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1.
Cell Mol Life Sci ; 77(5): 885-901, 2020 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-31278420

RESUMO

Purinergic P2 receptors are critical regulators of several functions within the vascular system, including platelet aggregation, vascular inflammation, and vascular tone. However, a role for ATP release and P2Y receptor signalling in angiogenesis remains poorly defined. Here, we demonstrate that blood vessel growth is controlled by P2Y2 receptors. Endothelial sprouting and vascular tube formation were significantly dependent on P2Y2 expression and inhibition of P2Y2 using a selective antagonist blocked microvascular network generation. Mechanistically, overexpression of P2Y2 in endothelial cells induced the expression of the proangiogenic molecules CXCR4, CD34, and angiopoietin-2, while expression of VEGFR-2 was decreased. Interestingly, elevated P2Y2 expression caused constitutive phosphorylation of ERK1/2 and VEGFR-2. However, stimulation of cells with the P2Y2 agonist UTP did not influence sprouting unless P2Y2 was constitutively expressed. Finally, inhibition of VEGFR-2 impaired spontaneous vascular network formation induced by P2Y2 overexpression. Our data suggest that P2Y2 receptors have an essential function in angiogenesis, and that P2Y2 receptors present a therapeutic target to regulate blood vessel growth.


Assuntos
Células Endoteliais/metabolismo , Endotélio Vascular/crescimento & desenvolvimento , Neovascularização Fisiológica/fisiologia , Receptores Purinérgicos P2Y2/metabolismo , Angiopoietina-2/biossíntese , Antígenos CD34/biossíntese , Células Cultivadas , Humanos , Proteína Quinase 1 Ativada por Mitógeno/biossíntese , Proteína Quinase 3 Ativada por Mitógeno/biossíntese , Fosforilação/fisiologia , Agregação Plaquetária/fisiologia , Antagonistas do Receptor Purinérgico P2Y/farmacologia , Interferência de RNA , RNA Interferente Pequeno/genética , Receptores CXCR4/biossíntese , Receptores Purinérgicos P2Y2/genética , Receptor 2 de Fatores de Crescimento do Endotélio Vascular/biossíntese
2.
Platelets ; 31(1): 3-14, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-30744477

RESUMO

Cigarette smoking is an important cardiovascular risk factor, causing morbidity and mortality. There are many original studies on the impact of smoking, but its influence on platelet ADP-P2Y12 receptor inhibitors lack consistency. Thus, we conducted a systematic review and meta-analysis of already existing data/studies to further explore this issue. PubMed, Web of science, EMBASE, Clinical Trials, and the Cochrane Library were searched from inception to March 2018. Studies investigating the residual platelet reactivity categorized by smoking status and patients treated with platelet ADP-P2Y12 receptor inhibitors qualified the inclusion criteria. The primary outcome was P2Y12 reaction unit (PRU) value measured by VerifyNow P2Y12 assay, compared with different smoking status in ADP-P2Y12 receptor inhibitors treatment groups. Secondary outcome was post-treatment with 5 µmol/L ADP-inhibition of platelet aggregation (ADP-IPA) measured by light transmittance aggregometry (LTA). Of the 4954 citations retrieved, 12 studies involving 16 296 patients with acute coronary syndrome and/or stent deployment using platelet ADP-P2Y12 receptor inhibitors were included for meta-analysis. Pooled analysis revealed that PRU values of current smokers were 25.70 lower than nonsmokers (95% CI -38.81 to -12.60, p = 0.0001), getting better effects of antiplatelet treatment. In the smoking extent subgroup analysis, patients smoking >10 cigarettes/day shown about 46.49 lower of PRU values than patients smoking <10 cigarettes/day (p < 0.00001). Racial subgroup analyses found that smokers had increased platelet inhibition in the Caucasian population. Further, pooled analysis of ADP-IPA values for 1658 patients from five studies showed a significantly lower residual platelet reactivity in current smokers compared to that in nonsmokers (MD = -4.19; 95% CI -6.55 to -1.83; p = 0.0005). This systematic review and meta-analysis suggested that smokers have increased platelet inhibition and lower aggregation in response to clopidogrel than nonsmokers. These residual platelet reactivity observations may help to explain differential clinical outcomes in smokers vs. nonsmokers in large scale clinical trials.


Assuntos
Plaquetas/efeitos dos fármacos , Plaquetas/metabolismo , Clopidogrel/farmacologia , Ativação Plaquetária/efeitos dos fármacos , Inibidores da Agregação de Plaquetas/farmacologia , Antagonistas do Receptor Purinérgico P2Y/farmacologia , Fumar/efeitos adversos , Adulto , Idoso , Biomarcadores , Humanos , Pessoa de Meia-Idade , Testes de Função Plaquetária , Viés de Publicação , Receptores Purinérgicos P2Y12/metabolismo
3.
J Neuroinflammation ; 16(1): 217, 2019 Nov 13.
Artigo em Inglês | MEDLINE | ID: mdl-31722730

RESUMO

BACKGROUND: Microglial activation contributes to the development of chronic migraine (CM). The P2Y12 receptor (P2Y12R), a metabolic purinoceptor that is expressed on microglia in the central nervous system (CNS), has been indicated to play a critical role in the pathogenesis of chronic pain. However, whether it contributes to the mechanism of CM remains unknown. Thus, the present study investigated the precise details of microglial P2Y12R involvement in CM. METHODS: Mice subjected to recurrent nitroglycerin (NTG) treatment were used as the CM model. Hyperalgesia were assessed by mechanical withdrawal threshold to electronic von Frey and thermal withdrawal latency to radiant heat. Western blot and immunohistochemical analyses were employed to detect the expression of P2Y12R, Iba-1, RhoA, and ROCK2 in the trigeminal nucleus caudalis (TNC). To confirm the role of P2Y12R and RhoA/ROCK in CM, we systemically administered P2Y12R antagonists (MRS2395 and clopidogrel) and a ROCK2 inhibitor (fasudil) and investigated their effects on microglial activation, c-fos, and calcitonin gene-related peptide (CGRP) expression in the TNC. To further confirm the effect of P2Y12R on microglial activation, we preincubated lipopolysaccharide (LPS)-treated BV-2 microglia with MRS2395 and clopidogrel. ELISA was used to evaluate the levels of inflammatory cytokines. RESULTS: The protein levels of P2Y12R, GTP-RhoA, ROCK2, CGRP, c-fos, and inducible nitric oxide synthase (iNOS) in the TNC were increased after recurrent NTG injection. A double labeling study showed that P2Y12R was restricted to microglia in the TNC. MRS2395 and clopidogrel attenuated the development of tactile allodynia and suppressed the expression of CGRP, c-fos, and GTP-RhoA/ROCK2 in the TNC. Furthermore, fasudil also prevented hyperalgesia and suppressed the expression of CGRP in the TNC. In addition, inhibiting P2Y12R and ROCK2 activities suppressed NTG-induced microglial morphological changes (process retraction) and iNOS production in the TNC. In vitro, a double labeling study showed that P2Y12R was colocalized with BV-2 cells, and the levels of iNOS, IL-1ß, and TNF-α in LPS-stimulated BV-2 microglia were reduced by P2Y12R inhibitors. CONCLUSIONS: These data demonstrate that microglial P2Y12R in the TNC plays a critical role in the pathogenesis of CM by regulating microglial activation in the TNC via RhoA/ROCK pathway.


Assuntos
Microglia/metabolismo , Transtornos de Enxaqueca/metabolismo , Receptores Purinérgicos P2Y12/metabolismo , Núcleos do Trigêmeo/metabolismo , Quinases Associadas a rho/metabolismo , Proteína rhoA de Ligação ao GTP/metabolismo , Adenina/análogos & derivados , Adenina/farmacologia , Animais , Clopidogrel/farmacologia , Modelos Animais de Doenças , Camundongos , Microglia/efeitos dos fármacos , Antagonistas do Receptor Purinérgico P2Y/farmacologia , Transdução de Sinais/efeitos dos fármacos , Transdução de Sinais/fisiologia , Núcleos do Trigêmeo/efeitos dos fármacos , Valeratos/farmacologia
4.
PLoS One ; 14(11): e0225771, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-31774869

RESUMO

BACKGROUND: Gender influences platelet biology. Women have a larger platelet count, but gender-based differences in platelet function remain debated. We performed a study addressing gender-based differences in platelet function using point-of-care platelet function tests (PFT). METHODS: The patient population consisted of 760 cardiac surgery patients where preoperative PFT (multiple-electrode aggregometry [MEA]) were available. Platelet count and function at the ADPtest and TRAPtest were compared in the overall population and separately in patients with or without residual effects of P2Y12 inhibitors. RESULTS: Women had a significantly (P = 0.001) higher platelet count but a non-significantly higher platelet reactivity to ADP. In clopidogrel-treated patients, the platelets ADP reactivity was significantly (P = 0.031) higher in women, and platelet count was the main determinant of platelet hyper-reactivity. Within patients under full clopidogrel effects, women with a platelet count ≥ 200,000 cells/µL had a significantly (P = 0.023) higher rate of high-on-treatment platelet reactivity (HTPR, 45.5%) with respect to males with a platelet count < 200,000 cells/µL (11.9%), with a relative risk of 6.2 (95% confidence interval 1.4-29). CONCLUSIONS: Our findings confirm that women have a larger platelet count than men, and that this is associated to a trend towards a higher platelet reactivity. HTPR is largely represented in women with a high platelet count. This generates the hypothesis that women requiring P2Y12 inhibitors could potentially benefit from larger doses of drug or should be treated with anti-platelet agents with a low rate of HTPR.


Assuntos
Difosfato de Adenosina/metabolismo , Plaquetas/fisiologia , Agregação Plaquetária/efeitos dos fármacos , Antagonistas do Receptor Purinérgico P2Y/farmacologia , Idoso , Plaquetas/efeitos dos fármacos , Procedimentos Cirúrgicos Cardíacos , Feminino , Humanos , Masculino , Pessoa de Meia-Idade , Contagem de Plaquetas , Testes de Função Plaquetária , Fatores Sexuais
5.
Mol Med Rep ; 20(5): 4713-4722, 2019 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-31545497

RESUMO

Ticagrelor and prasugrel are widely used in the treatment of acute coronary syndrome. The co­administration of ticagrelor or prasugrel with statins in the clinic has already drawn a great deal of attention. The aims of the present study were to evaluate the safety and effectiveness, and guide the rational clinical use of, co­administration of ticagrelor or prasugrel with statins by exploring potential drug interactions. The activity of cytochrome P450 family 3 subfamily A member 4 (CYP3A4) was detected, and its protein and mRNA expression levels were measured in a rat model and liver microsomes to evaluate the effect of the drug combinations on CYP3A4. High performance liquid chromatography, western blotting and reverse transcription­quantitative PCR were used to perform these investigations. The in vitro experiments suggested that ticagrelor inhibited CYP3A4 activity, with IC50 and inhibitor constant (Ki) values of 68.74 and 26.47 µM, respectively; prasugrel also inhibited CYP3A4, activity with IC50 and Ki values of 16.24 and 10.84 µM, respectively. When different dosages of the antagonists were combined with simvastatin or atorvastatin, the metabolic rate was reduced more effectively at higher dosages when compared with lower dosages. An in vivo pharmacokinetic study demonstrated that the co­administration of ticagrelor or prasugrel with simvastatin caused an increase in the principal pharmacokinetic parameters of the probe drug dapsone [area under the concentration/time curve (AUC)0­t, AUC0­∞ and t1/2] and a decrease in clearance compared with ticagrelor, prasugrel or simvastatin alone. Additional studies confirmed that the two investigated P2Y12 inhibitors were able to decrease the protein level of CYP3A4 by promoting protein degradation through the proteasomal pathway, and combination with statins such as simvastatin had a synergistic inhibitory effect on CYP3A4 activity. These results demonstrated that the co­administration of P2Y12 inhibitors with simvastatin could markedly inhibit the activity of CYP3A4, and these findings will further influence the assessment of the clinical effectiveness (reduced or enhanced efficacy) and safety (bleeding and rhabdomyolysis) in the clinic.


Assuntos
Citocromo P-450 CYP3A/genética , Inibidores de Hidroximetilglutaril-CoA Redutases/farmacologia , Antagonistas do Receptor Purinérgico P2Y/farmacologia , Animais , Biomarcadores , Citocromo P-450 CYP3A/metabolismo , Relação Dose-Resposta a Droga , Interações Medicamentosas , Ativação Enzimática/efeitos dos fármacos , Expressão Gênica , Humanos , Concentração Inibidora 50 , Masculino , Microssomos Hepáticos/efeitos dos fármacos , Microssomos Hepáticos/metabolismo , RNA Mensageiro/genética , Ratos
6.
Drug Metab Pers Ther ; 34(3)2019 09 27.
Artigo em Inglês | MEDLINE | ID: mdl-31560647

RESUMO

Background The aim of this study was to evaluate the association of the carriage of the rs2244613 polymorphism of the CES1 gene with clopidogrel resistance as well as to evaluate the effectiveness of antiplatelet therapy in the carriers of this marker who have had acute coronary syndrome (ACS). This study also analyzes the procedure of percutaneous coronary intervention and compares the rs2244613 carrier rate between patients with ACS and healthy participants. Methods The study involved 81 patients diagnosed with ACS and 136 conditionally healthy participants. The optical detection of platelet agglutination by VerifyNow was employed to measure residual platelet reactivity in patients with ACS. The rs2244613 polymorphism was determined using real-time polymerase chain reaction. Results According to the results, the AA genotype of the rs2244613 polymorphism of the CES1 gene was detected in 37 patients (45.6%), the CA genotype in 42 patients (51.8%) and the CC genotype in 2 patients (2.6%). The level of residual platelet reactivity in rs2244613 carriers was higher compared with patients who did not have this allelic variant: 183.23 PRU ± 37.24 vs. 154.3 PRU ± 60.36 (p = 0.01). The frequencies of the minor allele C were 28.4% and 28.3% in patients with ACS and healthy participants, respectively. The results of the linear statistical model PRU due to CES1 genotype were as follows: df = 1, F = 6.96, p = 0.01). The standardized beta was 0.285 (p = 0.01) and R2 was 0.081. However, we also added CYP2C19*2 and *17 into the linear regression model. The results of the model were as follows: df = 3, F = 5.1, p = 0.003) and R2 was 0.166. Conclusions We identified a statistically significant correlation between the carriage of the rs2244613 polymorphism of the CES1 gene and the level of residual platelet aggregation among patients with ACS and the procedure of percutaneous coronary intervention.


Assuntos
Síndrome Coronariana Aguda/tratamento farmacológico , Hidrolases de Éster Carboxílico/genética , Clopidogrel/farmacologia , Inibidores da Agregação de Plaquetas/farmacologia , Polimorfismo Genético/genética , Antagonistas do Receptor Purinérgico P2Y/farmacologia , Receptores Purinérgicos P2Y12/metabolismo , Síndrome Coronariana Aguda/diagnóstico , Síndrome Coronariana Aguda/genética , Adulto , Hidrolases de Éster Carboxílico/sangue , Feminino , Genótipo , Humanos , Masculino , Pessoa de Meia-Idade , Agregação Plaquetária/efeitos dos fármacos
7.
Thromb Res ; 181: 92-98, 2019 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-31376607

RESUMO

INTRODUCTION: P2Y12 receptor antagonists reduce risk of thrombotic complications after stent implantation but increase bleeding risk. Activation of P2Y12 receptors by ADP causes Gi-protein-mediated inhibition of adenylate cyclase (AC), thus limiting platelet response to anti-aggregatory effect of prostacyclin (PGI2). However, P2Y12 blockade reverses this ADP-induced suppression of the platelet PGI2/AC signaling pathway. We previously demonstrated that impairment of this pathway predicts poor response to clopidogrel. OBJECTIVES: To identify clinical correlates of variability in PGI2/AC signaling, and to assess the impact of such variability on individual responses to the direct P2Y12 receptor antagonists ticagrelor (in vivo) and 2-methyl-thioadenosine-monophosphate (2MeSAMP) (in vitro). PATIENTS/METHODS: We compared the inhibitory effects of prostaglandin E1 (PGE1) and the PGI2 analog Iloprost (Ilt) on platelet aggregation in whole blood samples from healthy control subjects (n = 17), and patients with stable angina pectoris (SAP; n = 35) or acute coronary syndromes (ACS; n = 23), with or without associated diabetes/hyperglycemia. RESULTS: Compared to control subjects, patients with ACS and - to a lesser extent - those with SAP, exhibited impaired responses to PGE1, accentuated in the presence of hyperglycemia. Efficacy of ticagrelor treatment, measured as change in platelet reactivity index, was directly related to pre-treatment PGE1 response, both at univariate and multivariate analysis. There was a strong correlation between extent of inhibition of platelet aggregation, whether by PGE1 or Ilt, and the anti-aggregatory effect of 2MeSAMP in vitro. CONCLUSIONS: The integrity of PGI2/AC signaling, which is impaired in the presence of ACS and hyperglycemia, predetermines the anti-aggregatory efficiency of P2Y12 receptor antagonists.


Assuntos
Síndrome Coronariana Aguda/sangue , Síndrome Coronariana Aguda/tratamento farmacológico , Adenilil Ciclases/metabolismo , Agregação Plaquetária/efeitos dos fármacos , Antagonistas do Receptor Purinérgico P2Y/uso terapêutico , Síndrome Coronariana Aguda/patologia , Idoso , Feminino , Humanos , Masculino , Pessoa de Meia-Idade , Antagonistas do Receptor Purinérgico P2Y/farmacologia , Transdução de Sinais
8.
Thromb Haemost ; 19(10): 1606-1616, 2019 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-31408901

RESUMO

There are clinical scenarios where the balance between the risk of ischemic and bleeding events leads to the clinical decision to reverse the antiplatelet effect of P2Y12 receptor inhibitors. These scenarios comprise emergency situations such as active severe bleeding, urgent procedures with presumed high bleeding risk, or major trauma with (anticipated) bleeding. Supplementation of platelets has been investigated in ex vivo as well as in in vivo studies. These studies indicate that the inhibition of adenosine diphosphate-induced aggregation by the irreversibly binding thienopyridine derivatives clopidogrel and prasugrel can be reversed by administration of platelet concentrates. Supplementation of platelets in patients on prasugrel is more effective if this can be transfused > 6 hours after last dosing. Studies on the reversal effect obtained by administration of platelet concentrates in patients on ticagrelor show conflicting results. Experimental data suggest that administration of serum albumin might increase the reversal effect. A monoclonal antibody fragment (PB2452) for neutralizing ticagrelor is currently in clinical development. A recently published first in man study shows that reversal of platelet inhibition occurs within 5 minutes after start of administration and the effect is maintained for 20 to 24 hours after a 16-hour infusion which is by far the most effective approach for reversal of ticagrelor.


Assuntos
Plaquetas/citologia , Cardiologia/tendências , Clopidogrel/farmacologia , Cloridrato de Prasugrel/farmacologia , Antagonistas do Receptor Purinérgico P2Y/farmacologia , Ticagrelor/antagonistas & inibidores , Administração Oral , Anticorpos Monoclonais/farmacologia , Anticorpos Amplamente Neutralizantes/farmacologia , Cardiologia/métodos , Hemadsorção , Hemorragia , Hemostasia , Humanos , Agregação Plaquetária/efeitos dos fármacos , Inibidores da Agregação de Plaquetas/farmacologia , Testes de Função Plaquetária , Ensaios Clínicos Controlados Aleatórios como Assunto , Risco , Transdução de Sinais , Trombose/tratamento farmacológico , Ticagrelor/farmacologia
9.
PLoS One ; 14(8): e0222010, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-31465489

RESUMO

For endovascular research pigs are an established animal model. However, experiences regarding analyses of platelet inhibition in pigs using the Multiplate® Analyzer are limited. The aims of the present study were to investigate if (1) the Multiplate® Analyzer is a suitable method for examination of porcine platelet function using manufacturers' recommendations for human blood, and (2) platelet inhibition can be induced with acetylsalicylic acid (ASA) and clopidogrel in pigs reliably, and if (3) non-responders to one of the drug can be detected. Additionally we examined differences in (4) the effectiveness of ASA between oral administration and intravenous application, and (5) between domestic pigs (German Landrace; GL) and miniature pigs (MP). We investigated platelet function of 36 unmedicated pigs (GL n = 28; MP n = 8). In addition, 32 blood samples taken from medicated pigs (GL n = 15; MP n = 17) were analysed. Platelet inhibition was induced in four different ways: (1) 500 mg ASA intravenously (n = 11), (2) 500 mg ASA intravenously and 450 mg clopidogrel orally (n = 5), (3) 250 mg ASA orally (n = 11), (4) 250 mg ASA orally and 75 mg clopidogrel orally (n = 5). Results of the ASPI and ADP test of the Multiplate® Analyzer subtests in unmedicated and medicated pigs were in a comparable range to results known from humans. Application of ASA decreased the mean values of the ASPI test significantly regardless of the application method. Joined administration of ASA and clopidogrel also decreased the mean values of the ADP test significantly. Both, oral and intravenous administrations of ASA as well as oral administration of clopidogrel effectively inhibited platelet function in pigs. One pig did not respond to clopidogrel. We found no differences between domestic and miniature pigs regarding reference values in unmedicated pigs and the effectiveness of ASA and clopidogrel.


Assuntos
Testes de Função Plaquetária/métodos , Animais , Aspirina/farmacologia , Plaquetas/efeitos dos fármacos , Plaquetas/imunologia , Plaquetas/metabolismo , Agregação Plaquetária/efeitos dos fármacos , Inibidores da Agregação de Plaquetas/farmacologia , Antagonistas do Receptor Purinérgico P2Y/farmacologia , Suínos
10.
Pharmacol Res Perspect ; 7(4): e00509, 2019 08.
Artigo em Inglês | MEDLINE | ID: mdl-31372229

RESUMO

The novel clopidogrel conjugate, DT-678, is an effective inhibitor of platelets and thrombosis in preclinical studies. However, a comparison of the bleeding risk with DT-678 and currently approved P2Y12 antagonists has yet to be determined. The objective of this study was to evaluate the bleeding tendency of animals treated with clopidogrel, ticagrelor, and DT-678. Ninety-one New Zealand white rabbits were randomized to one of 13 treatment groups (n = 7). Platelet activation was assessed by flow cytometry and light transmission aggregometry before and after the administration of various doses of DT-678, clopidogrel, and ticagrelor. Tongue template bleeding times were also measured before and after drug treatment. Treatment with P2Y12 receptor antagonists caused a dose-dependent reduction in markers of platelet activation (P-selectin and integrin αIIbß3) and aggregation in response to adenosine diphosphate stimulation. At the same doses required for platelet inhibition, clopidogrel and ticagrelor significantly prolonged bleeding times, while DT-678 did not. DT-678 and the FDA-approved P2Y12 antagonists clopidogrel and ticagrelor are effective inhibitors of platelet activation and aggregation. However, unlike clopidogrel and ticagrelor, DT-678 did not prolong bleeding times at equally effective antiplatelet doses. The results suggest a more favorable benefit/risk ratio for DT-678 and potential utility as part of a dual antiplatelet therapy regimen.


Assuntos
Dissulfetos/administração & dosagem , Ativação Plaquetária/efeitos dos fármacos , Antagonistas do Receptor Purinérgico P2Y/administração & dosagem , Animais , Tempo de Sangramento , Clopidogrel/administração & dosagem , Clopidogrel/química , Clopidogrel/farmacologia , Dissulfetos/química , Dissulfetos/farmacologia , Relação Dose-Resposta a Droga , Antagonistas do Receptor Purinérgico P2Y/farmacologia , Coelhos , Distribuição Aleatória , Ticagrelor/administração & dosagem , Ticagrelor/farmacologia
11.
IUBMB Life ; 71(10): 1552-1560, 2019 10.
Artigo em Inglês | MEDLINE | ID: mdl-31301116

RESUMO

Rheumatoid arthritis is a common chronic inflammatory joint disease. Fibroblast-like synoviocytes-mediated inflammation is closely associated with the development of rheumatoid arthritis. In this study, we report that P2Y11 receptor activity is required for cytokine-induced inflammation in primary fibroblast-like synoviocytes (FLS). P2Y11R is fairly expressed in primary FLS isolated from healthy subjects and is elevated to around three- to four-fold in rheumatoid arthritis-derived FLS. The expression of P2Y11R is inducible upon IL-1ß treatment. Blockage of P2Y11R by its antagonist suppresses IL-1ß-induced TNF-α and IL-6 induction and ameliorates oxidative stress as determined by levels of cellular ROS and the oxidative byproduct 4-HNE. Moreover, blockage of P2Y11R by NF340 inhibits IL-1ß-induced matrix metalloproteinase protein expression as indicated by the levels of MMP-1, MMP-3, and MMP-13. Mechanistically, blockage of P2Y11R mitigates IL-1ß-activated NFκB signaling, which was revealed by reduced IκBα phosphorylation, nuclear p65 accumulation, and NFκB promoter activity. Our study provides evidence of a protective mechanism of P2Y11R antagonist NF340 against cytokine-induced inflammation. Therefore, targeting P2Y11R could have potential therapeutic implication in the treatment of RA.


Assuntos
Artrite Reumatoide/tratamento farmacológico , Inflamação/tratamento farmacológico , Antagonistas do Receptor Purinérgico P2Y/farmacologia , Receptores Purinérgicos P2/genética , Animais , Artrite Reumatoide/genética , Artrite Reumatoide/patologia , Fibroblastos/efeitos dos fármacos , Regulação da Expressão Gênica/efeitos dos fármacos , Humanos , Inflamação/genética , Inflamação/patologia , Mediadores da Inflamação/farmacologia , Interleucina-1beta/genética , Metaloproteinase 1 da Matriz/genética , Metaloproteinase 13 da Matriz/genética , Metaloproteinase 3 da Matriz/genética , Inibidor de NF-kappaB alfa/genética , NF-kappa B/genética , Estresse Oxidativo/efeitos dos fármacos , Fosforilação/efeitos dos fármacos , Regiões Promotoras Genéticas/efeitos dos fármacos , Receptores Purinérgicos P2/efeitos dos fármacos , Sinoviócitos/efeitos dos fármacos
12.
Purinergic Signal ; 15(3): 287-298, 2019 09.
Artigo em Inglês | MEDLINE | ID: mdl-31270713

RESUMO

Extracellular nucleotides mediate multiple physiological effects such as proliferation, differentiation, or induction of apoptosis through G protein-coupled P2Y receptors or P2X ion channels. Evaluation of the complete physiological role of nucleotides has long been hampered by a lack of potent and selective ligands for all P2 subtypes. Meanwhile, for most of the P2 receptors, selective ligands are available, but only a few potent and selective P2Y2 receptor antagonists are described. This limits the understanding of the role of P2Y2 receptors. The purpose of this study was to search for P2Y2 receptor antagonists by a combinatorial screening of a library of around 415 suramin-derived compounds. Calcium fluorescence measurements at P2Y2 receptors recombinantly expressed in human 1321N1 astrocytoma cells identified NF272 [8-(4-methyl-3-(3-phenoxycarbonylimino-benzamido)benzamido)-naphthalene-1,3,5-trisulfonic acid trisodium salt] as a competitive P2Y2 receptor antagonist with a Ki of 19 µM which is 14-fold more potent than suramin at this receptor subtype. The SCHILD analysis of competitive inhibition resulted in a pA2 value of 5.03 ± 0.22 (mean ± SEM) with a slope not significantly different from unity. Among uracil-nucleotide-preferring P2Y receptors, NF272 shows a moderate selectivity over P2Y4 (3.6-fold) and P2Y6 (5.7-fold). However, NF272 is equipotent at P2Y1, and even more potent at P2Y11 and P2Y12 receptors. Up to 250 µM, NF272 showed no cytotoxicity in MTT cell viability assays in 1321N1, HEK293, and OVCAR-3 cells. Further, NF272 was able to inhibit the ATP-induced calcium signal in OVCAR-3 cells demonstrated to express P2Y2 receptors. In conclusion, NF272 is a competitive but non-selective P2Y2 receptor antagonist with 14-fold higher potency than suramin lacking cytotoxic effects. Therefore, NF272 may serve as a lead structure for further development of P2Y2 receptor antagonists.


Assuntos
Descoberta de Drogas , Naftalenos/farmacologia , Antagonistas do Receptor Purinérgico P2Y/farmacologia , Receptores Purinérgicos P2Y2/efeitos dos fármacos , Animais , Humanos , Naftalenos/química , Antagonistas do Receptor Purinérgico P2Y/química , Suramina/análogos & derivados
13.
PLoS One ; 14(6): e0218934, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-31242230

RESUMO

Ticagrelor (TIC), a P2Y purinoceptor 12 (P2Y12)-receptor antagonist, has been widely used to treat patients with acute coronary syndrome. Although animal studies suggest that TIC protects against atherosclerosis, it remains unknown whether it does so through its potent platelet inhibition or through other pathways. Here, we placed hypercholesterolemic Ldlr-/-Apobec1-/- mice on a high-fat diet and treated them with either 25 mg/kg/day of clopidogrel (CLO) or 180 mg/kg/day of TIC for 16 weeks and evaluated the extent of atherosclerosis. Both treatments equally inhibited platelets as determined by ex vivo platelet aggregation assays. The extent of atherosclerosis, however, was significantly less in the TIC group than in the CLO group. Immunohistochemical staining and ELISA showed that TIC treatment was associated with less macrophage infiltration to the atherosclerotic intima and lower serum levels of CCL4, CXCL10, and TNFα, respectively, than CLO treatment. Treatment with TIC, but not CLO, was associated with higher serum activity and tissue level of paraoxonase-1 (PON1), an anti-atherosclerotic molecule, suggesting that TIC might exert greater anti-atherosclerotic activity, compared with CLO, through its unique ability to induce PON1. Although further studies are needed, TIC may prove to be a viable strategy in the prevention and treatment of chronic stable human atherosclerosis.


Assuntos
Arildialquilfosfatase/metabolismo , Aterosclerose/tratamento farmacológico , Clopidogrel/farmacologia , Hipercolesterolemia/tratamento farmacológico , Ticagrelor/farmacologia , Síndrome Coronariana Aguda/tratamento farmacológico , Síndrome Coronariana Aguda/metabolismo , Animais , Aterosclerose/metabolismo , Plaquetas/efeitos dos fármacos , Plaquetas/metabolismo , Estudos Transversais , Hipercolesterolemia/metabolismo , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Agregação Plaquetária/efeitos dos fármacos , Inibidores da Agregação de Plaquetas/farmacologia , Testes de Função Plaquetária/métodos , Antagonistas do Receptor Purinérgico P2Y/farmacologia , Receptores de LDL/metabolismo
14.
Fundam Clin Pharmacol ; 33(6): 604-611, 2019 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-31206802

RESUMO

In a previous publication when we studied the purinergic receptor with which ATP interacted in mouse brain bEND.3 endothelial cells, we observed addition of 3 µm ARC 118925XX (ARC; selective P2Y2 antagonist) strongly suppressed ATP-triggered Ca2+ release, suggesting the response was mediated via P2Y2 receptors. We here report ARC unexpectedly promoted substantial Ca2+ influx even when ATP-triggered Ca2+ release was largely inhibited. Since this large Ca2+ influx may have important pharmacological significance, we proceeded to investigate its mechanism. ARC did not trigger intracellular Ca2+ release thus suggesting Ca2+ influx triggered by ARC was not store-operated. ARC-triggered Ca2+ influx could be blocked by 1 mm Ni2+ , a general Ca2+ channel blocker, but not by SK&F 96365, a nonselective TRP channel blocker. Unexpectedly, ARC promoted influx of Na+ and La3+ , but not Mn2+ . This is a surprising finding, since Mn2+ is conventionally used as a Ca2+ surrogate ion (as it permeates Ca2+ channel), and La3+ is classically used as a potent Ca2+ channel antagonist. Electrophysiological examination showed ARC did not stimulate any cation currents. Therefore, ARC opened, rather than a cation channel pore, an unidentified Ca2+ influx pathway which was Na+ - and La3+ -permeable but Mn2+ -impermeable.


Assuntos
Células Endoteliais/metabolismo , Antagonistas do Receptor Purinérgico P2Y/farmacologia , Animais , Cálcio/metabolismo , Células Cultivadas , Manganês/metabolismo , Camundongos , Sódio/metabolismo
15.
Eur J Pharmacol ; 855: 160-166, 2019 Jul 15.
Artigo em Inglês | MEDLINE | ID: mdl-31063775

RESUMO

P2Y1 receptors play an essential role in inhibitory neuromuscular transmission in the gastrointestinal tract. The signalling pathway involves the opening of small conductance calcium activated potassium-channels (Kca2 family) that results in smooth muscle hyperpolarization and relaxation. Inorganic polyphosphates and dinucleotidic polyphosphates are putative neurotransmitters that potentially act on P2Y1 receptors. A pharmacological approach using both orthosteric (MRS2500) and allosteric (BPTU) blockers of the P2Y1 receptor and openers (CyPPA) and blockers (apamin) of Kca2 channels was used to pharmacologically characterise the effect of these neurotransmitters. Organ bath and microelectrodes were used to evaluate the effect of P1,P4-Di (adenosine-5') tetraphosphate ammonium salt (Ap4A), inorganic polyphosphates (PolyP) and CyPPA on spontaneous contractions and membrane potential of mouse colonic smooth muscle cells. PolyP neither modified contractions nor membrane potential. In contrast, Ap4A caused a concentration-dependent inhibition of spontaneous contractions reaching a maximum effect at 100 µM Ap4A response was antagonised by MRS2500 (1 µM), BPTU (3 µM) and apamin (1 µM). CyPPA (10 µM) inhibited spontaneous contractions and this response was antagonised by apamin but it was not affected by MRS2500 or BPTU. Both CyPPA and Ap4A caused smooth muscle hyperpolarization that was blocked by apamin and MRS2500 respectively. We conclude that Ap4A but not PolyP activates P2Y1 receptors causing smooth muscle hyperpolarization and relaxation. Ap4A signalling causes activation of Kca2 channels through activation of P2Y1 receptors. In contrast, CyPPA acts directly on Kca2 channels. Further studies are needed to evaluate if dinucleotidic polyphosphates are released from inhibitory motor neurons.


Assuntos
Colo/efeitos dos fármacos , Fosfatos de Dinucleosídeos/farmacologia , Relaxamento Muscular/efeitos dos fármacos , Músculo Liso/efeitos dos fármacos , Músculo Liso/fisiologia , Receptores Purinérgicos P2Y1/metabolismo , Regulação Alostérica/efeitos dos fármacos , Animais , Colo/fisiologia , Nucleotídeos de Desoxiadenina/farmacologia , Feminino , Ativação do Canal Iônico/efeitos dos fármacos , Potenciais da Membrana/efeitos dos fármacos , Camundongos , Músculo Liso/citologia , Músculo Liso/metabolismo , Bloqueadores dos Canais de Potássio/farmacologia , Antagonistas do Receptor Purinérgico P2Y/farmacologia , Transdução de Sinais/efeitos dos fármacos , Canais de Potássio Ativados por Cálcio de Condutância Baixa/metabolismo
16.
Thromb Haemost ; 119(7): 1037-1047, 2019 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-31079416

RESUMO

Oral P2Y12 receptor inhibitors represent a mainstay treatment in patients with acute coronary syndrome and those undergoing percutaneous coronary intervention. In the setting of ST-elevation myocardial infarction, when early platelet inhibition is highly desirable, the onset of action of oral P2Y12 receptor inhibitors is, however, delayed, likely due to delayed drug absorption. Crushing the tablets, which are to be used for patient loading with an oral P2Y12 receptor inhibitor, has been shown to provide earlier platelet inhibition than standard, integral tablets administration. Chewed ticagrelor tablets may also result in a similar effect. Such findings should be interpreted with caution, mainly due to the small number of patients enrolled and the nature (pharmacodynamic/pharmacokinetic) of the respective studies. Furthermore, in patients with out-of-hospital cardiac arrest, who remain comatose, crushing tablets is commonly applied in clinical practice for platelet P2Y12 receptor inhibition. In this review, we focus on current evidence regarding the role of crushed P2Y12 receptor inhibitor pills, analyzing clinical scenarios where most of the promise exists along with future expectations from this type of formulation. Large randomized studies are needed to draw firm conclusions regarding the clinical benefit of 'crushing' over the usual 'not-crushing' practice.


Assuntos
Síndrome Coronariana Aguda/tratamento farmacológico , Plaquetas/fisiologia , Composição de Medicamentos/métodos , Intervenção Coronária Percutânea , Antagonistas do Receptor Purinérgico P2Y/uso terapêutico , Ticagrelor/uso terapêutico , Síndrome Coronariana Aguda/cirurgia , Plaquetas/efeitos dos fármacos , Humanos , Antagonistas do Receptor Purinérgico P2Y/farmacologia , Comprimidos , Ticagrelor/farmacologia
17.
Am J Physiol Endocrinol Metab ; 317(1): E25-E41, 2019 07 01.
Artigo em Inglês | MEDLINE | ID: mdl-30912960

RESUMO

Fructose is widely used as a sweetener in processed food and is also associated with metabolic disorders, such as obesity. However, the underlying cellular mechanisms remain unclear, in particular, regarding the pancreatic ß-cell. Here, we investigated the effects of chronic exposure to fructose on the function of insulinoma cells and isolated mouse and human pancreatic islets. Although fructose per se did not acutely stimulate insulin exocytosis, our data show that chronic fructose rendered rodent and human ß-cells hyper-responsive to intermediate physiological glucose concentrations. Fructose exposure reduced intracellular ATP levels without affecting mitochondrial function, induced AMP-activated protein kinase activation, and favored ATP release from the ß-cells upon acute glucose stimulation. The resulting increase in extracellular ATP, mediated by pannexin1 (Panx1) channels, activated the calcium-mobilizer P2Y purinergic receptors. Immunodetection revealed the presence of both Panx1 channels and P2Y1 receptors in ß-cells. Addition of an ectonucleotidase inhibitor or P2Y1 agonists to naïve ß-cells potentiated insulin secretion stimulated by intermediate glucose, mimicking the fructose treatment. Conversely, the P2Y1 antagonist and Panx1 inhibitor reversed the effects of fructose, as confirmed using Panx1-null islets and by the clearance of extracellular ATP by apyrase. These results reveal an important function of ATP signaling in pancreatic ß-cells mediating fructose-induced hyper-responsiveness.


Assuntos
Trifosfato de Adenosina/fisiologia , Frutose/farmacologia , Glucose/farmacologia , Secreção de Insulina/efeitos dos fármacos , Células Secretoras de Insulina/efeitos dos fármacos , Células Secretoras de Insulina/metabolismo , Transdução de Sinais/efeitos dos fármacos , Proteínas Quinases Ativadas por AMP/genética , Proteínas Quinases Ativadas por AMP/metabolismo , Trifosfato de Adenosina/metabolismo , Animais , Apirase/metabolismo , Conexinas/genética , Conexinas/metabolismo , Humanos , Ilhotas Pancreáticas/efeitos dos fármacos , Ilhotas Pancreáticas/metabolismo , Camundongos , Camundongos Endogâmicos C57BL , Proteínas do Tecido Nervoso/genética , Proteínas do Tecido Nervoso/metabolismo , Agonistas do Receptor Purinérgico P2Y/farmacologia , Antagonistas do Receptor Purinérgico P2Y/farmacologia , Receptores Purinérgicos P2Y/efeitos dos fármacos , Receptores Purinérgicos P2Y/metabolismo , Receptores Purinérgicos P2Y1/efeitos dos fármacos , Receptores Purinérgicos P2Y1/metabolismo
18.
J Med Chem ; 62(6): 3088-3106, 2019 03 28.
Artigo em Inglês | MEDLINE | ID: mdl-30843696

RESUMO

P2Y12 antagonists are widely used as antiplatelet agents for the prevention and treatment of arterial thrombosis. Based on the scaffold of a known P2Y12 antagonist AZD1283, a series of novel bicyclic pyridine derivatives were designed and synthesized. The cyclization of the ester substituent on the pyridine ring to the ortho-methyl group led to lactone analogues of AZD1283 that showed significantly enhanced metabolic stability in subsequent structure-pharmacokinetic relationship studies. The metabolic stability was further enhanced by adding a 4-methyl substituent to the piperidinyl moiety. Compound 58l displayed potent inhibition of platelet aggregation in vitro as well as antithrombotic efficacy in a rat ferric chloride model. Moreover, 58l showed a safety profile that was superior to what was observed for clopidogrel in a rat tail-bleeding model. These results support the further evaluation of compound 58l as a promising drug candidate.


Assuntos
Descoberta de Drogas , Niacina/análogos & derivados , Inibidores da Agregação de Plaquetas/farmacologia , Antagonistas do Receptor Purinérgico P2Y/farmacologia , Receptores Purinérgicos P2Y12/efeitos dos fármacos , Sulfonamidas/farmacologia , Animais , Humanos , Masculino , Niacina/química , Niacina/farmacocinética , Niacina/farmacologia , Inibidores da Agregação de Plaquetas/química , Inibidores da Agregação de Plaquetas/farmacocinética , Antagonistas do Receptor Purinérgico P2Y/química , Antagonistas do Receptor Purinérgico P2Y/farmacocinética , Ratos , Ratos Sprague-Dawley , Ratos Wistar , Relação Estrutura-Atividade , Sulfonamidas/química , Sulfonamidas/farmacocinética
19.
J Cardiovasc Pharmacol Ther ; 24(4): 371-376, 2019 07.
Artigo em Inglês | MEDLINE | ID: mdl-30744423

RESUMO

P2Y12 receptor-blocking drugs given at reperfusion offer protection against myocardial infarction in animal models of transient coronary occlusion. Two recent reports concluded that ticagrelor was more cardioprotective than clopidogrel and attributed this to ticagrelor's unique ability to raise tissue adenosine by blocking the equilibrative nucleoside transporter 1. Indeed, an adenosine receptor blocker attenuated ticagrelor's protection. The related P2Y12 inhibitor cangrelor, which does not block the transporter, protects hearts only when platelets are in the perfusate, while adenosine is known to protect equally in situ blood-perfused and crystalloid-perfused isolated hearts. We, therefore, tested whether ticagrelor liberates a sufficient amount of adenosine to protect a Krebs buffer-perfused isolated rat heart subjected to 40 minutes of global ischemia followed by 2 hours of reperfusion. In untreated hearts, 77.6% ± 4.0% of the ventricle was infarcted as measured by triphenyltetrazolium staining. Ischemically preconditioned hearts had only 32.7% ± 3.6% infarction ( P < .001 vs untreated), indicating that our model could be protected by preconditioning which is known to involve adenosine. Strikingly, hearts treated with 10 µmol/L ticagrelor in the buffer throughout the reperfusion period had 77.5% ± 2.4% infarction comparable to unprotected controls ( P = NS vs untreated). These data strongly suggest that ticagrelor was unable to release sufficient adenosine from the crystalloid-perfused rat heart to protect it against infarction. Our previous studies have found no difference in the anti-infarct potency among clopidogrel, cangrelor, and ticagrelor in open-chest rats and rabbits, and surprisingly adenosine receptor antagonists block protection from all 3 drugs. We have no explanation why ticagrelor is more protective in the pig than clopidogrel but suspect a species or perhaps a treatment schedule difference that may or may not involve adenosine.


Assuntos
Adenosina/metabolismo , Transportador Equilibrativo 1 de Nucleosídeo/metabolismo , Infarto do Miocárdio/prevenção & controle , Traumatismo por Reperfusão Miocárdica/prevenção & controle , Antagonistas do Receptor Purinérgico P2Y/farmacologia , Ticagrelor/farmacologia , Animais , Modelos Animais de Doenças , Precondicionamento Isquêmico Miocárdico , Preparação de Coração Isolado , Masculino , Infarto do Miocárdio/metabolismo , Infarto do Miocárdio/patologia , Traumatismo por Reperfusão Miocárdica/metabolismo , Traumatismo por Reperfusão Miocárdica/patologia , Ratos Sprague-Dawley
20.
Int J Med Sci ; 16(2): 264-275, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-30745807

RESUMO

Background: Platelet-derived microvesicles (PMVs), shed from platelet surface membranes, constitute the majority of circulating microvesicles and have been implicated in procoagulant, pro-inflammatory and pro-atherosclerotic effects. Our aim was to compare plasma PMVs numbers in relation to platelet reactivity during dual antiplatelet therapy (DAPT) with various P2Y12 adenosine diphosphate (ADP) receptor antagonists. Methods: In pre-discharge men treated with DAPT for an acute coronary syndrome, plasma PMVs were quantified by flow cytometry on the basis of CD62P (P-selectin) and CD42 (glycoprotein Ib) positivity, putative indices of PMVs release from activated and all platelets, respectively. ADP-induced platelet aggregation was measured by multiple-electrode aggregometry. Results: Clinical characteristics were similar in patients on clopidogrel (n=16), prasugrel (n=10) and ticagrelor (n=12). Platelet reactivity was comparably reduced on ticagrelor or prasugrel versus clopidogrel (p<0.01). Compared to clopidogrel-treated patients, CD42+/CD62P+ PMVs counts were 3-4-fold lower in subjects receiving ticagrelor (p=0.001) or prasugrel (p<0.05), while CD42+ PMVs were significantly reduced on ticagrelor (by about 6-fold, p<0.001), but not prasugrel (p=0.3). CD42+/CD62P+ PMVs numbers correlated positively to the ADP-induced aggregation on clopidogrel (p<0.01) or prasugrel (p<0.05), which was absent in ticagrelor users (p=0.8). CD42+ PMVs counts were unrelated to platelet reactivity (p>0.5). Conclusions: Higher antiplatelet potency of prasugrel and ticagrelor versus clopidogrel is associated with decreased plasma CD42+/CD62P+ PMVs numbers. However, in contrast to thienopyridines, the association of reduced CD42+/CD62P+ PMVs counts with ticagrelor use appears independent of its anti-aggregatory effect. Despite similar platelet-inhibitory activity of ticagrelor and prasugrel, only the treatment with ticagrelor seems associated with lower total PMVs release. Our preliminary findings may suggest a novel pleiotropic effect of ticagrelor extending beyond pure anti-aggregatory properties of the drug.


Assuntos
Síndrome Coronariana Aguda/tratamento farmacológico , Plaquetas/efeitos dos fármacos , Micropartículas Derivadas de Células/efeitos dos fármacos , Antagonistas do Receptor Purinérgico P2Y/farmacologia , Piridinas/farmacologia , Idoso , Aspirina/uso terapêutico , Plaquetas/metabolismo , Micropartículas Derivadas de Células/metabolismo , Humanos , Masculino , Pessoa de Meia-Idade , Antagonistas do Receptor Purinérgico P2Y/uso terapêutico , Piridinas/uso terapêutico , Ticagrelor/farmacologia , Ticagrelor/uso terapêutico
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