RESUMEN
Coenzyme Q10 (CoQ10) exists in a wide variety of foods and has promising cardiovascular benefits. However, its effects on platelets and integrin αIIbß3 signaling during atherosclerosis have not been previously explored. Here, apolipoprotein E-deficient (ApoE-/-) mice were fed a standard diet, high-fat diet (HFD) or CoQ10-supplemented HFD for 12 weeks. We found that CoQ10 supplementation in ApoE-/- mice significantly alleviated formation of HFD-induced atherosclerotic lesions, and attenuated platelet hyper-aggregation and granule secretion, including CD62P, CD63 and CD40 ligand (CD40L) expression and platelet factor-4, ß-thromboglobulin and activation normal T cell expressed and secreted (CCL5) release. CoQ10 supplementation decreased soluble fibrinogen and JON/A binding to αIIbß3 on activated platelets, indicating that αIIbß3-mediated inside-out signaling was attenuated. Additionally, CoQ10 down-regulated platelet αIIbß3 outside-in signaling including decreasing phosphorylation of the ß3 intracellular tail, cellular and sarcoma tyrosine-protein kinase (c-Src), and myosin light chain (MLC), and consistently attenuating platelet spreading and clot retraction. Importantly, platelet-monocyte aggregation that was primarily mediated by αIIbß3 and can be blocked using an αIIbß3-specific antagonist tirofiban was also markedly diminished by CoQ10. Thus, CoQ10 supplementation attenuates platelet hyper-reactivity via down-regulating both αIIbß3 inside-out and outside-in signaling, which may play important preventive roles in atherothrombosis.
Asunto(s)
Aterosclerosis/tratamiento farmacológico , Agregación Plaquetaria/efectos de los fármacos , Complejo GPIIb-IIIa de Glicoproteína Plaquetaria/metabolismo , Transducción de Señal/efectos de los fármacos , Ubiquinona/análogos & derivados , Animales , Retracción del Coagulo , Masculino , Ratones , Ratones Noqueados para ApoE , Ubiquinona/uso terapéuticoRESUMEN
Platelets play a crucial role in the development and progression of atherosclerosis-thrombosis and, therefore, antiplatelet drugs are widely used in the treatment of coronary artery disease. Moreover, advances in understanding the biological functions of natural plant products can provide new pharmacological strategies aimed at promoting cardiovascular health. Atractylenolide I (ATL-1), ATL-2, and ATL-3 are the major bioactive components of a Qi tonifying medicinal herb Rhizoma Atractylodis Macrocephalae (Atractylodes macrocephala), which is commonly used in traditional Chinese medicine (TCM). These components possess well-documented anti-inflammatory and anticancer activities, but their effects on platelet activation are still unknown. In this study, the effects of ATL on platelet function in vitro and in vivo were investigated, and the underlying mechanism was explored. We found that ATL-2 and ATL-3 but not ATL-1 diminished agonist-induced platelet aggregation and diminished adenosine triphosphate (ATP) release from dense granules. The levels of phospho-Akt (Ser473) and phospho-p38 MAPK were downregulated in the presence of ATL-2 and ATL-3. We also found that ATL-2 and ATL-3 have a similar inhibitory effect on platelet activation as acetylsalicylic acid in response to agonists. Furthermore, ATL-2 and ATL-3 diminished the spreading of human platelets on immobilized fibrinogen (Fg), delayed clot retraction in platelet-depleted plasma containing human platelets, extended first occlusion time in a mouse model of ferric chloride (FeCl3)-induced carotid arterial thrombosis, and prolonged the bleeding time. These observations suggest that ATL-2 and ATL-3 are potential candidate therapeutic drugs for the prevention of thrombosis.
Asunto(s)
Atractylodes/química , Plaquetas/efectos de los fármacos , Lactonas/farmacología , Extractos Vegetales/farmacología , Activación Plaquetaria/efectos de los fármacos , Inhibidores de Agregación Plaquetaria/farmacología , Adenosina Trifosfato/metabolismo , Animales , Tiempo de Sangría , Plaquetas/metabolismo , Retracción del Coagulo , GTP Fosfohidrolasas/metabolismo , GTP Fosfohidrolasas/farmacología , Hemostasis/efectos de los fármacos , Lactonas/química , Ratones , Fosforilación , Extractos Vegetales/química , Agregación Plaquetaria/efectos de los fármacos , Inhibidores de Agregación Plaquetaria/química , Proteínas Proto-Oncogénicas c-akt/metabolismo , Trombosis/sangre , Trombosis/tratamiento farmacológico , Proteínas Quinasas p38 Activadas por Mitógenos/metabolismoRESUMEN
Fermentation studies of batch-mode cultivation in 4-L fermenters were carried out to obtain an active recombinant DNA-derived tissue plasminogen activator (t-PA) deletion mutant, lekteplase, secreted and correctly folded from Escherichia coli. The OmpA signal sequence was used to deliver the heterologous product composed of kringle 2 plus serine protease domain (K2S) to the medium. Supplementing the complex medium with 10% glycerol and 20 mmol/l magnesium chloride led to an increase in cell numbers with final cell density reaching an OD600 of 24. The expression level of lekteplase in the medium detected by sandwich ELISA was 100 mg/L. Enzymatic activity of lysine-sepharose purified product was demonstrated by amidolytic assay, in vitro fibrin clot lysis, and copolymerization PAGE.
Asunto(s)
Escherichia coli/metabolismo , Activador de Tejido Plasminógeno/farmacología , Secuencia de Bases , Reactores Biológicos , Western Blotting , Retracción del Coagulo , Electroforesis en Gel de Poliacrilamida , Escherichia coli/efectos de los fármacos , Escherichia coli/genética , Fermentación , Glicerol/farmacología , Inmunoensayo , Cloruro de Magnesio/farmacología , Datos de Secuencia Molecular , Plásmidos/genética , Serina Endopeptidasas/metabolismo , Activador de Tejido Plasminógeno/metabolismoRESUMEN
OBJECTIVES: To determine the molecular and genetic basis for thrombasthenic thrombopathia in Otterhounds and establish whether the defect would be best classified as type-I Glanzmann's thrombasthenia. ANIMALS: 57 dogs, including 13 affected Otterhounds, 23 carrier Otterhounds, 17 unaffected Otterhounds, and 4 clinically normal unrelated dogs of other breeds. PROCEDURE: Functional (platelet aggregation, clot retraction, buccal mucosa bleeding time) and biochemical (electrophoresis, flow cytometry, fibrinogen content) analyses were conducted. In addition, first-strand cDNA synthesis from platelet total RNA was performed. Exons of the genes encoding for glycoproteins (GP) IIb and IIIa were amplified in overlapping fashion. The resulting products were excised from agarose gels and sequenced. The sequences obtained were compared with known cDNA sequences for canine GPIIb and GPIIIa. RESULTS: A single nucleotide change at position G1193 (1100) was detected in exon 12 of the gene encoding for platelet GPIIb in 2 affected Otterhounds. Carrier Otterhounds were heterozygous at this position, and 2 unaffected Otterhounds were unchanged. This nucleotide change would result in substitution of histidine for aspartic acid at position 398 (367) within the third calcium-binding domain of GPIIb. CONCLUSIONS AND CLINICAL RELEVANCE: These studies suggest that thrombasthenic thrombopathia of Otterhounds is homologous phenotypically and has a similar molecular basis to type-I Glanzmann's thrombasthenia in humans.
Asunto(s)
Enfermedades de los Perros/genética , Trombastenia/veterinaria , Secuencia de Aminoácidos , Animales , Secuencia de Bases , Plaquetas/patología , Plaquetas/fisiología , Antígenos CD36/genética , Antígenos CD36/metabolismo , Portador Sano , Retracción del Coagulo , ADN Complementario/genética , Enfermedades de los Perros/sangre , Perros , Femenino , Fibrinógeno/metabolismo , Integrina alfa2 , Masculino , Glicoproteínas de Membrana/genética , Glicoproteínas de Membrana/metabolismo , Datos de Secuencia Molecular , Mucosa Bucal/patología , Linaje , Agregación Plaquetaria/genética , ARN/genética , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa/veterinaria , Homología de Secuencia de Ácido Nucleico , Trombastenia/sangre , Trombastenia/genéticaRESUMEN
Fungicide, phenylmercuric acetate (PMA) dose-dependently inhibits in vitro ADP-induced rat platelet primary aggregation. During ex vivo experiments, after oral application of different doses of PMA, the ADP-induced aggregation, clot retraction, bleeding time and clotting time were estimated. Inhibition of platelet activity was found only after a single administration with the high dose of PMA (0.5 LD50). Administration to the rats five times a low dose of PMA (0.2 LD50) and exposure during 5 weeks to the dose of 0.05 LD50 produced hypercoagulation (statistically significant reduction of clotting time) with simultaneous stimulation of platelet activity (stimulation of aggregation, clot retraction and shortening of bleeding time).