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2.
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 de Plaquetas/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 de Plaquetas/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
3.
Arterioscler Thromb Vasc Biol ; 40(9): 2127-2142, 2020 09.
Artigo em Inglês | MEDLINE | ID: mdl-32698684

RESUMO

OBJECTIVE: Atherothrombosis occurs upon rupture of an atherosclerotic plaque and leads to the formation of a mural thrombus. Computational fluid dynamics and numerical models indicated that the mechanical stress applied to a thrombus increases dramatically as a thrombus grows, and that strong inter-platelet interactions are essential to maintain its stability. We investigated whether GPVI (glycoprotein VI)-mediated platelet activation helps to maintain thrombus stability by using real-time video-microscopy. Approach and Results: We showed that GPVI blockade with 2 distinct Fab fragments promoted efficient disaggregation of human thrombi preformed on collagen or on human atherosclerotic plaque material in the absence of thrombin. ACT017-induced disaggregation was achieved under arterial blood flow conditions, and its effect increased with wall shear rate. GPVI regulated platelet activation within a growing thrombus as evidenced by the loss in thrombus contraction when GPVI was blocked, and the absence of the disaggregating effect of an anti-GPVI agent when the thrombi were fully activated with soluble agonists. The GPVI-dependent thrombus stabilizing effect was further supported by the fact that inhibition of any of the 4 key immunoreceptor tyrosine-based motif signalling molecules, src-kinases, Syk, PI3Kß, or phospholipase C, resulted in kinetics of thrombus disaggregation similar to ACT017. The absence of ACT017-induced disaggregation of thrombi from 2 afibrinogenemic patients suggests that the role of GPVI requires interaction with fibrinogen. Finally, platelet disaggregation of fibrin-rich thrombi was also promoted by ACT017 in combination with r-tPA (recombinant tissue plasminogen activator). CONCLUSIONS: This work identifies an unrecognized role for GPVI in maintaining thrombus stability and suggests that targeting GPVI could dissolve platelet aggregates with a poor fibrin content.


Assuntos
Afibrinogenemia/sangue , Plaquetas/efeitos dos fármacos , Fibrinogênio/metabolismo , Fragmentos Fab das Imunoglobulinas/farmacologia , Inibidores da Agregação de Plaquetas/farmacologia , Agregação Plaquetária/efeitos dos fármacos , Glicoproteínas da Membrana de Plaquetas/antagonistas & inibidores , Trombose/tratamento farmacológico , Afibrinogenemia/diagnóstico , Afibrinogenemia/genética , Plaquetas/metabolismo , Simulação por Computador , Fibrinogênio/genética , Fibrinolíticos/farmacologia , Humanos , Cinética , Microscopia de Vídeo , Modelos Biológicos , Glicoproteínas da Membrana de Plaquetas/metabolismo , Transdução de Sinais , Estresse Mecânico , Trombina/metabolismo , Trombose/sangue , Trombose/diagnóstico , Trombose/genética
4.
Platelets ; 31(2): 187-197, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-30849265

RESUMO

Losartan and honokiol are small molecules which have been described to inhibit aggregation of platelets by collagen. Losartan has been proposed to block clustering of GPVI but not to affect binding of collagen. Honokiol has been reported to bind directly to GPVI but only at a concentration that is three orders of magnitude higher than that needed for inhibition of aggregation. The mechanism of action of both inhibitors is so far unclear. In the present study, we confirm the inhibitory effects of both agents on platelet aggregation by collagen and show that both also block the aggregation induced by the activation of CLEC-2 or the low affinity immune receptor FcγRIIa at similar concentrations. For GPVI and CLEC-2, this inhibition is associated with a reduction in protein tyrosine phosphorylation of multiple proteins including Syk. In contrast, on a collagen surface, spreading of platelets and clustering of GPVI (measured by single molecule localisation microscopy) was not altered by losartan or honokiol. Furthermore, in flow whole-blood, both inhibitors suppressed the formation of multi-layered platelet thrombi at arteriolar shear rates at concentrations that hardly affect collagen-induced platelet aggregation in platelet rich plasma. Together, these results demonstrate that losartan and honokiol have multiple effects on platelets which should be considered in the use of these compounds as anti-platelet agents.


Assuntos
Compostos de Bifenilo/farmacologia , Lignanas/farmacologia , Losartan/farmacologia , Inibidores da Agregação de Plaquetas/farmacologia , Agregação Plaquetária/efeitos dos fármacos , Glicoproteínas da Membrana de Plaquetas/antagonistas & inibidores , Plaquetas/metabolismo , Colágeno/farmacologia , Humanos , Lectinas Tipo C/metabolismo , Glicoproteínas de Membrana/metabolismo , Fosforilação , Glicoproteínas da Membrana de Plaquetas/metabolismo , Plasma Rico em Plaquetas/efeitos dos fármacos , Plasma Rico em Plaquetas/enzimologia , Plasma Rico em Plaquetas/metabolismo , Receptores de IgG/metabolismo , Quinase Syk/metabolismo , Trombose
5.
Molecules ; 24(19)2019 Sep 26.
Artigo em Inglês | MEDLINE | ID: mdl-31561469

RESUMO

Atroxlysin-III (Atr-III) was purified from the venom of Bothrops atrox. This 56-kDa protein bears N-linked glycoconjugates and is a P-III hemorrhagic metalloproteinase. Its cDNA-deduced amino acid sequence reveals a multidomain structure including a proprotein, a metalloproteinase, a disintegrin-like and a cysteine-rich domain. Its identity with bothropasin and jararhagin from Bothrops jararaca is 97% and 95%, respectively. Its enzymatic activity is metal ion-dependent. The divalent cations, Mg2+ and Ca2+, enhance its activity, whereas excess Zn2+ inhibits it. Chemical modification of the Zn2+-complexing histidine residues within the active site by using diethylpyrocarbonate (DEPC) inactivates it. Atr-III degrades plasma fibronectin, type I-collagen, and mainly the α-chains of fibrinogen and fibrin. The von Willebrand factor (vWF) A1-domain, which harbors the binding site for GPIb, is not hydrolyzed. Platelets interact with collagen via receptors for collagen, glycoprotein VI (GPVI), and α2ß1 integrin. Neither the α2ß1 integrin nor its collagen-binding A-domain is fragmented by Atr-III. In contrast, Atr-III cleaves glycoprotein VI (GPVI) into a soluble ~55-kDa fragment (sGPVI). Thereby, it inhibits aggregation of platelets which had been stimulated by convulxin, a GPVI agonist. Selectively, Atr-III targets GPVI antagonistically and thus contributes to the antithrombotic effect of envenomation by Bothrops atrox.


Assuntos
Plaquetas/efeitos dos fármacos , Plaquetas/metabolismo , Venenos de Crotalídeos/enzimologia , Crotalinae , Metaloproteases/farmacologia , Glicoproteínas da Membrana de Plaquetas/biossíntese , Sequência de Aminoácidos , Animais , Crotalinae/metabolismo , Matriz Extracelular , Metaloproteases/química , Metaloproteases/genética , Metaloproteases/isolamento & purificação , Modelos Moleculares , Filogenia , Glicoproteínas da Membrana de Plaquetas/antagonistas & inibidores , Glicoproteínas da Membrana de Plaquetas/química , Conformação Proteica , Proteólise , Relação Estrutura-Atividade
6.
Thromb Haemost ; 119(11): 1720-1739, 2019 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-31564053

RESUMO

Despite significant advances in the treatment of thrombogenic diseases, antiplatelet therapies are still associated with a high bleeding risk. Consequently, potential benefits of preventing thromboembolic events by pharmacological agents need to be balanced with the potential harm of inducing hemorrhage. Glycoprotein VI (GPVI) is a platelet-specific receptor, which plays a crucial role in thrombus formation. GPVI deficiency has been identified in patients who suffer from significant reduction of collagen-induced thrombus formation, with a slight tendency for mild bleeding. However, an isolated GPVI deficiency can reduce thrombus formation while not resulting in severe bleeding. Together, these observations strongly suggest that physiological hemostasis does not require GPVI, but pharmacological GPVI modulation may provide novel "bleeding-free" antithrombotic therapies. In this review, we discuss recent findings regarding the biological role of GPVI in platelet-related disorders and highlight the efforts to develop potential therapeutic strategies based on its structure, signaling pathways, and biological effects.


Assuntos
Plaquetas/efeitos dos fármacos , Fibrinolíticos/uso terapêutico , Hemostasia/efeitos dos fármacos , Inibidores da Agregação de Plaquetas/uso terapêutico , Glicoproteínas da Membrana de Plaquetas/antagonistas & inibidores , Trombose/tratamento farmacológico , Animais , Plaquetas/metabolismo , Fibrinolíticos/efeitos adversos , Hemorragia/induzido quimicamente , Humanos , Inibidores da Agregação de Plaquetas/efeitos adversos , Glicoproteínas da Membrana de Plaquetas/metabolismo , Medição de Risco , Transdução de Sinais , Trombose/sangue , Resultado do Tratamento
7.
Blood ; 133(25): 2696-2706, 2019 06 20.
Artigo em Inglês | MEDLINE | ID: mdl-30952674

RESUMO

Maintenance of tumor vasculature integrity is indispensable for tumor growth and thus affects tumor progression. Previous studies have identified platelets as major regulators of tumor vascular integrity, as their depletion selectively rendered tumor vessels highly permeable and caused massive intratumoral hemorrhage. While these results established platelets as potential targets for antitumor therapy, their depletion is not a treatment option due to their essential role in hemostasis. Thus, a detailed understanding of how platelets safeguard vascular integrity in tumors is urgently demanded. Here, we show for the first time that functional inhibition of glycoprotein VI (GPVI) on the platelet surface with an antibody (JAQ1) F(ab)2 fragment rapidly induces tumor hemorrhage and diminishes tumor growth similar to complete platelet depletion while not inducing systemic bleeding complications. The intratumor bleeding and tumor growth arrest could be reverted by depletion of Ly6G+ cells, confirming them to be responsible for the induction of bleeding and necrosis within the tumor. In addition, JAQ1 F(ab)2-mediated GPVI inhibition increased intratumoral accumulation of coadministered chemotherapeutic agents, such as Doxil and paclitaxel, thereby resulting in a profound antitumor effect. In summary, our findings identify platelet GPVI as a key regulator of vascular integrity specifically in growing tumors and could serve as a basis for the development of antitumor strategies based on the interference with platelet function.


Assuntos
Fragmentos Fab das Imunoglobulinas/farmacologia , Neoplasias Experimentais/patologia , Glicoproteínas da Membrana de Plaquetas/antagonistas & inibidores , Animais , Feminino , Hemorragia/patologia , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Neovascularização Patológica
8.
Int J Mol Sci ; 20(8)2019 Apr 24.
Artigo em Inglês | MEDLINE | ID: mdl-31022936

RESUMO

Platelet collagen interactions at sites of vascular injuries predominantly involve glycoprotein VI (GPVI) and the integrin α2ß1. Both proteins are primarily expressed on platelets and megakaryocytes whereas GPVI expression is also shown on endothelial and integrin α2ß1 expression on epithelial cells. We recently showed that depletion of GPVI improves stroke outcome without increasing the risk of cerebral hemorrhage. Genetic variants associated with higher platelet surface integrin α2 (ITGA2) receptor levels have frequently been found to correlate with an increased risk of ischemic stroke in patients. However until now, no preclinical stroke study has addressed whether platelet integrin α2ß1 contributes to the pathophysiology of ischemia/reperfusion (I/R) injury. Focal cerebral ischemia was induced in C57BL/6 and Itga2-/- mice by a 60 min transient middle cerebral artery occlusion (tMCAO). Additionally, wild-type animals were pretreated with anti-GPVI antibody (JAQ1) or Fab fragments of a function blocking antibody against integrin α2ß1 (LEN/B). In anti-GPVI treated animals, intravenous (IV) recombinant tissue plasminogen activator (rt-PA) treatment was applied immediately prior to reperfusion. Stroke outcome, including infarct size and neurological scoring was determined on day 1 after tMCAO. We demonstrate that targeting the integrin α2ß1 (pharmacologic; genetic) did neither reduce stroke size nor improve functional outcome on day 1 after tMCAO. In contrast, depletion of platelet GPVI prior to stroke was safe and effective, even when combined with rt-PA treatment. Our results underscore that GPVI, but not ITGA2, is a promising and safe target in the setting of ischemic stroke.


Assuntos
Anticorpos/uso terapêutico , Encéfalo/efeitos dos fármacos , Infarto da Artéria Cerebral Média/prevenção & controle , Integrina alfa2beta1/antagonistas & inibidores , Glicoproteínas da Membrana de Plaquetas/antagonistas & inibidores , Substâncias Protetoras/uso terapêutico , Ativador de Plasminogênio Tecidual/uso terapêutico , Animais , Encéfalo/metabolismo , Encéfalo/patologia , Colágeno/metabolismo , Infarto da Artéria Cerebral Média/metabolismo , Infarto da Artéria Cerebral Média/patologia , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Proteínas Recombinantes/uso terapêutico , Acidente Vascular Cerebral/metabolismo , Acidente Vascular Cerebral/patologia , Acidente Vascular Cerebral/prevenção & controle
9.
Biochem Pharmacol ; 163: 111-118, 2019 05.
Artigo em Inglês | MEDLINE | ID: mdl-30771281

RESUMO

Factor Xa (FXa) has been reported to activate platelet via interaction with glycoprotein (GP) VI but the underlying mechanism has not been fully elucidated. We investigated if Nox2-derived oxidative stress is implicated in FXa-induced platelet aggregation (PA), and the effect of a FXa inhibitor, namely rivaroxaban, with or without aspirin (ASA), on PA. We performed an in vitro study measuring convulxin-induced PA, thromboxane (Tx) B2 and isoprostanes biosynthesis, soluble Nox2-dp (sNox2-dp), a marker of Nox2 activation, soluble GPVI (sGPVI) and PLA2 activation in platelets from healthy subjects (n = 5) added with and without a Nox2 inhibitor. The same variables were also examined in platelets treated with rivaroxaban (15-60 ng/ml), combined or less with ASA (25 µM). Convulxin-stimulated platelets increased sGPVI, sNox2-dp, H2O2, eicosanoid biosynthesis and PLA2 phosphorylation, which were all inhibited by a Nox2 inhibitor. Rivaroxaban alone significantly reduced PA, sGPVI, TxB2 and isoprostanes biosynthesis, concomitantly with Syk, sNox2-dp and PLA2 activation in a dose-dependent fashion; a significant effect was achieved with 30 ng/ml rivaroxaban. Docking simulation analysis showed that rivaroxaban interacts with GPVI. In platelets co-incubated with ASA, rivaroxaban amplified the ASA antiplatelet effect, which was achieved with 30 ng/ml and prevalently attributable to Nox2 inhibition and impaired isoprostane biosynthesis. Here we show that rivaroxaban, at concentrations achievable in human circulation, inhibits PA via GPVI interaction and eventually Nox2-mediated isoprostanes biosynthesis and amplifies the ASA antiplatelet effect.


Assuntos
Aspirina/administração & dosagem , NADPH Oxidase 2/fisiologia , Ativação Plaquetária/efeitos dos fármacos , Glicoproteínas da Membrana de Plaquetas/antagonistas & inibidores , Glicoproteínas da Membrana de Plaquetas/metabolismo , Rivaroxabana/administração & dosagem , Adulto , Relação Dose-Resposta a Droga , Combinação de Medicamentos , Inibidores do Fator Xa/administração & dosagem , Feminino , Humanos , Masculino , Pessoa de Meia-Idade , Ativação Plaquetária/fisiologia , Inibidores da Agregação de Plaquetas/administração & dosagem
10.
Physiol Rep ; 7(3): e13981, 2019 02.
Artigo em Inglês | MEDLINE | ID: mdl-30756528

RESUMO

Cigarette smoking is the number one risk factor for bladder cancer development and epidemiological data suggest that nearly half of all bladder cancer patients have a history of smoking. In addition to stimulating the growth of a primary tumor, it has been shown that there is a correlation between smoking and tumor metastasis. Platelet activating factor (PAF) is expressed on the cell surface of the activated endothelium and, through binding with the PAF-receptor (PAF-R), facilitates transendothelial migration of cells in the circulation (McHowat et al. Biochemistry 40:14921-14931; 2001). In this study, we show that the exposure of bladder cancer cells to cigarette smoke extract (CSE) results in increased PAF accumulation and increased expression of the PAF-R. Furthermore, treatment with CSE increases adherence of bladder cancer cells to bladder endothelial cells and could be abrogated by pretreatment with ginkgolide B. Immunohistochemical analysis of tumor biopsy samples from bladder cancer patients who smoked revealed increased PAF and the PAF-R in tumor regions when compared to normal tissue. These data highlight a pathway in bladder cancer that is influenced by CSE which could facilitate primary tumor growth and increase metastatic potential. Targeting of the PAF-PAFR interaction could serve as a beneficial therapeutic target for managing further growth of a developing tumor.


Assuntos
Fator de Ativação de Plaquetas/metabolismo , Fumaça/efeitos adversos , Produtos do Tabaco/toxicidade , Neoplasias da Bexiga Urinária/etiologia , Bexiga Urinária/efeitos dos fármacos , Urotélio/efeitos dos fármacos , Antineoplásicos Fitogênicos/farmacologia , Adesão Celular/efeitos dos fármacos , Linhagem Celular Tumoral , Técnicas de Cocultura , Células Endoteliais/efeitos dos fármacos , Células Endoteliais/metabolismo , Ginkgolídeos/farmacologia , Humanos , Lactonas/farmacologia , Glicoproteínas da Membrana de Plaquetas/antagonistas & inibidores , Glicoproteínas da Membrana de Plaquetas/metabolismo , Receptores Acoplados a Proteínas-G/antagonistas & inibidores , Receptores Acoplados a Proteínas-G/metabolismo , Transdução de Sinais , Regulação para Cima , Bexiga Urinária/metabolismo , Bexiga Urinária/patologia , Neoplasias da Bexiga Urinária/tratamento farmacológico , Neoplasias da Bexiga Urinária/metabolismo , Neoplasias da Bexiga Urinária/patologia , Urotélio/metabolismo , Urotélio/patologia
11.
Thromb Haemost ; 119(3): 397-406, 2019 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-30685871

RESUMO

Ibrutinib and acalabrutinib are approved for B cell malignancies and novel Bruton's tyrosine kinase (Btk) inhibitors undergo clinical testing also in B cell-driven autoimmune disorders. Btk in platelets mediates platelet activation via glycoprotein (GP) VI, which is crucial for atherosclerotic plaque-induced platelet thrombus formation. This can be selectively inhibited by Btk inhibitors. Since patients on second-generation Btk inhibitors apparently show less bleeding than patients on ibrutinib, we compared the effects of ibrutinib and four novel irreversible Btk inhibitors on GPVI-dependent platelet aggregation in blood and in vitro bleeding time. Low concentrations of collagen which induced the same low degree of GPVI-mediated platelet aggregation as atherosclerotic plaque material were applied. IC50 values for collagen (0.2-0.5 µg/mL)-induced platelet aggregation after 15-minute pre-incubation were: ibrutinib 0.12 µM, BGB-3111 0.51 µM, acalabrutinib 1.21 µM, ONO/GS-4059 1.20 µM and evobrutinib 5.84 µM. Peak venous plasma concentrations of ibrutinib (0.5 µM), acalabrutinib (2 µM) and ONO/GS-4059 (2 µM) measured after anti-proliferative dosage inhibited collagen-induced platelet aggregation, but did not increase PFA-200 closure time on collagen/epinephrine. Closure times were moderately increased by 2- to 2.5-fold higher concentrations of these inhibitors, but not by BGB-3111 (1 µM) and evobrutinib (10 µM). Prolonging platelet drug exposure to 60 minutes lowered IC50 values of any Btk inhibitor for GPVI-mediated aggregation by several fold, and 5- to 10-fold below anti-proliferative therapeutic drug plasma levels. In conclusion, low blood concentrations of ibrutinib and the novel Btk inhibitors suffice for GPVI selective platelet inhibition relevant for atherothrombosis but do not impair primary haemostasis.


Assuntos
Tirosina Quinase da Agamaglobulinemia/antagonistas & inibidores , Benzamidas/farmacologia , Plaquetas/efeitos dos fármacos , Imidazóis/farmacologia , Piperidinas/farmacologia , Inibidores da Agregação de Plaquetas/farmacologia , Glicoproteínas da Membrana de Plaquetas/antagonistas & inibidores , Inibidores de Proteínas Quinases/farmacologia , Pirazinas/farmacologia , Pirazóis/farmacologia , Pirimidinas/farmacologia , Tirosina Quinase da Agamaglobulinemia/sangue , Benzamidas/toxicidade , Plaquetas/metabolismo , Relação Dose-Resposta a Droga , Hemorragia/sangue , Hemorragia/induzido quimicamente , Hemostasia/efeitos dos fármacos , Humanos , Imidazóis/toxicidade , Concentração Inibidora 50 , Piperidinas/toxicidade , Agregação Plaquetária/efeitos dos fármacos , Inibidores da Agregação de Plaquetas/toxicidade , Glicoproteínas da Membrana de Plaquetas/metabolismo , Pirazinas/toxicidade , Pirazóis/toxicidade , Pirimidinas/toxicidade
12.
Hellenic J Cardiol ; 60(4): 211-215, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-30639352

RESUMO

The management of ST-segment elevation myocardial infarction (STEMI) has evolved significantly over the last decades. STEMI treatment includes reperfusion therapy, ideally by primary percutaneous coronary intervention (pPCI), modern antithrombotic therapy and secondary prevention measures. Even though many areas in the management of STEMI are well studied and analyzed in the guidelines, there are still challenges and unanswered questions on how to improve outcomes. This review aims to offer an insight in areas that need to be explored.


Assuntos
Reperfusão Miocárdica/métodos , Intervenção Coronária Percutânea/métodos , Infarto do Miocárdio com Supradesnível do Segmento ST/terapia , Terapia Trombolítica/métodos , Síndrome Coronariana Aguda/mortalidade , Idoso , Idoso de 80 Anos ou mais , Ciclosporina/farmacologia , Ciclosporina/uso terapêutico , Feminino , Humanos , Hipotermia , Masculino , Pessoa de Meia-Idade , Mitocôndrias Cardíacas/efeitos dos fármacos , Glicoproteínas da Membrana de Plaquetas/antagonistas & inibidores , Guias de Prática Clínica como Assunto , Antagonistas do Receptor Purinérgico P2Y/uso terapêutico , Infarto do Miocárdio com Supradesnível do Segmento ST/epidemiologia , Prevenção Secundária/métodos , Fatores de Tempo , Resultado do Tratamento , Vasodilatadores/uso terapêutico
13.
Biomed Pharmacother ; 109: 563-572, 2019 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-30399592

RESUMO

Gemcitabine resistance will occur by time after the initial response in pancreatic cancer. Ginkgolide B (GB), a major terpene lactone component of Ginkgo biloba leaves, is a highly selective and competitive inhibitor for platelet-activating factor (PAF) receptor. In the present study, we evaluated the effect of GB on gemcitabine sensitivity in pancreatic cancer cell lines in vitro and in vivo. Cell viability assay, flow cytometry, dual luciferase reporter assay and tumor xenograft model were used to evaluate cell proliferation, apoptosis, nuclear factor kappa b (NF-кB) activity in vitro and tumor growth in vivo. Western blot, immunohistochemistry (IHC) and immunofluorescence were used to shown different protein expression levels. We found the half maximal inhibitory concentration (IC50) of gemcitabine was significantly downregulated by GB in a dose-dependent manner. Furthermore, GB could suppress cell proliferation, increase cell apoptosis and repress tumor growth when combined with gemcitabine, but had no effect when treated alone. Gemcitabine could upregulate PAFR and phosphorylated NF-кB/p65 expression, and increase NF-кB activity, but this was largely suppressed in combination with GB. GB could suppress PAFR expression in a dose-dependent manner. Knockout of PAFR significantly decreased phosphorylated NF-кB/p65 expression, inhibited NF-кB activity, increased gemcitabine sensitivity and cell apoptosis. Besides, GB had no influence on gemcitabine IC50 in IκBα-SR stably expressed BxPC-3 and CAPAN1. Our results suggested that GB could enhance gemcitabine sensitivity in pancreatic cancer cell lines by suppressing PAFR/NF-кB pathway. Thus GB may have therapeutic potential when used in combination with gemcitabine in pancreatic cancer.


Assuntos
Antimetabólitos Antineoplásicos/administração & dosagem , Desoxicitidina/análogos & derivados , Ginkgolídeos/administração & dosagem , Lactonas/administração & dosagem , NF-kappa B/metabolismo , Neoplasias Pancreáticas/metabolismo , Glicoproteínas da Membrana de Plaquetas/metabolismo , Receptores Acoplados a Proteínas-G/metabolismo , Animais , Protocolos de Quimioterapia Combinada Antineoplásica/administração & dosagem , Linhagem Celular Tumoral , Desoxicitidina/administração & dosagem , Relação Dose-Resposta a Droga , Sinergismo Farmacológico , Fibrinolíticos/administração & dosagem , Células HEK293 , Humanos , Camundongos , Camundongos Nus , NF-kappa B/antagonistas & inibidores , Glicoproteínas da Membrana de Plaquetas/antagonistas & inibidores , Receptores Acoplados a Proteínas-G/antagonistas & inibidores , Ensaios Antitumorais Modelo de Xenoenxerto/métodos
14.
Toxicol Appl Pharmacol ; 364: 106-113, 2019 02 01.
Artigo em Inglês | MEDLINE | ID: mdl-30592962

RESUMO

Collagen and convulxin induce platelet aggregation through glycoprotein VI (GPVI)-FcRγ-Syk signaling pathway. In addition, fibrinogen induces platelet activation through integrin αIIbß3-FcγRIIa-Syk signaling pathway. We previously reported that high concentrations of selective serotonin reuptake inhibitors (SSRI) reduce platelet aggregation induced by collagen. We further investigated the effects of SSRI on GPVI- and αIIbß3-mediated signaling pathway. Citalopram and escitalopram, two relatively pure SSRI, were used in this study. Both citalopram and escitalopram concentration-dependently inhibited convulxin-induced platelet aggregation, serotonin (5-HT) release and the activation of αIIbß3. 5-HT concentration in washed platelets was unchanged after short-term treatment with citalopram. The additional 5-HT failed to fully rescue the inhibitory effect of citalopram on convulxin-induced aggregation. Convulxin-induced phosphorylation of Syk, LAT, and Akt was inhibited by citalopram and escitalopram. Citalopram inhibited the interaction between FcRγ and Syk, whereas the phosphorylation of FcRγ in response to convulxin remained unaltered. Further, citalopram inhibited the increase of the interaction between serotonin transporter and Syk induced by convulxin. In the presence of Mn2+, escitalopram inhibited the formation of lamellipodia on immobilized fibrinogen. Escitalopram did not influence the binding of fibrinogen to platelets. It inhibited the phosphorylation of Syk and PAK triggered by the adhesion on fibrinogen. Our data demonstrate that micromolar concentrations of citalopram and escitalopram inhibit GPVI- and αIIbß3-mediated platelet functions. The mechanism of the inhibitory effect of citalopram or escitalopram is not the influence on the activation of GPVI or the interaction between fibrinogen and αIIbß3, but the interaction between Syk and its upstream molecules.


Assuntos
Plaquetas/efeitos dos fármacos , Citalopram/farmacologia , Inibidores da Agregação de Plaquetas/farmacologia , Agregação Plaquetária/efeitos dos fármacos , Complexo Glicoproteico GPIIb-IIIa de Plaquetas/antagonistas & inibidores , Glicoproteínas da Membrana de Plaquetas/antagonistas & inibidores , Inibidores de Captação de Serotonina/farmacologia , Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Plaquetas/enzimologia , Venenos de Crotalídeos/farmacologia , Relação Dose-Resposta a Droga , Fibrinogênio/metabolismo , Humanos , Lectinas Tipo C , Proteínas de Membrana/metabolismo , Fosforilação , Adesividade Plaquetária/efeitos dos fármacos , Complexo Glicoproteico GPIIb-IIIa de Plaquetas/metabolismo , Glicoproteínas da Membrana de Plaquetas/metabolismo , Proteínas Proto-Oncogênicas c-akt/metabolismo , Pseudópodes/efeitos dos fármacos , Pseudópodes/enzimologia , Receptores de IgG/metabolismo , Serotonina/metabolismo , Transdução de Sinais/efeitos dos fármacos , Quinase Syk/metabolismo , Quinases Ativadas por p21/metabolismo
15.
Auris Nasus Larynx ; 46(4): 513-519, 2019 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-30503566

RESUMO

OBJECTIVE: Phosphorylcholine (PC) is a structural component of Streptococcus pneumoniae (Spn) and nontypeable Haemophilus influenzae (NTHi), and is known to be associated with adherence through the platelet activating factor receptor (PAF-R). Furthermore, high PC expression is considered to be involved in Spn and NTHi virulence. In this study, we examined the influence of PC expression on the adherence of Spn and NTHi to epithelial cells in order to clarify the potential effectiveness of a vaccine targeting PC. METHODS: Twenty-seven strains of Spn and twenty-two strains of NTHi were used, cultured overnight, and PC expression was evaluated by fluorescence activated cell sorting; the strains were divided into two groups: PC low expression (PC-low) and PC high expression (PC-high) groups. Bacterial adherence was then examined using Detroit 562 cells and BALB/c mice. Bacterial invasion was then examined in Detroit 562 cells. RESULTS: The adherence of Spn and NTHi and invasion of NTHi in the PC-high group was significantly reduced by pretreatment with a monoclonal anti-PC antibody (TEPC-15), PAF-R antagonist (ABT-491), and PC-keyhole limpet hemocyanin (PC-KLH). However, such findings were not observed in the PC-low group. CONCLUSION: The present study suggests that PC is involved in the mucosal adhesion of Spn and NTHi, and the mucosal invasion of NTHi with PC-high strains, but not PC-low strains. These results suggest that a PC-targeting mucosal vaccine only affects PC-high Spn and NTHi strains and does not disturb commensal bacterial flora in the upper respiratory tract, which comprises nonpathogenic PC-low bacteria.


Assuntos
Aderência Bacteriana/fisiologia , Células Epiteliais/metabolismo , Haemophilus influenzae/metabolismo , Mucosa Nasal/metabolismo , Fosforilcolina/metabolismo , Streptococcus pneumoniae/metabolismo , Animais , Linhagem Celular , Citometria de Fluxo , Hemocianinas/farmacologia , Humanos , Imidazóis/farmacologia , Indóis/farmacologia , Camundongos , Camundongos Endogâmicos BALB C , Fosforilcolina/antagonistas & inibidores , Glicoproteínas da Membrana de Plaquetas/antagonistas & inibidores , Glicoproteínas da Membrana de Plaquetas/metabolismo , Receptores Acoplados a Proteínas-G/antagonistas & inibidores , Receptores Acoplados a Proteínas-G/metabolismo , Mucosa Respiratória/metabolismo , Fatores de Virulência
16.
Int J Mol Sci ; 20(1)2018 Dec 21.
Artigo em Inglês | MEDLINE | ID: mdl-30577630

RESUMO

Studies, including ours, have shown that pro-oxidative stressors, such as chemotherapeutic agents, generate oxidized lipids with agonistic platelet-activating factor (PAF) activity. Importantly, recent reports have implicated that these PAF-agonists are transported extracellularly via microvesicle particles (MVPs). While the role of PAF-receptor (PAF-R) has been implicated in mediating chemotherapy effects, its significance in chemotherapy-mediated MVP release in pancreatic cancer has not been studied. The current studies determined the functional significance of PAF-R in gemcitabine chemotherapy-mediated MVP release in human pancreatic cancer cells. Using PAF-R-expressing (PANC-1) and PAF-R-deficient (Hs766T) cells, we demonstrate that gemcitabine induces MVP release in a PAF-R-dependent manner. Blocking of PAF-R via PAF-R antagonist or inhibition of MVP generation via inhibitor of acid sphingomyelinase (aSMase) enzyme, significantly attenuated gemcitabine-mediated MVP release from PANC-1 cells, however, exerted no effects in Hs766T cells. Notably, MVPs from gemcitabine-treated PANC-1 cells, contained a measurable amount of PAF-agonists. Mechanistically, pretreatment with ERK1/2 or p38 inhibitors significantly abrogated gemcitabine-mediated MVP release, indicating the involvement of mitogen-activated protein kinase (MAPK) pathway in PAF-R-dependent gemcitabine-mediated MVP release. These findings demonstrate the significance of PAF-R in gemcitabine-mediated MVP release, as well as the rationale of evaluating PAF-R targeting agents with gemcitabine against pancreatic cancer.


Assuntos
Antimetabólitos Antineoplásicos/farmacologia , Micropartículas Derivadas de Células/metabolismo , Desoxicitidina/análogos & derivados , Sistema de Sinalização das MAP Quinases/efeitos dos fármacos , Neoplasias Pancreáticas/metabolismo , Glicoproteínas da Membrana de Plaquetas/metabolismo , Receptores Acoplados a Proteínas-G/metabolismo , Linhagem Celular Tumoral , Desoxicitidina/farmacologia , Humanos , Glicoproteínas da Membrana de Plaquetas/agonistas , Glicoproteínas da Membrana de Plaquetas/antagonistas & inibidores , Receptores Acoplados a Proteínas-G/agonistas , Receptores Acoplados a Proteínas-G/antagonistas & inibidores , Esfingomielina Fosfodiesterase/metabolismo
17.
Clinics (Sao Paulo) ; 73(suppl 1): e792s, 2018 10 11.
Artigo em Inglês | MEDLINE | ID: mdl-30328954

RESUMO

Platelet activating factor is a lipid mediator of inflammation, and in recent decades, it has emerged as an important factor in tumor outcomes. Platelet activating factor acts by specific binding to its receptor, which is present in both tumor cells and cells that infiltrate tumors. Pro-tumorigenic effects of platelet activating factor receptor in tumors includes promotion of tumor cell proliferation, production of survival signals, migration of vascular cells and formation of new vessels and stimulation of dendritic cells and macrophages suppressor phenotype. In experimental models, blocking of platelet activating factor receptor reduced tumor growth and increased animal survival. During chemotherapy and radiotherapy, tumor cells that survive treatment undergo accelerated proliferation, a phenomenon known as tumor cell repopulation. Work from our group and others showed that these treatments induce overproduction of platelet activating factor-like molecules and increase expression of its receptor in tumor cells. In this scenario, antagonists of platelet activating factor markedly reduced tumor repopulation. Here, we note that combining chemo- and radiotherapy with platelet activating factor antagonists could be a promising strategy for cancer treatment.


Assuntos
Proliferação de Células , Neoplasias Experimentais/terapia , Glicoproteínas da Membrana de Plaquetas/antagonistas & inibidores , Receptores Acoplados a Proteínas-G/antagonistas & inibidores , Animais , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Proliferação de Células/efeitos da radiação , Terapia Combinada/métodos , Neoplasias Experimentais/patologia , Neovascularização Patológica/tratamento farmacológico , Neovascularização Patológica/terapia
18.
BMC Cancer ; 18(1): 999, 2018 Oct 19.
Artigo em Inglês | MEDLINE | ID: mdl-30340558

RESUMO

BACKGROUND: The tumour microenvironment conferred by mesenchymal stem cells (MSCs) plays a key role in tumour development and progression. We previously determined that platelet-activating factor receptor (PAFR) was overexpressed in ovarian cancer cells (OCCs) and that PAF can promote ovarian cancer progression via PAF/PAFR-mediated inflammatory signalling pathways. Evidence suggests that MSCs can secrete high concentrations of PAF. Here, we investigated the role of PAF/PAFR signalling in the microenvironment mediated by MSCs and OCCs and its effect on cancer progression. METHODS: The PAF concentrations in the culture media of MSCs, OCCs and co-cultured MSCs and OCCs were determined by ELISA. The effects of MSCs on OCCs in vitro were assessed on cells treated with conditioned medium (CM). The expression and phosphorylation of key proteins in the PAF/PAFR signalling pathway were evaluated. In vivo, MSCs/RFP and SKOV3 cells were co-administered at different proportions to nude mice by interscapular injection. Mice in the WEB2086 group were intraperitoneally injected with the PAFR antagonist WEB2086 at a dose of 1 mg/kg.d for the duration of the animal experiments. Tumour progression was observed, and the weight and survival time of mice were measured. The PAF concentration in peripheral and tumour site blood was determined by ELISA. RESULTS: High concentrations of PAF were detected in CM from MSCs and MSCs co-cultured with OCCs. Both types of medium promoted non-mucinous OCC proliferation and migration but had no effect on mucinous-type OCCs. These effects could be blocked by PAFR inhibitors. The expression and phosphorylation of key proteins in the PAF/PAFR pathway significantly increased upon treatment with PAF and MSC-CM. In vivo, the tumour volume was larger following co-injection of SKOV3 cells and MSCs/RFP than following injection of SKOV3 cells alone. The tumour-promoting effect of MSCs/RFP was blocked by the PAFR antagonist WEB2086. Serum PAF concentrations significantly increased in co-injected mice. CONCLUSION: Our results suggest that the tumour-promoting effect of MSCs on OCCs via their cross-talk in the tumour microenvironment was, at least in part, mediated by the PAF/PAFR pathway, suggesting a new target for the treatment of ovarian cancer.


Assuntos
Progressão da Doença , Células-Tronco Mesenquimais/metabolismo , Neoplasias Ovarianas/metabolismo , Fator de Ativação de Plaquetas/fisiologia , Glicoproteínas da Membrana de Plaquetas/fisiologia , Receptores Acoplados a Proteínas-G/fisiologia , Microambiente Tumoral/efeitos dos fármacos , Animais , Azepinas/farmacologia , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Proliferação de Células/fisiologia , Técnicas de Cocultura , Feminino , Humanos , Células-Tronco Mesenquimais/efeitos dos fármacos , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Nus , Neoplasias Ovarianas/patologia , Fator de Ativação de Plaquetas/antagonistas & inibidores , Inibidores da Agregação de Plaquetas/farmacologia , Glicoproteínas da Membrana de Plaquetas/antagonistas & inibidores , Receptores Acoplados a Proteínas-G/antagonistas & inibidores , Triazóis/farmacologia , Microambiente Tumoral/fisiologia
19.
J Lipid Res ; 59(11): 2063-2074, 2018 11.
Artigo em Inglês | MEDLINE | ID: mdl-30139761

RESUMO

Platelet-activating factor (PAF) is a potent inflammatory mediator that exerts its actions via the single PAF receptor (PAF-R). Cells that biosynthesize alkyl-PAF also make abundant amounts of the less potent PAF analogue acyl-PAF, which competes for PAF-R. Both PAF species are degraded by the plasma form of PAF acetylhydrolase (PAF-AH). We examined whether cogenerated acyl-PAF protects alkyl-PAF from systemic degradation by acting as a sacrificial substrate to enhance inflammatory stimulation or as an inhibitor to dampen PAF-R signaling. In ex vivo experiments both PAF species are prothrombotic in isolation, but acyl-PAF reduced the alkyl-PAF-induced stimulation of human platelets that express canonical PAF-R. In Swiss albino mice, alkyl-PAF causes sudden death, but this effect can also be suppressed by simultaneously administering boluses of acyl-PAF. When PAF-AH levels were incrementally elevated, the protective effect of acyl-PAF on alkyl-PAF-induced death was serially decreased. We conclude that, although acyl-PAF in isolation is mildly proinflammatory, in a pathophysiological setting abundant acyl-PAF suppresses the action of alkyl-PAF. These studies provide evidence for a previously unrecognized role for acyl-PAF as an inflammatory set-point modulator that regulates both PAF-R signaling and hydrolysis.


Assuntos
1-Alquil-2-acetilglicerofosfocolina Esterase/metabolismo , Fator de Ativação de Plaquetas/metabolismo , Glicoproteínas da Membrana de Plaquetas/metabolismo , Receptores Acoplados a Proteínas-G/metabolismo , 1-Alquil-2-acetilglicerofosfocolina Esterase/genética , Animais , Azepinas/farmacologia , Cromatografia Líquida , Feminino , Voluntários Saudáveis , Lisofosfatidilcolinas/metabolismo , Masculino , Espectrometria de Massas , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Fosfolipídeos/sangue , Fosfolipídeos/metabolismo , Agregação Plaquetária/efeitos dos fármacos , Agregação Plaquetária/genética , Glicoproteínas da Membrana de Plaquetas/antagonistas & inibidores , Glicoproteínas da Membrana de Plaquetas/genética , Receptores Acoplados a Proteínas-G/antagonistas & inibidores , Receptores Acoplados a Proteínas-G/genética , Triazóis/farmacologia
20.
Nat Chem ; 10(9): 938-945, 2018 09.
Artigo em Inglês | MEDLINE | ID: mdl-30061613

RESUMO

Studies of secondary metabolites (natural products) that cover their isolation, chemical synthesis and bioactivity investigation present myriad opportunities for discovery. For example, the isolation of novel secondary metabolites can inspire advances in chemical synthesis strategies to achieve their practical preparation for biological evaluation. In the process, chemical synthesis can also provide unambiguous structural characterization of the natural products. Although the isolation, chemical synthesis and bioactivity studies of natural products are mutually beneficial, they are often conducted independently. Here, we demonstrate the benefits of a collaborative study of the phomactins, diterpenoid fungal metabolites that serve as antagonists of the platelet activating factor receptor. Our isolation of novel phomactins has spurred the development of a bioinspired, unified approach that achieves the total syntheses of six congeners. We also demonstrate in vitro the beneficial effects of several phomactins in suppressing the rate of repopulation of tumour cells following gamma radiation therapy.


Assuntos
Produtos Biológicos/síntese química , Terpenos/química , Produtos Biológicos/isolamento & purificação , Produtos Biológicos/farmacologia , Linhagem Celular Tumoral , Sobrevivência Celular/efeitos dos fármacos , Sobrevivência Celular/efeitos da radiação , Fungos/química , Fungos/metabolismo , Raios gama , Humanos , Concentração Inibidora 50 , Glicoproteínas da Membrana de Plaquetas/antagonistas & inibidores , Glicoproteínas da Membrana de Plaquetas/metabolismo , Receptores Acoplados a Proteínas-G/antagonistas & inibidores , Receptores Acoplados a Proteínas-G/metabolismo , Estereoisomerismo , Relação Estrutura-Atividade , Terpenos/isolamento & purificação , Terpenos/farmacologia
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