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1.
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
2.
Ther Adv Cardiovasc Dis ; 13: 1753944719893274, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-31823688

RESUMO

Oral antiplatelet drugs are crucially important for patients with acute coronary syndrome or stable coronary artery disease undergoing percutaneous coronary intervention (PCI). In recent decades, several clinical trials have focused on reducing periprocedural ischemic events in patients undergoing PCI by means of more rapid platelet inhibition with the use of intravenous antiplatelet drugs. Glycoprotein IIb/IIIa receptor inhibitors (GPIs) block the final common pathway of platelet aggregation and enable potent inhibition in the peri-PCI period. In recent years, however, the use of GPIs has decreased due to bleeding concerns and the availability of more potent oral P2Y12 inhibitors. Cangrelor is an intravenous P2Y12 receptor antagonist. In a large-scale regulatory trial, cangrelor administration during PCI allowed for rapid, potent and rapidly reversible inhibition of platelet aggregation, with an anti-ischemic benefit and no increase in major bleeding. This article aims to provide an overview of general pharmacology, supporting evidence and current status of intravenous antiplatelet therapies (GPIs and cangrelor), with a focus on contemporary indications for their clinical use.


Assuntos
Síndrome Coronariana Aguda/terapia , Monofosfato de Adenosina/análogos & derivados , Plaquetas/efeitos dos fármacos , Doença da Artéria Coronariana/terapia , Intervenção Coronária Percutânea , Inibidores da Agregação de Plaquetas/administração & dosagem , Agregação Plaquetária/efeitos dos fármacos , Complexo Glicoproteico GPIIb-IIIa de Plaquetas/antagonistas & inibidores , Antagonistas do Receptor Purinérgico P2Y/administração & dosagem , Síndrome Coronariana Aguda/sangue , Síndrome Coronariana Aguda/diagnóstico , Monofosfato de Adenosina/administração & dosagem , Monofosfato de Adenosina/efeitos adversos , Administração Intravenosa , Animais , Plaquetas/metabolismo , Doença da Artéria Coronariana/sangue , Doença da Artéria Coronariana/diagnóstico , Hemorragia/induzido quimicamente , Humanos , Intervenção Coronária Percutânea/efeitos adversos , Inibidores da Agregação de Plaquetas/efeitos adversos , Complexo Glicoproteico GPIIb-IIIa de Plaquetas/metabolismo , Antagonistas do Receptor Purinérgico P2Y/efeitos adversos , Medição de Risco , Fatores de Risco , Fatores de Tempo , Resultado do Tratamento
3.
Int J Mol Sci ; 20(22)2019 Nov 08.
Artigo em Inglês | MEDLINE | ID: mdl-31717348

RESUMO

Auraptene is the most abundant coumarin derivative from plants. The pharmacological value of this compound has been well demonstrated, especially in the prevention of cancer and neurodegenerative diseases. Platelet activation is a major factor contributing to arterial thrombosis. Thus, this study evaluated the influence of auraptene in platelet aggregation and thrombotic formation. Auraptene inhibited platelet aggregation in human platelets stimulated with collagen only. However, auraptene was not effective in inhibiting platelet aggregation stimulated with thrombin, arachidonic acid, and U46619. Auraptene also repressed ATP release, [Ca2+]i mobilization, and P-selectin expression. Moreover, it markedly blocked PAC-1 binding to integrin αIIbß3. However, it had no influence on properties related to integrin αIIbß3-mediated outside-in signaling, such as the adhesion number, spreading area of platelets, and fibrin clot retraction. Auraptene inhibited the phosphorylation of Lyn-Fyn-Syk, phospholipase Cγ2 (PLCγ2), protein kinase C (PKC), Akt, and mitogen-activated protein kinases (MAPKs; extracellular-signal-regulated kinase (ERK1/2), and c-Jun N-terminal kinase (JNK1/2), but not p38 MAPK). Neither SQ22536, an adenylate cyclase inhibitor, nor ODQ, a guanylate cyclase inhibitor, reversed the auraptene-mediated inhibition of platelet aggregation. Auraptene reduced mortality caused by adenosine diphosphate (ADP)-induced pulmonary thromboembolism. In conclusion, this study provides definite evidence that auraptene signifies a potential therapeutic agent for preventing thromboembolic disorders.


Assuntos
Cumarínicos/uso terapêutico , Ativação Plaquetária/efeitos dos fármacos , Complexo Glicoproteico GPIIb-IIIa de Plaquetas/metabolismo , Embolia Pulmonar/tratamento farmacológico , Embolia Pulmonar/mortalidade , Transdução de Sinais , Trifosfato de Adenosina/metabolismo , Animais , Plaquetas/efeitos dos fármacos , Cálcio/metabolismo , Cumarínicos/química , Cumarínicos/farmacologia , Humanos , Camundongos , Nucleotídeos Cíclicos/metabolismo , Selectina-P/metabolismo , Fosforilação/efeitos dos fármacos , Embolia Pulmonar/sangue , Transdução de Sinais/efeitos dos fármacos
4.
Eur J Pharmacol ; 865: 172734, 2019 Dec 15.
Artigo em Inglês | MEDLINE | ID: mdl-31614139

RESUMO

Morin hydrate is an active constituent of Morus alba L, Prunus dulcis, and Cudrania tricuspidata and has been reported to inhibit platelet activation in vivo and in vitro, but no reports have been issued on its regulation of αIIbß3, a platelet-specific integrin and thromboxane A2 (TXA2), positive feedback molecule. In this study, we investigated the anti-platelet activity of morin hydrate in collagen- and thrombin-induced human platelets and attempted to identify the mechanism responsible for integrin αIIbß3 activation and TXA2 generation. Our results demonstrated that morin hydrate (25-100 µM) inhibited collagen- and thrombin-induced platelet aggregation, granule secretion (P-selectin expression, ATP, and serotonin release), calcium mobilization, TXA2 production, integrin αIIbß3 activation, and clot retraction. Additionally, morin hydrate attenuated the phosphorylations of phospholipase Cγ2 (PLCγ2), cytosolic phospholipase A2 (cPLA2), phosphoinositide 3-kinase (PI3K), Akt, c-Jun N-terminal kinase (JNK), and extracellular signal-regulated kinase (ERK), and enhanced the phosphorylations of inositol trisphosphate receptor (IP3 receptor) and cyclic adenosine monophosphate (cAMP) generation. However, it had no effect on the coagulation pathway. Taken together, these observations indicate morin hydrate inhibits platelet-mediated thrombosis by down-regulating TXA2 production and integrin αIIbß3 activation, and by upregulating cAMP generation, and thus, inhibits clot retraction. These results suggest morin hydrate may have therapeutic potential as a treatment for platelet-activation-related diseases.


Assuntos
Retração do Coágulo/efeitos dos fármacos , AMP Cíclico/metabolismo , Flavonoides/farmacologia , Ativação Plaquetária/efeitos dos fármacos , Complexo Glicoproteico GPIIb-IIIa de Plaquetas/metabolismo , Tromboxano A2/metabolismo , Coagulação Sanguínea/efeitos dos fármacos , Colágeno/farmacologia , Humanos , Receptores de Inositol 1,4,5-Trifosfato/metabolismo , Fosforilação/efeitos dos fármacos , Agregação Plaquetária/efeitos dos fármacos , Trombina/farmacologia
5.
Int J Nanomedicine ; 14: 7399-7417, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-31571858

RESUMO

Purpose: We studied the effects of silver nanoparticles (AgNPs) on human blood platelet function. We hypothesized that AgNPs, a known antimicrobial agent, can be used as blood-compatible, "ideal material'' in medical devices or as a drug delivery system. Therefore, the aim of the current study was to investigate if functionalized AgNPs affect platelet function and platelets as well as endothelial cell viability in vitro. Methods: AgNPs, functionalized with reduced glutathione (GSH), polyethylene glycol (PEG) and lipoic acid (LA) were synthesized. Quartz crystal microbalance with dissipation was used to measure the effect of AgNPs on platelet aggregation. Platelet aggregation was measured by changes in frequency and dissipation, and the presence of platelets on the sensor surface was confirmed and imaged by phase contrast microscopy. Flow cytometry was used to detect surface abundance of platelet receptors. Lactate dehydrogenase test was used to assess the potential cytotoxicity of AgNPs on human blood platelets, endothelial cells, and fibroblasts. Commercially available ELISA tests were used to measure the levels of thromboxane B2 and metalloproteinases (MMP-1, MMP-2) released by platelets as markers of platelet activation. Results: 2 nm AgNPs-GSH, 3.7 nm AgNPs-PEG both at 50 and 100 µg/mL, and 2.5 nm AgNPs-LA at 100 µg/mL reduced platelet aggregation, inhibited collagen-mediated increase in total P-selectin and GPIIb/IIIa, TXB2 formation, MMP-1, and MMP-2 release. The tested AgNPs concentrations were not cytotoxic as they did not affect, platelet, endothelial cell, or fibroblast viability. Conclusion: All tested functionalized AgNPs inhibited platelet aggregation at nontoxic concentrations. Therefore, functionalized AgNPs can be used as an antiplatelet agent or in design and manufacturing of blood-facing medical devices, such as vascular grafts, stents, heart valves, and catheters.


Assuntos
Plaquetas/efeitos dos fármacos , Nanopartículas Metálicas/química , Agregação Plaquetária/efeitos dos fármacos , Prata/farmacologia , Colágeno/metabolismo , Fibroblastos/efeitos dos fármacos , Citometria de Fluxo , Células Endoteliais da Veia Umbilical Humana/efeitos dos fármacos , Células Endoteliais da Veia Umbilical Humana/metabolismo , Humanos , L-Lactato Desidrogenase/metabolismo , Ligantes , Metaloproteinases da Matriz/metabolismo , Nanopartículas Metálicas/ultraestrutura , Selectina-P/metabolismo , Tamanho da Partícula , Complexo Glicoproteico GPIIb-IIIa de Plaquetas/metabolismo , Polietilenoglicóis/química , Técnicas de Microbalança de Cristal de Quartzo , Espectroscopia de Infravermelho com Transformada de Fourier , Tromboxano B2/metabolismo
6.
Eur J Pharmacol ; 862: 172627, 2019 Nov 05.
Artigo em Inglês | MEDLINE | ID: mdl-31461638

RESUMO

Isorhapontigenin is a polyphenolic compound found in Chinese herbs and grapes. It is a methoxylated analogue of a stilbenoid, resveratrol, which is well-known for its various beneficial effects including anti-platelet activity. Isorhapontigenin possesses greater oral bioavailability than resveratrol and has also been identified to possess anti-cancer and anti-inflammatory properties. However, its effects on platelet function have not been reported previously. In this study, we report the effects of isorhapontigenin on the modulation of platelet function. Isorhapontigenin was found to selectively inhibit ADP-induced platelet aggregation with an IC50 of 1.85 µM although it displayed marginal inhibition on platelet aggregation induced by other platelet agonists at 100 µM. However, resveratrol exhibited weaker inhibition on ADP-induced platelet aggregation (IC50 > 100 µM) but inhibited collagen induced platelet aggregation at 50 µM and 100 µM. Isorhapontigenin also inhibited integrin αIIbß3 mediated inside-out and outside-in signalling and dense granule secretion in ADP-induced platelet activation but interestingly, no effect was observed on α-granule secretion. Isorhapontigenin did not exert any cytotoxicity on platelets at the concentrations of up to 100 µM. Furthermore, it did not affect haemostasis in mice at the IC50 concentration (1.85 µM). In addition, the mechanistic studies demonstrated that isorhapontigenin increased cAMP levels and VASP phosphorylation at Ser157 and decreased Akt phosphorylation. This suggests that isorhapontigenin may interfere with cAMP and PI3K signalling pathways that are associated with the P2Y12 receptor. Molecular docking studies emphasised that isorhapontigenin has greater binding affinity to P2Y12 receptor than resveratrol. Our results demonstrate that isorhapontigenin has selective inhibitory effects on ADP-stimulated platelet activation possibly via P2Y12 receptor.


Assuntos
Plaquetas/efeitos dos fármacos , Inibidores da Agregação de Plaquetas/farmacologia , Agregação Plaquetária/efeitos dos fármacos , Estilbenos/farmacologia , Difosfato de Adenosina/farmacologia , Animais , Plaquetas/metabolismo , Avaliação Pré-Clínica de Medicamentos , Feminino , Voluntários Saudáveis , Humanos , Concentração Inibidora 50 , Masculino , Camundongos , Modelos Animais , Simulação de Acoplamento Molecular , Inibidores da Agregação de Plaquetas/uso terapêutico , Testes de Função Plaquetária , Complexo Glicoproteico GPIIb-IIIa de Plaquetas/antagonistas & inibidores , Complexo Glicoproteico GPIIb-IIIa de Plaquetas/metabolismo , Receptores Purinérgicos P2Y12/química , Receptores Purinérgicos P2Y12/metabolismo , Resveratrol/análogos & derivados , Resveratrol/farmacologia , Transdução de Sinais/efeitos dos fármacos , Estilbenos/química , Estilbenos/uso terapêutico , Trombose/tratamento farmacológico
7.
Nucl Med Biol ; 72-73: 45-48, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-31330411

RESUMO

PURPOSE: 4-(3S)-3-[5-(2-[18F]-fluoroethoxy)pyridin-3-yl]-3-[({(3R)-1-[3-(piperidin-4-yl)propanoyl]-piperidin-3-yl}carbonyl)amino]propanoic acid ([18F]GP1) is a radiotracer developed for targeted imaging of activated platelet glycoprotein IIb/IIIa receptors with positron emission tomography/computed tomography (PET/CT) in acute thromboembolism. We evaluated here radiation dosimetry of [18F]GP1 in humans. PROCEDURES: We studied 30 subjects (10 with deep vein thrombosis, 10 with pulmonary embolism, and 10 with arterial thromboembolism) who had signs or symptoms of acute thromboembolism, and were confirmed to have thromboembolic foci by imaging studies. Dynamic whole-body PET/CT images were acquired for up to 140 min after injection of 250 MBq of [18F]GP1. Radiation absorbed dose and effective dose were calculated using the OLINDA/EXM software. RESULTS: [18F]GP1 PET images showed high initial uptake of the tracer in the heart, spleen, kidney, and liver. [18F]GP1 activity was cleared by hepatobiliary and urinary excretion. The organ receiving the highest radiation absorbed dose (mGy/MBq) was the urinary bladder (0.0884 ±â€¯0.0458), followed by upper large intestine (0.0498 ±â€¯0.0189), small intestine (0.0454 ±â€¯0.0166), and kidneys (0.0350 ±â€¯0.0231). The effective dose (mSv/MBq) was 0.0212 ±â€¯0.0027 (ICRP 103). ED was not significantly different between the three disease groups (p = 0.94). A 45-minute voiding reduced the urinary bladder wall radiation dose to 0.0495 ±â€¯0.0140 mGy/MBq, and effective dose (ICRP 103) to 0.0186 ±â€¯0.0030. CONCLUSIONS: [18F]GP1 has favorable radiation dosimetry profile for clinical PET/CT imaging. The ED is comparable to commonly used 18F PET tracers.


Assuntos
Radioisótopos de Flúor/farmacocinética , Complexo Glicoproteico GPIIb-IIIa de Plaquetas/metabolismo , Tomografia por Emissão de Pósitrons/métodos , Compostos Radiofarmacêuticos/farmacocinética , Tromboembolia/metabolismo , Doença Aguda , Adulto , Idoso , Idoso de 80 Anos ou mais , Feminino , Seguimentos , Humanos , Masculino , Pessoa de Meia-Idade , Ácidos Nipecóticos/química , Complexo Glicoproteico GPIIb-IIIa de Plaquetas/antagonistas & inibidores , Estudos Prospectivos , Piridinas/química , Traçadores Radioativos , Radiometria , Tromboembolia/diagnóstico por imagem , Tromboembolia/patologia , Distribuição Tecidual , Adulto Jovem
8.
Int J Nanomedicine ; 14: 4817-4831, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-31308660

RESUMO

Background: In vitro (1R,3S)-1-methyl-1,2,3,4-tetrahydro-ß-carboline-3-carboxyl-Lys(Pro-Ala-Lys)-Arg-Gly-Asp-Val (MTCA-KKV) adheres activated platelets, targets P-selectin and GPIIb/IIIa. This led to the development of MTCA-KKV as thrombus targeting nano-medicine. Methods: MTCA-KKV was characterized by nano-feature, anti-thrombotic activity, thrombolytic activity, thrombus target and targeting release. Results: In vivo 0.01 µmol/kg of MTCA-KKV formed nano-particles less than 100 nm in diameter, targeted thrombus, released anti-thrombotic and thrombolytic pharmacophores, prevented thrombosis and dissolved blood clots. Conclusion: Based on the profiles of targeting thrombus, targeting release, inhibiting thrombosis and dissolving blood clots MTCA-KKV is a promising nano-medicine.


Assuntos
Coagulação Sanguínea , Carbolinas/química , Carbolinas/uso terapêutico , Nanopartículas/química , Peptídeos/uso terapêutico , Trombose/sangue , Trombose/tratamento farmacológico , Administração Oral , Animais , Coagulação Sanguínea/efeitos dos fármacos , Plaquetas/efeitos dos fármacos , Carbolinas/administração & dosagem , Eritrócitos/efeitos dos fármacos , Humanos , Leucócitos/efeitos dos fármacos , Espectroscopia de Ressonância Magnética , Masculino , Nanopartículas/ultraestrutura , Selectina-P/metabolismo , Peptídeos/farmacologia , Adesividade Plaquetária/efeitos dos fármacos , Complexo Glicoproteico GPIIb-IIIa de Plaquetas/metabolismo , Ratos Sprague-Dawley , Espectroscopia de Infravermelho com Transformada de Fourier
9.
Proc Natl Acad Sci U S A ; 116(25): 12295-12300, 2019 06 18.
Artigo em Inglês | MEDLINE | ID: mdl-31160446

RESUMO

Lateral transmembrane (TM) helix-helix interactions between single-span membrane proteins play an important role in the assembly and signaling of many cell-surface receptors. Often, these helices contain two highly conserved yet distinct interaction motifs, arranged such that the motifs cannot be engaged simultaneously. However, there is sparse experimental evidence that dual-engagement mechanisms play a role in biological signaling. Here, we investigate the function of the two conserved interaction motifs in the TM domain of the integrin ß3-subunit. The first motif uses reciprocating "large-large-small" amino acid packing to mediate the interaction of the ß3 and αIIb TM domains and maintain the inactive resting conformation of the platelet integrin αIIbß3. The second motif, S-x3-A-x3-I, is a variant of the classical "G-x3-G" motif. Using site-directed mutagenesis, optical trap-based force spectroscopy, and molecular modeling, we show that S-x3-A-x3-I does not engage αIIb but rather mediates the interaction of the ß3 TM domain with the TM domain of the αv-subunit of the integrin αvß3. Like αIIbß3, αvß3 on circulating platelets is inactive, and in the absence of platelet stimulation is unable to interact with components of the subendothelial matrix. However, disrupting any residue in the ß3 S-x3-A-x3-I motif by site-directed mutations is sufficient to induce αvß3 binding to the αvß3 ligand osteopontin and to the monoclonal antibody WOW-1. Thus, the ß3-integrin TM domain is able to engage in two mutually exclusive interactions that produce alternate α-subunit pairing, creating two integrins with distinct biological functions.


Assuntos
Integrina alfaVbeta3/metabolismo , Proteínas de Membrana/metabolismo , Complexo Glicoproteico GPIIb-IIIa de Plaquetas/metabolismo , Animais , Plaquetas/metabolismo , Células CHO , Membrana Celular/metabolismo , Sequência Conservada , Cricetulus , Humanos , Integrina alfaVbeta3/genética , Complexo Glicoproteico GPIIb-IIIa de Plaquetas/genética , Domínios Proteicos
10.
Clin Appl Thromb Hemost ; 25: 1076029619845056, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-31185733

RESUMO

Type 2 diabetes causes a significant risk of cardiovascular diseases, leading to 70% of deaths in patients with diabetes. The effective treatment of diabetes significantly reduces the risk of requiring the involvement of specialists from various fields of medicine. This research aimed to assess the risk of cardiovascular events based on selected biochemical parameters (glycoprotein [GP] IIb/IIIa, von Willebrand factor [vWf], fibrinogen) and their changes in response to physical exercise. The research group consisted of 52 patients with type 2 diabetes with micro- or macro-angiopathy at a mean age of 63.80 years (8.79). The control group consisted of 50 healthy volunteers (17 women and 33 men) at a mean age of 51.16 years (6.39). All the patients consented to have their venous blood tested to measure complete blood counts. Activated GP IIb/IIIa receptors were labeled and analyzed by flow cytometry. Mean values of vWF factor were higher when compared with the control group (196.59% [80.32%] vs 148.06% [90.34%], respectively). The GP IIb/IIIa receptor expression was much higher in test patients than in the control group (3.91% [2.91%] vs 2.79% [2.51%]). Physical exercise had a positive influence on GP IIb/IIIa receptor expression and vWF, decreasing their baseline percentage values.


Assuntos
Doenças Cardiovasculares/etiologia , Complexo Glicoproteico GPIIb-IIIa de Plaquetas/metabolismo , Medição de Risco , Adulto , Estudos de Casos e Controles , Diabetes Mellitus Tipo 2/complicações , Exercício Físico/fisiologia , Feminino , Fibrinogênio/metabolismo , Humanos , Masculino , Pessoa de Meia-Idade , Fator de von Willebrand/metabolismo
11.
Redox Biol ; 26: 101250, 2019 09.
Artigo em Inglês | MEDLINE | ID: mdl-31226648

RESUMO

BACKGROUND: Bilirubin, a by-product of haem catabolism, possesses potent endogenous antioxidant and platelet inhibitory properties. These properties may be useful in inhibiting inappropriate platelet activation and ROS production; for example, during storage for transfusion. Given the hydrophobicity of unconjugated bilirubin (UCB), we investigated the acute platelet inhibitory and ROS scavenging ability of a water-soluble bilirubin analogue, bilirubin ditaurate (BRT) on ex vivo platelet function to ascertain its potential suitability for inclusion during platelet storage. METHODS: The inhibitory potential of BRT (10-100 µM) was assessed using agonist induced platelet aggregation, dense granule exocytosis and flow cytometric analysis of P-selectin and GPIIb/IIIa expression. ROS production was investigated by analysis of H2DCFDA fluorescence following agonist simulation while mitochondrial ROS production investigated using MitoSOX™ Red. Platelet mitochondrial membrane potential and viability was assessed using TMRE and Zombie Green™ respectively. RESULTS: Our data shows ≤35 µM BRT significantly inhibits both dense and alpha granule exocytosis as measured by ATP release and P-selectin surface expression, respectively. Significant inhibition of GPIIb/IIIa expression was also reported upon ≤35 µM BRT exposure. Furthermore, platelet exposure to ≤10 µM BRT significantly reduces platelet mitochondrial ROS production. Despite the inhibitory effect of BRT, platelet viability, mitochondrial membrane potential and agonist induced aggregation were not perturbed. CONCLUSIONS: These data indicate, for the first time, that BRT, a water-soluble bilirubin analogue, inhibits platelet activation and reduces platelet ROS production ex vivo and may, therefore, may be of use in preserving platelet function during storage.


Assuntos
Antioxidantes/farmacologia , Bilirrubina/análogos & derivados , Plaquetas/efeitos dos fármacos , Ativação Plaquetária/efeitos dos fármacos , Espécies Reativas de Oxigênio/metabolismo , Taurina/análogos & derivados , Adolescente , Adulto , Alprostadil/farmacologia , Bilirrubina/farmacologia , Plaquetas/citologia , Plaquetas/metabolismo , Sobrevivência Celular/efeitos dos fármacos , Grânulos Citoplasmáticos/efeitos dos fármacos , Grânulos Citoplasmáticos/metabolismo , Exocitose/efeitos dos fármacos , Feminino , Expressão Gênica/efeitos dos fármacos , Humanos , Masculino , Potencial da Membrana Mitocondrial/efeitos dos fármacos , Pessoa de Meia-Idade , Mitocôndrias/efeitos dos fármacos , Mitocôndrias/metabolismo , Selectina-P/genética , Selectina-P/metabolismo , Fragmentos de Peptídeos/antagonistas & inibidores , Fragmentos de Peptídeos/farmacologia , Agregação Plaquetária/efeitos dos fármacos , Complexo Glicoproteico GPIIb-IIIa de Plaquetas/genética , Complexo Glicoproteico GPIIb-IIIa de Plaquetas/metabolismo , Cultura Primária de Células , Espécies Reativas de Oxigênio/agonistas , Espécies Reativas de Oxigênio/antagonistas & inibidores , Taurina/farmacologia
12.
Transfus Apher Sci ; 58(3): 337-340, 2019 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-31105058

RESUMO

Irradiation of platelets with filtered xenon (fXe) flash for pathogen inactivation increases PAC-1 binding, but does not cause discernible aggregation. We aimed to determine whether PAC-1-positive platelets irradiated with fXe flash can bind to fibrinogen. Apheresis-collected platelets (Aph-PLTs) were irradiated with fXe flash (fXe-PLTs). Activation of integrin αIIbß3 was determined by the binding of PAC-1, fibrinogen Alexa Fluor 488-conjugate (fibrinogen-AF488), and anti-fibrinogen antibody (clone 9F9) using flow cytometry under resting or ADP-stimulated conditions. PAC-1 binding to fXe-PLTs increased in an fXe flash dose-dependent manner. The fraction of PAC-1 binding to fXe-PLTs suspended in either phosphate-buffered saline (PBS) or plasma was larger than that to Aph-PLTs in a resting state. However, no difference of fibrinogen-AF488 binding between fXe-PLTs and Aph-PLTs in PBS was observed under either resting or ADP-stimulated conditions. The binding of anti-fibrinogen to fXe-PLTs in plasma was marginally increased compared with Aph-PLTs, but the magnitude of the positive fraction was significantly smaller than that of PAC-1. Pathogen-inactivating fXe irradiation per se did not induce fibrinogen binding to platelets, while PAC-1 binding was increased in a resting state. On the other hand, the fraction of fibrinogen binding was equivalent to that of PAC-1 under ADP-stimulated conditions.


Assuntos
Plaquetas/metabolismo , Desinfecção , Fibrinogênio/metabolismo , Complexo Glicoproteico GPIIb-IIIa de Plaquetas/metabolismo , Raios Ultravioleta , Plaquetas/citologia , Humanos , Plaquetoferese , Xenônio
13.
Phytomedicine ; 61: 152859, 2019 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-31039534

RESUMO

BACKGROUND: Curdione, a sesquiterpene compound isolated from the essential oil of Curcuma aromatica Salisb. inhibits platelet aggregation, suggesting its significant anticoagulant and antithrombotic effects. However, the mechanisms have not been fully elucidated. HYPOTHESIS: We hypothesized that curdione inhibits thrombin-induced platelet aggregation via regulating the AMP-activated protein kinase-vinculin/talin-integrin αIIbß3 signaling pathway. STUDY DESIGN: We performed in vitro assays to evaluate the effect of curdione on thrombin-induced expression levels of the AMPK signaling molecule and integrin αIIbß3 signaling pathway components. METHODS: Platelet proteins were extracted from washed human platelets, and the effects of curdione on thrombin-induced platelet aggregation were evaluated. The expression levels of the AMPK signaling molecule and integrin αIIbß3 signaling pathway-related proteins were examined using western blot and RT-PCR. The binding of vinculin and talin were studied using immunoprecipitation, double immunofluorescence staining and microscale thermophoresis. RESULTS: Platelet aggregation analysis showed that 0.02 U/ml thrombin significantly induces platelet aggregation. Western blot and RT-PCR analysis revealed that AMPK inhibits the vinculin/talin-mediated integrin αIIbß3 signaling pathway, and curdione downregulates the thrombin-induced expression of phosphorylated AMPK (P-AMPK) and P-integrin at both the protein and mRNA levels and downregulates vinculin and talin at the protein level. Furthermore, microscale thermophoresis experiments showed that curdione inhibits the binding of vinculin and talin. The results from the immunoprecipitation and double immunofluorescence staining were consistent with the results of the microscale thermophoresis experiments. CONCLUSION: Curdione inhibits thrombin-induced platelet aggregation via regulating the AMP-activated protein kinase-vinculin/talin-integrin αIIbß3 signaling pathway, which suggests its therapeutic potential in ethnomedicinal applications as an anti-platelet and anti-thrombotic compound to prevent thrombotic diseases.


Assuntos
Inibidores da Agregação de Plaquetas/farmacologia , Agregação Plaquetária/efeitos dos fármacos , Sesquiterpenos de Germacrano/farmacologia , Trombina/efeitos adversos , Proteínas Quinases Ativadas por AMP/genética , Proteínas Quinases Ativadas por AMP/metabolismo , Curcuma/química , Avaliação Pré-Clínica de Medicamentos , Humanos , Complexo Glicoproteico GPIIb-IIIa de Plaquetas/genética , Complexo Glicoproteico GPIIb-IIIa de Plaquetas/metabolismo , Transdução de Sinais/efeitos dos fármacos , Talina/metabolismo , Vinculina/metabolismo
14.
Thromb Haemost ; 119(6): 906-915, 2019 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-30934104

RESUMO

Factor XIIIa (fXIIIa) is a transglutaminase that plays a crucial role in fibrin clot stabilization and regulation of fibrinolysis. It is known to bind to procoagulant platelets. In contrast, the zymogen fXIII interaction with platelets is not well characterized. We investigated the interaction of zymogen fXIII with activated platelet subpopulations. Confocal microscopy and flow cytometry using fluorescently labelled factors and antibodies. Phosphatidylserine (PS)-positive activated platelets bound 700 to 800 molecules/cell of fXIII at 100 nM, while both PS-negative activated platelets and resting platelets bound 200 to 400 molecules/cell. The binding was reversible, calcium-independent and linear within the fXIII concentration range of up to 1,000 nM. fXIII predominantly bound to the caps of procoagulant platelets and co-localized with fibrinogen. Exogenous fibrinogen promoted fXIII binding by activated PS-negative platelets; this effect was abolished by the integrin αIIbß3 antagonist monafram. The fXIII binding was 1.5- to 3-fold decreased for platelets from four patients with grey platelet syndrome, and was variable for platelets from six patients with Glanzmann's thrombasthenia. Strong platelet stimulation, fibrinogen and αIIbß3 play essential roles in fXIII binding, without any of them fXIII does not bind to platelets. The preferential binding in the cap-like structures might be important for increasing local fXIII concentration in platelet thrombi.


Assuntos
Afibrinogenemia/metabolismo , Plaquetas/fisiologia , Fator VIII/metabolismo , Fibrinogênio/metabolismo , Complexo Glicoproteico GPIIb-IIIa de Plaquetas/metabolismo , Trombastenia/metabolismo , Afibrinogenemia/genética , Coagulação Sanguínea , Precursores Enzimáticos , Fibrinólise , Humanos , Ativação Plaquetária , Complexo Glicoproteico GPIIb-IIIa de Plaquetas/genética , Ligação Proteica
15.
PLoS One ; 14(4): e0214969, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-30978226

RESUMO

Integrins are transmembrane proteins involved in hemostasis, wound healing, immunity and cancer. In response to intracellular signals and ligand binding, integrins adopt different conformations: the bent (resting) form; the intermediate extended form; and the ligand-occupied active form. An integrin undergoing such conformational dynamics is the heterodimeric platelet receptor αIIbß3. Although the dramatic rearrangement of the overall structure of αIIbß3 during the activation process is potentially related to changes in the protein secondary structure, this has not been investigated so far in a membrane environment. Here we examine the Mn2+- and drug-induced activation of αIIbß3 and the impact on the structure of this protein reconstituted into liposomes. By quartz crystal microbalance with dissipation monitoring and activation assays we show that Mn2+ induces binding of the conformation-specific antibody PAC-1, which only recognizes the extended, active integrin. Circular dichroism spectroscopy reveals, however, that Mn2+-treatment does not induce major secondary structural changes of αIIbß3. Similarly, we found that treatment with clinically relevant drugs (e.g. quinine) led to the activation of αIIbß3 without significant changes in protein secondary structure. Molecular dynamics simulation studies revealed minor local changes in the beta-sheet probability of several extracellular domains of the integrin. Our experimental setup represents a new approach to study transmembrane proteins, especially integrins, in a membrane environment and opens a new way for testing drug binding to integrins under clinically relevant conditions.


Assuntos
Complexo Glicoproteico GPIIb-IIIa de Plaquetas/química , Quinina/química , Anticorpos/química , Dicroísmo Circular , Humanos , Lipossomos , Manganês/química , Manganês/metabolismo , Complexo Glicoproteico GPIIb-IIIa de Plaquetas/metabolismo , Domínios Proteicos , Estrutura Quaternária de Proteína , Estrutura Secundária de Proteína , Técnicas de Microbalança de Cristal de Quartzo
16.
Thromb Haemost ; 119(7): 1102-1111, 2019 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-31035301

RESUMO

The platelet receptor glycoprotein (GP) IIb/IIIa, formed by integrins αIIb and ß3, plays an important role in platelet adhesion and aggregation. Its major binding site is the arginine-glycine-aspartic acid (RGD) sequence present in several adhesive proteins. Upon platelet activation, inside-out signaling activates the complex permitting binding to RGD motif containing proteins, such as von Willebrand factor (VWF). VWF is a large multidomain plasma GP essential to primary hemostasis, which can directly interact with platelets because it exhibits binding sites for GPIbα and GPIIb/IIIa in its A1 and C4 domain, respectively. A vast variety of VWF variants have been identified in which domain-specific mutations affect distinct functions of VWF but reduced GPIIb/IIIa binding has barely been studied so far. Here, we strived to investigate the influence of C domain mutations, which have been identified in patients diagnosed with von Willebrand disease (VWD), on VWF-GPIIb/IIIa interaction. To determine binding to membrane-incorporated GPIIb/IIIa in the absence of GPIbα, we developed and validated a cell-based binding assay which uses HEK293 cells stably expressing a constitutively active form of the GPIIb/IIIa receptor complex on their plasma membrane. By employing this assay, we measured GPIIb/IIIa binding of 14 VWF C domain mutants identified in VWD patients. Mutants p.Cys2257Arg, p.Gly2441Cys, p.Cys2477Tyr, and p.Pro2722Ala exhibited significantly reduced binding. Summarizing, we have developed a useful research tool to specifically investigate GPIIb/IIIa interaction with its protein binding partners and identified four VWF variants that exhibit impaired GPIIb/IIIa binding. At least in the homozygous state, this defect could contribute to the VWD phenotype.


Assuntos
Plaquetas/fisiologia , Mutação de Sentido Incorreto/genética , Complexo Glicoproteico GPIIb-IIIa de Plaquetas/metabolismo , Domínios Proteicos/genética , Doenças de von Willebrand/genética , Fator de von Willebrand/genética , Anticorpos Monoclonais/metabolismo , Fibrinogênio/metabolismo , Variação Genética , Células HEK293 , Humanos , Ativação Plaquetária , Ligação Proteica , Engenharia de Proteínas , Transdução de Sinais , Doenças de von Willebrand/metabolismo
17.
Thromb Haemost ; 119(7): 1147-1153, 2019 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-31018220

RESUMO

Antiphospholipid antibodies (aPL) have been reported to activate platelets. This is considered to be one of the pathogenic properties of aPL. Even though aPL heterogeneity is quite well established, little is known, if the ability to activate platelets is common to all aPL or depends on antigen specificity. To further study this issue, we analyzed the ability of three human monoclonal aPL with distinctly different antigenic specificities to activate platelets in vitro. The results obtained with human monoclonal aPL were validated with immunoglobulin G (IgG) fractions obtained from patients with antiphospholipid syndrome (APS). A co-factor-independent human monoclonal anticardiolipin aPL had no discernible effect on human platelets. Two monoclonal aPL reactive against ß2 glycoprotein I (ß2GPI) induced platelet aggregation, integrin αIIbß3 activation and P-selectin surface expression. These data could be confirmed with patient IgG fractions which could only induce aggregation, if they had anti-ß2GPI activity. Anti-ß2GPI aPL-induced platelet activation depended on interaction of aPL with the low affinity Fcγ-receptor IIa on the platelet surface. It was completely abolished by pretreatment of platelet-rich plasma with the mechanistic target of rapamycin (mTOR) inhibitors rapamycin or everolimus. This extends previous data showing that mTOR is involved in signaling of anti-ß2GPI in monocytes and endothelial cells. In conclusion, anti-ß2GPI aPL activate platelets while co-factor-independent anticardiolipin aPL have no effect. mTOR is involved in this signaling process which has implications beyond APS, because so far the role of mTOR signaling in platelets is incompletely explored and requires further study.


Assuntos
Síndrome Antifosfolipídica/imunologia , Plaquetas/fisiologia , Epitopos de Linfócito B/metabolismo , Epitopos/metabolismo , Agregação Plaquetária/efeitos dos fármacos , Anticorpos Antifosfolipídeos/metabolismo , Síndrome Antifosfolipídica/tratamento farmacológico , Autoanticorpos/metabolismo , Células Cultivadas , Everolimo/farmacologia , Everolimo/uso terapêutico , Humanos , Complexo Glicoproteico GPIIb-IIIa de Plaquetas/metabolismo , Transdução de Sinais , Sirolimo/farmacologia , Sirolimo/uso terapêutico , Serina-Treonina Quinases TOR/antagonistas & inibidores , beta 2-Glicoproteína I/imunologia
18.
Oxid Med Cell Longev ; 2019: 1050476, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-31007831

RESUMO

The progression of Alzheimer's dementia is associated with neurovasculature impairment, which includes inflammation, microthromboses, and reduced cerebral blood flow. Here, we investigate the effects of ß amyloid peptides on the function of platelets, the cells driving haemostasis. Amyloid peptide ß1-42 (Aß1-42), Aß1-40, and Aß25-35 were tested in static adhesion experiments, and it was found that platelets preferentially adhere to Aß1-42 compared to other Aß peptides. In addition, significant platelet spreading was observed over Aß1-42, while Aß1-40, Aß25-35, and the scAß1-42 control did not seem to induce any platelet spreading, which suggested that only Aß1-42 activates platelet signalling in our experimental conditions. Aß1-42 also induced significant platelet adhesion and thrombus formation in whole blood under venous flow condition, while other Aß peptides did not. The molecular mechanism of Aß1-42 was investigated by flow cytometry, which revealed that this peptide induces a significant activation of integrin αIIbß3, but does not induce platelet degranulation (as measured by P-selectin membrane translocation). Finally, Aß1-42 treatment of human platelets led to detectable levels of protein kinase C (PKC) activation and tyrosine phosphorylation, which are hallmarks of platelet signalling. Interestingly, the NADPH oxidase (NOX) inhibitor VAS2870 completely abolished Aß1-42-dependent platelet adhesion in static conditions, thrombus formation in physiological flow conditions, integrin αIIbß3 activation, and tyrosine- and PKC-dependent platelet signalling. In summary, this study highlights the importance of NOXs in the activation of platelets in response to amyloid peptide ß1-42. The molecular mechanisms described in this manuscript may play an important role in the neurovascular impairment observed in Alzheimer's patients.


Assuntos
Peptídeos beta-Amiloides/toxicidade , NADPH Oxidases/metabolismo , Fragmentos de Peptídeos/toxicidade , Adesividade Plaquetária/efeitos dos fármacos , Trombose/patologia , Benzoxazóis/farmacologia , Humanos , Complexo Glicoproteico GPIIb-IIIa de Plaquetas/metabolismo , Transdução de Sinais/efeitos dos fármacos , Triazóis/farmacologia
19.
Nat Mater ; 18(7): 760-769, 2019 07.
Artigo em Inglês | MEDLINE | ID: mdl-30911119

RESUMO

Integrins are membrane receptors that mediate cell adhesion and mechanosensing. The structure-function relationship of integrins remains incompletely understood, despite the extensive studies carried out because of its importance to basic cell biology and translational medicine. Using a fluorescence dual biomembrane force probe, microfluidics and cone-and-plate rheometry, we applied precisely controlled mechanical stimulations to platelets and identified an intermediate state of integrin αIIbß3 that is characterized by an ectodomain conformation, ligand affinity and bond lifetimes that are all intermediate between the well-known inactive and active states. This intermediate state is induced by ligand engagement of glycoprotein (GP) Ibα via a mechanosignalling pathway and potentiates the outside-in mechanosignalling of αIIbß3 for further transition to the active state during integrin mechanical affinity maturation. Our work reveals distinct αIIbß3 state transitions in response to biomechanical and biochemical stimuli, and identifies a role for the αIIbß3 intermediate state in promoting biomechanical platelet aggregation.


Assuntos
Fenômenos Mecânicos , Agregação Plaquetária , Complexo Glicoproteico GPIIb-IIIa de Plaquetas/metabolismo , Fenômenos Biomecânicos , Humanos , Ligantes , Transdução de Sinais
20.
Nat Commun ; 10(1): 1204, 2019 03 13.
Artigo em Inglês | MEDLINE | ID: mdl-30867419

RESUMO

Platelets contract forcefully after their activation, contributing to the strength and stability of platelet aggregates and fibrin clots during blood coagulation. Viscoelastic approaches can be used to assess platelet-induced clot strengthening, but they require thrombin and fibrin generation and are unable to measure platelet forces directly. Here, we report a rapid, microfluidic approach for measuring the contractile force of platelet aggregates for the detection of platelet dysfunction. We find that platelet forces are significantly reduced when blood samples are treated with inhibitors of myosin, GPIb-IX-V, integrin αIIbß3, P2Y12, or thromboxane generation. Clinically, we find that platelet forces are measurably lower in cardiology patients taking aspirin. We also find that measuring platelet forces can identify Emergency Department trauma patients who subsequently require blood transfusions. Together, these findings indicate that microfluidic quantification of platelet forces may be a rapid and useful approach for monitoring both antiplatelet therapy and traumatic bleeding risk.


Assuntos
Plaquetas/fisiologia , Hemorragia/diagnóstico , Microfluídica/métodos , Inibidores da Agregação de Plaquetas/farmacologia , Agregação Plaquetária/fisiologia , Ferimentos e Lesões/complicações , Adulto , Aspirina/farmacologia , Aspirina/uso terapêutico , Coagulação Sanguínea/efeitos dos fármacos , Plaquetas/efeitos dos fármacos , Simulação por Computador , Estudos Transversais , Monitoramento de Medicamentos/métodos , Feminino , Hemorragia/etiologia , Hemorragia/terapia , Humanos , Masculino , Pessoa de Meia-Idade , Miosinas/antagonistas & inibidores , Miosinas/metabolismo , Agregação Plaquetária/efeitos dos fármacos , Inibidores da Agregação de Plaquetas/uso terapêutico , 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 , Prognóstico , Tromboxano A2/metabolismo , Ferimentos e Lesões/sangue , Ferimentos e Lesões/terapia
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