the control of thromboembolic diseases.
Assuntos
Monoterpenos Cicloexânicos , Simulação de Acoplamento Molecular , Inibidores da Agregação Plaquetária , Agregação Plaquetária , Ratos Endogâmicos SHR , Animais , Camundongos , Agregação Plaquetária/efeitos dos fármacos , Monoterpenos Cicloexânicos/farmacologia , Inibidores da Agregação Plaquetária/farmacologia , Masculino , Linhagem Celular , Ratos , Ratos Wistar , Sobrevivência Celular/efeitos dos fármacos , Plaquetas/efeitos dos fármacos , Plaquetas/metabolismo , Monoterpenos/farmacologiaRESUMO
Platelets are the critical target for preventing and treating pathological thrombus formation. However, despite current antiplatelet therapy, cardiovascular mortality remains high, and cardiovascular events continue in prescribed patients. In this study, first results were obtained with ortho-carbonyl hydroquinones as antiplatelet agents; we found that linking triphenylphosphonium cation to a bicyclic ortho-carbonyl hydroquinone moiety by a short alkyl chain significantly improved their antiplatelet effect by affecting the mitochondrial functioning. The mechanism of action involves uncoupling OXPHOS, which leads to an increase in mitochondrial ROS production and a decrease in the mitochondrial membrane potential and OCR. This alteration disrupts the energy production by mitochondrial function necessary for the platelet activation process. These effects are responsive to the complete structure of the compounds and not to isolated parts of the compounds tested. The results obtained in this research can be used as the basis for developing new antiplatelet agents that target mitochondria.
Assuntos
Plaquetas , Hidroquinonas , Potencial da Membrana Mitocondrial , Compostos Organofosforados , Inibidores da Agregação Plaquetária , Humanos , Plaquetas/metabolismo , Plaquetas/efeitos dos fármacos , Hidroquinonas/farmacologia , Hidroquinonas/química , Potencial da Membrana Mitocondrial/efeitos dos fármacos , Mitocôndrias/metabolismo , Mitocôndrias/efeitos dos fármacos , Compostos Organofosforados/farmacologia , Compostos Organofosforados/química , Fosforilação Oxidativa/efeitos dos fármacos , Ativação Plaquetária/efeitos dos fármacos , Agregação Plaquetária/efeitos dos fármacos , Inibidores da Agregação Plaquetária/farmacologia , Inibidores da Agregação Plaquetária/química , Espécies Reativas de Oxigênio/metabolismoRESUMO
Metastasis is the leading cause of cancer-related deaths. Despite this relevance, there is no specific therapy targeting metastasis. The interaction of the tumor cell with platelets, forming microemboli is crucial for successful hematogenous dissemination. Heparin disrupts it by a P-selectin-mediated event. However, its clinical use for this purpose is hindered by the requirement of high doses, leading to anticoagulant-related side effects. In this study, we obtained a low-anticoagulant heparin through the fractionation of a pharmaceutical bovine heparin. This derivative was referred to as LA-hep and we investigated its efficacy in inhibiting metastases and explored its capacity of suppressing the interaction between tumor cells and platelets. Our data revealed that LA-hep is as efficient as porcine unfractionated heparin in attenuating lung metastases from melanoma and colon adenocarcinoma cells in an assay with a single intravenous administration. It also prevents platelet arrest shortly after cell injection in wild-type mice and suppresses melanoma-platelets interaction in vitro. Moreover, LA-hep blocks P-selectin's direct binding to tumor cells and platelet aggregation, providing further evidence for the role of P-selectin as a molecular target. Even in P-selectin-depleted mice which developed a reduced number of metastatic foci, both porcine heparin and LA-hep further inhibited metastasis burden. This suggests evidence of an additional mechanism of antimetastatic action. Therefore, our results indicate a dissociation between the heparin anticoagulant and antimetastatic effects. Considering the simple and highly reproducible methodology used to purify LA-hep along with the data presented here, LA-hep emerges as a promising drug for future use in preventing metastasis in cancer patients.
Assuntos
Adenocarcinoma , Neoplasias do Colo , Melanoma , Humanos , Animais , Bovinos , Camundongos , Heparina/farmacologia , Anticoagulantes/farmacologia , Selectina-P/metabolismo , Melanoma/patologia , Adenocarcinoma/patologia , Neoplasias do Colo/patologia , Plaquetas/metabolismo , Preparações Farmacêuticas/metabolismo , Metástase Neoplásica/patologiaRESUMO
The aims of this study are to characterize the antiplatelet activity of StSBTc-3, a potato serine protease with fibrino (geno) lytic activity, and to provide information on its mechanism of action. The results obtained show that StSBTc-3 inhibits clot retraction and prevents platelet aggregation induced by thrombin, convulxin, and A23187. Platelet aggregation inhibition occurs in a dose-dependent manner and is not affected by inactivation of StSBTc-3 with the inhibitor of serine proteases phenylmethylsulfonyl fluoride (PMSF). In addition, StSBTc-3 reduces fibrinogen binding onto platelets. In-silico calculations show a high binding affinity between StSBTc-3 and human α2bß3 integrin suggesting that the antiplatelet activity of StSBTc-3 could be associated with the fibronectin type III domain present in its amino acid sequence. Binding experiments show that StSBTc-3 binds to α2bß3 preventing the interaction between α2bß3 and fibrinogen and, consequently, inhibiting platelet aggregation. StSBTc-3 represents a promising compound to be considered as an alternative to commercially available drugs used in cardiovascular therapies.
Assuntos
Solanum tuberosum , Humanos , Serina/metabolismo , Plaquetas/metabolismo , Agregação Plaquetária , Serina Endopeptidases/metabolismo , Fibrinogênio/metabolismo , Subtilisinas/metabolismoRESUMO
BACKGROUND: Multiple blood cell abnormalities participate in the development of inflammation in systemic lupus erythematosus (SLE). Although platelets have been suggested as one of these contributors through the release of their content during activation, there are limited specific data about their role as immune players in SLE. MATERIALS AND METHODS: Thirteen SLE patients were included. Flow cytometry was used to measure Toll-like receptors (TLR) 2, 4, and 9 in resting platelets, platelet-activation markers (PAC-1 binding, P-selectin, CD63, and CD40 ligand -L) and platelet-leukocyte aggregates before and after specific TLR stimulation. Soluble CD40L and von Willebrand factor (vWf) release from stimulated platelets was measured using ELISA. RESULTS: In resting conditions, SLE platelets showed normal expression levels of TLR 2, 4 and 9. Platelet surface activation markers, soluble CD40L, and vWf release were normal at baseline and after TLR stimulation. Platelet-monocyte aggregates were elevated in resting conditions in SLE samples and showed only a marginal increase after TLR stimulation, while baseline and stimulated platelet-neutrophil and platelet-lymphocyte aggregates were normal. C-reactive protein levels positively correlated with platelet-monocyte aggregates both at baseline and after stimulation with the TLR-2 agonist PAM3CSK4, suggesting these complexes could reflect the inflammatory activity in SLE. In our cohort, 12 of 13 patients received treatment with hydroxychloroquine (HCQ), a known inhibitor of endosomal activity and a potential inhibitor of platelet activation. The fact that SLE platelets showed an adequate response to TLR agonists suggests that, despite this treatment, they retain the ability to respond to the increased levels of damage-associated molecular patterns (DAMPs), which represent known TLR ligands, present in the circulation of SLE patients. Interestingly, elevated plasma levels of high mobility group box 1 (HMGB1), a classical DAMP, correlated with vWf release from TLR-stimulated platelets, suggesting that HMGB1 may also be released by platelets, thereby creating a positive feedback loop for platelet activation that contributes to inflammation. CONCLUSION: Our study demonstrates normal platelet TLR expression and function together with increased circulating platelet-monocyte aggregates. In addition, a direct correlation was observed between plasma HMGB1 levels and platelet vWf release following TLR2 stimulation. This platelet behavior in a group of patients undergoing HCQ treatment suggests that platelets could play a role in the inflammatory state of SLE.
Assuntos
Proteína HMGB1 , Lúpus Eritematoso Sistêmico , Humanos , Proteína HMGB1/metabolismo , Ligante de CD40 , Fator de von Willebrand/metabolismo , Receptores Toll-Like/metabolismo , Plaquetas/metabolismo , Inflamação/metabolismo , Receptor Toll-Like 9RESUMO
AIMS: Melatonin is known to inhibit platelet aggregation induced by arachidonic acid (AA). In the present study we investigated whether agomelatine (Ago), an antidepressant with agonist activity at melatonin receptor 1 (MT1) and MT2 could reduce platelets aggregation and adhesion. MAIN METHODS: Human platelets from healthy donors were used to test the in vitro effects of Ago in the presence of different platelet activators. We performed aggregation and adhesion assays, thromboxane B2 (TxB2), cAMP and cGMP measurements, intra-platelet calcium registration and flow cytometry assays. KEY FINDINGS: Our data revealed that different concentrations of Ago reduced AA- and collagen-induced human platelet aggregation in vitro. Ago also reduced AA-induced increase in thromboxane B2 (TxB2) production, intracellular calcium levels and P-selectin expression at plasma membrane. The effects of Ago in AA-activated platelets were likely dependent on MT1 as they were blocked by luzindole (a MT1/MT2 antagonist) and mimicked by the MT1 agonist UCM871 in a luzindole-sensitive manner. The MT2 agonist UCM924 was also able to inhibit platelet aggregation, but this response was not affected by luzindole. On the other hand, although UCM871 and UCM924 reduced collagen-induced platelet aggregation and adhesion, inhibition of collagen-induced platelet aggregation by Ago was not mediated by melatonin receptors because it was not affected by luzindole. SIGNIFICANCE: The present data show that Ago suppresses human platelet aggregation and suggest that this antidepressant may have the potential to prevent atherothrombotic ischemic events by reducing thrombus formation and vessel occlusion.
Assuntos
Cálcio , Agregação Plaquetária , Humanos , Receptores de Melatonina/metabolismo , Cálcio/metabolismo , Plaquetas/metabolismo , Colágeno/metabolismo , Antidepressivos/farmacologia , Tromboxanos/metabolismo , Tromboxano B2/metabolismo , Tromboxano B2/farmacologiaRESUMO
The tumor microenvironment (TME) is constituted by a great diversity of highly dynamic cell populations, each of which contributes ligands, receptors, soluble proteins, mRNAs, and miRNAs, in order to regulate cellular activities within the TME and even promote processes such as angiogenesis or metastasis. Intravasated platelets (PLT) undergo changes in the TME that convert them into tumor-educated platelets (TEP), which supports the development of cancer, angiogenesis, and metastasis through the degranulation and release of biomolecules. Several authors have reported that the deregulation of PF4, VEGF, PDGF, ANG-1, WASF3, LAPTM4B, TPM3, and TAC1 genes participates in breast cancer progression, angiogenesis, and metastasis. The present work aimed to analyze the expression levels of this set of genes in tumor tissues and platelets derived from breast cancer patients by reverse transcription-quantitative polymerase chain reaction (RTqPCR) assays, in order to determine if there was an expression correlation between these sources and to take advantage of the new information to be used in possible diagnosis by liquid biopsy. Data from these assays showed that platelets and breast cancer tumors present similar expression levels of a subset of these genes' mRNAs, depending on the molecular subtype, comorbidities, and metastasis presence.
Assuntos
Neoplasias da Mama , MicroRNAs , Humanos , Feminino , Neoplasias da Mama/metabolismo , Plaquetas/metabolismo , MicroRNAs/genética , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Expressão Gênica , Microambiente Tumoral/genética , Proteínas de Membrana/metabolismo , Proteínas Oncogênicas/genética , Família de Proteínas da Síndrome de Wiskott-Aldrich/metabolismoRESUMO
Reduced expression of a platelet protein protects against thrombosis during chronic immobilization.
Assuntos
Plaquetas , Proteínas de Choque Térmico HSP47 , Hipocinesia , Ursidae , Trombose Venosa , Animais , Humanos , Plaquetas/metabolismo , Agregação Plaquetária , Ursidae/metabolismo , Trombose Venosa/etiologia , Trombose Venosa/metabolismo , Hipocinesia/complicações , Proteínas de Choque Térmico HSP47/metabolismoRESUMO
Objective: Platelet-rich plasma (PRP) is used in multiple coagulation disorders. Its therapeutic effectiveness relies on technical procedures related to PRP procurement and preservation because free radicals induce platelet activation and aging. This work aims to elucidate the oxidative mechanisms involved in activation of platelets obtained from PRP during storage. Materials and Methods: One hundred ten PRP batches were obtained from healthy donors and kept under stirring at a temperature of 20-24 °C. Protein extraction was performed from platelet homogenates and plasma at different times of storage from day 1 to 20. The activities of antioxidant markers such as catalase (CAT), superoxide dismutase, and ceruloplasmin, as well as fibrinolytic protein activity metalloproteases 2 and 3, plasmin, and urokinase plasminogen activator, were analyzed by zymography assays. Oxidized proteins were also determined. Results: Significant activity of antioxidant enzymes and fibrinolytic molecules was observed on day 5 of storage in PRP homogenates, which increased over time and was concomitantly correlated with oxidized protein levels. Reverse enzymatic activity patterns were observed in plasma, except for CAT, which remained unchanged. Conclusion: Storage conditions of platelets from PRP for up to 5 days induced in vitro platelet activation by oxidative damage and proteolysis. This finding confirms the need for proper management of these blood products to preserve their viability and functionality.