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1.
Int J Mol Sci ; 22(7)2021 Mar 31.
Artigo em Inglês | MEDLINE | ID: mdl-33807403

RESUMO

Platelets play a crucial role in the physiology of primary hemostasis and pathological processes such as arterial thrombosis; thus, developing a therapeutic target that prevents platelet activation can reduce arterial thrombosis. Pterostilbene (PTE) has remarkable pharmacological activities, including anticancer and neuroprotection. Few studies have reported the effects of pterostilbene on platelet activation. Thus, we examined the inhibitory mechanisms of pterostilbene in human platelets and its role in vascular thrombosis prevention in mice. At low concentrations (2-8 µM), pterostilbene strongly inhibited collagen-induced platelet aggregation. Furthermore, pterostilbene markedly diminished Lyn, Fyn, and Syk phosphorylation and hydroxyl radical formation stimulated by collagen. Moreover, PTE directly hindered integrin αIIbß3 activation through interfering with PAC-1 binding stimulated by collagen. In addition, pterostilbene affected integrin αIIbß3-mediated outside-in signaling, such as integrin ß3, Src, and FAK phosphorylation, and reduced the number of adherent platelets and the single platelet spreading area on immobilized fibrinogen as well as thrombin-stimulated fibrin clot retraction. Furthermore, pterostilbene substantially prolonged the occlusion time of thrombotic platelet plug formation in mice. This study demonstrated that pterostilbene exhibits a strong activity against platelet activation through the inhibition of integrin αIIbß3-mediated inside-out and outside-in signaling, suggesting that pterostilbene can serve as a therapeutic agent for thromboembolic disorders.


Assuntos
Plaquetas/metabolismo , Complexo Glicoproteico GPIIb-IIIa de Plaquetas/efeitos dos fármacos , Estilbenos/metabolismo , Animais , Coagulação Sanguínea/efeitos dos fármacos , Plaquetas/efeitos dos fármacos , Retração do Coágulo/efeitos dos fármacos , Colágeno , Fibrinogênio/metabolismo , Hemostasia/efeitos dos fármacos , Humanos , Integrina alfa2/efeitos dos fármacos , Integrina alfa2/metabolismo , Integrina beta3/efeitos dos fármacos , Integrina beta3/metabolismo , Integrinas/efeitos dos fármacos , Integrinas/metabolismo , Camundongos , Selectina-P/metabolismo , Fosforilação , Ativação Plaquetária/efeitos dos fármacos , Agregação Plaquetária/efeitos dos fármacos , Complexo Glicoproteico GPIIb-IIIa de Plaquetas/metabolismo , Transdução de Sinais/efeitos dos fármacos , Estilbenos/farmacologia , Trombose/metabolismo
2.
Int J Mol Sci ; 22(7)2021 Mar 25.
Artigo em Inglês | MEDLINE | ID: mdl-33806083

RESUMO

The integrin αIIbß3 is the most abundant integrin on platelets. Upon platelet activation, the integrin changes its conformation (inside-out signalling) and outside-in signalling takes place leading to platelet spreading, platelet aggregation and thrombus formation. Bloodsucking parasites such as mosquitoes, leeches and ticks express anticoagulant and antiplatelet proteins, which represent major sources of lead compounds for the development of useful therapeutic agents for the treatment of haemostatic disorders or cardiovascular diseases. In addition to hematophagous parasites, snakes also possess anticoagulant and antiplatelet proteins in their salivary glands. Two snake venom proteins have been developed into two antiplatelet drugs that are currently used in the clinic. The group of proteins discussed in this review are disintegrins, low molecular weight integrin-binding cysteine-rich proteins, found in snakes, ticks, leeches, worms and horseflies. Finally, we highlight various oral antagonists, which have been tested in clinical trials but were discontinued due to an increase in mortality. No new αIIbß3 inhibitors are developed since the approval of current platelet antagonists, and structure-function analysis of exogenous disintegrins could help find platelet antagonists with fewer adverse side effects.


Assuntos
Ativação Plaquetária/efeitos dos fármacos , Inibidores da Agregação Plaquetária/farmacologia , Agregação Plaquetária/efeitos dos fármacos , Complexo Glicoproteico GPIIb-IIIa de Plaquetas/metabolismo , Trombose/terapia , Actinas/química , Ancylostoma , Animais , Sítios de Ligação , Plaquetas/metabolismo , Dípteros , Desintegrinas/química , Desenho de Fármacos , Fibrinolíticos/farmacologia , Humanos , Ligantes , Testes de Função Plaquetária , Complexo Glicoproteico GPIIb-IIIa de Plaquetas/antagonistas & inibidores , Transdução de Sinais , Venenos de Serpentes/metabolismo , Serpentes
5.
PLoS One ; 16(1): e0244736, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-33411760

RESUMO

Galectin-1 (gal-1) is a carbohydrate-binding lectin with important functions in angiogenesis, immune response, hemostasis and inflammation. Comparable functions are exerted by platelet factor 4 (CXCL4), a chemokine stored in the α-granules of platelets. Previously, gal-1 was found to activate platelets through integrin αIIbß3. Both gal-1 and CXCL4 have high affinities for polysaccharides, and thus may mutually influence their functions. The aim of this study was to investigate a possible synergism of gal-1 and CXCL4 in platelet activation. Platelets were treated with increasing concentrations of gal-1, CXCL4 or both, and aggregation, integrin activation, P-selectin and phosphatidyl serine (PS) exposure were determined by light transmission aggregometry and by flow cytometry. To investigate the influence of cell surface sialic acid, platelets were treated with neuraminidase prior to stimulation. Gal-1 and CXCL4 were found to colocalize on the platelet surface. Stimulation with gal-1 led to integrin αIIbß3 activation and to robust platelet aggregation, while CXCL4 weakly triggered aggregation and primarily induced P-selectin expression. Co-incubation of gal-1 and CXCL4 potentiated platelet aggregation compared with gal-1 alone. Whereas neither gal-1 and CXCL4 induced PS-exposure on platelets, prior removal of surface sialic acid strongly potentiated PS exposure. In addition, neuraminidase treatment increased the binding of gal-1 to platelets and lowered the activation threshold for gal-1. However, CXCL4 did not affect binding of gal-1 to platelets. Taken together, stimulation of platelets with gal-1 and CXCL4 led to distinct and complementary activation profiles, with additive rather than synergistic effects.


Assuntos
Plaquetas/efeitos dos fármacos , Galectina 1/farmacologia , Ativação Plaquetária/efeitos dos fármacos , Fator Plaquetário 4/farmacologia , Plaquetas/metabolismo , Humanos , Ácido N-Acetilneuramínico/metabolismo , Agregação Plaquetária/efeitos dos fármacos , Complexo Glicoproteico GPIIb-IIIa de Plaquetas/metabolismo , Transdução de Sinais/efeitos dos fármacos
6.
Cell Death Dis ; 12(1): 50, 2021 01 05.
Artigo em Inglês | MEDLINE | ID: mdl-33414384

RESUMO

Novel coronavirus disease 2019 (COVID-19) is associated with a hypercoagulable state, characterized by abnormal coagulation parameters and by increased incidence of cardiovascular complications. With this study, we aimed to investigate the activation state and the expression of transmembrane proteins in platelets of hospitalized COVID-19 patients. We investigated transmembrane proteins expression with a customized mass cytometry panel of 21 antibodies. Platelets of 8 hospitalized COVID-19 patients not requiring intensive care support and without pre-existing conditions were compared to platelets of healthy controls (11 donors) with and without in vitro stimulation with thrombin receptor-activating peptide (TRAP). Mass cytometry of non-stimulated platelets detected an increased surface expression of activation markers P-Selectin (0.67 vs. 1.87 median signal intensity for controls vs. patients, p = 0.0015) and LAMP-3 (CD63, 0.37 vs. 0.81, p = 0.0004), the GPIIb/IIIa complex (4.58 vs. 5.03, p < 0.0001) and other adhesion molecules involved in platelet activation and platelet-leukocyte interactions. Upon TRAP stimulation, mass cytometry detected a higher expression of P-selectin in COVID-19 samples compared to controls (p < 0.0001). However, we observed a significantly reduced capacity of COVID-19 platelets to increase the expression of activation markers LAMP-3 and P-Selectin upon stimulation with TRAP. We detected a hyperactivated phenotype in platelets during SARS-CoV-2 infection, consisting of highly expressed platelet activation markers, which might contribute to the hypercoagulopathy observed in COVID-19. In addition, several transmembrane proteins were more highly expressed compared to healthy controls. These findings support research projects investigating antithrombotic and antiplatelet treatment regimes in COVID-19 patients, and provide new insights on the phenotypical platelet expression during SARS-CoV-2 infection.


Assuntos
Plaquetas/patologia , COVID-19/complicações , Leucócitos/patologia , SARS-CoV-2/isolamento & purificação , Trombose/epidemiologia , Adulto , Plaquetas/metabolismo , Plaquetas/virologia , COVID-19/transmissão , COVID-19/virologia , Estudos de Casos e Controles , Feminino , Alemanha/epidemiologia , Humanos , Leucócitos/metabolismo , Leucócitos/virologia , Masculino , Pessoa de Meia-Idade , Selectina-P/metabolismo , Fragmentos de Peptídeos/metabolismo , Fenótipo , Complexo Glicoproteico GPIIb-IIIa de Plaquetas/metabolismo , Trombose/virologia
7.
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
8.
Phytomedicine ; 80: 153363, 2021 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-33070081

RESUMO

BACKGROUND: The ingestion of flavonoids has been reported to be associated with reduced cardiovascular disease risk. Quercitrin is a common flavonoid in nature, and it exhibits antioxidant properties. Although the process of thrombogenesis is intimately related to cardiovascular disease risk, it is unclear whether quercitrin plays a role in thrombogenesis. PURPOSE: The aim of this study was to examine the antiplatelet effect of quercitrin in platelet activation. METHODS: Platelet aggregation, granule secretion, calcium mobilization, and integrin activation were used to assess the antiplatelet activity of quercitrin. Antithrombotic effect was determined in mouse using ferric chloride (FeCl3)-induced arterial thrombus formation in vivo and thrombus formation on collagen-coated surfaces under arteriolar shear in vitro. Transection tail bleeding time was used to evaluate whether quercitrin inhibited primary hemostasis. RESULTS: Quercitrin significantly impaired collagen-related peptide-induced platelet aggregation, granule secretion, reactive oxygen species generation, and intracellular calcium mobilization. Outside-in signaling of αIIbß3 integrin was significantly inhibited by quercitrin in a concentration-dependent manner. The inhibitory effect of quercitrin resulted from inhibition of the glycoprotein VI-mediated platelet signal transduction during cell activation. Further, the antioxidant effect is derived from decreased phosphorylation of components of the TNF receptor-associated factor 4/p47phox/Hic5 axis signalosome. Oral administration of quercitrin efficiently blocked FeCl3-induced arterial thrombus formation in vivo and thrombus formation on collagen-coated surfaces under arteriolar shear in vitro, without prolonging bleeding time. Studies using a mouse model of ischemia/reperfusion-induced stroke indicated that treatment with quercitrin reduced the infarct volume in stroke. CONCLUSIONS: Our results demonstrated that quercitrin could be an effective therapeutic agent for the treatment of thrombotic diseases.


Assuntos
Fibrinolíticos/farmacologia , Hemostasia/efeitos dos fármacos , Quercetina/análogos & derivados , Trombose/tratamento farmacológico , Trifosfato de Adenosina/metabolismo , Animais , Artérias , Cálcio/metabolismo , Relação Dose-Resposta a Droga , Humanos , Masculino , Camundongos Endogâmicos C57BL , Fosforilação/efeitos dos fármacos , Ativação Plaquetária/efeitos dos fármacos , Agregação Plaquetária/efeitos dos fármacos , Inibidores da Agregação Plaquetária/farmacologia , Complexo Glicoproteico GPIIb-IIIa de Plaquetas/metabolismo , Glicoproteínas da Membrana de Plaquetas/metabolismo , Quercetina/efeitos adversos , Quercetina/farmacologia , Traumatismo por Reperfusão/induzido quimicamente , Trombose/induzido quimicamente , Trombose/metabolismo
9.
Arterioscler Thromb Vasc Biol ; 40(10): 2391-2403, 2020 10.
Artigo em Inglês | MEDLINE | ID: mdl-32787521

RESUMO

OBJECTIVE: Reelin, a secreted glycoprotein, was originally identified in the central nervous system, where it plays an important role in brain development and maintenance. In the cardiovascular system, reelin plays a role in atherosclerosis by enhancing vascular inflammation and in arterial thrombosis by promoting platelet adhesion, activation, and thrombus formation via APP (amyloid precursor protein) and GP (glycoprotein) Ib. However, the role of reelin in hemostasis and arterial thrombosis is not fully understood to date. Approach and Results: In the present study, we analyzed the importance of reelin for cytoskeletal reorganization of platelets and thrombus formation in more detail. Platelets release reelin to amplify alphaIIb beta3 integrin outside-in signaling by promoting platelet adhesion, cytoskeletal reorganization, and clot retraction via activation of Rho GTPases RAC1 (Ras-related C3 botulinum toxin substrate) and RhoA (Ras homolog family member A). Reelin interacts with the collagen receptor GP (glycoprotein) VI with subnanomolar affinity, induces tyrosine phosphorylation in a GPVI-dependent manner, and supports platelet binding to collagen and GPVI-dependent RAC1 activation, PLC gamma 2 (1-phosphatidylinositol-4,5-bisphosphate phosphodiesterase gamma-2) phosphorylation, platelet activation, and aggregation. When GPVI was deleted from the platelet surface by antibody treatment in reelin-deficient mice, thrombus formation was completely abolished after injury of the carotid artery while being only reduced in either GPVI-depleted or reelin-deficient mice. CONCLUSIONS: Our study identified a novel signaling pathway that involves reelin-induced GPVI activation and alphaIIb beta3 integrin outside-in signaling in platelets. Loss of both, GPVI and reelin, completely prevents stable arterial thrombus formation in vivo suggesting that inhibiting reelin-platelet-interaction might represent a novel strategy to avoid arterial thrombosis in cardiovascular disease.


Assuntos
Plaquetas/enzimologia , Lesões das Artérias Carótidas/enzimologia , Moléculas de Adesão Celular Neuronais/sangue , Proteínas da Matriz Extracelular/sangue , Proteínas do Tecido Nervoso/sangue , Neuropeptídeos/sangue , Fosfolipase C gama/sangue , Complexo Glicoproteico GPIIb-IIIa de Plaquetas/metabolismo , Glicoproteínas da Membrana de Plaquetas/metabolismo , Serina Endopeptidases/sangue , Trombose/enzimologia , Proteínas rac1 de Ligação ao GTP/sangue , Proteína rhoA de Ligação ao GTP/sangue , Precursor de Proteína beta-Amiloide/genética , Precursor de Proteína beta-Amiloide/metabolismo , Animais , Coagulação Sanguínea , Lesões das Artérias Carótidas/sangue , Lesões das Artérias Carótidas/etiologia , Moléculas de Adesão Celular Neuronais/deficiência , Moléculas de Adesão Celular Neuronais/genética , Retração do Coágulo , Citoesqueleto/enzimologia , Modelos Animais de Doenças , Proteínas da Matriz Extracelular/deficiência , Proteínas da Matriz Extracelular/genética , Camundongos da Linhagem 129 , Camundongos Endogâmicos C3H , Camundongos Endogâmicos C57BL , Proteínas do Tecido Nervoso/deficiência , Proteínas do Tecido Nervoso/genética , Ativação Plaquetária , Serina Endopeptidases/deficiência , Serina Endopeptidases/genética , Transdução de Sinais , Trombose/sangue , Trombose/etiologia
10.
J Cardiovasc Pharmacol Ther ; 25(6): 578-586, 2020 11.
Artigo em Inglês | MEDLINE | ID: mdl-32691614

RESUMO

BACKGROUND: Smoking is the main preventable cause of death in the United States and worldwide and is associated with serious cardiovascular health consequences, including thrombotic diseases. Recently, electronic cigarettes (e-cigarettes) and, in particular JUUL, have attained wide popularity among smokers, nonsmokers, pregnant females, and even the youth, which is alarming. Interestingly, there is/are no information/studies regarding the effect of JUUL on cardiovascular diseases, specifically in the context of modulation of platelet activation. Thus, it is important to discern the cardiovascular disease health risks associated with JUUL. METHODS AND RESULTS: We used a passive e-vape vapor inhalation system where C57BL/6J mice (10-12 weeks old) were exposed to JUUL e-cigarette vape. Menthol flavored JUUL pods containing 5% nicotine by weight were used as the e-liquid. Mice were exposed to a total of 70 puffs daily for 2 weeks; 3-second puff duration, and 25-second puff interval. The effects of JUUL relative to clean air were analyzed, on mouse platelet function in vitro (eg, aggregation) and in vivo (eg, FeCl3-induced carotid artery injury thrombosis model). Our results indicate that short-term exposure to JUUL e-cigarette causes hyperactivation of platelets and shortens the thrombus occlusion as well as hemostasis/bleeding times, relative to clean air (medians of 14 vs. 200 seconds, P < .01 and 35 vs. 295 seconds, P < .001, respectively). CONCLUSION: Our findings document-for the first time-that short-term exposure to the JUUL e-cigarette increases the risk of thrombotic events, in part by modulating platelet function, such as aggregation and secretion, in mice.


Assuntos
Plaquetas/metabolismo , Trombose das Artérias Carótidas/etiologia , Vapor do Cigarro Eletrônico/efeitos adversos , Sistemas Eletrônicos de Liberação de Nicotina , Ativação Plaquetária , Vaping/efeitos adversos , Animais , Trombose das Artérias Carótidas/sangue , Modelos Animais de Doenças , Masculino , Camundongos Endogâmicos C57BL , Fosfatidilserinas/sangue , Agregação Plaquetária , Complexo Glicoproteico GPIIb-IIIa de Plaquetas/metabolismo , Transdução de Sinais , Vaping/sangue
11.
F1000Res ; 92020.
Artigo em Inglês | MEDLINE | ID: mdl-32566134

RESUMO

Background: Upon wound formation, platelets adhere to the neighboring extracellular matrix and spread on it, a process which is critical for physiological wound healing. Multiple external factors, such as the molecular composition of the environment and its mechanical properties, play a key role in this process and direct its speed and outcome. Methods: We combined live cell imaging, quantitative interference reflection microscopy and cryo-electron tomography to characterize, at a single platelet level, the differential spatiotemporal dynamics of the adhesion process to fibrinogen- and collagen IV-functionalized surfaces. Results: Initially, platelets sense both substrates by transient rapid extensions of filopodia. On collagen IV, a short-term phase of filopodial extension is followed by lamellipodia-based spreading. This transition is preceded by the extension of a single or couple of microtubules into the platelet's periphery and their apparent insertion into the core of the filopodia. On fibrinogen surfaces, the filopodia-to-lamellipodia transition was partial and microtubule extension was not observed leading to limited spreading, which could be restored by manganese or thrombin. Conclusions: Based on these results, we propose that interaction with collagen IV stimulate platelets to extend microtubules to peripheral filopodia, which in turn, enhances filopodial-to-lamellipodial transition and overall lamellipodia-based spreading. Fibrinogen, on the other hand, fails to induce these early microtubule extensions, leading to full lamellipodia spreading in only a fraction of the seeded platelets. We further suggest that activation of integrin αIIbß3 is essential for filopodial-to-lamellipodial transition, based on the capacity of integrin activators to enhance lamellipodia spreading on fibrinogen.


Assuntos
Plaquetas/citologia , Colágeno Tipo IV/química , Fibrinogênio/química , Adesividade Plaquetária , Células Cultivadas , Humanos , Microtúbulos , Complexo Glicoproteico GPIIb-IIIa de Plaquetas/metabolismo , Pseudópodes
12.
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
13.
J Biomed Sci ; 27(1): 60, 2020 May 06.
Artigo em Inglês | MEDLINE | ID: mdl-32375785

RESUMO

BACKGROUND: Columbianadin (CBN) is one of the main coumarin constituents isolated from Angelica pubescens. The pharmacological value of CBN is well demonstrated, especially in the prevention of several cancers and analgesic activity. A striking therapeutic target for arterial thrombosis is inhibition of platelet activation because platelet activation significantly contributes to these diseases. The current study examined the influence of CBN on human platelet activation in vitro and vascular thrombotic formation in vivo. METHODS: Aggregometry, immunoblotting, immunoprecipitation, confocal microscopic analysis, fibrin clot retraction, and thrombogenic animals were used in this study. RESULTS: CBN markedly inhibited platelet aggregation in washed human platelets stimulated only by collagen, but was not effective in platelets stimulated by other agonists such as thrombin, arachidonic acid, and U46619. CBN evidently inhibited ATP release, intracellular ([Ca2+]i) mobilization, and P-selectin expression. It also inhibited the phosphorylation of phospholipase C (PLC)γ2, protein kinase C (PKC), Akt (protein kinase B), and mitogen-activated protein kinases (MAPKs; extracellular signal-regulated kinase [ERK] 1/2 and c-Jun N-terminal kinase [JNK] 1/2, but not p38 MAPK) in collagen-activated platelets. Neither SQ22536, an adenylate cyclase inhibitor, nor ODQ, a guanylate cyclase inhibitor, reversed the CBN-mediated inhibition of platelet aggregation. CBN had no significant effect in triggering vasodilator-stimulated phosphoprotein phosphorylation. Moreover, it markedly hindered integrin αIIbß3 activation by interfering with the binding of PAC-1; nevertheless, it had no influences on integrin αIIbß3-mediated outside-in signaling such as adhesion number and spreading area of platelets on immobilized fibrinogen as well as thrombin-stimulated fibrin clot retraction. Additionally, CBN did not attenuate FITC-triflavin binding or phosphorylation of proteins, such as integrin ß3, Src, and focal adhesion kinase, in platelets spreading on immobilized fibrinogen. In experimental mice, CBN increased the occlusion time of thrombotic platelet plug formation. CONCLUSION: This study demonstrated that CBN exhibits an exceptional activity against platelet activation through inhibition of the PLCγ2-PKC cascade, subsequently suppressing the activation of Akt and ERKs/JNKs and influencing platelet aggregation. Consequently, this work provides solid evidence and considers that CBN has the potential to serve as a therapeutic agent for the treatment of thromboembolic disorders.


Assuntos
Cumarínicos/farmacologia , Ativação Plaquetária/efeitos dos fármacos , Complexo Glicoproteico GPIIb-IIIa de Plaquetas/metabolismo , Substâncias Protetoras/farmacologia , Transdução de Sinais/efeitos dos fármacos , Trombose/tratamento farmacológico , Animais , Humanos , Camundongos
14.
Thromb Haemost ; 120(5): 768-775, 2020 May.
Artigo em Inglês | MEDLINE | ID: mdl-32369848

RESUMO

BACKGROUND: Cardiovascular events are associated with low circulating vitamin D concentrations, although the underlying mechanisms are poorly understood. This study investigated associations between 25-hydroxyvitamin D concentrations, platelet function, and single-nucleotide polymorphisms (SNPs) in genes influencing vitamin D biology in the 500 Functional Genomics (500FG) cohort. METHODS: In this observational study, platelet activation and function were measured by flow cytometry by binding of fibrinogen to the activated fibrinogen receptor integrin αIIbß3 and expression of P-selectin, markers of platelet aggregation and degranulation, respectively. These parameters were correlated to serum 25-hydroxyvitamin D and genotyping was performed to investigate SNPs in genes important for vitamin D biology. RESULTS: Circulating 25-hydroxyvitamin D concentrations correlated inversely with baseline platelet binding of fibrinogen to integrin αIIbß3 (Pearson's r= -0.172, p = 0.002) and platelet responses to platelet agonist cross-linked collagen-related peptide (CRP-XL) (Pearson's r= -0.196,p = 0.002). This effect was due to circulating vitamin D levels ≤50nmol/L, since no differences in platelet fibrinogen binding were observed between subjects with normal 25-hydroxyvitamin D concentrations (>75nmol/L) and a 25-hydroxyvitamin D insufficiency (50-75 nmol/L). No correlations between 25-hydroxyvitamin D concentrations and platelet P-selectin expression were found. Several SNPs in the GC region of the vitamin D binding proteingene were associated with platelet responses to CRP-XL. CONCLUSION: Low circulating vitamin D concentrations are associated with increased platelet fibrinogen binding to integrin αIIbß3 in unstimulated samples and after stimulation with CRP-XL. These findings may contribute to the increased incidence of cardiovascular events in vitamin D deficient adults and its seasonal variation. Further studies are needed to investigate causality.


Assuntos
Plaquetas/metabolismo , Ativação Plaquetária , Complexo Glicoproteico GPIIb-IIIa de Plaquetas/metabolismo , Deficiência de Vitamina D/sangue , Adolescente , Adulto , Biomarcadores/sangue , Degranulação Celular , Feminino , Fibrinogênio/metabolismo , Voluntários Saudáveis , Humanos , Masculino , Selectina-P/sangue , Agregação Plaquetária , Polimorfismo de Nucleotídeo Único , Receptores de Calcitriol/genética , Receptores de Calcitriol/metabolismo , Transdução de Sinais , Vitamina D/análogos & derivados , Vitamina D/sangue , Deficiência de Vitamina D/diagnóstico , Deficiência de Vitamina D/genética , Adulto Jovem
15.
Thromb Haemost ; 120(5): 776-792, 2020 May.
Artigo em Inglês | MEDLINE | ID: mdl-32369849

RESUMO

BACKGROUND: Implantable cardiovascular therapeutic devices, while hemodynamically effective, remain limited by thrombosis. A driver of device-associated thrombosis is shear-mediated platelet activation (SMPA). Underlying mechanisms of SMPA, as well as useful biomarkers able to detect and discriminate mechanical versus biochemical platelet activation, are poorly defined. We hypothesized that SMPA induces a differing pattern of biomarkers compared with biochemical agonists. METHODS: Gel-filtered human platelets were subjected to mechanical activation via either uniform constant or dynamic shear; or to biochemical activation by adenosine diphosphate (ADP), thrombin receptor-activating peptide 6 (TRAP-6), thrombin, collagen, epinephrine, or arachidonic acid. Markers of platelet activation (P-selectin, integrin αIIbß3 activation) and apoptosis (mitochondrial membrane potential, caspase 3 activation, and phosphatidylserine externalization [PSE]) were examined using flow cytometry. Platelet procoagulant activity was detected by chromogenic assay measuring thrombin generation. Contribution of platelet calcium flux in SMPA was tested employing calcium chelators, ethylenediaminetetraacetic acid (EDTA), and BAPTA-AM. RESULTS: Platelet exposure to continuous shear stress, but not biochemical agonists, resulted in a dramatic increase of PSE and procoagulant activity, while no integrin αIIbß3 activation occurred, and P-selectin levels remained barely elevated. SMPA was associated with dissipation of mitochondrial membrane potential, but no caspase 3 activation was observed. Shear-mediated PSE was significantly decreased by chelation of extracellular calcium with EDTA, while intracellular calcium depletion with BAPTA-AM had no significant effect. In contrast, biochemical agonists ADP, TRAP-6, arachidonic acid, and thrombin were potent inducers of αIIbß3 activation and/or P-selectin exposure. This differing pattern of biomarkers seen for SMPA for continuous uniform shear was replicated in platelets exposed to dynamic shear stress via circulation through a ventricular assist device-propelled circulatory loop. CONCLUSION: Elevated shear stress, but not biochemical agonists, induces a differing pattern of platelet biomarkers-with enhanced PSE and thrombin generation on the platelet surface. This differential biomarker phenotype of SMPA offers the potential for early detection and discrimination from that mediated by biochemical agonists.


Assuntos
Plaquetas/efeitos dos fármacos , Sinalização do Cálcio/efeitos dos fármacos , Mecanotransdução Celular , Ativação Plaquetária/efeitos dos fármacos , Apoptose/efeitos dos fármacos , Biomarcadores/sangue , Coagulação Sanguínea/efeitos dos fármacos , Plaquetas/metabolismo , Plaquetas/patologia , Caspase 3/sangue , Humanos , Potencial da Membrana Mitocondrial/efeitos dos fármacos , Selectina-P/sangue , Fosfatidilserinas/sangue , Complexo Glicoproteico GPIIb-IIIa de Plaquetas/metabolismo , Estresse Mecânico
16.
Cardiovasc Drugs Ther ; 34(1): 53-63, 2020 02.
Artigo em Inglês | MEDLINE | ID: mdl-32062795

RESUMO

PURPOSE: Since ticagrelor inhibits the cellular uptake of adenosine, thereby increasing extracellular adenosine concentration and biological activity, we hypothesized that ticagrelor has adenosine-dependent antiplatelet properties. In the current study, we compared the effects of ticagrelor and prasugrel on platelet activation in acute coronary syndrome (ACS). METHODS: Platelet surface expression of P-selectin and activated glycoprotein (GP) IIb/IIIa in response to adenosine diphosphate (ADP), the toll-like receptor (TLR)-1/2 agonist Pam3CSK4, the TLR-4 agonist lipopolysaccharide (LPS), the protease-activated receptor (PAR)-1 agonist SFLLRN, and the PAR-4 agonist AYPGKF were measured by flow cytometry in blood from 80 ticagrelor- and 80 prasugrel-treated ACS patients on day 3 after percutaneous coronary intervention. Residual platelet aggregation to arachidonic acid (AA) and ADP were assessed by multiple electrode aggregometry and light transmission aggregometry. RESULTS: ADP-induced platelet activation and aggregation, and AA-induced platelet aggregation were similar in patients on ticagrelor and prasugrel, respectively (all p ≥ 0.3). Further, LPS-induced platelet surface expression of P-selectin and activated GPIIb/IIIa did not differ significantly between ticagrelor- and prasugrel-treated patients (both p > 0.4). In contrast, Pam3CSK4-induced platelet surface expression of P-selectin and activated GPIIb/IIIa were significantly lower in ticagrelor-treated patients (both p ≤ 0.005). Moreover, SFLLRN-induced platelet surface expression of P-selectin and activated GPIIb/IIIa were significantly less pronounced in patients on ticagrelor therapy compared to prasugrel-treated patients (both p < 0.03). Finally, PAR-4 mediated platelet activation as assessed by platelet surface expression of activated GPIIb/IIIa following stimulation with AYPGKF was significantly lower in patients receiving ticagrelor (p = 0.02). CONCLUSION: Ticagrelor inhibits TLR-1/2 and PAR mediated platelet activation in ACS patients more strongly than prasugrel.


Assuntos
Síndrome Coronariana Aguda/terapia , Plaquetas/efeitos dos fármacos , Intervenção Coronária Percutânea , Inibidores da Agregação Plaquetária/uso terapêutico , Agregação Plaquetária/efeitos dos fármacos , Cloridrato de Prasugrel/uso terapêutico , Antagonistas do Receptor Purinérgico P2Y/uso terapêutico , Receptores Ativados por Proteinase/metabolismo , Ticagrelor/uso terapêutico , Receptores Toll-Like/metabolismo , Síndrome Coronariana Aguda/sangue , Síndrome Coronariana Aguda/diagnóstico , Idoso , Plaquetas/metabolismo , Feminino , Humanos , Masculino , Pessoa de Meia-Idade , Selectina-P/metabolismo , Intervenção Coronária Percutânea/efeitos adversos , Complexo Glicoproteico GPIIb-IIIa de Plaquetas/metabolismo , Transdução de Sinais , Resultado do Tratamento
17.
Nanoscale ; 12(7): 4676-4685, 2020 Feb 20.
Artigo em Inglês | MEDLINE | ID: mdl-32048702

RESUMO

Platelets play an important role in the early stage of arterial remodeling after injury. Integrin GPIIb/IIIα (αIIbß3) regulates platelet activation in the inside-out and outside-in signaling pathways. The use of tirofiban, an integrin αIIbß3 inhibitor, in clinical therapy is limited by its short in vivo circulation time. Herein, a controlled drug-release system was formulated using CuS@mSiO2-PEG core-shell nanoparticles as near-infrared-triggered nanocarriers to release tirofiban on demand. The nanocarriers possessed good colloidal stability and very high loading efficiency for the integrin αIIbß3 inhibitor (14.5 wt% for tirofiban). Local application of αIIbß3 antagonist-tirofiban on an injured arterial wall inhibited platelet activation, which was accelerated by laser irradiation. Ex vivo platelet-promoted monocyte transmigration trans-well assays revealed decreased monocyte transmigration after platelet activation was inhibited by tirofiban. Two weeks after the wire-induced injury, the intimal area and cellular content were analyzed. The neointimal area was decreased in ApoE-/- mice with CuS@mSiO2-PEG/tirofiban and laser irradiation-promoted tirofiban release, which had limited the neointima formation. The lesions showed a decreased content of macrophages and smooth muscle cells compared with ApoE-/- mice without tirofiban inhibition. Therefore, the action of platelet-integrin αIIbß3 in neointima formation after vascular injury was successfully inhibited in vivo through the controlled release of tirofiban using a near-infrared-triggered nanocarrier, leading to the decrease of early-stage neointima formation. This study also emphasizes the role of platelets in vascular remodeling and provides a new target, namely integrin αIIbß3, for the inhibition of neointimal hyperplasia during vascular inflammation.


Assuntos
Plaquetas/metabolismo , Portadores de Fármacos , Raios Infravermelhos , Nanopartículas , Neointima/tratamento farmacológico , Complexo Glicoproteico GPIIb-IIIa de Plaquetas/antagonistas & inibidores , Tirofibana , Animais , Plaquetas/patologia , Portadores de Fármacos/química , Portadores de Fármacos/farmacocinética , Portadores de Fármacos/farmacologia , Camundongos , Camundongos Knockout para ApoE , Nanopartículas/química , Nanopartículas/uso terapêutico , Neointima/metabolismo , Neointima/patologia , Complexo Glicoproteico GPIIb-IIIa de Plaquetas/metabolismo , Células RAW 264.7 , Tirofibana/química , Tirofibana/farmacocinética , Tirofibana/farmacologia
18.
Int J Nanomedicine ; 15: 465-481, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32021191

RESUMO

Background: 1-(4-isopropylphenyl)-ß-carboline-3-carboxylic acid (ICCA) was modified by Trp-Phe-Phe to form 1-(4-isopropylphenyl)-ß-carboline-3-carbonyl-Trp-Phe-Phe (ICCA-WFF). Purpose: The object of preparing ICCA-WFF was to enhance the in vivo efficacy of ICCA, to explore the possible targeting action, and to visualize the nano-feature. Methods: The advantages of ICCA-WFF over ICCA were demonstrated by a series of in vivo assays, such as anti-tumor assay, anti-arterial thrombosis assay, anti-venous thrombosis assay, P-selectin expression assay, and GPIIb/IIIa expression assay. The nano-features of ICCA-WFF were visualized by TEM, SEM and AFM images. The thrombus targeting and tumor-targeting actions were evidenced by FT-MS spectrum analysis. Results: The minimal effective dose of ICCA-WFF slowing tumor growth and inhibiting thrombosis was 10-fold lower than that of ICCA. ICCA-WFF, but not ICCA, formed nano-particles capable of safe delivery in blood circulation. In vivo ICCA-WFF, but not ICCA, can target thrombus and tumor. In thrombus and tumor, ICCA-WFF released Trp-Phe-Phe and/or ICCA. Conclusion: Modifying ICCA with Trp-Phe-Phe successfully enhanced the anti-tumor activity, improved the anti-thrombotic action, formed nano-particles, targeted tumor tissue and thrombus, and provided an oligopeptide modification strategy for heterocyclic compounds.


Assuntos
Antineoplásicos/química , Antineoplásicos/farmacologia , Carbolinas/química , Carbolinas/farmacologia , Fibrinolíticos/farmacologia , Peptídeos/química , Animais , Domínio Catalítico , Linhagem Celular Tumoral , Ensaios de Seleção de Medicamentos Antitumorais , Fibrinolíticos/química , Humanos , Masculino , Camundongos Endogâmicos ICR , Simulação de Acoplamento Molecular , Nanopartículas/química , Selectina-P/química , Selectina-P/metabolismo , Peptídeos/farmacologia , Complexo Glicoproteico GPIIb-IIIa de Plaquetas/metabolismo , Ratos Sprague-Dawley , Trombose/sangue , Trombose/tratamento farmacológico
19.
Int J Mol Sci ; 21(3)2020 Feb 06.
Artigo em Inglês | MEDLINE | ID: mdl-32041177

RESUMO

RasGRP2 is calcium and diacylglycerol-regulated guanine nucleotide exchange factor I that activates Rap1, which is an essential signaling-knot in "inside-out" αIIbß3 integrin activation in platelets. Inherited platelet function disorder caused by variants of RASGRP2 represents a new congenital bleeding disorder referred to as platelet-type bleeding disorder-18 (BDPLT18). We review here the structure of RasGRP2 and its functions in the pathophysiology of platelets and of the other cellular types that express it. We will also examine the different pathogenic variants reported so far as well as strategies for the diagnosis and management of patients with BDPLT18.


Assuntos
Transtornos Plaquetários/genética , Plaquetas/patologia , Fatores de Troca do Nucleotídeo Guanina/genética , Hemorragia/genética , Transtornos Plaquetários/congênito , Pré-Escolar , Feminino , Fatores de Troca do Nucleotídeo Guanina/metabolismo , Hemorragia/congênito , Humanos , Lactente , Masculino , Complexo Glicoproteico GPIIb-IIIa de Plaquetas/metabolismo , Transdução de Sinais/genética , Proteínas de Ligação a Telômeros/metabolismo
20.
Biochim Biophys Acta Biomembr ; 1862(5): 183198, 2020 05 01.
Artigo em Inglês | MEDLINE | ID: mdl-31958436

RESUMO

Binding of integrin alphaIIbbeta3 (αiibß3) to its ligands is a highly restricted and regulated mechanism. Any modification of the protein structure yields a dysfunctional role, especially in a redox environment. Here, we examine the effect of nitrosative stress on the αiibß3 reconstituted into nanodiscs. Using single molecule force spectroscopy, we measured the interaction between αiibß3 and its ligand RGD and found that in the presence of exogenous nitric oxide (NO) two force regimes are generated: a low force regime of ~100pN indicating the presence of integrin in a normal status, and a broad spectrum of high force regime (~210-450pN) suggesting the protein modification/aggregation. By high resolution atomic force microscopy imaging, we demonstrate that both NO and nitrite (a stable product formed from NO) are involved in destabilizing the transmembrane protein complex leading to release of αiibß3 from the lipid bilayer and protein aggregation. Our experimental setup opens new ways for testing in a membrane environment the effect of radical species on integrins under clinically relevant conditions.


Assuntos
Estresse Nitrosativo/fisiologia , Complexo Glicoproteico GPIIb-IIIa de Plaquetas/química , Complexo Glicoproteico GPIIb-IIIa de Plaquetas/metabolismo , Integrinas/química , Integrinas/metabolismo , Ligantes , Bicamadas Lipídicas/química , Bicamadas Lipídicas/metabolismo , Proteínas de Membrana/química , Proteínas de Membrana/metabolismo , Microscopia de Força Atômica/métodos , Nitratos/metabolismo , Óxido Nítrico/química , Óxido Nítrico/metabolismo , Oligopeptídeos , Complexo Glicoproteico GPIIb-IIIa de Plaquetas/fisiologia , Ligação Proteica , Espécies Reativas de Nitrogênio/química , Espécies Reativas de Nitrogênio/metabolismo
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