RESUMEN
BACKGROUND: Antrodia cinnamomea is an indigenous medicinal mushroom in Taiwan, commonly used for the treatment of cancers and inflammatory disorders. 4-acetylantroquinonol B (4AAQB) is one of the active component isolated from the mycelium of A. cinnamomea. However, whether 4AAQB exhibits anti-inflammatory effect is not clear. METHODS: The anti-inflammatory activity of 4AAQB was examined by ELISA to measure the pro-inflammatory cytokines production in lipopolysaccharide (LPS)-simulated RAW264.7 cells, peritoneal macrophages and in mice. The effect of 4AAQB for MAPK kinase molecules phosphorylation in LPS-stimulated RAW264.7 macrophage including ERK, JNK and p38 were evaluated. The in vivo efficacy of 4AAQB was also demonstrated. RESULTS: In the present study, we found that 4AAQB exhibits anti-inflammatory effects inhibit tumor necrosis factor-α (TNF-α)/interleukin-6 (IL-6) releasing and LPS-stimulated phagocytes migration without affect cell growth. In addition, the MAPK kinase molecules phosphorylation in LPS-stimulated RAW264.7 macrophage including ERK, JNK and p38 was inhibited by 4AAQB. The phosphorylation of NFκB subunit p65 and IkBα were also decreased after 4AAQB treatment. Furthermore, 4AAQB attenuates the cytokine production in LPS-induced and CLP-induced septic mice. CONCLUSION: These results showed that 4AAQB exhibited anti-inflammatory property both in vitro and in vivo, suggesting that 4AAQB may be a therapeutic candidate which used in inflammatory disorders treatment.
Asunto(s)
4-Butirolactona/análogos & derivados , Ciclohexanonas/farmacología , Lipopolisacáridos/efectos adversos , Sistema de Señalización de MAP Quinasas/efectos de los fármacos , FN-kappa B/metabolismo , Sepsis/metabolismo , 4-Butirolactona/farmacología , Animales , Supervivencia Celular/efectos de los fármacos , Citocinas/metabolismo , Masculino , Ratones , Ratones Endogámicos BALB C , Ratones Endogámicos ICR , Proteínas Quinasas Activadas por Mitógenos/metabolismo , Células RAW 264.7RESUMEN
OBJECTIVE: Currently prescribed antiplatelet drugs have 1 common side effect-an increased risk of hemorrhage and thrombocytopenia. On the contrary, bleeding defects associated with glycoprotein VI (GPVI) expression deficiency are usually slightly prolonged bleeding times. However, GPVI antagonists are lacking in clinic. APPROACH AND RESULTS: Using reverse-phase high-performance liquid chromatography and sequencing, we revealed the partial sequence of trowaglerix α subunit, a potent specific GPVI-targeting snaclec (snake venom C-type lectin protein). Hexapeptide (Troα6 [trowaglerix a chain hexapeptide, CKWMNV]) and decapeptide (Troα10) derived from trowaglerix specifically inhibited collagen-induced platelet aggregation through blocking platelet GPVI receptor. Computational peptide design helped to design a series of Troα6/Troα10 peptides. Protein docking studies on these decapeptides and GPVI suggest that Troα10 was bound at the lower surface of D1 domain and outer surface of D2 domain, which was at the different place of the collagen-binding site and the scFv (single-chain variable fragment) D2-binding site. The newly discovered site was confirmed by inhibitory effects of polyclonal antibodies on collagen-induced platelet aggregation. This indicates that D2 domain of GPVI is a novel and important binding epitope on GPVI-mediated platelet aggregation. Troα6/Troα10 displayed prominent inhibitory effect of thrombus formation in fluorescein sodium-induced platelet thrombus formation of mesenteric venules and ferric chloride-induced carotid artery injury thrombosis model without prolonging the in vivo bleeding time. CONCLUSIONS: We develop a novel antithrombotic peptides derived from trowaglerix that acts through GPVI antagonism with greater safety-no severe bleeding. The binding epitope of polypeptides on GPVI is novel and important. These hexa/decapeptides have therapeutic potential for developing ideal small-mass GPVI antagonists for arterial thrombogenic diseases.
Asunto(s)
Plaquetas/efectos de los fármacos , Traumatismos de las Arterias Carótidas/tratamiento farmacológico , Venenos de Crotálidos/farmacología , Fibrinolíticos/farmacología , Fragmentos de Péptidos/farmacología , Inhibidores de Agregación Plaquetaria/farmacología , Agregación Plaquetaria/efectos de los fármacos , Glicoproteínas de Membrana Plaquetaria/antagonistas & inhibidores , Trombosis/prevención & control , Animales , Sitios de Unión , Plaquetas/metabolismo , Traumatismos de las Arterias Carótidas/sangre , Traumatismos de las Arterias Carótidas/inducido químicamente , Cloruros , Diseño Asistido por Computadora , Venenos de Crotálidos/metabolismo , Venenos de Crotálidos/toxicidad , Modelos Animales de Enfermedad , Relación Dosis-Respuesta a Droga , Diseño de Fármacos , Compuestos Férricos , Fibrinolíticos/metabolismo , Fibrinolíticos/toxicidad , Fluoresceína , Hemorragia/inducido químicamente , Humanos , Lectinas Tipo C/metabolismo , Masculino , Ratones Endogámicos ICR , Simulación del Acoplamiento Molecular , Fragmentos de Péptidos/metabolismo , Fragmentos de Péptidos/toxicidad , Inhibidores de Agregación Plaquetaria/metabolismo , Inhibidores de Agregación Plaquetaria/toxicidad , Glicoproteínas de Membrana Plaquetaria/metabolismo , Unión Proteica , Dominios y Motivos de Interacción de Proteínas , Transducción de Señal/efectos de los fármacos , Trombosis/sangre , Trombosis/inducido químicamenteRESUMEN
VEGF and VEGFR antibodies have been used as a therapeutic strategy to inhibit angiogenesis in many diseases; however, frequent and repeated administration of these antibodies to patients induces immunogenicity. In previous studies, we demonstrated that aggretin, a heterodimeric snake venom C-type lectin, exhibits pro-angiogenic activities via integrin α2ß1 ligation. We hypothesised that small-mass aggretin fragments may bind integrin α2ß1 and act as antagonists of angiogenesis. In this study, the anti-angiogenic efficacy of a synthesised aggretin α-chain C-terminus (AACT, residue 106-136) was evaluated in both in vitro and in vivo angiogenesis models. The AACT demonstrated inhibitory effects on collagen-induced platelet aggregation and HUVEC adhesion to immobilised collagen. These results indicated that AACT may block integrin α2ß1-collagen interaction. AACT also inhibited HUVEC migration and tube formation. Aortic ring sprouting and Matrigel implant models demonstrated that AACT markedly inhibited VEGF-induced neovascularisation. In addition, induction of FAK/PI3K/ERK1/2 tyrosine phosphorylation and talin 1/2 associated with integrin ß1 which are induced by VEGF were blocked by AACT. Similarly, tyrosine phosphorylation of VEFGR2 and ERK1/2 induced by VEGF was diminished in integrin α2-silenced endothelial cells. Our results demonstrate that AACT is a potential therapeutic candidate for angiogenesis related-diseases via integrin α2ß1 blockade.
Asunto(s)
Inhibidores de la Angiogénesis/farmacología , Integrina alfa2beta1/antagonistas & inhibidores , Péptidos/farmacología , Venenos de Serpiente/farmacología , Inhibidores de la Angiogénesis/química , Movimiento Celular/efectos de los fármacos , Proliferación Celular/efectos de los fármacos , Supervivencia Celular/efectos de los fármacos , Células Cultivadas , Colágeno/metabolismo , Combinación de Medicamentos , Quinasa 1 de Adhesión Focal/metabolismo , Células Endoteliales de la Vena Umbilical Humana/metabolismo , Humanos , Laminina , Lectinas Tipo C , Neovascularización Fisiológica/efectos de los fármacos , Péptidos/química , Fosfatidilinositol 3-Quinasas/metabolismo , Agregación Plaquetaria/efectos de los fármacos , Unión Proteica , Proteoglicanos , Transducción de Señal/efectos de los fármacos , Venenos de Serpiente/química , Factor A de Crecimiento Endotelial Vascular/metabolismoRESUMEN
INTRODUCTION: The applicability of protein drugs is confined by protein degradation and rapid elimination. PEGylation of polypeptides improves protein stability by sterically obstructing the degradation by serum proteases and reduces renal clearance by the increased mass. EXPERIMENTAL APPROACH: We compared the antithrombotic activities of intact rhodostomin (Rn) and PEGylated rhodostomin (PRn) both in vitro and in vivo systems. In addition, the functional half-life in inhibiting platelet aggregation and the tendency in causing bleeding side effect were investigated. RESULTS: Rn and PRn both potently inhibited human and mouse platelet aggregation induced by collagen, thrombin or ADP in vitro with a similar IC50 around 60-100nM. Rotational thromboelastometry assay indicated that PRn was more effective than Rn in preventing clot formation in human whole blood. In platelet-rich plasma from mice injected with Rn or PRn, the inhibitory effects on collagen-induced platelet aggregation were also comparable, but Rn caused higher bleeding tendency. In ferric chloride-induced arterial thrombosis, Rn and PRn significantly prolonged occlusion time at high dosage (0.2µg/g). However, PRn obviously prolonged the occlusion time even given at a lower dosage (0.06µg/g). The functional half-life assay revealed that PEGylation prolonged the in vivo half-life of Rn. CONCLUSIONS: PRn exhibits higher antithrombotic potency and longer half-life in vivo as compared with native Rn on a molar basis. In addition, PRn exhibits a better safety profile at an efficacious antithrombotic dose in vivo. Therefore, PEGylation may be one of the ideal options in modifying disintegrin derivatives as the safe therapeutic agents for integrin-related diseases.
Asunto(s)
Hemorragia/inducido químicamente , Péptidos/efectos adversos , Péptidos/uso terapéutico , Inhibidores de Agregación Plaquetaria/efectos adversos , Inhibidores de Agregación Plaquetaria/uso terapéutico , Complejo GPIIb-IIIa de Glicoproteína Plaquetaria/antagonistas & inhibidores , Trombosis/tratamiento farmacológico , Animales , Plaquetas/efectos de los fármacos , Desintegrinas/efectos adversos , Desintegrinas/química , Desintegrinas/farmacocinética , Desintegrinas/uso terapéutico , Estabilidad de Medicamentos , Semivida , Hemorragia/sangre , Humanos , Masculino , Ratones , Ratones Endogámicos ICR , Péptidos/química , Péptidos/farmacocinética , Agregación Plaquetaria/efectos de los fármacos , Inhibidores de Agregación Plaquetaria/química , Inhibidores de Agregación Plaquetaria/farmacocinética , Complejo GPIIb-IIIa de Glicoproteína Plaquetaria/metabolismo , Polietilenglicoles/química , Estabilidad Proteica , Trombosis/sangreRESUMEN
A novel benzimidazole derivative, nstpbp5185, was discovered through in vitro and in vivo evaluations for antiplatelet activity. Thromaboxane receptor (TP) is important in vascular physiology, haemostasis and pathophysiological thrombosis. Nstpbp5185 concentration-dependently inhibited human platelet aggregation caused by collagen, arachidonic acid and U46619. Nstpbp5185 caused a right-shift of the concentration-response curve of U46619 and competitively inhibited the binding of 3H-SQ-29548 to TP receptor expressed on HEK-293 cells, with an IC50 of 0.1 µM, indicating that nstpbp5185 is a TP antagonist. In murine thrombosis models, nstpbp5185 significantly prolonged the latent period in triggering platelet plug formation in mesenteric and FeCl3-induced thrombi formation, and increased the survival rate in pulmonary embolism model with less bleeding than aspirin. This study suggests nstpbp5185, an orally selective anti-thrombotic agent, acting through blockade of TXA2 receptor, may be efficacious for prevention or treatment of pathologic thrombosis.
Asunto(s)
Bencimidazoles/farmacología , Agregación Plaquetaria/efectos de los fármacos , Receptores de Tromboxano A2 y Prostaglandina H2/antagonistas & inhibidores , Trombosis/prevención & control , Ácido 15-Hidroxi-11 alfa,9 alfa-(epoximetano)prosta-5,13-dienoico/farmacología , Animales , Ácido Araquidónico/farmacología , Aspirina/farmacología , Plaquetas/efectos de los fármacos , Plaquetas/metabolismo , Calcio/sangre , Colágeno/farmacología , Humanos , Técnicas In Vitro , Masculino , Ratones , Ratones Endogámicos ICR , Microvasos/efectos de los fármacos , Selectina-P/sangre , Embolia Pulmonar/prevención & control , Tromboxano A2/sangreRESUMEN
Bacterial infection-induced sepsis is the leading cause of septic inflammatory disease. Rhodostomin (Rn), a snake venom disintegrin, was previously reported to interact with the αVß3 integrin and the TLR4 on phagocyte in attenuating LPS-induced endotoxemia. In this report, we further evaluated the effects of Rn on TLR2-activated monocytes and its in vivo efficacy. Rn effectively suppressed the adhesion, migration, and cytokine release of Pam3CSK4-activated THP-1 cells. Rn specifically bound to integrin αVß3 of TLR2-activated THP-1. Integrin αV and Akt siRNA transfection both restrained Pam3CSK4-elicited cytokine release. Rn decreased the Pam3CSK4-induced phosporylation of MAPKs, degradation of IκB and activation of FAK, Akt, c-Src and Syk. The Pam3CSK4-induced translocation of MyD88, a central adaptor of TLR2, to the cell membrane was also inhibited by Rn treatment. In the polymicrobial inflammatory caecal ligation and puncture model, Rn significantly reduced pro-inflammatory cytokine and chemokine release, alleviated tissue injury and elevated survival rate in vivo. Taken together, in addition to inhibiting the activation of TLR4, Rn exhibits anti-inflammatory activity through antagonizing the activation of phagocytes and interrupting the crosstalk between αVß3 and TLR2-dependent signaling pathways.
Asunto(s)
Integrina alfaVbeta3/metabolismo , Monocitos/efectos de los fármacos , Péptidos/administración & dosificación , Sepsis/tratamiento farmacológico , Receptor Toll-Like 2/metabolismo , Animales , Adhesión Celular/efectos de los fármacos , Línea Celular , Movimiento Celular/efectos de los fármacos , Quimiocinas/metabolismo , Citocinas/metabolismo , Modelos Animales de Enfermedad , Regulación de la Expresión Génica/efectos de los fármacos , Humanos , Masculino , Ratones , Monocitos/citología , Péptidos/farmacología , Sepsis/metabolismo , Transducción de Señal/efectos de los fármacosRESUMEN
Hepatocellular carcinoma (HCC) has become one of most common malignancies and a leading cause of cancer mortality worldwide. Previous study has shown that 4-acetylantroquinonol B (4AAQB) isolated from Antrodia cinnamomea (or niu-chang-chih) was observed to inhibit HepG2 cell proliferation via affecting cell cycle. However, the in vivo effects and antimetastatic activity of 4AAQB have not yet been addressed. This study found that 4AAQB inhibited HepG2 and HuH-7 hepatoma cell growth in both in vitro and in vivo models and exhibited pronounced inhibitory effects on HuH-7 tumor growth in xenograft and orthotopic models. 4AAQB efficiently inhibited the phosphorylation of mTOR and its upstream kinases and the downstream effectors and decreased the production of VEGF and activity of Rho GTPases in HuH-7 cells. Furthermore, 4AAQB inhibited in vitro HuH-7 cell migration and in vivo pulmonary metastasis. The results suggested that 4AAQB is a potential candidate for HCC therapy.
Asunto(s)
4-Butirolactona/análogos & derivados , Antineoplásicos/administración & dosificación , Antrodia/química , Carcinoma Hepatocelular/tratamiento farmacológico , Proliferación Celular/efectos de los fármacos , Ciclohexanonas/administración & dosificación , Neoplasias Hepáticas/tratamiento farmacológico , Factor A de Crecimiento Endotelial Vascular/metabolismo , 4-Butirolactona/administración & dosificación , Animales , Carcinoma Hepatocelular/metabolismo , Carcinoma Hepatocelular/patología , Carcinoma Hepatocelular/fisiopatología , Movimiento Celular/efectos de los fármacos , Regulación hacia Abajo/efectos de los fármacos , Células Hep G2 , Humanos , Neoplasias Hepáticas/metabolismo , Neoplasias Hepáticas/patología , Neoplasias Hepáticas/fisiopatología , Masculino , Huesos Metatarsianos , Ratones Desnudos , Ratones SCID , Fosforilación/efectos de los fármacos , Biosíntesis de Proteínas/efectos de los fármacos , Transducción de Señal/efectos de los fármacos , Serina-Treonina Quinasas TOR/genética , Serina-Treonina Quinasas TOR/metabolismo , Factor A de Crecimiento Endotelial Vascular/genéticaRESUMEN
The development of restenosis involves migration and hyperproliferation of vascular smooth muscle cells (VSMCs). Platelet-derived growth factor (PDGF) is one of the major factors. Butein modulates inflammatory pathways and affects the proliferation and invasion of the tumor. We investigated the hypothesis that butein might prevent the restenosis process via a similar pathway. Our results demonstrated that butein inhibited PDGF-induced VSMC proliferation and migration as determined by BrdU proliferation and two-dimensional migration scratch assay. Butein also concentration-dependently repressed PDGF-induced phosphorylation of PDGF-receptor ß, mitogen-activated protein kinases, phosphoinositide 3-kinase/Akt, and phopholipase Cγ/c-Src in VSMCs. In addition, in vivo results showed that butein attenuated neointima formation in balloon-injured rat carotid arteries. These results indicate that butein may inhibit PDGF-induced VSMC proliferation and migration, resulting in attenuation of neointima formation after percutaneous transluminal coronary angioplasty. Our study demonstrates for the first time that systemic administration of butein is able to reduce neointima formation after vascular injury.
Asunto(s)
Chalconas/farmacología , Reestenosis Coronaria/tratamiento farmacológico , Neointima/tratamiento farmacológico , Animales , Movimiento Celular , Proliferación Celular , Células Cultivadas , Reestenosis Coronaria/genética , Reestenosis Coronaria/metabolismo , Reestenosis Coronaria/fisiopatología , Humanos , Masculino , Proteínas Quinasas Activadas por Mitógenos/genética , Proteínas Quinasas Activadas por Mitógenos/metabolismo , Miocitos del Músculo Liso/citología , Miocitos del Músculo Liso/efectos de los fármacos , Miocitos del Músculo Liso/metabolismo , Neointima/genética , Neointima/metabolismo , Neointima/fisiopatología , Fosforilación , Factor de Crecimiento Derivado de Plaquetas/metabolismo , Proteínas Proto-Oncogénicas c-akt/genética , Proteínas Proto-Oncogénicas c-akt/metabolismo , Ratas , Ratas Wistar , Transducción de Señal/efectos de los fármacosRESUMEN
BACKGROUND AND PURPOSE: 1,4-Naphthoquinones exhibit antiplatelet activity both in vivo and in vitro. In the present study, we investigated the antiplatelet effect of a novel naphthoquinone derivative NP-313, 2-acetylamino-3-chloro-1,4-naphthoquinone and its mechanism of action. EXPERIMENTAL APPROACH: We measured platelet aggregation, Ca(2+) mobilization, thromboxane B2 formation and P-selectin expression and examined several enzymatic activities. Furthermore, we used the irradiated mesenteric venules in fluorescein sodium-treated mice to monitor the antithrombotic effect of NP-313 in vivo. KEY RESULTS: NP-313 concentration-dependently inhibited human platelet aggregation induced by collagen, arachidonic acid, thapsigargin, thrombin and A23187. NP-313 also inhibited P-selectin expression, thromboxane B(2) formation and [Ca(2+) ](i) elevation in platelets stimulated by thrombin and collagen. NP-313 at 10 µM inhibited cyclooxygenase, thromboxane A(2) synthase, and protein kinase Cα, whereas it did not affect phospholipase A(2) or phospholipase C activity. In the presence of indomethacin and an adenosine 5-diphosphate scavenger, NP-313 concentration-dependently inhibited thrombin- and A23187-induced [Ca(2+)](i) increase through its inhibitory effects on Ca(2+) influx, rather than blocking Ca(2+) release from intracellular stores. NP-313 also inhibited thapsigargin-mediated Ca(2+) influx through store-operated calcium channel but had no effect on Ca(2+) influx through store-independent calcium channel evoked by the diacylglycerol analogue 1-oleoyl-2-acetyl-sn-glycerol. Nevertheless, it had little effect on cyclic AMP and cyclic GMP levels. Also, intravenously administered NP-313 dose-dependently inhibited the thrombus occlusion of the irradiated mesenteric vessels of fluorescein-pretreated mice. CONCLUSIONS AND IMPLICATIONS: Taken together, these results indicate that NP-313 exerts its antithrombotic activity through dual inhibition of thromboxane A(2) synthesis and Ca(2+) influx through SOCC.
Asunto(s)
Fibrinolíticos/farmacología , Naftoquinonas/farmacología , Inhibidores de Agregación Plaquetaria/farmacología , Agregación Plaquetaria/efectos de los fármacos , Animales , Calcio/metabolismo , Canales de Calcio/metabolismo , Relación Dosis-Respuesta a Droga , Fibrinolíticos/administración & dosificación , Masculino , Ratones , Ratones Endogámicos ICR , Naftoquinonas/administración & dosificación , Selectina-P/genética , Inhibidores de Agregación Plaquetaria/administración & dosificación , Tromboxano A2/biosíntesis , Tromboxano B2/biosíntesisRESUMEN
Pulmonary aspergilloma and pulmonary actinomycosis are rare pulmonary infectious diseases. Clinical manifestations of pulmonary aspergilloma and pulmonary actinomycosis include chronic cough, fever, chest pain, haemoptysis and other pathologies, but some patients may be asymptomatic. We report a case of a healthy 33-year-old woman without any underlying diseases, who was admitted to Zhongxing Branch of Taipei City Hospital, Taiwan, for intermittent haemoptysis and right upper chest pain, which had persisted for several months. A chest radiograph revealed a focal consolidation in the right upper lobe (RUL) of the lung, which grew in size over time. A sputum study and bronchoscopy revealed no positive findings, although malignancy could not be ruled out. Thus, the patient received a wedge resection of the RUL lesion. Subsequent, pathological examination demonstrated the presence of pulmonary aspergilloma and pulmonary actinomycosis. The patient's symptoms resolved after resection of the RUL lesion.
Asunto(s)
Actinomicosis/diagnóstico , Enfermedades Pulmonares/microbiología , Aspergilosis Pulmonar/diagnóstico , Actinomyces/aislamiento & purificación , Actinomicosis/patología , Actinomicosis/cirugía , Adulto , Aspergillus/aislamiento & purificación , Broncoscopía , Femenino , Histocitoquímica , Humanos , Pulmón/diagnóstico por imagen , Pulmón/patología , Enfermedades Pulmonares/patología , Enfermedades Pulmonares/cirugía , Aspergilosis Pulmonar/patología , Aspergilosis Pulmonar/cirugía , Radiografía Torácica , Esputo , Taiwán , Tomografía Computarizada por Rayos XRESUMEN
Pleomorphic hyalinizing angiectatic tumor (PHAT) of soft parts is a rare, nonmetastasizing tumor of uncertain lineage which was first reported in 1996. Here, we report a case of PHAT and review the literature. A 49-year-old man presented with a soft and progressively enlarging mass over the right buttock for several years. On suspicion that the mass was a right gluteal lipoma, he underwent surgical excision. The excised lesion measured 14 x 6 x 3.5 cm. It had a variegated appearance with a white-tan to yellowish color on the cut surface. Some punctate hemorrhage and vessel thrombosis were seen. Microscopically, the tumor was a PHAT characterized by clusters of ectatic, fibrin-lined, thin-walled vessels, which were surrounded by a mitotically inert, spindled, pleomorphic, neoplastic stroma that contained a variable inflammatory component. Immunohistochemical study showed that the tumor cells were positive for CD34, and negative for S-100, HMB45 and actin. The patient experienced local recurrence 6 months later. The recurrent tumor was widely excised. No evidence of metastasis was found during the 18 months after the second operation. The recurrent lesion had a microscopic appearance that was similar to the initial lesion.
Asunto(s)
Recurrencia Local de Neoplasia/patología , Neoplasias de los Tejidos Blandos/patología , Células del Estroma/patología , Antígenos CD34/análisis , Humanos , Inmunohistoquímica , Masculino , Persona de Mediana Edad , Recurrencia Local de Neoplasia/cirugía , Neoplasias de los Tejidos Blandos/cirugía , Resultado del TratamientoRESUMEN
The established antiplatelet and anticoagulant agents show beneficial effects in the treatment of thromboembolic diseases; however, these drugs still have considerable limitations. The effects of NP-184, a synthetic compound, on platelet functions, plasma coagulant activity, and mesenteric venule thrombosis in mice were investigated. NP-184 concentration-dependently inhibited the human platelet aggregation induced by collagen, arachidonic acid (AA), and U46619, a thromboxane (TX)A(2) mimic, with IC(50) values of 4.5 +/- 0.2, 3.9 +/- 0.1, and 9.3 +/- 0.5 microM, respectively. Moreover, NP-184 concentration-dependently suppressed TXA(2) formations caused by collagen and AA. In exploring effects of NP-184 on enzymes involved in TXA(2) synthesis, we found that NP-184 selectively inhibited TXA(2) synthase activity with an IC(50) value of 4.3 +/- 0.2 microM. Furthermore, NP-184 produced a right shift of the concentration-response curve of U46619, indicating a competitive antagonism on TXA(2)/prostaglandin H(2) receptor. Intriguingly, NP-184 also caused a concentration-dependent prolongation of the activated partial thromboplastin time (aPTT) with no changes in the prothrombin and thrombin time, indicating that it selectively impairs the intrinsic coagulation pathway. Oral administration of NP-184 significantly inhibited thrombus formation of the irradiated mesenteric venules in fluorescein sodium-treated mice without affecting the bleeding time induced by tail transection. However, after oral administration, NP-184 inhibited the ex vivo mouse platelet aggregation triggered by collagen and U46619 and also prolonged aPTT. Taken together, the dual antiplatelet and anticoagulant activities of NP-184 may have therapeutic potential as an oral antithrombotic agent in the treatment of thromboembolic disorders.
Asunto(s)
Bencimidazoles/farmacología , Fibrinolíticos/farmacología , Administración Oral , Animales , Anticoagulantes/administración & dosificación , Anticoagulantes/farmacología , Bencimidazoles/administración & dosificación , Coagulación Sanguínea/efectos de los fármacos , Relación Dosis-Respuesta a Droga , Fibrinolíticos/administración & dosificación , Humanos , Concentración 50 Inhibidora , Masculino , Ratones , Ratones Endogámicos ICR , Agregación Plaquetaria/efectos de los fármacos , Inhibidores de Agregación Plaquetaria/administración & dosificación , Inhibidores de Agregación Plaquetaria/farmacología , Trombosis de la Vena/tratamiento farmacológicoRESUMEN
Subendothelial collagen supports platelet adhesion, activation, and thrombus growth at sites of vascular damage. Previous studies have shown that chalcones possess antiplatelet activity, but their mechanism of action is not fully understood. In this study, a recently synthesized chalcone, 2'-ethoxy-5'-methoxy-2-(5-methylthienyl)chalcone (EMMTC), was used to investigate chalcone effects on platelet aggregation and adhesion. We found that EMMTC potently inhibited collagen-induced platelet aggregation with an IC(50) of 1.01 micromol/l. In contrast, it did not inhibit thrombin- and ADP-induced platelet aggregation. EMMTC could inhibit arachidonic acid (AA)-induced platelet aggregation and collagen- and AA-induced thromboxane B(2) (TXB(2)) formation, suggesting that cyclooxygenase and/or thromboxane synthase were affected during this process. Moreover, EMMTC suppressed collagen-induced protein tyrosine phosphorylation, including phospholipase C-gamma2 (PLC-gamma2), phosphatidylinositol-3 kinase (PI-3K), Src, Fyn and LAT. Strikingly, EMMTC also blocked platelet adhesion to immobilized collagen and convulxin (a snake venom-derived protein that activates platelet glycoprotein VI receptor). The attenuation of phosphorylation of focal adhesion kinase (FAK) was observed during adhesion. Taken together, our results presented here demonstrate that the chalcone derivative EMMTC affects collagen-induced protein tyrosine phosphorylation and TXB(2) formation and functionally blocks collagen-induced platelet aggregation and adhesion.
Asunto(s)
Chalcona/análogos & derivados , Colágeno/metabolismo , Adhesividad Plaquetaria/efectos de los fármacos , Agregación Plaquetaria/efectos de los fármacos , Tiofenos/farmacología , Animales , Chalcona/administración & dosificación , Chalcona/farmacología , Colágeno/farmacología , Venenos de Crotálidos/metabolismo , Proteína-Tirosina Quinasas de Adhesión Focal/efectos de los fármacos , Proteína-Tirosina Quinasas de Adhesión Focal/metabolismo , Humanos , Concentración 50 Inhibidora , Lectinas Tipo C/metabolismo , Fosforilación/efectos de los fármacos , Prostaglandina-Endoperóxido Sintasas/metabolismo , Tiofenos/administración & dosificación , Tromboxano B2/biosíntesis , Tromboxano-A Sintasa/metabolismo , Tirosina/efectos de los fármacos , Tirosina/metabolismoRESUMEN
BACKGROUND AND PURPOSE: During the development of atherosclerotic plaques, vascular smooth muscle cells (VSMCs) migrate from the media to the intima through the basement membrane and interstitial collagenous matrix, and proliferate to form neointima. Here, we investigate the mechanism of VSMC migration and proliferation caused by aggretin, a snake venom integrin alpha2beta1 agonist. EXPERIMENTAL APPROACH: Cultures of rat and human VSMCs were treated with aggretin and the signal transduction pathways induced by this agonist were examined by Western blotting, immunoprecipitation and electrophoretic mobility shift assay techniques. KEY RESULTS: Aggretin-induced VSMC proliferation was blocked by a monoclonal antibody (mAb) against integrin alpha2 (AII2E10) or against the platelet-derived growth factor receptor (PDGFR)-beta. Proliferation was also blocked by inhibition of the tyrosine kinase Src with PP2, phospholipase C (PLC) with U73122, extracellular signal-regulated kinase (ERK) with PD98059 or nuclear factor-kappa B (NF-kB) activation with pyrrolidine dithiocarbamate (PDTC). VSMC migration towards immobilized aggretin was increased in a modified Boyden chamber and this effect was blocked by alpha2beta1-Src-PLC-MAPK axis inhibitors, but not by PDTC, PDGFR-beta mAb, or a phosphoinositide-3 kinase inhibitor, LY294002. Aggretin stimulated the phosphorylation of PDGFR-beta, Src and ERK in a time-dependent manner. NF-kB translocation and platelet-derived growth factor (PDGF)-BB production were also observed. The ERK activation, NF-kB translocation and PDGF-BB production were blocked by PP2, U73122 and PD98059. CONCLUSIONS AND IMPLICATIONS: Aggretin induces VSMC proliferation and migration mainly through binding to integrin alpha2beta1, and subsequently activates Src, PLC and ERK pathways, inducing NF-kB activation and PDGF production.
Asunto(s)
Integrina alfa2beta1/agonistas , Músculo Liso Vascular/citología , Miocitos del Músculo Liso/efectos de los fármacos , FN-kappa B/metabolismo , Factor de Crecimiento Derivado de Plaquetas/biosíntesis , Venenos de Víboras/farmacología , Animales , Becaplermina , Movimiento Celular , Proliferación Celular , Células Cultivadas , Activación Enzimática , Quinasas MAP Reguladas por Señal Extracelular/metabolismo , Humanos , Integrina alfa2beta1/fisiología , Lectinas Tipo C , Miocitos del Músculo Liso/citología , Miocitos del Músculo Liso/fisiología , Fosforilación , Transporte de Proteínas , Proteínas Proto-Oncogénicas c-sis , Ratas , Receptor beta de Factor de Crecimiento Derivado de Plaquetas/agonistas , Transducción de Señal , Fosfolipasas de Tipo C/fisiología , Familia-src Quinasas/metabolismoRESUMEN
Platelet microparticles (MPs) are membrane vesicles shed by platelets after activation, and carry antigens characteristic of intact platelets, such as glycoprotein (GP) IIb/IIIa, GPIb and P-selectin. Elevated platelet MPs have been observed in many disorders in which platelet activation is documented. Recently, platelet GPIb has been implicated in the mediation of platelet-leukocyte interaction via binding to its ligand Mac-1 on leukocyte. The role of GPIb for mediating adhesion-activation interactions between platelet MPs and leukocytes has not been clarified. In this study we investigate the role of GPIb in the interplay between platelet MPs and neutrophils. Platelet MPs were obtained from collagen-stimulated platelet-rich plasma (PRP). In a study model of neutrophil aggregation, platelet MPs can serve a bridge to support neutrophil aggregation under venous level shear stress, suggesting that platelet MPs may enhance leukocyte aggregation, which would bear clinical relevance in diseases where the platelet MPs are elevated. The level of aggregation can be reduced by GPIb blocking antibodies, AP1 and SZ2, but not by anti-CD18 mAb. The GPIb blocking antibodies also decreased platelet MP-mediated neutrophil activation, including beta2 integrin expression, adherence-dependent superoxide release and platelet MP-mediated neutrophil adherence to immobilized fibrinogen. Our data provide the evidence for the involvement of GPIb-Mac-1 interaction in the cross-talk between platelet MPs and neutrophils.
Asunto(s)
Plaquetas/fisiología , Activación Neutrófila , Complejo GPIb-IX de Glicoproteína Plaquetaria/fisiología , Adhesión Celular , Citometría de Flujo , Humanos , Neutrófilos/citología , Neutrófilos/metabolismo , Especies Reactivas de Oxígeno/metabolismoRESUMEN
The metastasis of tumor cells to bone involves migration, invasion and adhesion to bone. Breast and prostate cancer cells have predilection for spreading to bone. Snake venom-derived arginine-glycine-aspartic acid (RGD)-containing disintegrins (e.g. rhodostomin) have been demonstrated to inhibit cell adhesion. Here, we found that rhodostomin inhibited the adhesion of breast and prostate carcinoma cells to both unmineralized and mineralized bone extracellular matrices in a dose-dependent manner, without affecting the viability of tumor cells. In addition, rhodostomin also inhibited the migration and invasion of breast and prostate carcinoma cells. It specifically inhibited the binding of monoclonal antibody (MoAb) 7E3, which recognizes integrin alphavbeta3, to tumor cells, but not those of other MoAbs against other integrin subunits such as alpha2, alpha3, alpha5 and beta1. As breast cancer cells MDA-MB-231 were locally injected into tibia in nude mice, histological examination of the tibia of control group revealed that most of the cancellous bone had been replaced by the breast cancer cells after 28 days' inoculation. In contrast, co-administration of trigramin with cancer cells markedly inhibited tumor growth and bone destruction. Taken together, disintegrins strongly inhibit the adhesion, migration, invasion of tumor cells and also tumor growth of human breast cancer cells in bone as well. Therefore, disintegrins may be developed as alternate therapy for bone metastasis of cancer cells.
Asunto(s)
Neoplasias Óseas/secundario , Neoplasias de la Mama/fisiopatología , Adhesión Celular/efectos de los fármacos , Desintegrinas/farmacología , Neoplasias de la Próstata/fisiopatología , Animales , Anticuerpos Monoclonales/metabolismo , Apoptosis/efectos de los fármacos , Neoplasias Óseas/tratamiento farmacológico , Movimiento Celular/efectos de los fármacos , Desintegrinas/uso terapéutico , Relación Dosis-Respuesta a Droga , Matriz Extracelular/metabolismo , Femenino , Citometría de Flujo , Fluorescencia , Humanos , Péptidos y Proteínas de Señalización Intercelular , Masculino , Ratones , Ratones Desnudos , Invasividad Neoplásica/prevención & control , Péptidos/farmacología , Ratas , Tibia/patología , Células Tumorales CultivadasRESUMEN
Injury to peripheral nerves may result in severe and intractable neuropathic pain. Many efforts have been focused on the elucidation of the mechanisms of neuropathic pain. It was found here that integrin plays an important role in the induction of neuropathic pain and treatment of disintegrin is able to attenuate neuropathic pain. The rats were induced hyperalgesia by tightly ligating the L5 spinal nerve and cut just distal to the ligature on one side. Mechanical and thermal stimuli were applied in the middle dermatome of the hind paw. Epidural administration of triflavin (TFV), an arginine-glycine-aspartic acid (RGD) containing disintegrin, inhibited hyperalgesia induced by either mechanical or thermal stimulation. Immunohistochemistry showed that the sprouting of sympathetic nerves into DRG by neuropathic surgery was markedly inhibited by TFV. Beta 1 integrin mRNA of L5 DRG increased immediately 1 day after tight ligation and cut of L5 spinal nerve. However, beta 1 integrin mRNA in uninjured L4 DRG increased later on Day 3 after surgery. On the other hand, alpha-CGRP precursor mRNA decreased in ipsilateral L5 DRG but increased in L4 DRG after neuropathic surgery. Immunohistochemistry shows that beta 3 integrins of L5 as well as L4 increased in response to neuropathic surgery and administration of triflavin antagonized the increasing action. These results suggest that there is interaction between injured and uninjured neurons and the induction of neuropathic pain is related to neuronal sprouting. Disintegrin is able to inhibit neuronal sprouting and the induction of hyperalgesia induced by peripheral nerve injury and may thus be a new category of drugs to be developed for the treatment of neuropathic pain.
Asunto(s)
Venenos de Crotálidos/uso terapéutico , Desintegrinas/uso terapéutico , Dolor/prevención & control , Péptidos/uso terapéutico , Animales , Venenos de Crotálidos/farmacología , Desintegrinas/farmacología , Integrinas/biosíntesis , Integrinas/fisiología , Dolor/metabolismo , Dimensión del Dolor/efectos de los fármacos , Dimensión del Dolor/métodos , Péptidos/farmacología , Ratas , Ratas Sprague-Dawley , Nervios Espinales/efectos de los fármacos , Nervios Espinales/lesiones , Nervios Espinales/metabolismoRESUMEN
Disintegrins are a group of Arg (or Lys)-Gly-Asp-containing snake venom proteins which inhibit platelet aggregation via the blockade of alpha(IIb)beta(3) integrin. Here, we studied the effect of rhodostomin, a disintegrin purified from the venom of Calloselasma rhodostoma, on the functions of neutrophils. By flow cytometric analysis of whole blood, we found that rhodostomin interacted with leukocytes of the myeloid and monocytic lineage as well as with platelets. The binding of rhodostomin to neutrophils could reach saturation in a dose-dependent manner, and its binding was increased in neutrophils stimulated with phorbol 12-myristate 13-acetate (PMA) and N-formyl-Met-Leu-Phe. EDTA did not inhibit the binding of rhodostomin. In addition, bound rhodostomin was not internalized. Soluble fibrinogen, a natural ligand of Mac-1 (CD11b/CD18, alpha(M)beta(2)), and the peptide, GRGDS, inhibited the binding of rhodostomin to PMA-activated neutrophils, while 7E3, a monoclonal antibody (mAb) raised against beta(3) integrin, or mAbs raised against alpha(M) and beta(2) integrin did not. Rhodostomin blocked the Mac-1-dependent adhesion of neutrophils to immobilized fibrinogen, in parallel with decreasing the production of superoxide from adherent neutrophils. Taken together, our results indicate that rhodostomin binds to activated neutrophils in an RGD-dependent manner, blocks the adhesion of activated neutrophils to fibrinogen and attenuates superoxide production, suggesting that rhodostomin may have anti-inflammatory properties.
Asunto(s)
Adhesión Celular/efectos de los fármacos , Fibrinógeno/farmacología , Neutrófilos/fisiología , Péptidos/farmacología , Inhibidores de Agregación Plaquetaria/farmacología , Superóxidos/sangre , Humanos , Cinética , Neutrófilos/efectos de los fármacos , Valores de ReferenciaRESUMEN
Triflamp, a 28 kDa snake venom metalloproteinase purified from the venom of Trimeresurus flavoviridis, possesses the proteolytic activities toward P-selectin glycoprotein ligand-1 (PSGL-1), glycoprotein Ib (GPIb) and fibrinogen. In human whole blood preparation, however, triflamp (6 microg/ml) failed to cleave neutrophil PSGL-1 and platelet GPIb. Human alpha2-macroglobulin (alpha2M) was mainly responsible for the neutralization of the proteolytic effects of triflamp on PSGL-1, GPIb and fibrinogen. Human alpha2M neutralized triflamp at a stoichiometry about 1.1:1 (molar basis) determined by azocaseinolysis. SDS-PAGE analysis revealed that triflamp cleaved the bait-region of alpha2M. Western blot demonstrated that triflamp interacted with the C-terminal half-subunits of truncated alpha2M resulting in the formation of high-molecular-weight species of alpha2M-triflamp complexes. In the presence of competing nucleophile, 0.2 M methylamine, the proteolytic activity of triflamp was conserved. In vivo we found that mice neutrophils were resistant to the cleavage of PSGL-1 by triflamp. However, mouse PSGL-1 and GPIb were susceptible to be cleaved by triflamp in washed mouse neutrophil and platelet preparation, respectively. Similarly, mouse serum was also responsible for the inactivation of the proteolytic activity of triflamp. This study provides direct evidences for the reasonable explanation regarding the reduced proteolytic activity of triflamp toward its substrates in whole blood preparation and in vivo model.