Your browser doesn't support javascript.
loading
Mostrar: 20 | 50 | 100
Resultados 1 - 20 de 5.690
Filtrar
1.
Biochemistry (Mosc) ; 86(6): 773-783, 2021 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-34225599

RESUMO

The review summarizes the results of our own studies and published data on the biological markers of psychiatric disorders, with special emphasis on the activity of platelet monoamine oxidase. Pharmacotherapy studies in patients with the mixed anxiety-depressive disorder and first episode of schizophrenia have shown that the activity of platelet monoamine oxidase could serve as a potential biomarker of the efficacy of therapeutic interventions in these diseases.


Assuntos
Plaquetas/enzimologia , Transtornos Mentais/sangue , Monoaminoxidase/sangue , Transtorno Depressivo/sangue , Transtorno Depressivo/tratamento farmacológico , Humanos , Transtornos Mentais/tratamento farmacológico , Esquizofrenia/sangue , Esquizofrenia/tratamento farmacológico , Resultado do Tratamento
2.
Molecules ; 26(10)2021 May 19.
Artigo em Inglês | MEDLINE | ID: mdl-34069658

RESUMO

Atherosclerotic cardiovascular disease is the leading cause of death in developed countries. Therefore, there is an increasing interest in developing new potent and safe antiplatelet agents. Coumarins are a family of polyphenolic compounds with several pharmacological activities, including platelet aggregation inhibition. However, their antiplatelet mechanism of action needs to be further elucidated. The aim of this study is to provide insight into the biochemical mechanisms involved in this activity, as well as to establish a structure-activity relationship for these compounds. With this purpose, the antiplatelet aggregation activities of coumarin, esculetin and esculin were determined in vitro in human whole blood and platelet-rich plasma, to set the potential interference with the arachidonic acid cascade. Here, the platelet COX activity was evaluated from 0.75 mM to 6.5 mM concentration by measuring the levels of metabolites derived from its activity (MDA and TXB2), together with colorimetric assays performed with the pure recombinant enzyme. Our results evidenced that the coumarin aglycones present the greatest antiplatelet activity at 5 mM and 6.5 mM on aggregometry experiments and inhibiting MDA levels.


Assuntos
Plaquetas/efeitos dos fármacos , Cumarínicos/farmacologia , Ciclo-Oxigenase 1/metabolismo , Inibidores da Agregação Plaquetária/farmacologia , Plaquetas/enzimologia , Plaquetas/metabolismo , Humanos , Técnicas In Vitro , Malondialdeído/metabolismo
3.
Int J Mol Sci ; 22(11)2021 May 30.
Artigo em Inglês | MEDLINE | ID: mdl-34070931

RESUMO

It is now about 20 years since the first case of a gain-of-function mutation involving the as-yet-unknown actor in cholesterol homeostasis, proprotein convertase subtilisin/kexin type 9 (PCSK9), was described. It was soon clear that this protein would have been of huge scientific and clinical value as a therapeutic strategy for dyslipidemia and atherosclerosis-associated cardiovascular disease (CVD) management. Indeed, PCSK9 is a serine protease belonging to the proprotein convertase family, mainly produced by the liver, and essential for metabolism of LDL particles by inhibiting LDL receptor (LDLR) recirculation to the cell surface with the consequent upregulation of LDLR-dependent LDL-C levels. Beyond its effects on LDL metabolism, several studies revealed the existence of additional roles of PCSK9 in different stages of atherosclerosis, also for its ability to target other members of the LDLR family. PCSK9 from plasma and vascular cells can contribute to the development of atherosclerotic plaque and thrombosis by promoting platelet activation, leukocyte recruitment and clot formation, also through mechanisms not related to systemic lipid changes. These results further supported the value for the potential cardiovascular benefits of therapies based on PCSK9 inhibition. Actually, the passive immunization with anti-PCSK9 antibodies, evolocumab and alirocumab, is shown to be effective in dramatically reducing the LDL-C levels and attenuating CVD. While monoclonal antibodies sequester circulating PCSK9, inclisiran, a small interfering RNA, is a new drug that inhibits PCSK9 synthesis with the important advantage, compared with PCSK9 mAbs, to preserve its pharmacodynamic effects when administrated every 6 months. Here, we will focus on the major understandings related to PCSK9, from its discovery to its role in lipoprotein metabolism, involvement in atherothrombosis and a brief excursus on approved current therapies used to inhibit its action.


Assuntos
Aterosclerose/genética , LDL-Colesterol/metabolismo , Dislipidemias/genética , Placa Aterosclerótica/genética , Pró-Proteína Convertase 9/genética , Trombose/genética , Anticorpos Monoclonais Humanizados/uso terapêutico , Aterosclerose/tratamento farmacológico , Aterosclerose/enzimologia , Aterosclerose/patologia , Plaquetas/efeitos dos fármacos , Plaquetas/enzimologia , Plaquetas/patologia , LDL-Colesterol/antagonistas & inibidores , Dislipidemias/tratamento farmacológico , Dislipidemias/enzimologia , Dislipidemias/patologia , Fibrinolíticos/uso terapêutico , Regulação da Expressão Gênica , Humanos , Hipolipemiantes/uso terapêutico , Metabolismo dos Lipídeos/efeitos dos fármacos , Metabolismo dos Lipídeos/genética , Placa Aterosclerótica/tratamento farmacológico , Placa Aterosclerótica/enzimologia , Placa Aterosclerótica/patologia , Ativação Plaquetária/efeitos dos fármacos , Pró-Proteína Convertase 9/antagonistas & inibidores , Pró-Proteína Convertase 9/biossíntese , RNA Interferente Pequeno/uso terapêutico , Receptores de LDL/genética , Receptores de LDL/metabolismo , Transdução de Sinais , Trombose/enzimologia , Trombose/patologia , Trombose/prevenção & controle
4.
Fish Physiol Biochem ; 47(4): 1105-1117, 2021 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-34052972

RESUMO

Activation of the cAMP pathway by ß-adrenergic stimulation and cGMP pathway by activation of guanylate cyclase substantially affects red blood cell (RBC) membrane properties in mammals. However, whether similar mechanisms are involved in RBC regulation of lower vertebrates, especially teleosts, is not elucidated yet. In this study, we evaluated the effects of adenylate cyclase activation by epinephrine and forskolin, guanylate cyclase activation by sodium nitroprusside, and the role of Na+/H+-exchanger in the changes of osmotic fragility and regulatory volume decrease (RVD) response in crucian carp RBCs. Western blot analysis of protein kinase A and protein kinase G substrate phosphorylation revealed that changes in osmotic fragility were regulated via the protein kinase A, but not protein kinase G signaling pathway. At the same time, the RVD response in crucian carp RBCs was not affected either by activation of adenylate or guanylate cyclase. Adenylate cyclase/protein kinase A activation significantly decreased RBC osmotic fragility, i.e., increased cell rigidity. Inhibition of Na+/H+-exchanger by amiloride had no effect on the epinephrine-mediated decrease of RBC osmotic fragility. NO donor SNP did not activate guanylate cyclase, however affected RBCs osmotic fragility by protein kinase G-independent mechanisms. Taken together, our data demonstrated that the cAMP/PKA signaling pathway and NO are involved in the regulation of crucian carp RBC osmotic fragility, but not in RVD response. The authors confirm that the study has no clinical trial.


Assuntos
Carpas/sangue , Carpas/metabolismo , Proteínas Quinases Dependentes de AMP Cíclico/metabolismo , Proteínas de Peixes/metabolismo , Óxido Nítrico/metabolismo , Adenilil Ciclases/metabolismo , Animais , Plaquetas/enzimologia , Humanos , Fragilidade Osmótica
5.
Cardiovasc Diabetol ; 20(1): 77, 2021 04 03.
Artigo em Inglês | MEDLINE | ID: mdl-33812377

RESUMO

BACKGROUND: Patients with type 2 diabetes (T2DM) have a prothrombotic state that needs to be fully clarified; microparticles (MPs) have emerged as mediators and markers of this condition. Thus, we investigate, in vivo, in T2DM either with good (HbA1c ≤ 7.0%; GGC) or poor (HbA1c > 7.0%; PGC) glycemic control, the circulating levels of MPs, and in vitro, the molecular pathways involved in the release of MPs from platelets (PMP) and tested their pro-inflammatory effects on THP-1 transformed macrophages. METHODS: In 59 T2DM, and 23 control subjects with normal glucose tolerance (NGT), circulating levels of CD62E+, CD62P+, CD142+, CD45+ MPs were determined by flow cytometry, while plasma levels of ICAM-1, VCAM-1, IL-6 by ELISA. In vitro, PMP release and activation of isolated platelets from GGC and PGC were investigated, along with their effect on IL-6 secretion in THP-1 transformed macrophages. RESULTS: We found that MPs CD62P+ (PMP) and CD142+ (tissue factor-bearing MP) were significantly higher in PGC T2DM than GGC T2DM and NGT. Among MPs, PMP were also correlated with HbA1c and IL-6. In vitro, we showed that acute thrombin exposure stimulated a significantly higher PMP release in PGC T2DM than GGC T2DM through a more robust activation of PAR-4 receptor than PAR-1 receptor. Treatment with PAR-4 agonist induced an increased release of PMP in PGC with a Ca2+-calpain dependent mechanism since this effect was blunted by calpain inhibitor. Finally, the uptake of PMP derived from PAR-4 treated PGC platelets into THP-1 transformed macrophages promoted a marked increase of IL-6 release compared to PMP derived from GGC through the activation of the NF-kB pathway. CONCLUSIONS: These results identify PAR-4 as a mediator of platelet activation, microparticle release, and inflammation, in poorly controlled T2DM.


Assuntos
Glicemia/metabolismo , Plaquetas/enzimologia , Cálcio/metabolismo , Calpaína/metabolismo , Micropartículas Derivadas de Células/enzimologia , Diabetes Mellitus Tipo 2/enzimologia , Macrófagos/metabolismo , Ativação Plaquetária , Receptores de Trombina/metabolismo , Biomarcadores/sangue , Plaquetas/efeitos dos fármacos , Estudos de Casos e Controles , Micropartículas Derivadas de Células/efeitos dos fármacos , Diabetes Mellitus Tipo 2/sangue , Feminino , Hemoglobina A Glicada/metabolismo , Humanos , Interleucina-6/metabolismo , Masculino , Pessoa de Meia-Idade , Ativação Plaquetária/efeitos dos fármacos , Receptores de Trombina/agonistas , Células THP-1 , Trombina/farmacologia
6.
Biomed Pharmacother ; 139: 111525, 2021 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-33882412

RESUMO

3-Hydroxytyrosol (HXT) is a natural polyphenol present in extra virgin olive oil. It is a key component of Mediterranean diet and is known for its strong antioxidant activity. The present study evaluated the potential of HXT as an anti-parkinsonian molecule in terms of its ability to inhibit MAO-B and thereby maintaining dopamine (DA) levels in Parkinson's disease (PD). In-silico molecular docking study followed by MMGBSA binding free energy calculation revealed that HXT has a strong binding affinity for MAO-B in comparison to MAO-A. Moreover, rasagiline and HXT interacted with the similar binding sites and modes of interactions. Additionally, molecular dynamics simulation studies revealed stable nature of HXT-MAO-B interaction and also provided information about the amino acid residues involved in binding. Moreover, in vitro studies revealed that HXT inhibited MAO-B in human platelets with IC50 value of 7.78 µM. In vivo studies using MPTP-induced mouse model of PD revealed increase in DA levels with concomitant decrease in DA metabolites (DOPAC and HVA) on HXT treatment. Furthermore, MAO-B activity was also inhibited on HXT administration to PD mice. In addition, HXT treatment prevented MPTP-induced loss of DA neurons in substantia nigra and their nerve terminals in the striatum. HXT also attenuated motor impairments in PD mice assessed by catalepsy bar, narrow beam walk and open field tests. Thus, the present findings reveal HXT as a potential inhibitor of MAO-B, which may be used as a lead molecule for the development of therapeutics for PD.


Assuntos
Antiparkinsonianos/farmacologia , Intoxicação por MPTP/tratamento farmacológico , Doença de Parkinson Secundária/induzido quimicamente , Doença de Parkinson Secundária/tratamento farmacológico , Álcool Feniletílico/análogos & derivados , Animais , Plaquetas/efeitos dos fármacos , Plaquetas/enzimologia , Simulação por Computador , Dopamina/metabolismo , Neurônios Dopaminérgicos/efeitos dos fármacos , Neurônios Dopaminérgicos/patologia , Humanos , Isoenzimas/metabolismo , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Simulação de Acoplamento Molecular , Simulação de Dinâmica Molecular , Monoaminoxidase/metabolismo , Neostriado/efeitos dos fármacos , Neostriado/patologia , Azeite de Oliva/química , Doença de Parkinson Secundária/patologia , Álcool Feniletílico/farmacologia , Substância Negra/efeitos dos fármacos , Substância Negra/patologia
8.
Am J Physiol Cell Physiol ; 320(5): C902-C915, 2021 05 01.
Artigo em Inglês | MEDLINE | ID: mdl-33689480

RESUMO

Spleen tyrosine kinase (Syk) and Bruton's tyrosine kinase (BTK) play critical roles in platelet physiology, facilitating intracellular immunoreceptor tyrosine-based activation motif (ITAM)-mediated signaling downstream of platelet glycoprotein VI (GPVI) and GPIIb/IIIa receptors. Small molecule tyrosine kinase inhibitors (TKIs) targeting Syk and BTK have been developed as antineoplastic and anti-inflammatory therapeutics and have also gained interest as antiplatelet agents. Here, we investigate the effects of 12 different Syk and BTK inhibitors on GPVI-mediated platelet signaling and function. These inhibitors include four Syk inhibitors, Bay 61-3606, R406 (fostamatinib), entospletinib, TAK-659; four irreversible BTK inhibitors, ibrutinib, acalabrutinib, ONO-4059 (tirabrutinib), AVL-292 (spebrutinib); and four reversible BTK inhibitors, CG-806, BMS-935177, BMS-986195, and fenebrutinib. In vitro, TKIs targeting Syk or BTK reduced platelet adhesion to collagen, dense granule secretion, and alpha granule secretion in response to the GPVI agonist cross-linked collagen-related peptide (CRP-XL). Similarly, these TKIs reduced the percentage of activated integrin αIIbß3 on the platelet surface in response to CRP-XL, as determined by PAC-1 binding. Although all TKIs tested inhibited phospholipase C γ2 (PLCγ2) phosphorylation following GPVI-mediated activation, other downstream signaling events proximal to phosphoinositide 3-kinase (PI3K) and PKC were differentially affected. In addition, reversible BTK inhibitors had less pronounced effects on GPIIb/IIIa-mediated platelet spreading on fibrinogen and differentially altered the organization of PI3K around microtubules during platelets spreading on fibrinogen. Select TKIs also inhibited platelet aggregate formation on collagen under physiological flow conditions. Together, our results suggest that TKIs targeting Syk or BTK inhibit central platelet functional responses but may differentially affect protein activities and organization in critical systems downstream of Syk and BTK in platelets.


Assuntos
Tirosina Quinase da Agamaglobulinemia/antagonistas & inibidores , Plaquetas/efeitos dos fármacos , Ativação Plaquetária/efeitos dos fármacos , Inibidores da Agregação Plaquetária/farmacologia , Glicoproteínas da Membrana de Plaquetas/metabolismo , Inibidores de Proteínas Quinases/farmacologia , Quinase Syk/antagonistas & inibidores , Tirosina Quinase da Agamaglobulinemia/metabolismo , Plaquetas/enzimologia , Feminino , Humanos , Masculino , Terapia de Alvo Molecular , Adesividade Plaquetária/efeitos dos fármacos , Agregação Plaquetária/efeitos dos fármacos , Transdução de Sinais , Quinase Syk/metabolismo
9.
J Thromb Haemost ; 19(5): 1319-1330, 2021 05.
Artigo em Inglês | MEDLINE | ID: mdl-33587773

RESUMO

BACKGROUND: Heparanase (HPSE) is the only known mammalian enzyme that can degrade heparan sulfate. Heparan sulfate proteoglycans are essential components of the glycocalyx, and maintain physiological barriers between the blood and endothelial cells. HPSE increases during sepsis, which contributes to injurious glyocalyx degradation, loss of endothelial barrier function, and mortality. OBJECTIVES: As platelets are one of the most abundant cellular sources of HPSE, we sought to determine whether HPSE expression and activity increases in human platelets during clinical sepsis. We also examined associations between platelet HPSE expression and clinical outcomes. PATIENTS/METHODS: Expression and activity of HPSE was determined in platelets isolated from septic patients (n = 59) and, for comparison, sex-matched healthy donors (n = 46) using complementary transcriptomic, proteomic, and functional enzymatic assays. Septic patients were followed for the primary outcome of mortality, and clinical data were captured prospectively for septic patients. RESULTS: The mRNA expression of HPSE was significantly increased in platelets isolated from septic patients. Ribosomal footprint profiling, followed by [S35] methionine labeling assays, demonstrated that HPSE mRNA translation and HPSE protein synthesis were significantly upregulated in platelets during sepsis. While both the pro- and active forms of HPSE protein increased in platelets during sepsis, only the active form of HPSE protein significantly correlated with sepsis-associated mortality. Consistent with transcriptomic and proteomic upregulation, HPSE enzymatic activity was also increased in platelets during sepsis. CONCLUSIONS: During clinical sepsis HPSE, translation, and enzymatic activity are increased in platelets. Increased expression of the active form of HPSE protein is associated with sepsis-associated mortality.


Assuntos
Plaquetas/enzimologia , Glucuronidase/metabolismo , Sepse , Células Endoteliais , Glucuronidase/genética , Humanos , Proteômica
10.
Arterioscler Thromb Vasc Biol ; 41(2): 683-697, 2021 02.
Artigo em Inglês | MEDLINE | ID: mdl-33267663

RESUMO

OBJECTIVE: Using 3KO (triple NOX [NADPH oxidase] knockout) mice (ie, NOX1-/-/NOX2-/-/NOX4-/-), we aimed to clarify the role of this family of enzymes in the regulation of platelets in vitro and hemostasis in vivo. Approach and Results: 3KO mice displayed significantly reduced platelet superoxide radical generation, which was associated with impaired platelet aggregation, adhesion, and thrombus formation in response to the key agonists collagen and thrombin. A comparison with single-gene knockouts suggested that the phenotype of 3KO platelets is the combination of the effects of the genetic deletion of NOX1 and NOX2, while NOX4 does not show any significant function in platelet regulation. 3KO platelets displayed significantly higher levels of cGMP-a negative platelet regulator that activates PKG (protein kinase G). The inhibition of PKG substantially but only partially rescued the defective phenotype of 3KO platelets, which are responsive to both collagen and thrombin in the presence of the PKG inhibitors KT5823 or Rp-8-pCPT-cGMPs, but not in the presence of the NOS (NO synthase) inhibitor L-NG-monomethyl arginine. In vivo, triple NOX deficiency protected against ferric chloride-driven carotid artery thrombosis and experimental pulmonary embolism, while hemostasis tested in a tail-tip transection assay was not affected. Procoagulatory activity of platelets (ie, phosphatidylserine surface exposure) and the coagulation cascade in platelet-free plasma were normal. CONCLUSIONS: This study indicates that inhibiting NOXs has strong antithrombotic effects partially caused by increased intracellular cGMP but spares hemostasis. NOXs are, therefore, pharmacotherapeutic targets to develop new antithrombotic drugs without bleeding side effects.


Assuntos
Coagulação Sanguínea , Plaquetas/enzimologia , Trombose das Artérias Carótidas/enzimologia , NADPH Oxidases/sangue , Ativação Plaquetária , Embolia Pulmonar/enzimologia , Animais , Coagulação Sanguínea/efeitos dos fármacos , Plaquetas/efeitos dos fármacos , Trombose das Artérias Carótidas/sangue , Trombose das Artérias Carótidas/genética , Trombose das Artérias Carótidas/prevenção & controle , GMP Cíclico/sangue , Proteínas Quinases Dependentes de GMP Cíclico/sangue , Modelos Animais de Doenças , Inibidores Enzimáticos/farmacologia , Feminino , Fibrinolíticos/farmacologia , Humanos , Masculino , Camundongos Endogâmicos BALB C , Camundongos Endogâmicos C57BL , Camundongos Knockout , NADPH Oxidase 1 , NADPH Oxidase 2 , NADPH Oxidase 4 , NADPH Oxidases/antagonistas & inibidores , NADPH Oxidases/genética , Ativação Plaquetária/efeitos dos fármacos , Embolia Pulmonar/sangue , Embolia Pulmonar/genética , Embolia Pulmonar/prevenção & controle , Transdução de Sinais , Superóxidos/sangue
11.
Arterioscler Thromb Vasc Biol ; 41(2): 668-682, 2021 02.
Artigo em Inglês | MEDLINE | ID: mdl-33297751

RESUMO

OBJECTIVE: Current antiplatelet medications increase the risk of bleeding, which leads to a clear clinical need in developing novel mechanism-based antiplatelet drugs. TYMP (Thymidine phosphorylase), a cytoplasm protein that is highly expressed in platelets, facilitates multiple agonist-induced platelet activation, and enhances thrombosis. Tipiracil hydrochloride (TPI), a selective TYMP inhibitor, has been approved by the Food and Drug Administration for clinical use. We tested the hypothesis that TPI is a safe antithrombotic medication. Approach and Results: By coexpression of TYMP and Lyn, GST (glutathione S-transferase) tagged Lyn-SH3 domain or Lyn-SH2 domain, we showed the direct evidence that TYMP binds to Lyn through both SH3 and SH2 domains, and TPI diminished the binding. TYMP deficiency significantly inhibits thrombosis in vivo in both sexes. Pretreatment of platelets with TPI rapidly inhibited collagen- and ADP-induced platelet aggregation. Under either normal or hyperlipidemic conditions, treating wild-type mice with TPI via intraperitoneal injection, intravenous injection, or gavage feeding dramatically inhibited thrombosis without inducing significant bleeding. Even at high doses, TPI has a lower bleeding side effect compared with aspirin and clopidogrel. Intravenous delivery of TPI alone or combined with tissue plasminogen activator dramatically inhibited thrombosis. Dual administration of a very low dose of aspirin and TPI, which had no antithrombotic effects when used alone, significantly inhibited thrombosis without disturbing hemostasis. CONCLUSIONS: This study demonstrated that inhibition of TYMP, a cytoplasmic protein, attenuated multiple signaling pathways that mediate platelet activation, aggregation, and thrombosis. TPI can be used as a novel antithrombotic medication without the increase in risk of bleeding.


Assuntos
Plaquetas/efeitos dos fármacos , Trombose das Artérias Carótidas/prevenção & controle , Inibidores Enzimáticos/farmacologia , Fibrinolíticos/farmacologia , Ativação Plaquetária/efeitos dos fármacos , Inibidores da Agregação Plaquetária/farmacologia , Pirrolidinas/farmacologia , Timidina Fosforilase/antagonistas & inibidores , Timina/farmacologia , Animais , Aspirina/farmacologia , Plaquetas/enzimologia , Células COS , Trombose das Artérias Carótidas/sangue , Trombose das Artérias Carótidas/enzimologia , Trombose das Artérias Carótidas/genética , Chlorocebus aethiops , Modelos Animais de Doenças , Terapia Antiplaquetária Dupla , Inibidores Enzimáticos/toxicidade , Feminino , Fibrinolíticos/toxicidade , Hemorragia/induzido quimicamente , Masculino , Camundongos Endogâmicos C57BL , Camundongos Knockout , Adesividade Plaquetária/efeitos dos fármacos , Agregação Plaquetária/efeitos dos fármacos , Inibidores da Agregação Plaquetária/toxicidade , Ligação Proteica , Pirrolidinas/toxicidade , Transdução de Sinais , Timidina Fosforilase/genética , Timidina Fosforilase/metabolismo , Timina/toxicidade , Domínios de Homologia de src , Quinases da Família src/genética , Quinases da Família src/metabolismo
12.
Biochem J ; 477(22): 4327-4342, 2020 11 27.
Artigo em Inglês | MEDLINE | ID: mdl-33242335

RESUMO

Our knowledge on the expression, regulation and roles of the different phosphoinositide 3-kinases (PI3Ks) in platelet signaling and functions has greatly expanded these last twenty years. Much progress has been made in understanding the roles and regulations of class I PI3Ks which produce the lipid second messenger phosphatidylinositol 3,4,5 trisphosphate (PtdIns(3,4,5)P3). Selective pharmacological inhibitors and genetic approaches have allowed researchers to generate an impressive amount of data on the role of class I PI3Kα, ß, δ and γ in platelet activation and in thrombosis. Furthermore, platelets do also express two class II PI3Ks (PI3KC2α and PI3KC2ß), thought to generate PtdIns(3,4)P2 and PtdIns3P, and the sole class III PI3K (Vps34), known to synthesize PtdIns3P. Recent studies have started to reveal the importance of PI3KC2α and Vps34 in megakaryocytes and platelets, opening new perspective in our comprehension of platelet biology and thrombosis. In this review, we will summarize previous and recent advances on platelet PI3Ks isoforms. The implication of these kinases and their lipid products in fundamental platelet biological processes and thrombosis will be discussed. Finally, the relevance of developing potential antithrombotic strategies by targeting PI3Ks will be examined.


Assuntos
Plaquetas/enzimologia , Classe II de Fosfatidilinositol 3-Quinases/metabolismo , Classe III de Fosfatidilinositol 3-Quinases/metabolismo , Trombose/enzimologia , Trombose/terapia , Animais , Plaquetas/patologia , Humanos , Isoenzimas/metabolismo , Fosfatos de Fosfatidilinositol/metabolismo , Trombose/patologia
13.
Oxid Med Cell Longev ; 2020: 4165358, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-33194001

RESUMO

Background: Although preclinical studies highlighted the potential role of NADPH oxidase (NOX) in sepsis, only few studies evaluated the oxidative stress in patients with sepsis and septic shock. The objective of the study is to appraise the oxidative stress status and platelet function in patients with sepsis and septic shock compared to healthy controls. Methods and Results: Patients with sepsis or septic shock admitted to the hospital Policlinico Umberto I (Sapienza University, Rome) underwent a blood sample collection within 1 hour from admission. Platelet aggregation, serum thromboxane B2 (TxB2), soluble NOX2-derived peptides (sNox2-dp), and hydrogen peroxide breakdown activity (HBA) were measured and compared to those of healthy volunteers. Overall, 33 patients were enrolled; of these, 20 (60.6%) had sepsis and 13 (39.4%) septic shock. Compared to healthy controls (n = 10, age 67.8 ± 3.2, male 50%), patients with sepsis and septic shock had higher platelet aggregation (49% (IQR 45-55), 60% (55.75-67.25), and 73% (IQR 69-80), respectively, p < 0.001), higher serum TxB2 (77.5 (56.5-86.25), 122.5 (114-131.5), and 210 (195-230) pmol/L, respectively, p < 0.001), higher sNox2-dp (10 (7.75-12), 19.5 (17.25-21), and 33 (29.5-39) pg/mL, respectively, p < 0.001), and lower HBA (75% (67.25-81.5), 50% (45-54.75), and 27% (21.5-32.5), respectively, p < 0.001). Although not statistically significant, a trend in higher levels of serum TxB2 and sNox2-dp in patients who died was observed. Conclusions: Patients with septic shock exhibit higher Nox2 activity and platelet activation than patients with sepsis. These insights joined to better knowledge of these mechanisms could guide the identification of future prognostic biomarkers and new therapeutic strategies in the scenario of septic shock.


Assuntos
Plaquetas/enzimologia , NADPH Oxidase 2/sangue , Ativação Plaquetária , Choque Séptico/sangue , Idoso , Idoso de 80 Anos ou mais , Intervalo Livre de Doença , Feminino , Humanos , Masculino , Pessoa de Meia-Idade , Estudos Prospectivos , Choque Séptico/mortalidade , Taxa de Sobrevida , Tromboxano B2/sangue
14.
Exp Cell Res ; 396(1): 112282, 2020 11 01.
Artigo em Inglês | MEDLINE | ID: mdl-32919954

RESUMO

In the present study, we have explored the prognostic value of the Phosphofructokinase Platelet-type (PFKP) expression and its therapeutic relevance in metastatic breast cancer. PFKP immunohistochemistry was performed on Invasive ductal carcinomas (IDCs; n = 87) of breast, and its association with clinicopathological parameters were evaluated. Using online meta-analysis tools, PFKP's prognostic value was investigated in overall breast cancer as well as in triple negative subtype (TNBCs). For in vitro analysis, MDA-MB-231 cells model was used in order to elucidate mechanisms behind PFKP regulated glycolysis and its impact on cancer cell physiology. Therapeutic relevance of PFKP was further evaluated using PFKP siRNA and Quercetin. PFKP protein expression was found to be positively associated with nodal invasion (p = 0.009), receptor (ER & PR) negative status (p = 0.005 & p = 0.028) and reduced overall survival in breast cancer patients (p = 0.014). In MDA-MB-231 cells, quercetin treatment impaired PFKP-LDHA signaling axis thereby inhibiting aerobic glycolysis mediated increased migration of cancer cells. Our present study demonstrates that elevated PFKP levels are associated with basal cells/TNBC subtypes and might serve as prognostic indicator for TNBC patients. Ability of quercetin to inhibit aerobic glycolysis, cell migration and clonogenic potential of malignant breast cancer cells advocates possibility of quercetin in aggressive breast cancer treatment.


Assuntos
Neoplasias da Mama/genética , Carcinoma Ductal de Mama/genética , Regulação Neoplásica da Expressão Gênica , L-Lactato Desidrogenase/genética , Fosfofrutoquinase-1 Tipo C/genética , Neoplasias de Mama Triplo Negativas/genética , Adulto , Idoso , Antineoplásicos Fitogênicos/farmacologia , Biomarcadores Tumorais/genética , Biomarcadores Tumorais/metabolismo , Plaquetas/efeitos dos fármacos , Plaquetas/enzimologia , Plaquetas/patologia , Neoplasias da Mama/diagnóstico , Neoplasias da Mama/mortalidade , Neoplasias da Mama/patologia , Carcinoma Ductal de Mama/diagnóstico , Carcinoma Ductal de Mama/mortalidade , Carcinoma Ductal de Mama/patologia , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Receptor alfa de Estrogênio/genética , Receptor alfa de Estrogênio/metabolismo , Feminino , Glucose/metabolismo , Glicólise/efeitos dos fármacos , Glicólise/genética , Humanos , L-Lactato Desidrogenase/antagonistas & inibidores , L-Lactato Desidrogenase/metabolismo , Pessoa de Meia-Idade , Estadiamento de Neoplasias , Fosfofrutoquinase-1 Tipo C/antagonistas & inibidores , Fosfofrutoquinase-1 Tipo C/metabolismo , Prognóstico , Quercetina/farmacologia , RNA Interferente Pequeno/genética , RNA Interferente Pequeno/metabolismo , Receptor ErbB-2/genética , Receptor ErbB-2/metabolismo , Transdução de Sinais , Análise de Sobrevida , Neoplasias de Mama Triplo Negativas/diagnóstico , Neoplasias de Mama Triplo Negativas/mortalidade , Neoplasias de Mama Triplo Negativas/patologia
15.
Arterioscler Thromb Vasc Biol ; 40(10): 2391-2403, 2020 10.
Artigo em Inglês | MEDLINE | ID: mdl-32787521

RESUMO

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


Assuntos
Plaquetas/enzimologia , Lesões das Artérias Carótidas/enzimologia , Moléculas de Adesão Celular Neuronais/sangue , Proteínas da Matriz Extracelular/sangue , Proteínas do Tecido Nervoso/sangue , Neuropeptídeos/sangue , Fosfolipase C gama/sangue , Complexo Glicoproteico GPIIb-IIIa de Plaquetas/metabolismo , Glicoproteínas da Membrana de Plaquetas/metabolismo , Serina Endopeptidases/sangue , Trombose/enzimologia , Proteínas rac1 de Ligação ao GTP/sangue , Proteína rhoA de Ligação ao GTP/sangue , Precursor de Proteína beta-Amiloide/genética , Precursor de Proteína beta-Amiloide/metabolismo , Animais , Coagulação Sanguínea , Lesões das Artérias Carótidas/sangue , Lesões das Artérias Carótidas/etiologia , Moléculas de Adesão Celular Neuronais/deficiência , Moléculas de Adesão Celular Neuronais/genética , Retração do Coágulo , Citoesqueleto/enzimologia , Modelos Animais de Doenças , Proteínas da Matriz Extracelular/deficiência , Proteínas da Matriz Extracelular/genética , Camundongos da Linhagem 129 , Camundongos Endogâmicos C3H , Camundongos Endogâmicos C57BL , Proteínas do Tecido Nervoso/deficiência , Proteínas do Tecido Nervoso/genética , Ativação Plaquetária , Serina Endopeptidases/deficiência , Serina Endopeptidases/genética , Transdução de Sinais , Trombose/sangue , Trombose/etiologia
16.
Arterioscler Thromb Vasc Biol ; 40(10): e262-e272, 2020 10.
Artigo em Inglês | MEDLINE | ID: mdl-32814440

RESUMO

OBJECTIVE: The risk of thrombosis in myeloproliferative neoplasms, such as primary myelofibrosis varies depending on the type of key driving mutation (JAK2 [janus kinase 2], CALR [calreticulin], and MPL [myeloproliferative leukemia protein or thrombopoietin receptor]) and the accompanying mutations in other genes. In the current study, we sought to examine the propensity for thrombosis, as well as platelet activation properties in a mouse model of primary myelofibrosis induced by JAK2V617F (janus kinase 2 with valine to phenylalanine substitution on codon 617) mutation. Approach and Results: Vav1-hJAK2V617F transgenic mice show hallmarks of primary myelofibrosis, including significant megakaryocytosis and bone marrow fibrosis, with a moderate increase in red blood cells and platelet number. This mouse model was used to study responses to 2 models of vascular injury and to investigate platelet properties. Platelets derived from the mutated mice have reduced aggregation in response to collagen, reduced thrombus formation and thrombus size, as demonstrated using laser-induced or FeCl3-induced vascular injury models, and increased bleeding time. Strikingly, the mutated platelets had a significantly reduced number of dense granules, which could explain impaired ADP secretion upon platelet activation, and a diminished second wave of activation. CONCLUSIONS: Together, our study highlights for the first time the influence of a hyperactive JAK2 on platelet activation-induced ADP secretion and dense granule homeostasis, with consequent effects on platelet activation properties.


Assuntos
Coagulação Sanguínea , Plaquetas/enzimologia , Lesões das Artérias Carótidas/enzimologia , Janus Quinase 2/sangue , Megacariócitos/enzimologia , Ativação Plaquetária , Mielofibrose Primária/enzimologia , Trombose/enzimologia , Animais , Lesões das Artérias Carótidas/sangue , Lesões das Artérias Carótidas/genética , Modelos Animais de Doenças , Janus Quinase 2/genética , Camundongos Transgênicos , Mutação , Agregação Plaquetária , Mielofibrose Primária/sangue , Mielofibrose Primária/genética , Trombopoese , Trombose/sangue , Trombose/genética
17.
Int J Mol Sci ; 21(14)2020 Jul 18.
Artigo em Inglês | MEDLINE | ID: mdl-32708482

RESUMO

According to a widespread theory, thrombotic masses are not formed in the pulmonary artery (PA) but result from migration of blood clots from the venous system. This concept has prevailed in clinical practice for more than a century. However, a new technologic era has brought forth more diagnostic possibilities, and it has been shown that thrombotic masses in the PA could, in many cases, be found without any obvious source of emboli. Chronic obstructive pulmonary disease, asthma, sickle cell anemia, emergency and elective surgery, viral pneumonia, and other conditions could be complicated by PA thrombosis development without concomitant deep vein thrombosis (DVT). Different pathologies have different causes for local PA thrombotic process. As evidenced by experimental results and clinical observations, endothelial and platelet activation are the crucial mechanisms of this process. Endothelial dysfunction can impair antithrombotic function of the arterial wall through downregulation of endothelial nitric oxide synthase (eNOS) or via stimulation of adhesion receptor expression. Hypoxia, proinflammatory cytokines, or genetic mutations may underlie the procoagulant phenotype of the PA endothelium. Both endotheliocytes and platelets could be activated by protease mediated receptor (PAR)- and receptors for advanced glycation end (RAGE)-dependent mechanisms. Hypoxia, in particular induced by high altitudes, could play a role in thrombotic complications as a trigger of platelet activity. In this review, we discuss potential mechanisms of PA thrombosis in situ.


Assuntos
Plaquetas/metabolismo , Micropartículas Derivadas de Células/metabolismo , Endotélio Vascular/metabolismo , Ativação Plaquetária/imunologia , Artéria Pulmonar/metabolismo , Embolia Pulmonar/metabolismo , Receptor para Produtos Finais de Glicação Avançada/metabolismo , Trombose/metabolismo , Plaquetas/enzimologia , Plaquetas/imunologia , Hipóxia Celular , Micropartículas Derivadas de Células/patologia , Citocinas/metabolismo , Endotélio Vascular/enzimologia , Endotélio Vascular/imunologia , Proteína HMGB1/metabolismo , Humanos , Óxido Nítrico Sintase Tipo III/metabolismo , Artéria Pulmonar/enzimologia , Artéria Pulmonar/imunologia , Artéria Pulmonar/patologia , Embolia Pulmonar/genética , Embolia Pulmonar/fisiopatologia , Embolia Pulmonar/virologia , Receptor PAR-1/metabolismo , Fatores de Risco
18.
Circ Res ; 127(7): e166-e183, 2020 09 11.
Artigo em Inglês | MEDLINE | ID: mdl-32588751

RESUMO

RATIONALE: Ca2+ signaling is a key and ubiquitous actor of cell organization and its modulation controls many cellular responses. SERCAs (sarco-endoplasmic reticulum Ca2+-ATPases) pump Ca2+ into internal stores that play a major role in the cytosolic Ca2+ concentration rise upon cell activation. Platelets exhibit 2 types of SERCAs, SERCA2b and SERCA3 (SERCA3 deficient mice), which may exert specific roles, yet ill-defined. We have recently shown that Ca2+ mobilization from SERCA3-dependent stores was required for full platelet activation in weak stimulation conditions. OBJECTIVE: To uncover the signaling mechanisms associated with Ca2+ mobilization from SERCA3-dependent stores leading to ADP secretion. METHODS AND RESULTS: Using platelets from wild-type or Serca3-deficient mice, we demonstrated that an early (within 5-10 s following stimulation) secretion of ADP specifically dependent on SERCA3 stored Ca2+ is exclusively mobilized by nicotinic acid adenosine dinucleotide-phosphate (NAADP): both Ca2+ mobilization from SERCA3-dependent stores and primary ADP secretion are blocked by the NAADP receptor antagonist Ned-19, and reciprocally both are stimulated by permeant NAADP. In contrast, Ca2+ mobilization from SERCA3-dependent stores and primary ADP secretion were unaffected by inhibition of the production of IP3 (inositol-1,4,5-trisphosphate) by phospholipase-C and accordingly were not stimulated by permeant IP3. CONCLUSIONS: Upon activation, an NAADP/SERCA3 Ca2+ mobilization pathway initiates an early ADP secretion, potentiating platelet activation, and a secondary wave of ADP secretion driven by both an IP3/SERCA2b-dependent Ca2+ stores pathway and the NAADP/SERCA3 pathway. This does not exclude that Ca2+ mobilized from SERCA3 stores may also enhance platelet global reactivity to agonists. Because of its modulating effect on platelet activation, this NAADP-SERCA3 pathway may be a relevant target for anti-thrombotic therapy. Graphic Abstract: A graphic abstract is available for this article.


Assuntos
Difosfato de Adenosina/sangue , Comunicação Autócrina , Plaquetas/enzimologia , Sinalização do Cálcio , NADP/análogos & derivados , Ativação Plaquetária , ATPases Transportadoras de Cálcio do Retículo Sarcoplasmático/sangue , Animais , Comunicação Autócrina/efeitos dos fármacos , Plaquetas/efeitos dos fármacos , Sinalização do Cálcio/efeitos dos fármacos , Humanos , Inositol 1,4,5-Trifosfato/sangue , Camundongos Endogâmicos C57BL , Camundongos Knockout , NADP/sangue , Ativação Plaquetária/efeitos dos fármacos , ATPases Transportadoras de Cálcio do Retículo Sarcoplasmático/genética , Via Secretória , Trombina/farmacologia , Tromboxano A2/sangue , Fatores de Tempo
20.
Arterioscler Thromb Vasc Biol ; 40(5): 1340-1351, 2020 05.
Artigo em Inglês | MEDLINE | ID: mdl-32131611

RESUMO

OBJECTIVE: Patients with psoriasis have impaired vascular health and increased cardiovascular disease (CVD). Platelets are key players in the pathogenesis of vascular dysfunction in cardiovascular disease and represent therapeutic targets in cardiovascular prevention. The object of this study was to define the platelet phenotype and effector cell properties on vascular health in psoriasis and evaluate whether aspirin modulates the platelet-induced phenotype. Approach and Results: Platelets from psoriasis patients (n=45) exhibited increased platelet activation (relative to age- and gender-matched controls, n=18), which correlated with psoriasis skin severity. Isolated platelets from psoriasis patients demonstrated a 2- to 3-fold (P<0.01) increased adhesion to human aortic endothelial cells and induced proinflammatory transcriptional changes, including upregulation of IL 8 (interleukin 8), IL1ß, and Cox (cyclooxygenase)-2 Platelet RNA sequencing revealed an interferon signature and elevated expression of COX-1, which correlated with psoriasis disease severity (r=0.83, P=0.01). In a randomized trial of patients with psoriasis, 2 weeks of 81 mg low-dose aspirin, a COX-1 inhibitor, reduced serum thromboxane (Tx) B2 and reduced brachial vein endothelial proinflammatory transcript expression >70% compared with the no-treatment group (P<0.01). Improvement in brachial vein endothelial cell inflammation significantly correlated with change in serum TxB2 (r=0.48, P=0.02). CONCLUSIONS: In patients with psoriasis, platelets are activated and induce endothelial cell inflammation. Low-dose aspirin improved endothelial cell health in psoriasis via platelet COX-1 inhibition. These data demonstrate a previously unappreciated role of platelets in psoriasis and endothelial cell inflammation and suggests that aspirin may be effective in improving vascular health in patients with psoriasis. Registration: URL: http://www.clinicaltrials.gov. Unique identifier: NCT03228017.


Assuntos
Plaquetas/enzimologia , Ciclo-Oxigenase 1/sangue , Células Endoteliais/enzimologia , Ativação Plaquetária , Psoríase/sangue , Adulto , Aspirina/administração & dosagem , Plaquetas/efeitos dos fármacos , Células Cultivadas , Ciclo-Oxigenase 1/genética , Inibidores de Ciclo-Oxigenase/administração & dosagem , Células Endoteliais/efeitos dos fármacos , Feminino , Humanos , Masculino , Pessoa de Meia-Idade , Adesividade Plaquetária , Psoríase/diagnóstico , Psoríase/tratamento farmacológico , Psoríase/enzimologia , Índice de Gravidade de Doença , Transdução de Sinais , Tromboxano B2/sangue , Resultado do Tratamento
SELEÇÃO DE REFERÊNCIAS
DETALHE DA PESQUISA
...