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1.
Int J Mol Sci ; 22(19)2021 Sep 22.
Artigo em Inglês | MEDLINE | ID: mdl-34638529

RESUMO

Bernard-Soulier syndrome (BSS) is an autosomal-recessive bleeding disorder caused by biallelic variants in the GP1BA, GP1BB, and GP9 genes encoding the subunits GPIbα, GPIbß, and GPIX of the GPIb-IX complex. Pathogenic variants usually affect the extracellular or transmembrane domains of the receptor subunits. We investigated a family with BSS caused by the homozygous c.528_550del (p.Arg177Serfs*124) variant in GP1BB, which is the first mutation ever identified that affects the cytoplasmic domain of GPIbß. The loss of the intracytoplasmic tail of GPIbß results in a mild form of BSS, characterized by only a moderate reduction of the GPIb-IX complex expression and mild or absent bleeding tendency. The variant induces a decrease of the total platelet expression of GPIbß; however, all of the mutant subunit expressed in platelets is correctly assembled into the GPIb-IX complex in the plasma membrane, indicating that the cytoplasmic domain of GPIbß is not involved in assembly and trafficking of the GPIb-IX receptor. Finally, the c.528_550del mutation exerts a dominant effect and causes mild macrothrombocytopenia in heterozygous individuals, as also demonstrated by the investigation of a second unrelated pedigree. The study of this novel GP1BB variant provides new information on pathophysiology of BSS and the assembly mechanisms of the GPIb-IX receptor.


Assuntos
Síndrome de Bernard-Soulier/genética , Complexo Glicoproteico GPIb-IX de Plaquetas/genética , Trombocitopenia/genética , Adulto , Síndrome de Bernard-Soulier/sangue , Síndrome de Bernard-Soulier/patologia , Plaquetas/fisiologia , Feminino , Humanos , Masculino , Pessoa de Meia-Idade , Linhagem , Complexo Glicoproteico GPIb-IX de Plaquetas/metabolismo , Domínios Proteicos/genética , Trombocitopenia/patologia , Fator de von Willebrand/metabolismo
2.
Nutrients ; 13(9)2021 Aug 24.
Artigo em Inglês | MEDLINE | ID: mdl-34578790

RESUMO

(1) Background: In previous research, higher levels of urine heavy metals, especially lead and cadmium, have been associated with increased cardiovascular risk. However, there is no information linking exposure to heavy metal to endothelial and platelet microparticles (EMPs and PMPs), particularly in the younger population, which are novel biomarkers of endothelial dysfunction. (2) Methods: From a nationwide database, which was incepted in 1992-2000, screening for renal health among Taiwanese school children, a total of 789 subjects were recruited. Cross-sectional analysis was performed to evaluate the association between serum EMPs/PMPs and urine iron, nickel, copper, cadmium, lead, chromium, manganese, and zinc levels in the adolescent and young adult population. (3) Results: After we adjusted the conventional cardiovascular risk factors, CD31+/CD42a- and CD31+/CD42a+ counts, in subjects' serum, respective markers of EMP and PMP displayed a significant positive dose-response relationship with urinary lead and cadmium levels. Higher quartiles of urine lead and cadmium levels were associated with an increased risk of higher EMPs/PMPs (≥75th percentile) in a multivariate logistic regression model. (4) Conclusion: Higher urinary lead and cadmium concentrations are strongly associated with endothelium-platelet microparticles in this adolescent and young adult population, which could help explain, in part, the mechanism through which heavy metal exposure results in cardiotoxicity.


Assuntos
Plaquetas/metabolismo , Cádmio/urina , Doenças Cardiovasculares/epidemiologia , Micropartículas Derivadas de Células/metabolismo , Endotélio/metabolismo , Chumbo/urina , Adolescente , Adulto , Biomarcadores/urina , Doenças Cardiovasculares/metabolismo , Criança , Estudos Transversais , Feminino , Humanos , Modelos Logísticos , Masculino , Metais Pesados/urina , Complexo Glicoproteico GPIb-IX de Plaquetas/metabolismo , Fatores de Risco , Taiwan , Adulto Jovem
3.
Molecules ; 26(16)2021 Aug 13.
Artigo em Inglês | MEDLINE | ID: mdl-34443497

RESUMO

Soy diet is thought to help prevent cardiovascular diseases in humans. Isoflavone, which is abundant in soybean and other legumes, has been reported to possess antiplatelet activity and potential antithrombotic effect. Our study aims to elucidate the potential target of soy isoflavone in platelet. The anti-thrombosis formation effect of genistein and daidzein was evaluated in ex vivo perfusion chamber model under low (300 s-1) and high (1800 s-1) shear forces. The effect of genistein and daidzein on platelet aggregation and spreading was evaluated with platelets from both wildtype and GPIbα deficient mice. The interaction of these soy isoflavone with 14-3-3ζ was detected by surface plasmon resonance (SPR) and co-immunoprecipitation, and the effect of αIIbß3-mediated outside-in signaling transduction was evaluated by western blot. We found both genistein and daidzein showed inhibitory effect on thrombosis formation in perfusion chamber, especially under high shear force (1800 s-1). These soy isoflavone interact with 14-3-3ζ and inhibited both GPIb-IX and αIIbß3-mediated platelet aggregation, integrin-mediated platelet spreading and outside-in signaling transduction. Our findings indicate that 14-3-3ζ is a novel target of genistein and daidzein. 14-3-3ζ, an adaptor protein that regulates both GPIb-IX and αIIbß3-mediated platelet activation is involved in soy isoflavone mediated platelet inhibition.


Assuntos
Proteínas 14-3-3/metabolismo , Plaquetas/metabolismo , Isoflavonas/farmacologia , Complexo Glicoproteico GPIb-IX de Plaquetas/metabolismo , Transdução de Sinais , Soja/química , Animais , Fibrinogênio/metabolismo , Genisteína/química , Genisteína/farmacologia , Proteínas Imobilizadas/metabolismo , Isoflavonas/química , Masculino , Camundongos Endogâmicos C57BL , Agregação Plaquetária/efeitos dos fármacos , Trombose/patologia
4.
Curr Opin Hematol ; 28(5): 301-307, 2021 09 01.
Artigo em Inglês | MEDLINE | ID: mdl-34183536

RESUMO

PURPOSE OF REVIEW: In this review, we will describe how the combined ability of platelets and neutrophils to interact with each other drives ischemic stroke brain injury. RECENT FINDINGS: Neutrophils are one of the first cells to respond during ischemic stroke. Although animals stroke models have indicated targeting neutrophils improves outcomes, clinical trials have failed to yield successful strategies. Platelets play a critical role in recruiting neutrophils to sites of injury by acting as a bridge to the injured endothelium. After initial platelet adhesion, neutrophils can rapidly bind platelets through P-selectin and glycoprotein Ibα. In addition, recent data implicated platelet phosphatidylserine as a novel key regulator of platelet-neutrophil interactions in the setting of ischemic stroke. Inhibition of procoagulant platelets decreases circulating platelet-neutrophil aggregates and thereby reduces infarct size. Platelet binding alters neutrophil function, which contributes to the injury associated with ischemic stroke. This includes inducing the release of neutrophil extracellular traps, which are neurotoxic and pro-thrombotic, leading to impaired stroke outcomes. SUMMARY: Platelet-neutrophil interactions significantly contribute to the pathophysiology of ischemic stroke brain injury. Better understanding the mechanisms behind their formation and the downstream consequences of their interactions will lead to improved therapies for stroke patients.


Assuntos
Plaquetas/metabolismo , AVC Isquêmico/metabolismo , Ativação de Neutrófilo , Neutrófilos/metabolismo , Adesividade Plaquetária , Animais , Plaquetas/patologia , Armadilhas Extracelulares/metabolismo , Humanos , AVC Isquêmico/patologia , Neutrófilos/patologia , Selectina-P/metabolismo , Complexo Glicoproteico GPIb-IX de Plaquetas/metabolismo
5.
J Neuroinflammation ; 18(1): 46, 2021 Feb 18.
Artigo em Inglês | MEDLINE | ID: mdl-33602266

RESUMO

BACKGROUND: In acute ischemic stroke, cessation of blood flow causes immediate tissue necrosis within the center of the ischemic brain region accompanied by functional failure in the surrounding brain tissue designated the penumbra. The penumbra can be salvaged by timely thrombolysis/thrombectomy, the only available acute stroke treatment to date, but is progressively destroyed by the expansion of infarction. The underlying mechanisms of progressive infarction are not fully understood. METHODS: To address mechanisms, mice underwent filament occlusion of the middle cerebral artery (MCAO) for up to 4 h. Infarct development was compared between mice treated with antigen-binding fragments (Fab) against the platelet surface molecules GPIb (p0p/B Fab) or rat immunoglobulin G (IgG) Fab as control treatment. Moreover, Rag1-/- mice lacking T-cells underwent the same procedures. Infarct volumes as well as the local inflammatory response were determined during vessel occlusion. RESULTS: We show that blocking of the platelet adhesion receptor, glycoprotein (GP) Ibα in mice, delays cerebral infarct progression already during occlusion and thus before recanalization/reperfusion. This therapeutic effect was accompanied by decreased T-cell infiltration, particularly at the infarct border zone, which during occlusion is supplied by collateral blood flow. Accordingly, mice lacking T-cells were likewise protected from infarct progression under occlusion. CONCLUSIONS: Progressive brain infarction can be delayed by blocking detrimental lymphocyte/platelet responses already during occlusion paving the way for ultra-early treatment strategies in hyper-acute stroke before recanalization.


Assuntos
Plaquetas/metabolismo , Encéfalo/patologia , Progressão da Doença , Infarto da Artéria Cerebral Média/sangue , Infarto da Artéria Cerebral Média/patologia , Linfócitos/metabolismo , Animais , Encéfalo/metabolismo , Circulação Cerebrovascular/fisiologia , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Complexo Glicoproteico GPIb-IX de Plaquetas/metabolismo , Ratos
6.
Biochimie ; 184: 1-7, 2021 May.
Artigo em Inglês | MEDLINE | ID: mdl-33548391

RESUMO

Glycoprotein (GP)Ib that binds von Willebrand factor (vWF) and glycoprotein (GP)VI, that binds collagen play a significant role in platelet activation and aggregation, and are potential targets for antithrombotic treatment. They are targeted by snake venom proteinases. The effect of a such proteinase, mutalysin-II, on platelet aggregation was examined using washed human platelets and platelet-rich plasma. Its proteolytic activity on vWF, on its binding partner GPIbα, and on GPVI was analyzed by SDS-PAGE, and immunodetection with the corresponding antibodies after blotting. Dose- and time-dependently, mutalysin-II inhibits aggregation of washed platelets induced by vWF plus ristocetin and by convulxin, but with no significant effect on platelet-rich-plasma. Furthermore, mutalysin-II cleaves vWF into low molecular mass multimers of vWF and a rvWF-A1 domain to realease a ∼27-kDa fragment detectable by SDS-PAGE and blotting with mouse anti-rvWF-A1-domain IgG. Moreover, GPVI was cut by mutalysin-II into a soluble ∼55-kDa ectodomain and a fragment of ∼35-kDa. Thus, mutalysin-II inhibits vWF-induced platelet aggregation via cleavage of bound vWF-A1, and its receptor GPIbα. The additional cleavage of, GPVI, blocks collagen-induced platelets. Our data highlight mutalysin-II as an interesting platelet-directed tool targeting vWF-GPIbα binding and particularly GPVI. Thus, it might be suited for antithrombotic therapy as its combined inactivation of two receptors does not significantly compromise hemostasis, but shows high efficacy and safety. Studies are needed to further develop and demonstrate its potential benefits.


Assuntos
Plaquetas/química , Metaloendopeptidases/química , Inibidores da Agregação Plaquetária/química , Complexo Glicoproteico GPIb-IX de Plaquetas/química , Glicoproteínas da Membrana de Plaquetas/química , Venenos de Serpentes/química , Animais , Plaquetas/metabolismo , Feminino , Humanos , Camundongos , Camundongos Endogâmicos BALB C , Complexo Glicoproteico GPIb-IX de Plaquetas/metabolismo , Glicoproteínas da Membrana de Plaquetas/metabolismo
7.
Transfusion ; 61(3): 919-930, 2021 03.
Artigo em Inglês | MEDLINE | ID: mdl-33527430

RESUMO

BACKGROUND: Deterioration in quality of platelet concentrates (PCs) during storage results from the appearance of storage lesions affecting the hemostatic functions and posttransfusion survival of platelets. These lesions depend on the preparation and pathogen inactivation methods used, duration of storage, and platelet additive solutions (PASs) present in storage bags. METHODS: We investigated the effects of citrate contained in third-generation PAS (PAS-III) on storage lesions in buffy-coat PCs with or without photochemical (amotosalen-ultraviolet A) treatment over 7 days. RESULTS: Platelet counts were conserved in all groups during storage, as was platelet swirling without appearance of macroscopic aggregates. Glycoprotein (GP) IIbIIIa and GPVI expression remained stable, whereas GPIbα declined similarly in all groups during storage. Removal of citrate from PAS-III, resulting in global reduction of citrate from 11 to 5 mM, led to a significant decrease in glucose consumption, which largely countered a modest deleterious effect of photochemical treatment. Citrate reduction also resulted in decreased lactate generation and better maintenance of pH during storage, while photochemical treatment had no impact on these parameters. Moreover, citrate-free storage significantly reduced exposure of P-selectin and the apoptosis signal phosphatidylserine, thereby abolishing the activating effect of photochemical treatment on both parameters. Citrate reduction benefited platelet aggregation to various agonists up to Day 7, whereas PCT had no impact on these responses. CONCLUSION: Removal of citrate from PAS-III has a beneficial impact on platelet metabolism, spontaneous activation, and apoptosis, and improves platelet aggregation, irrespective of photochemical treatment, which should allow transfusion of platelets with better and longer-lasting functional properties.


Assuntos
Plaquetas/metabolismo , Preservação de Sangue/métodos , Ácido Cítrico/farmacologia , Agregação Plaquetária/efeitos dos fármacos , Apoptose/efeitos dos fármacos , Furocumarinas/farmacologia , Hemostasia/efeitos dos fármacos , Humanos , Concentração de Íons de Hidrogênio , Ácido Láctico/metabolismo , Selectina-P/metabolismo , Fosfatidilserinas , Contagem de Plaquetas , Testes de Função Plaquetária , Complexo Glicoproteico GPIb-IX de Plaquetas/metabolismo , Glicoproteínas da Membrana de Plaquetas/metabolismo
9.
Transfusion ; 61(1): 191-201, 2021 01.
Artigo em Inglês | MEDLINE | ID: mdl-33107611

RESUMO

BACKGROUND: We previously reported a flow path-ultraviolet C (UVC) irradiation system for platelet concentrates (PCs) with platelet additive solution (PAS) to minimize contamination by bacteria. Here, we investigated functionalities of irradiated platelets (PLTs) in in vitro thrombus formation and in vivo hemostasis. STUDY DESIGN AND METHODS: PAS-PCs were irradiated with flash UVC using the flow path system. Their variables (PLT count, mean platelet volume, pH, glucose, lactate, glycoprotein [GP] Ib, and activated integrin αIIbß3) were evaluated. Static adhesion to collagen or fibrinogen was analyzed using fluorescent microscopy. Thrombus formation under flow conditions was assessed using a collagen-coated bead column. Adenosine diphosphate (ADP)-induced Akt phosphorylation was determined by western blot. In vivo hemostasis and circulatory survival of PLTs were assessed with a rabbit bleeding model. RESULTS: All variables, except for GPIb expression, were slightly, but significantly, impaired after flash UVC irradiation throughout the 6-day storage period. No difference was observed in static adhesion to either collagen or fibrinogen between irradiated and nonirradiated PAS-PCs. In vitro thrombus formation of flash UVC-irradiated PAS-PCs was significantly greater than that of nonirradiated PAS-PCs. ADP-induced Akt phosphorylation was enhanced in irradiated PAS-PCs. In vivo hemostatic efficacy was comparable between the groups on Day 1. The efficacy declined in nonirradiated PAS-PCs on Day 5, while it was retained in flash UVC-irradiated PAS-PCs. Circulatory survival of PLTs was lower in irradiated PAS-PCs. CONCLUSIONS: PAS-PCs irradiated with UVC from xenon flash have favorable properties to achieve hemostasis compared with nonirradiated PAS-PCs.


Assuntos
Plaquetas/metabolismo , Hemostasia/fisiologia , Trombose/metabolismo , Raios Ultravioleta/efeitos adversos , Xenônio/efeitos adversos , Difosfato de Adenosina/metabolismo , Animais , Bactérias/efeitos da radiação , Plaquetas/efeitos da radiação , Colágeno/metabolismo , Colágeno/efeitos da radiação , Fibrinogênio/metabolismo , Fibrinogênio/efeitos da radiação , Hemostasia/efeitos da radiação , Humanos , Masculino , Volume Plaquetário Médio/estatística & dados numéricos , Microscopia de Fluorescência/métodos , Modelos Animais , Complexo Glicoproteico GPIIb-IIIa de Plaquetas/metabolismo , Complexo Glicoproteico GPIIb-IIIa de Plaquetas/efeitos da radiação , Complexo Glicoproteico GPIb-IX de Plaquetas/metabolismo , Complexo Glicoproteico GPIb-IX de Plaquetas/efeitos da radiação , Plaquetoferese/métodos , Coelhos , Xenônio/efeitos da radiação
10.
Blood ; 137(6): 844-847, 2021 02 11.
Artigo em Inglês | MEDLINE | ID: mdl-33181828

RESUMO

Agkisacucetin, a snake C-type lectin-like protein isolated from the venom of Deinagkistrodon acutus (formerly Agkistrodon acutus), is a novel antithrombotic drug candidate in phase 2 clinical trials. Agkisacucetin specifically recognizes the platelet surface receptor glycoprotein Ib α chain (GPIbα) to block GPIb and von Willebrand factor (VWF). In this study, we solved the crystal structure of the GPIbα N-terminal domain (residues 1-305) in complex with agkisacucetin to understand their molecular recognition mechanism. The crystal structure showed that agkisacucetin primarily contacts GPIbα at the C-terminal part of the conserved leucine-rich repeat (LRR) domain (LRR-6 to LRR-8) and the previously described "ß-switch" region through the ß chain. In addition, we found that agkisacucetin α chain contacts part of the GPIbα C-terminal peptide after the LRR domain through complementary charge interactions. This C-terminal peptide plays a key role in GPIbα and thrombin recognition. Therefore, our structure revealed that agkisacucetin can sterically block the interaction between the GPIb receptor and VWF and thrombin proteins to inhibit platelet function. Our structural work provides key molecular insights into how an antithrombotic drug candidate recognizes the GPIb receptor to modulate platelet function to inhibit thrombosis.


Assuntos
Venenos de Crotalídeos/metabolismo , Fibrinolíticos/metabolismo , Lectinas Tipo C/metabolismo , Complexo Glicoproteico GPIb-IX de Plaquetas/química , Cristalografia por Raios X , Humanos , Imunoprecipitação , Modelos Moleculares , Complexo Glicoproteico GPIb-IX de Plaquetas/metabolismo , Ligação Proteica/efeitos dos fármacos , Conformação Proteica , Domínios Proteicos , Mapeamento de Interação de Proteínas , Relação Estrutura-Atividade , Ressonância de Plasmônio de Superfície , Trombina/metabolismo , Fator de von Willebrand/metabolismo
11.
Transfusion ; 61(1): 202-211, 2021 01.
Artigo em Inglês | MEDLINE | ID: mdl-33166431

RESUMO

BACKGROUND: Therapeutic phlebotomy is the standard treatment of hereditary hemochromatosis (HH), the most common genetic disease in people of Northern European descent. Red cell concentrates from HH donors have been reported safe for transfusion, but little data is available on the storage properties of platelet concentrates from HH donors. STUDY DESIGN AND METHODS: Whole blood was collected from 10 healthy individuals and 10 newly diagnosed HH patients with elevated serum ferritin. Platelet-rich plasma (PRP) was prepared and split into four 20-mL units. Platelet quality tests were performed on days 0, 1, 3, 5, and 7 of storage, including platelet aggregation (ADP, arachidonic acid, collagen, and epinephrine agonists), blood gas analysis, flow cytometry (CD41, CD42b, and CD62P expression), and ELISA (sCD40L and sCD62p in supernatant). RESULTS: Mean serum ferritin levels were higher in HH patients than in controls (847.5 vs 45.8 ng/mL, P < .001). Overall, no difference in quality test results was observed between the two study groups over 7-day storage (P > .05), including blood gas analysis, platelet aggregation, and expression of surface (CD62p and CD42b) and secreted (sCD62P and sCD40L) activation markers. Expected alterations in metabolic (CO2 and glucose decrease, O2 and lactate increase, P < .001) and platelet activation markers (CD42b decrease, CD62P increase, P < .05) over time were observed in both groups. CONCLUSION: Although these findings indicate that platelets of individuals with HH are comparable to platelets from healthy donors, more extensive studies are needed before definite conclusions can be drawn.


Assuntos
Doadores de Sangue/estatística & dados numéricos , Plaquetas/citologia , Preservação de Sangue/métodos , Hemocromatose/diagnóstico , Adulto , Gasometria/métodos , Plaquetas/fisiologia , Preservação de Sangue/estatística & dados numéricos , Feminino , Ferritinas/sangue , Citometria de Fluxo/métodos , Voluntários Saudáveis , Hemocromatose/sangue , Hemocromatose/etnologia , Hemocromatose/terapia , Humanos , Masculino , Pessoa de Meia-Idade , Selectina-P/metabolismo , Flebotomia/métodos , Ativação Plaquetária/fisiologia , Agregação Plaquetária/fisiologia , Testes de Função Plaquetária/métodos , Complexo Glicoproteico GPIb-IX de Plaquetas/metabolismo , Plasma Rico em Plaquetas/metabolismo
12.
Platelets ; 32(1): 42-46, 2021 Jan 02.
Artigo em Inglês | MEDLINE | ID: mdl-32614622

RESUMO

Von Willebrand factor has a pivotal role in primary hemostasis. Its role in thrombotic microangiopathies (TMA), as well as cardiovascular disease, has been demonstrated. Thrombotic thrombocytopenic purpura (TTP), a thrombotic microangiopathy, is a life-threatening condition with a high mortality rate if untreated. Current management strategies comprise plasma exchange to remove autoantibodies and replenish ADAMTS13, along with immunosuppressive agents in immune TTP. This review focuses on novel antiplatelet strategies that target VWF and GPIb. The benefits of the nanobody caplacizumab in achieving faster normalization of platelet count, as well as reduced thromboembolic events were shown through TITAN and HERCULES trials, and these findings have been practice changing. The use of caplacizumab in patients with immune TTP (iTTP) has now become well established. Potential benefits of ARC1779 and N-acetylcysteine have also been shown on a small scale in iTTP, however these lack evidence through larger randomized controlled trials. Further therapies, some in early phase, others in clinical practice, target platelet aggregation within arteries and their utility is presented with cerebrovascular disorders.


Assuntos
Inibidores da Agregação Plaquetária/uso terapêutico , Complexo Glicoproteico GPIb-IX de Plaquetas/metabolismo , Fator de von Willebrand/metabolismo , Humanos , Inibidores da Agregação Plaquetária/farmacologia
13.
Methods Mol Biol ; 2217: 237-249, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-33215384

RESUMO

Platelets are small, anucleate cells that play oversized roles in hemostasis, immunity, and inflammation. An important mediator of platelet function is integrin αIIbß3, which is required for fibrinogen-dependent platelet aggregation during hemostasis. This platelet response is dependent on conformational changes in the integrin induced by "inside-out" biochemical signals that are triggered by platelet agonists. In turn, fibrinogen binding to αIIbß3 initiates "outside-in" biochemical and mechanical signals that regulate the platelet cytoskeleton and help to promote full platelet aggregation and secretory responses. Without a nucleus, there is a limited range of experimental manipulations that are possible with human platelets to study the molecular basis of integrin signaling in these primary cells. Consequently, many studies of αIIbß3 function use genetic approaches that rely on heterologous expression systems or platelets from gene-targeted mice, sometimes with uncertain applicability to human platelets. This chapter will detail a method for genetic manipulation of megakaryocytes and platelets derived from human induced pluripotent stem cells for molecular studies of αIIbß3 signaling and for modeling of human platelet functions potentially relevant to hemostasis, immunity, and inflammation.


Assuntos
Plaquetas/metabolismo , Engenharia Celular/métodos , Megacariócitos/metabolismo , Agregação Plaquetária/genética , Complexo Glicoproteico GPIIb-IIIa de Plaquetas/genética , Complexo Glicoproteico GPIb-IX de Plaquetas/genética , Glicoproteína IIb da Membrana de Plaquetas/genética , Plaquetas/citologia , Diferenciação Celular , Linhagem Celular , Citoesqueleto/metabolismo , Citoesqueleto/ultraestrutura , Fibrinogênio/genética , Fibrinogênio/metabolismo , Regulação da Expressão Gênica , Hemostasia/genética , Humanos , Células-Tronco Pluripotentes Induzidas/citologia , Células-Tronco Pluripotentes Induzidas/metabolismo , Megacariócitos/citologia , Complexo Glicoproteico GPIIb-IIIa de Plaquetas/metabolismo , Complexo Glicoproteico GPIb-IX de Plaquetas/metabolismo , Glicoproteína IIb da Membrana de Plaquetas/metabolismo , Ligação Proteica , RNA Interferente Pequeno/genética , RNA Interferente Pequeno/metabolismo , Transdução de Sinais , Ubiquitinas/antagonistas & inibidores , Ubiquitinas/genética , Ubiquitinas/metabolismo
14.
Int J Mol Sci ; 21(21)2020 Oct 24.
Artigo em Inglês | MEDLINE | ID: mdl-33114406

RESUMO

Cardiovascular diseases (CVDs) are the leading cause of death globally-partly a consequence of increased population size and ageing-and are major contributors to reduced quality of life. Platelets play a major role in hemostasis and thrombosis. While platelet activation and aggregation are essential for hemostasis at sites of vascular injury, uncontrolled platelet activation leads to pathological thrombus formation and provokes thrombosis leading to myocardial infarction or stroke. Platelet activation and thrombus formation is a multistage process with different signaling pathways involved to trigger platelet shape change, integrin activation, stable platelet adhesion, aggregation, and degranulation. Apart from thrombotic events, thrombo-inflammation contributes to organ damage and dysfunction in CVDs and is mediated by platelets and inflammatory cells. Therefore, in the past, many efforts have been made to investigate specific signaling pathways in platelets to identify innovative and promising approaches for novel antithrombotic and anti-thrombo-inflammatory strategies that do not interfere with hemostasis. In this review, we focus on some of the most recent data reported on different platelet receptors, including GPIb-vWF interactions, GPVI activation, platelet chemokine receptors, regulation of integrin signaling, and channel homeostasis of NMDAR and PANX1.


Assuntos
Plaquetas/metabolismo , Doenças Cardiovasculares/metabolismo , Redes Reguladoras de Genes , Anti-Inflamatórios/farmacologia , Doenças Cardiovasculares/sangue , Doenças Cardiovasculares/tratamento farmacológico , Fibrinolíticos/farmacologia , Redes Reguladoras de Genes/efeitos dos fármacos , Humanos , Terapia de Alvo Molecular , Ativação Plaquetária , Complexo Glicoproteico GPIb-IX de Plaquetas/metabolismo , Qualidade de Vida , Fator de von Willebrand/metabolismo
15.
Mol Immunol ; 128: 139-149, 2020 12.
Artigo em Inglês | MEDLINE | ID: mdl-33126082

RESUMO

Fever and inflammatory responses were observed in some subjects in early clinical trials of vaccines adjuvanted with muramyl dipeptide (MDP), a NOD2 agonist. Biosynthesis of Prostaglandin E2 (PGE2) that transmits febrile signals to the brain is controlled by an inducible enzyme, Cyclooxygenase 2 (COX-2). MDP alone was not sufficient to induce expression of COX-2 and PGE2 production in vitro. Conditioned medium prepared from Peripheral Blood Mononuclear Cells (PBMCs)-derived CD3-bead purified human T cells (TCM) dramatically increased COX2 gene transcription, COX-2 protein expression, and PGE2 production in MDP-treated monocytes. We explored epigenetic changes at the COX2 promoter using Chromatin Immunoprecipitation assay (ChIP). Increase in COX2 transcription correlated with increased recruitment of RNA polymerase II (Pol II) and p300 histone acetyl transferase (HAT) to the COX2 promoter in monocytes activated with MDP and TCM. The role of p300 HAT was confirmed by using C646, an inhibitor of p300, that reduced binding of acetylated H3 and H4 histones at the COX2 promoter, COX2 transcription, and PGE2 production in monocytes. Binding of p300, Nuclear Factor Kappa B (NF-κB), and Pol II to the COX2 promoter was also sensitive to inhibitors of Mitogen-Activated Protein Kinase (MAPK) pathway and to antibodies against Macrophage-1 (Mac-1) integrin in MDP/TCM-treated monocytes. Importantly, recombinant Glycoprotein Ib alfa (GPIbα), the recently identified factor in TCM, increased binding of NF-κB, p300, and of Pol II to the COX2 promoter and COX2 transcription in MDP-treated monocytes. Our findings suggest that a second signal through Mac-1 and MAPK is triggered by a T cell derived soluble GPIbα protein leading to the assembly of the transcription machinery at the COX2 promoter and production of PGE2 in human monocytes in response to MDP/NOD2 activation.


Assuntos
Dinoprostona/metabolismo , Proteína p300 Associada a E1A/metabolismo , Proteínas Quinases Ativadas por Mitógeno/metabolismo , Monócitos/metabolismo , Transdução de Sinais/fisiologia , Linfócitos T/metabolismo , Acetilmuramil-Alanil-Isoglutamina/farmacologia , Adjuvantes Imunológicos/farmacologia , Células Cultivadas , Ciclo-Oxigenase 2/metabolismo , Febre/metabolismo , Humanos , Leucócitos Mononucleares/efeitos dos fármacos , Leucócitos Mononucleares/metabolismo , Antígeno de Macrófago 1/metabolismo , Macrófagos/efeitos dos fármacos , Macrófagos/metabolismo , Monócitos/efeitos dos fármacos , NF-kappa B/metabolismo , Complexo Glicoproteico GPIb-IX de Plaquetas/metabolismo , Regiões Promotoras Genéticas/efeitos dos fármacos , Regiões Promotoras Genéticas/fisiologia , Transdução de Sinais/efeitos dos fármacos , Linfócitos T/efeitos dos fármacos , Células Th1/efeitos dos fármacos , Células Th1/metabolismo , Transcrição Genética/efeitos dos fármacos , Transcrição Genética/fisiologia
16.
BMC Mol Cell Biol ; 21(1): 64, 2020 Sep 11.
Artigo em Inglês | MEDLINE | ID: mdl-32917131

RESUMO

BACKGROUND: It has been demonstrated that von Willebrand factor (VWF) mediated platelet-endothelium and platelet-platelet interactions are shear dependent. The VWF's mobility under dynamic conditions (e.g. flow) is pivotal to platelet adhesion and VWF-mediated aggregate formation in the cascade of VWF-platelet interactions in haemostasis. RESULTS: Combining microfluidic tools with fluorescence and reflection interference contrast microscopy (RICM), here we show, that specific deletions in the A-domains of the biopolymer VWF affect both, adhesion and aggregation properties independently. Intuitively, the deletion of the A1-domain led to a significant decrease in both adhesion and aggregate formation of platelets. Nevertheless, the deletion of the A2-domain revealed a completely different picture, with a significant increase in formation of rolling aggregates (gain of function). We predict that the A2-domain effectively 'masks' the potential between the platelet glycoprotein (GP) Ib and the VWF A1-domain. Furthermore, the deletion of the A3-domain led to no significant variation in either of the two functional characteristics. CONCLUSIONS: These data demonstrate that the macroscopic functional properties i.e. adhesion and aggregate formation cannot simply be assigned to the properties of one particular domain, but have to be explained by cooperative phenomena. The absence or presence of molecular entities likewise affects the properties (thermodynamic phenomenology) of its neighbours, therefore altering the macromolecular function.


Assuntos
Plaquetas/metabolismo , Plaquetas/fisiologia , Adesividade Plaquetária/fisiologia , Agregação Plaquetária/fisiologia , Fator de von Willebrand/metabolismo , Biopolímeros/metabolismo , Linhagem Celular , Fluorescência , Células HEK293 , Hemostasia/fisiologia , Humanos , Microfluídica/métodos , Microscopia/métodos , Complexo Glicoproteico GPIb-IX de Plaquetas/metabolismo
17.
Nutrients ; 12(10)2020 Sep 25.
Artigo em Inglês | MEDLINE | ID: mdl-32992756

RESUMO

Microparticles play a role in cardiovascular disease pathology. The flavanol-like epicatechin is increasingly considered due to its cardioprotective effects. The aim of this study was to investigate the impact of epicatechin on microparticle generation, phenotype and procoagulant properties. Plasma samples from 15 healthy subjects were incubated with increasing concentrations of epicatechin (1 to 100 µM). Then, the expression of glycoprotein IIb, phosphatidylserine (PS), glycoprotein Ib (GPIb) and P-selectin was assessed by flow cytometry analysis after (or not) platelet stimulation. Microparticle procoagulant activity was determined using ZymuphenTM MP and ZymuphenTM MP-TF for phospholipid and tissue factor content, and with thrombin generation (TG) assays for procoagulant function. Platelet microparticles that express GPIb (/µL) decreased from 20,743 ± 24,985 (vehicle) to 14,939 ± 14,333 (p = 0.6), 21,366 ± 16,949 (p = 0.9) and 15,425 ± 9953 (p < 0.05) in samples incubated with 1, 10 and 100 µM epicatechin, respectively. Microparticle concentration (nM PS) decreased from 5.6 ± 2.0 (vehicle) to 5.1 ± 2.2 (p = 0.5), 4.5 ± 1.5 (p < 0.05) and 4.7 ± 2.0 (p < 0.05) in samples incubated with 1, 10 and 100µM epicatechin, respectively. Epicatechin had no impact on tissue factor-positive microparticle concentration. Epicatechin decreased TG (endogenous thrombin potential, nM.min) from 586 ± 302 to 509 ± 226 (p = 0.3), 512 ± 270 (p = 0.3) and 445 ± 283 (p < 0.05). These findings indicate that epicatechin affects microparticle release, phenotype and procoagulant properties.


Assuntos
Coagulação Sanguínea/efeitos dos fármacos , Catequina/farmacologia , Micropartículas Derivadas de Células/metabolismo , Adulto , Plaquetas/metabolismo , Doenças Cardiovasculares , Feminino , Citometria de Fluxo , Hemostasia , Humanos , Masculino , Selectina-P/metabolismo , Fosfatidilserinas/metabolismo , Fosfolipídeos , Complexo Glicoproteico GPIb-IX de Plaquetas/metabolismo , Trombina/biossíntese , Tromboplastina , Adulto Jovem
18.
Physiol Rep ; 8(15): e14534, 2020 08.
Artigo em Inglês | MEDLINE | ID: mdl-32748505

RESUMO

Concentrations of different circulating microparticles (MPs) may have clinical and physiological relevance to cardiovascular disease pathologies. PURPOSE: To quantify plasma concentrations of CD31+/CD42b-, CD62E+, and CD34+ MPs across healthy individuals and those with coronary artery disease (CAD) or acute cardiovascular events (non-ST elevation myocardial infarction (NSTEMI)). Fasted blood was obtained from CAD patients (n = 10), NSTEMI patients (n = 13), and healthy older men (n = 15) 60-75 years old. METHODS: CD31+/CD42b-, CD62E+, and CD34+ MPs were isolated from plasma and quantified using flow cytometry. Relationships between MP subtypes, fasting blood lipids, blood glucose, blood pressure, body mass index, and total number of medications were assessed. RESULTS: Concentrations of CD31+/CD42b- MPs were significantly lower in CAD and NSTEMI subjects compared with healthy individuals (p = .02 and .003, respectively). No differences between groups were found for CD62E+ or CD34+ MPs (p > .05 for both). Surprisingly, among all variables assessed, only CD62E+ MP concentrations were positively correlated with triglyceride levels (p = .012) and inversely correlated with SBP (p = .03). CONCLUSIONS: Our findings provide support for the use of different MP subtypes, specifically CD31+/CD42b- MPs, as a potential biomarker of cardiovascular disease. Importantly, results from this study should be looked at in adjunct to previous MP work in CVD conditions as a way of highlighting the complex interactions of variables such as comorbid conditions and medications on MP concentrations.


Assuntos
Micropartículas Derivadas de Células/metabolismo , Doença da Artéria Coronariana/sangue , Infarto do Miocárdio sem Supradesnível do Segmento ST/sangue , Idoso , Antígenos CD34/genética , Antígenos CD34/metabolismo , Biomarcadores/sangue , Doença da Artéria Coronariana/patologia , Endotélio Vascular/metabolismo , Endotélio Vascular/patologia , Humanos , Masculino , Pessoa de Meia-Idade , Infarto do Miocárdio sem Supradesnível do Segmento ST/patologia , Molécula-1 de Adesão Celular Endotelial a Plaquetas/genética , Molécula-1 de Adesão Celular Endotelial a Plaquetas/metabolismo , Complexo Glicoproteico GPIb-IX de Plaquetas/metabolismo
19.
Stroke Vasc Neurol ; 5(2): 185-197, 2020 06.
Artigo em Inglês | MEDLINE | ID: mdl-32606086

RESUMO

Arterial thrombosis is in part contributed by excessive platelet aggregation, which can lead to blood clotting and subsequent heart attack and stroke. Platelets are sensitive to the haemodynamic environment. Rapid haemodynamcis and disturbed blood flow, which occur in vessels with growing thrombi and atherosclerotic plaques or is caused by medical device implantation and intervention, promotes platelet aggregation and thrombus formation. In such situations, conventional antiplatelet drugs often have suboptimal efficacy and a serious side effect of excessive bleeding. Investigating the mechanisms of platelet biomechanical activation provides insights distinct from the classic views of agonist-stimulated platelet thrombus formation. In this work, we review the recent discoveries underlying haemodynamic force-reinforced platelet binding and mechanosensing primarily mediated by three platelet receptors: glycoprotein Ib (GPIb), glycoprotein IIb/IIIa (GPIIb/IIIa) and glycoprotein VI (GPVI), and their implications for development of antithrombotic 'mechano-medicine' .


Assuntos
Arteriopatias Oclusivas/tratamento farmacológico , Coagulação Sanguínea/efeitos dos fármacos , Plaquetas/efeitos dos fármacos , Fibrinolíticos/uso terapêutico , Hemodinâmica , Mecanotransdução Celular/efeitos dos fármacos , Ativação Plaquetária/efeitos dos fármacos , Inibidores da Agregação Plaquetária/uso terapêutico , Trombose/tratamento farmacológico , Animais , Arteriopatias Oclusivas/sangue , Arteriopatias Oclusivas/fisiopatologia , Plaquetas/metabolismo , Fibrinolíticos/efeitos adversos , Humanos , Terapia de Alvo Molecular , Inibidores da Agregação Plaquetária/efeitos adversos , Complexo Glicoproteico GPIIb-IIIa de Plaquetas/antagonistas & inibidores , Complexo Glicoproteico GPIIb-IIIa de Plaquetas/metabolismo , Complexo Glicoproteico GPIb-IX de Plaquetas/antagonistas & inibidores , Complexo Glicoproteico GPIb-IX de Plaquetas/metabolismo , Glicoproteínas da Membrana de Plaquetas/antagonistas & inibidores , Glicoproteínas da Membrana de Plaquetas/metabolismo , Estresse Mecânico , Trombose/sangue , Trombose/diagnóstico
20.
Eur J Pharmacol ; 883: 173385, 2020 Sep 15.
Artigo em Inglês | MEDLINE | ID: mdl-32710955

RESUMO

Fluoxetine is one of SSRIs commonly used as first-line antidepressants. It also induces adverse effects, including bleeding events. This study clarified the bleeding effect of fluoxetine and explored the action cascade of this drug leading to a longer bleeding time. A total of 48 male adult mice were evenly distributed into four groups and given fluoxetine in saline at 0, 4, 8, or 16 mg/kg, for 14 days. On day 15, tail bleeding time of 6 mice/group was measured, and their blood samples were collected for analyses of relevant platelet functions. The remained mice were allowed to survive for another 14 days without fluoxetine, and subjected to the same analyses on day 29. A significant effect of fluoxetine was reveled on bleeding time (F (3,20) = 16.842, P < 0.01) and intraplatelet serotonin (F (3,20) = 90.967, P < 0.01). Moreover, fluoxetine effectively inhibited platelet aggregation (F(3, 20) = 30.247, P < 0.01), decreased amount of GPIbα (F(3, 20) = 23.855, P < 0.01), suppressed GPIIb/IIIa activation (F(3, 20) = 89.441, P < 0.01), and lowered P-selectin (F(3, 20) = 7.960, P < 0.01) on platelet surface. Negative correlations existed between bleeding time and the aforementioned four indices, whereas correlations between intraplatelet serotonin and the same indices were positive. All changes returned to same levels as Control group after fluoxetine withdrawal. These data suggest an action pathway of fluoxetine starting at binding to serotonin transporter, followed by decreased intraplatelet serotonin, increased GPIbα shedding, suppressed GPIIb/IIIa activation, and inhibited α-granule release, and concluding with prolonged bleeding time in mice.


Assuntos
Antidepressivos de Segunda Geração/toxicidade , Plaquetas/efeitos dos fármacos , Fluoxetina/toxicidade , Ativação Plaquetária/efeitos dos fármacos , Inibidores de Captação de Serotonina/toxicidade , Animais , Tempo de Sangramento , Plaquetas/metabolismo , Grânulos Citoplasmáticos/efeitos dos fármacos , Grânulos Citoplasmáticos/metabolismo , Masculino , Camundongos Endogâmicos ICR , Agregação Plaquetária/efeitos dos fármacos , Complexo Glicoproteico GPIIb-IIIa de Plaquetas/metabolismo , Complexo Glicoproteico GPIb-IX de Plaquetas/metabolismo , Serotonina/sangue , Proteínas da Membrana Plasmática de Transporte de Serotonina/sangue , Proteínas da Membrana Plasmática de Transporte de Serotonina/efeitos dos fármacos , Fatores de Tempo
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