RESUMEN
BACKGROUND: Patients with symptomatic internal carotid artery (ICA) stenosis are at high risk of recurrent ischemic stroke and require early interventional treatment and antiplatelet therapy. Increased bleeding rates might counterbalance the periprocedural efficacy of intensified platelet inhibition. We aim to investigate, whether Revacept, a competitive antagonist of glycoprotein VI, adjunct to standard antiplatelet therapy reduces the occurrence of ischemic lesions in patients with symptomatic ICA stenosis. METHODS: International, multicenter (16 sites), 3-arm, randomized (1:1:1), double-blind, and placebo-controlled study with parallel groups, including patients with symptomatic ICA stenosis. A single infusion over 20 minutes of either placebo, 40 mg or 120 mg Revacept in addition to guideline-conform antiplatelet therapy was evaluated with regard to the exploratory efficacy end point: Number of new ischemic lesions on diffusion-weighted magnetic resonance imaging after treatment initiation. Main clinical outcome was the combined safety and efficacy end point including any stroke or death, transient ischemic attack, myocardial infarction, coronary intervention, and bleeding complications during follow-up. RESULTS: Out of 160 randomized patients, 158 patients (68±10.1 years, 24% female) received study medication (51 patients placebo, 54 patients 40 mg Revacept and 53 patients 120 mg Revacept) and were followed for 11.2±2.3 months. A total of 1.16 (95% CI, 0.88-1.53)/1.05 (95% CI, 0.78-1.42; P=0.629)/0.63 (95% CI, 0.43-0.93) new diffusion-weighted magnetic resonance imaging lesions per patient were detected in the placebo/40 mg/120 mg Revacept groups, without statistical evidence of a difference. A reduction of the combined safety and efficacy end point during the study period was observed in patients who received 120 mg (HR, 0.46 [95% CI, 0.21-0.99]; P=0.047), but not 40 mg Revacept compared with placebo (HR, 0.72 [95% CI, 0.37-1.42]; P=0.343). CONCLUSIONS: Revacept 120 mg reduced the combined safety and efficacy end point in patients with symptomatic ICA stenosis. REGISTRATION: URL: https://www. CLINICALTRIALS: gov; Unique Identifier: NCT01645306.
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Estenosis Carotídea , Glicoproteínas , Fragmentos Fc de Inmunoglobulinas , Inhibidores de Agregación Plaquetaria , Anciano , Estenosis Carotídea/tratamiento farmacológico , Constricción Patológica/complicaciones , Femenino , Glicoproteínas/efectos adversos , Humanos , Fragmentos Fc de Inmunoglobulinas/efectos adversos , Masculino , Persona de Mediana Edad , Inhibidores de Agregación Plaquetaria/efectos adversos , Accidente Cerebrovascular , Resultado del TratamientoRESUMEN
BACKGROUND/AIMS: Oxidative modifications of low-density lipoprotein (ox-LDL) play a key role in initial steps of atheroprogression possibly via specific scavenger receptors on inflammatory and endothelial cells. Amongst others, CD68 might play a crucial role in this leading to fatty streak formation. METHODS: Different CD68-Fc fusion proteins were cloned, expressed and tested in vitro for their oxLDL binding properties as a decoy for endogenous oxLDL. Physiological functions were tested in foam cell assays with human monocytes in culture and by binding oxLDL from human blood. The best suited candidate FcIgG2-FL-CD68 was injected twice weekly in LDL receptor and ApoBec deficient mice (LDLR-/-/Apobec-/-), and the oxLDL content was measured in peripheral blood, in different cell types of the spleen and aortic wall by specific oxLDL antibodies using flow cytometry. RESULTS: Different variants of the CD68-Fc bound to copper-oxided LDL (oxLDL), LDL and to a lesser extent HDL with different efficacy in an ELISA based binding assay in vitro. Native oxLDL content in human blood derived from patients with extended atherosclerosis was reduced after passage through a specific protein G column conjugated with the different CD68-Fc fusion proteins. Foam cell formation from human peripheral blood monocyte-platelet co-culture was reduced by the most effective CD68-Fc fusion proteins. oxLDL was not increased in the blood but markedly increased in the vessel wall from LDLR-/-/Apobec-/- mice at an early stage of atherosclerosis. Platelet-like cells in the vessel well contributed most to the increase in tissue oxLDL. FcIgG2-FL-CD68, reduced oxLDL content of aortic vessel wall cells from LDLR-/-/Apobec-/- mice. However a tissue specific reduction on the oxLDL content in peripheral blood, the spleen or cells from the aortic vessel by FcIgG2-FL-CD68 could not be shown. CONCLUSION: Platelets contribute to increased tissue oxLDL in the aortic wall but not in peripheral blood. CD68 seems to play a role in the oxLDL metabolism in the vessel wall at early stages of atherosclerosis. FcIgG2-FL-CD68 could serve as a novel therapeutic option to modify the oxLDL content in the vessel wall.
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Desaminasas APOBEC-1/genética , Antígenos CD/metabolismo , Antígenos de Diferenciación Mielomonocítica/metabolismo , Plaquetas/metabolismo , Lipoproteínas LDL/genética , Desaminasas APOBEC-1/deficiencia , Animales , Antígenos CD/genética , Antígenos de Diferenciación Mielomonocítica/genética , Aterosclerosis/metabolismo , Aterosclerosis/patología , Plaquetas/citología , Vasos Sanguíneos/metabolismo , Vasos Sanguíneos/patología , Modelos Animales de Enfermedad , Células Espumosas/citología , Células Espumosas/metabolismo , Humanos , Fragmentos Fc de Inmunoglobulinas/genética , Fragmentos Fc de Inmunoglobulinas/metabolismo , Lipoproteínas HDL/metabolismo , Lipoproteínas LDL/análisis , Lipoproteínas LDL/deficiencia , Lipoproteínas LDL/metabolismo , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Oxidación-Reducción , Unión Proteica , Proteínas Recombinantes de Fusión/biosíntesis , Proteínas Recombinantes de Fusión/genética , Proteínas Recombinantes de Fusión/aislamiento & purificaciónRESUMEN
RATIONALE: Autoantibodies directed against the second extracellular loop of the cardiac ß1-adrenergic receptor (ß1-AR) are thought to contribute to the pathogenesis of dilated cardiomyopathy (DCM) and Chagas heart disease. Various approaches have been used to detect such autoantibodies; however, the reported prevalence varies largely, depending on the detection method used. OBJECTIVE: We analyzed sera from 167 DCM patients (ejection fraction<45%) and from 110 age-matched volunteers who did not report any heart disease themselves, with an often used simple peptide-ELISA approach, and compared it with a novel whole cell-based ELISA, using cells expressing the full transgene for the human ß1-AR. Additionally, 35 patients with hypertensive heart disease with preserved ejection fraction were investigated. METHODS AND RESULTS: The novel assay was designed according to the currently most reliable anti-TSH receptor antibody-ELISA used to diagnose Graves disease ("third-generation assay") and also detects the target antibodies by competition with a specific monoclonal anti-ß1-AR antibody (ß1-AR MAb) directed against the functionally relevant ß1-AR epitope. Anti-ß1-AR antibodies were detected in ≈60% of DCM patients and in ≈8% of healthy volunteers using the same cutoff values. The prevalence of these antibodies was 17% in patients with hypertensive heart disease. Anti-ß1-AR antibody titers (defined as inhibition of ß1-AR MAb-binding) were no longer detected after depleting sera from IgG antibodies by protein G adsorption. In contrast, a previously used ELISA conducted with a linear 26-meric peptide derived from the second extracellular ß1-AR loop yielded a high number of false-positive results precluding any specific identification of DCM patients. CONCLUSIONS: We established a simple and efficient screening assay detecting disease-relevant ß1-AR autoantibodies in patient sera yielding a high reproducibility also in high throughput screening. The assay was validated according to "good laboratory practice" and can serve as a companion biodiagnostic assay for the development and evaluation of antibody-directed therapies in antibody-positive heart failure.
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Autoanticuerpos/inmunología , Cardiomiopatía Dilatada/inmunología , Ensayo de Inmunoadsorción Enzimática/métodos , Receptores Adrenérgicos beta 1/inmunología , Animales , Anticuerpos Monoclonales/inmunología , Afinidad de Anticuerpos/inmunología , Especificidad de Anticuerpos/inmunología , Autoanticuerpos/sangre , Secuencia de Bases , Cardiomiopatía Dilatada/sangre , Cardiomiopatía Dilatada/diagnóstico , Femenino , Humanos , Ratones , Ratones Endogámicos BALB C , Datos de Secuencia Molecular , Receptores Adrenérgicos beta 1/genética , Reproducibilidad de los Resultados , Sensibilidad y Especificidad , Células Sf9 , TransfecciónRESUMEN
BACKGROUND AND AIMS: Glycoprotein VI (GPVI) is the major platelet-specific collagen receptor. GPVI shedding with generation of soluble GPVI (sGPVI) is an endogenous feedback mechanism preventing platelet overstimulation. sGPVI has not been investigated in patients with chronic coronary syndrome (CCS) undergoing percutaneous coronary intervention (PCI), especially regarding its potential value as a predictor of ischemic and bleeding risk. METHODS: Baseline plasma sGPVI levels were available in 318 patients with CCS undergoing PCI. Platelet function was assessed by measuring both adenosine diphosphate (ADP) and collagen-induced platelet aggregation. Co-primary endpoints were a composite of death or myocardial injury at 48 hours after PCI, and Bleeding Academic Research Consortium (BARC) type 1 to 5 bleeding at 30 days. RESULTS: There was no significant correlation between sGPVI and platelet function at baseline or at 48 hours after PCI and loading with antiplatelet drugs. Baseline plasma sGPVI levels were not associated with the ischemic risk: the incidence of the ischemic endpoint was 25.0% in the lower, 22.9% in the middle, and 26.7% in the upper sGPVI tertile (p = 0.82). There was a significant nonlinear relationship between sGPVI and the risk of bleeding: the incidence of the bleeding endpoint was 11.8% in the lower, 12.6% in the middle, and 26.4% in the upper sGPVI tertile (p = 0.006). CONCLUSION: In patients with CCS undergoing PCI, plasma levels of sGPVI did not correlate with ADP- or collagen-induced platelet aggregation. Patients with higher baseline levels of sGPVI may carry an increased risk of bleeding at 30 days after PCI but no excess risk of ischemic events.
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Intervención Coronaria Percutánea , Humanos , Intervención Coronaria Percutánea/efectos adversos , Agregación Plaquetaria , Hemorragia/inducido químicamente , Inhibidores de Agregación Plaquetaria/efectos adversos , Glicoproteínas/farmacología , Colágeno/farmacología , Adenosina Difosfato/farmacología , Resultado del TratamientoRESUMEN
Cyclooxygenase (COX)-2-derived prostanoids can influence several processes that are linked to carcinogenesis. We aimed to address the hypothesis that platelets contribute to aberrant COX-2 expression in HT29 colon carcinoma cells and to reveal the role of platelet-induced COX-2 on the expression of proteins involved in malignancy and marker genes of epithelial-mesenchymal transition (EMT). Human platelets cocultured with HT29 cells rapidly adhered to cancer cells and induced COX-2 mRNA expression, but not protein synthesis, which required the late release of platelet-derived growth factor and COX-2 mRNA stabilization. Platelet-induced COX-2-dependent prostaglandin E2 (PGE2) synthesis in HT29 cells was involved in the downregulation of p21(WAF1/CIP1) and the upregulation of cyclinB1 since these effects were prevented by rofecoxib (a selective COX-2 inhibitor) and rescued by exogenous PGE2. Galectin-3, which is highly expressed in HT29 cells, is unique among galectins because it contains a collagen-like domain. Thus, we studied the role of galectin-3 and platelet collagen receptors in platelet-induced COX-2 overexpression. Inhibitors of galectin-3 function (ß-lactose, a dominant-negative form of galectin-3, Gal-3C, and anti-galectin-3 antibody M3/38) or collagen receptor-mediated platelet adhesion (revacept, a dimeric platelet collagen receptor GPVI-Fc) prevented aberrant COX-2 expression. Inhibition of platelet-cancer cell interaction by revacept was more effective than rofecoxib in preventing platelet-induced mRNA changes of EMT markers, suggesting that direct cell-cell contact and aberrant COX-2 expression synergistically induced gene expression modifications associated with EMT. In conclusion, our findings provide the rationale for testing blockers of collagen binding sites, such as revacept, and galectin-3 inhibitors in the prevention of colon cancer metastasis in animal models, followed by studies in patients.
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Plaquetas/efectos de los fármacos , Plaquetas/patología , Comunicación Celular/efectos de los fármacos , Neoplasias del Colon/sangre , Neoplasias del Colon/enzimología , Ciclooxigenasa 2/genética , Ciclooxigenasa 2/metabolismo , Sitios de Unión , Plaquetas/enzimología , Plaquetas/metabolismo , Comunicación Celular/genética , Línea Celular Tumoral , Neoplasias del Colon/genética , Neoplasias del Colon/metabolismo , Ciclina B1/genética , Ciclina B1/metabolismo , Inhibidores de la Ciclooxigenasa 2/farmacología , Dinoprostona/genética , Dinoprostona/metabolismo , Regulación hacia Abajo/efectos de los fármacos , Transición Epitelial-Mesenquimal/efectos de los fármacos , Transición Epitelial-Mesenquimal/genética , Galectina 3/antagonistas & inhibidores , Galectina 3/genética , Galectina 3/metabolismo , Expresión Génica/efectos de los fármacos , Glicoproteínas/farmacología , Células HT29 , Humanos , Fragmentos Fc de Inmunoglobulinas/farmacología , Lactonas/farmacología , Lactosa/farmacología , Factor de Crecimiento Derivado de Plaquetas/genética , Factor de Crecimiento Derivado de Plaquetas/metabolismo , ARN Mensajero/genética , Receptores de Colágeno/genética , Receptores de Colágeno/metabolismo , Sulfonas/farmacología , Regulación hacia Arriba/efectos de los fármacosRESUMEN
RATIONALE: There is strong evidence that oxidative modification of low-density lipoprotein (oxLDL) plays a critical role in atherogenesis and that oxLDL may profoundly influence the mechanical stability of atherosclerotic plaques. OBJECTIVE: To block oxLDL, we designed, expressed, and tested Fc-CD68, a soluble oxLDL binding protein consisting of human Fc and the extracellular domain of the human oxLDL-binding receptor CD68. METHODS AND RESULTS: Fc-CD68 bound with high specific affinity to oxLDL and strongly bound and colocalized with oxLDL in plaques. To study the effects of repeated administrations of Fc-CD68 on the progression of atherosclerosis and plaque vulnerability, 12- and 16-week old cholesterol-fed ApoE(-/-) mice received either Fc-CD68 (n = 6) or Fc control protein (n = 6 to 8) thrice weekly for 4 weeks. Macroscopic and histological analysis of Sudan red lipid staining showed strong and significant reduction of plaque extension in the aorta and in the aortic root, respectively. Histological analysis of pentachrome- and Sirius-stained sections of the brachiocephalic arteries of 20 week-old ApoE(-/-) mice revealed that Fc-CD68 significantly reduced the occurrence of spontaneous ruptures of established plaques by ≈20%, compared with Fc and drastically increased the collagen content of plaques. Furthermore, in immunostained sections of the brachiocephalic artery and the aortic root, Fc-CD68 reduced the infiltration of plaques with T lymphocytes, and macrophages by ≈50% and 30%, respectively. CONCLUSIONS: The oxLDL binding protein Fc-CD68 attenuates atherosclerosis and strengthens the stability of atherosclerotic plaques.
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Antígenos CD/genética , Antígenos de Diferenciación Mielomonocítica/genética , Aterosclerosis/patología , Tronco Braquiocefálico/efectos de los fármacos , Tronco Braquiocefálico/patología , Fragmentos Fc de Inmunoglobulinas/genética , Placa Aterosclerótica/patología , Proteínas Recombinantes de Fusión/farmacología , Animales , Aorta/efectos de los fármacos , Aorta/patología , Apolipoproteínas E/deficiencia , Aterosclerosis/metabolismo , Unión Competitiva , Proteínas Portadoras/metabolismo , Proteínas Portadoras/farmacología , Colágeno/metabolismo , Progresión de la Enfermedad , Humanos , Inflamación/metabolismo , Inflamación/patología , Metabolismo de los Lípidos , Lipoproteínas LDL/metabolismo , Macrófagos/patología , Masculino , Ratones , Ratones Noqueados , Placa Aterosclerótica/metabolismo , Proteínas Recombinantes de Fusión/metabolismo , Linfocitos T/patologíaRESUMEN
Glycoprotein VI (GPVI) is a platelet-specific receptor for collagen and fibrin, regulating important platelet functions such as platelet adhesion and thrombus growth. Although the blockade of GPVI function is widely recognized as a potent anti-thrombotic approach, there are limited studies focused on site-specific targeting of GPVI. Using computational modeling and bioinformatics, we analyzed collagen- and CRP-binding surfaces of GPVI monomers and dimers, and compared the interacting surfaces with other mammalian GPVI isoforms. We could predict a minimal collagen-binding epitope of GPVI dimer and designed an EA-20 antibody that recognizes a linear epitope of this surface. Using platelets and whole blood samples donated from wild-type and humanized GPVI transgenic mice and also humans, our experimental results show that the EA-20 antibody inhibits platelet adhesion and aggregation in response to collagen and CRP, but not to fibrin. The EA-20 antibody also prevents thrombus formation in whole blood, on the collagen-coated surface, in arterial flow conditions. We also show that EA-20 does not influence GPVI clustering or receptor shedding. Therefore, we propose that blockade of this minimal collagen-binding epitope of GPVI with the EA-20 antibody could represent a new anti-thrombotic approach by inhibiting specific interactions between GPVI and the collagen matrix.
RESUMEN
Platelets play a critical role in the pathophysiology of reperfusion, sepsis, and cardiovascular diseases. In a multiple step process, they adhere to activated endothelium and release proinflammatory cytokines thereby promoting the inflammatory process. Glycoprotein VI (GPVI) is the major collagen receptor on the platelet surface and triggers platelet activation and primary hemostasis. Activation of GPVI leads to stable platelet adhesion and degranulation of platelet granules. However, GPVI is critically involved in platelet adhesion to activated endothelium without exposure of subendothelial matrix. Earlier studies show that the soluble GPVI-Fc binds to collagen and protects mice from atherosclerosis and decreases neointima proliferation after arterial injury. Here, we show for the first time that recombinant GPVI-Fc binds to activated endothelium mainly via vitronectin and prevents platelet/endothelial interaction. Administration of GPVI-Fc reduced infarct size and preserved cardiac function in a mouse model of myocardial infarction. This process was associated with reduced GPVI-induced platelet degranulation and release of proinflammatory cytokines in vitro and in vivo. Taken together, administration of GPVI-Fc offers a novel strategy to control platelet-mediated inflammation and to preserve myocardial function following myocardial infarction.
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Endotelio Vascular/metabolismo , Corazón/fisiología , Fragmentos Fc de Inmunoglobulinas/metabolismo , Ataque Isquémico Transitorio/metabolismo , Adhesividad Plaquetaria/fisiología , Glicoproteínas de Membrana Plaquetaria/metabolismo , Animales , Células CHO , Bovinos , Cricetinae , Cricetulus , Endotelio Vascular/patología , Células Endoteliales de la Vena Umbilical Humana , Humanos , Ataque Isquémico Transitorio/patología , Masculino , Ratones , Ratones Endogámicos C57BL , Multimerización de Proteína/fisiologíaRESUMEN
BACKGROUND: Blocking of glycoprotein VI-dependent pathways by interfering in vascular collagen sites is commonly seen as an attractive target for an antiplatelet therapy of acute atherosclerotic diseases such as myocardial infarction or stroke. Revacept (soluble dimeric glycoprotein VI-Fc fusion protein) has been shown to reduce platelet adhesion by blocking vascular collagen in plaques or erosion and to be safe in preclinical studies. A dose-escalating clinical phase I study was performed to assess the safety, tolerability, pharmacokinetics, and pharmacodynamics of Revacept in humans. METHODS AND RESULTS: In a first-in-humans study, 30 healthy men received a single intravenous administration of 10, 20, 40, 80, or 160 mg Revacept. The serum concentration-time courses of each dosage of Revacept showed a narrow variation and a concentration and time dependence. Revacept did not significantly affect the bleeding time. Collagen-induced platelet aggregation was dose-dependently inhibited up to 48 hours at lower doses and for 7 days after higher dose levels. In contrast, ADP- or thrombin receptor activating peptide-dependent platelet aggregation remained unaltered. There were no relevant drug-related adverse events or drug-related changes in laboratory parameters (biochemistry, hematology, and coagulation parameters). There were no drug-related changes in blood pressure, pulse rate, or ECG parameters (including 24-hour Holter monitoring). No anti-Revacept antibodies were detected. CONCLUSION: This phase I study demonstrated that Revacept is a safe and well-tolerated new antiplatelet compound with a clear dose-dependent pharmacokinetic profile with specific, dose-related inhibition of platelet aggregation despite completely unaltered general hemostasis. CLINICAL TRIAL REGISTRATION: URL: www.clinicaltrials.gov. Unique identifier: NCT 01042964. URL: eudract.ema.europa.eu. Identifier: 2005-004656-12.
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Glicoproteínas/administración & dosificación , Fragmentos Fc de Inmunoglobulinas/administración & dosificación , Inhibidores de Agregación Plaquetaria/administración & dosificación , Agregación Plaquetaria/efectos de los fármacos , Glicoproteínas de Membrana Plaquetaria/administración & dosificación , Adulto , Animales , Células CHO , Colágeno/administración & dosificación , Cricetinae , Cricetulus , Relación Dosis-Respuesta a Droga , Hemostasis/efectos de los fármacos , Humanos , Fragmentos Fc de Inmunoglobulinas/efectos adversos , Inyecciones Intravenosas , Masculino , Inhibidores de Agregación Plaquetaria/efectos adversos , Inhibidores de Agregación Plaquetaria/farmacocinética , Recuento de Plaquetas , Glicoproteínas de Membrana Plaquetaria/efectos adversos , Glicoproteínas de Membrana Plaquetaria/farmacocinética , Proteínas Recombinantes de Fusión/administración & dosificación , Proteínas Recombinantes de Fusión/efectos adversos , Proteínas Recombinantes de Fusión/farmacocinética , Adulto JovenRESUMEN
Glycoprotein VI (GPVI) mediates binding of platelets to subendothelial collagen during acute arterial thrombosis. GPVI interactions with the activated atherosclerotic vascular endothelium during early atherosclerosis, however, are not well understood. In ApoE-/- mice, platelet adhesion to atherosclerotic arteries was increased, as measured by intravital microscopy. This platelet adhesion was significantly inhibited by IV injection of GPVI-Fc (1 mg/kg body weight). Atherosclerosis in ApoE-/- mice was attenuated both after 7 and 10 weeks of treatment with the anti-GPVI antibody JAQ1 (2 mg/kg body weight i.p. twice weekly). Binding of GPVI-Fc (1 mg/kg IV) occurred to deeper layers, but also to the luminal site of plaques in atherosclerotic rabbits, but not to the vessel wall of healthy littermates. Gene transfer of GPVI-Fc to the carotid vascular wall significantly attenuated athero-progression and endothelial dysfunction in atherosclerotic rabbits in vivo. Specific binding of the soluble GPVI receptor (GPVI-Fc) to fibronectin was found in vitro to coated ELISA plates. Platelet adhesion to fibronectin was significantly inhibited both by GPVI-Fc and by the anti-GPVI antibody 5C4 ex vivo in flow chamber experiments. GPVI plays a role in platelet adhesion to atherosclerotic endothelium in the absence of plaque rupture. Inhibition of GPVI both via GPVI-Fc and anti-GPVI-antibodies results in protection against atherosclerosis in both cholesterol-fed rabbits and ApoE-/- mice. This novel mechanism of GPVI-mediated platelet adhesion-possibly via fibronectin-could relevantly contribute to platelet-triggered atheroprogression.
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Aterosclerosis/patología , Endotelio Vascular/metabolismo , Fibronectinas/metabolismo , Adhesividad Plaquetaria/fisiología , Glicoproteínas de Membrana Plaquetaria/metabolismo , Adenoviridae/genética , Animales , Anticuerpos Monoclonales/farmacología , Apolipoproteínas E/fisiología , Aterosclerosis/metabolismo , Células CHO , Arterias Carótidas/metabolismo , Arterias Carótidas/patología , Cricetinae , Cricetulus , Endotelio Vascular/patología , Técnicas de Transferencia de Gen , Proteínas Fluorescentes Verdes , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Activación Plaquetaria , Glicoproteínas de Membrana Plaquetaria/genética , Glicoproteínas de Membrana Plaquetaria/inmunología , ConejosRESUMEN
Patients with stroke or transient ischemic attacks (TIAs) and internal carotid artery stenosis harbor an increased risk of recurrent stroke especially within 2 weeks after the first event. In addition, the revascularization procedure itself (carotid endarterectomy [CEA] or carotid artery stenting [CAS]) is associated with both clinically apparent and silent brain infarctions, mainly caused by the embolic nature of the ruptured carotid plaque. The glycoprotein VI (GPVI) fusion protein Revacept is a highly specific antithrombotic drug without direct inhibition of systemic platelet function that might reduce periprocedural distal embolization from the vulnerable ruptured plaque located at the internal carotid artery. By shielding collagen at the site of vascular injury, Revacept inhibits plaque-mediated platelet adhesion and aggregation, while not directly affecting systemic hemostasis. In this phase II study, 158 patients with symptomatic carotid artery stenosis with recent TIA or stroke were randomized to receive a single dose of either Revacept (40 or 120 mg) or placebo. All patients were on standard secondary preventive therapy (statins and platelet inhibition) and underwent CEA, CAS, or best medical therapy according to current guidelines. The efficacy of Revacept was evaluated by exploratory assessment of new diffusion-weighted imaging lesions on magnetic resonance imaging after the revascularization procedure; a combination of cardiovascular events (ischemic and hemorrhagic stroke, TIA, myocardial infarction, or coronary intervention) and bleeding complications served to assess clinically critical patients' outcome and safety. This exploratory phase II randomized, double-blind clinical trial provides valuable insights on the safety, tolerability, and efficacy of Revacept in patients with symptomatic carotid artery stenosis.
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Neointima hyperplasia is a crucial component of restenosis after coronary angioplasty. We have hypothesized that enhanced generation of platelet-derived thromboxane (TX)A2 in response to vascular damage plays a critical role in neointimal hyperplasia and that antiplatelet agents may mitigate it. In cocultures of human platelets and coronary artery smooth muscle cells (CASMC), we found that platelets induced morphologic changes and enhanced the migration of CASMC. The exposure of platelets to Aspirin [an inhibitor of cyclooxygenase (COX)-1] reduced the generation of TXA2 and prevented the morphological and functional changes induced by platelets in CASMC. Platelet-derived TXA2 induced COX-2 and enhanced prostaglandin (PG)E2 biosynthesis in CASMC, a known mechanism promoting neointimal hyperplasia. COX-2 induction was prevented by different antiplatelet agents, i.e., Aspirin, the TP antagonist SQ29,548, or Revacept (a dimeric soluble GPVI-Fc fusion protein). The administration of the novel antiplatelet agent Revacept to C57BL/6 mice, beginning three days before femoral artery denudation, and continuing up to seven days after injury, prevented the increase of the systemic biosynthesis di TXA2 and reduced femoral artery intima-to-media area and the levels of markers of cell proliferation and macrophage infiltration. Revacept might serve as a therapeutic agent for percutaneous coronary angioplasty and stent implantation.
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Plaquetas/citología , Vasos Coronarios/citología , Glicoproteínas/farmacología , Fragmentos Fc de Inmunoglobulinas/farmacología , Neointima/prevención & control , Inhibidores de Agregación Plaquetaria/farmacología , Tromboxano A2/biosíntesis , Orina/química , Adulto , Animales , Plaquetas/efectos de los fármacos , Plaquetas/metabolismo , Movimiento Celular/efectos de los fármacos , Proliferación Celular/efectos de los fármacos , Técnicas de Cocultivo , Vasos Coronarios/efectos de los fármacos , Vasos Coronarios/metabolismo , Ciclooxigenasa 2/metabolismo , Humanos , Hiperplasia , Masculino , Ratones , Miocitos del Músculo Liso/citología , Miocitos del Músculo Liso/efectos de los fármacos , Neointima/metabolismo , Neointima/patología , Adulto JovenRESUMEN
Glycoprotein VI (GPVI), a platelet collagen receptor, is crucial in mediating atherothrombosis. Besides collagen, injured plaques expose tissue factor (TF) that triggers fibrin formation. Previous studies reported that GPVI also is a platelet receptor for fibrinogen and fibrin. We studied the effect of anti-GPVI antibodies and inhibitors of GPVI signaling kinases (Syk and Btk) on platelet adhesion and aggregate formation onto immobilized fibrinogen and different types of fibrin under arterial flow conditions. Fibrin was prepared from isolated fibrinogen ("pure fibrin"), recombinant fibrinogen ("recombinant fibrin"), or generated more physiologically from endogenous fibrinogen in plasma ("plasma fibrin") or by exposing TF-coated surfaces to flowing blood ("blood fibrin"). Inhibition of GPVI and Syk did not inhibit platelet adhesion and aggregate formation onto fibrinogen. In contrast anti-GPVI antibodies, inhibitors of Syk and Btk and the anti-GPIb antibody 6B4 inhibited platelet aggregate formation onto pure and recombinant fibrin. However, inhibition of GPVI and GPVI signaling did not significantly reduce platelet coverage of plasma fibrin and blood fibrin. Plasma fibrin contained many proteins incorporated during clot formation. Advanced optical imaging revealed plasma fibrin as a spongiform cushion with thicker, knotty, and long fibers and little activation of adhering platelets. Albumin intercalated in plasma fibrin fibers left only little space for platelet attachment. Pure fibrin was different showing a dense mesh of thin fibers with strongly activated platelets. We conclude that fibrin formed in plasma and blood contains plasma proteins shielding GPVI-activating epitopes. Our findings do not support a role of GPVI for platelet activation by physiologic fibrin.
Asunto(s)
Plaquetas/metabolismo , Fibrina/metabolismo , Glicoproteínas de Membrana Plaquetaria/fisiología , Receptores de Péptidos/metabolismo , Agammaglobulinemia Tirosina Quinasa/sangre , Agammaglobulinemia Tirosina Quinasa/fisiología , Activación Enzimática , Fibrinógeno/metabolismo , Hemorreología , Humanos , Microscopía Confocal/métodos , Plasma , Adhesividad Plaquetaria , Agregación Plaquetaria , Complejo GPIb-IX de Glicoproteína Plaquetaria/metabolismo , Glicoproteínas de Membrana Plaquetaria/antagonistas & inhibidores , Glicoproteínas de Membrana Plaquetaria/inmunología , Unión Proteica , Proteínas Recombinantes/metabolismo , Quinasa Syk/antagonistas & inhibidores , Quinasa Syk/sangre , Quinasa Syk/fisiología , Tromboplastina/metabolismoRESUMEN
The Extracellular Matrix Metalloproteinase Inducer (EMMPRIN, CD147, basigin) is an immunoglobulin-like receptor expressed in various cell types. During cellular interactions homotypic EMMPRIN-EMMPRIN interactions are known to induce the synthesis of matrix metalloproteinases. Recently, we have identified EMMPRIN as a novel receptor on platelets. To our knowledge EMMPRIN has not been shown to serve as adhesion receptor, yet. Here we characterise platelet glycoprotein VI (GPVI) as a novel adhesion receptor for EMMPRIN. Human platelets were prestimulated with ADP and perfused over immobilised recombinant EMMPRIN-Fc or Fc-fragments under arterial shear conditions. ADP-stimulated platelets showed significantly enhanced rolling (but not enhanced firm adhesion) on immobilised EMMPRIN-Fc compared to Fc. Pretreatment of platelets with blocking mAbs anti-EMMPRIN or anti-GPVI leads to a significant reduction of rolling platelets on immobilised EMMPRIN-Fc, whereas pretreatment with blocking mAbs anti-p-selectin, anti-alpha4-integrin or anti-GPIIb/IIIa complex (20 microg/ml each) had no effect. Consistently, chinese hamster ovary (CHO) cells stably transfected with GPVI showed enhanced rolling (but not adhesion) on immobilised EMMPRIN-Fc in comparison to non-transfected CHO cells. Similarly, CHO cells stably transfected with EMMPRIN showed enhanced rolling on immobilised GPVI-Fc (or EMMPRIN-Fc) compared to non transfected CHO-cells. Finally, specific binding of EMMPRIN to GPVI was demonstrated by a modified ELISA and surface plasmon resonance technology with a dissociation constant of 88 nM. Platelet GPVI is a novel receptor for EMMPRIN and can mediate platelet rolling via GPVI-EMMPRIN interaction.
Asunto(s)
Basigina/metabolismo , Plaquetas/metabolismo , Antígenos CD36/metabolismo , Movimiento Celular , Activación Plaquetaria , Adenosina Difosfato/metabolismo , Animales , Anticuerpos Monoclonales , Basigina/genética , Basigina/inmunología , Antígenos CD36/genética , Antígenos CD36/inmunología , Células CHO , Adhesión Celular , Cricetinae , Cricetulus , Humanos , Ratones , Selectina-P/metabolismo , Adhesividad Plaquetaria , Unión Proteica , Proteínas Recombinantes de Fusión/metabolismo , Factores de Tiempo , TransfecciónRESUMEN
AIMS: Rupture of advanced atherosclerotic plaques initiates platelet activation and aggregation as subendothelial collagen is exposed. Platelet collagen receptor glycoprotein VI (GPVI) was found to bind preferentially to the core region of human plaques. Consequently, platelets contribute to inflammatory processes and trigger atherosclerotic lesion progression. In this study, we examined binding of soluble platelet collagen receptor GPVI-Fc to atherosclerotic lesions and its effect on platelet-triggered athero-progression and neointima formation after wire-induced carotid injury. METHODS AND RESULTS: For binding studies after ligation-induced arterial injury, the left common carotid artery of C57BL/6J mice was ligated. For binding studies at spontaneously formed atherosclerotic lesion sites, Apolipoprotein E-deficient (ApoE(-/-)) mice were fed a 0.25% cholesterol diet over 16 weeks. Binding of [(124)I]GPVI-Fc was monitored by autoradiography 48 h after intravenous injection and by immunostaining. To study the effect of GPVI-Fc on neointima formation vs. control-Fc, a wire-induced injury of the left A. carotis communis of ApoE(-/-)-mice was performed. Mice were treated intraperitoneally with GPVI-Fc for 8 days and neointima formation was assessed 4 weeks after intervention. [(124)I]GPVI-Fc preferentially bound to injury sites after carotid ligation in C57BL/6J mice and to lipid-rich atherosclerotic lesions of the carotid artery and aortic arch in uninjured ApoE(-/-)-mice. Histological examinations of wire-injured carotid arteries showed that neointima formation was significantly reduced in GPVI-Fc-treated ApoE(-/-) mice compared to ApoE(-/-) mice receiving control-Fc (P < 0.05). CONCLUSION: GPVI-Fc preferentially bound to sites of vascular injury and was able to inhibit neointima formation after wire-induced vascular injury in ApoE(-/-) mice. Thus, soluble GPVI-Fc might be also a promising compound to attenuate lesion progression after plaque rupture.
Asunto(s)
Aterosclerosis/metabolismo , Traumatismos de las Arterias Carótidas/metabolismo , Glicoproteínas de Membrana Plaquetaria/metabolismo , Animales , Apolipoproteínas E/genética , Aterosclerosis/fisiopatología , Autorradiografía , Traumatismos de las Arterias Carótidas/diagnóstico por imagen , Traumatismos de las Arterias Carótidas/patología , Arteria Carótida Común/diagnóstico por imagen , Arteria Carótida Común/patología , Colágeno/sangre , Humanos , Inmunohistoquímica , Masculino , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Radiografía , Proteínas Recombinantes de Fusión/metabolismoRESUMEN
RATIONALE: Despite advances in pharmacotherapy, heart failure still incurs significant morbidity and mortality. Stimulating antibodies directed against the secondextracellular loop of the human ß1-adrenergic receptor (anti-ß1EC2) cause myocyte damage and heart failure in rats. This receptor domain is 100% homologous between rats and humans. OBJECTIVE: ß1EC2-mimicking cyclopeptides (25-meric) markedly improved the development and/or course of anti-ß1EC2-mediated cardiomyopathy. Further developments should be investigated. METHODS AND RESULTS: The shortened 18-meric cyclic peptide COR-1, in which one of the two disulphide bonds was removed to enable reproducible GMP production, can also be used to treat cardiomyopathic rats. Echocardiography, catheterization and histopathology of the rat hearts revealed that monthly intravenous administrations of COR-1 almost fully reversed the cardiomyopathic phenotype within 6 months at doses of 1 to 4 mg/kg body weight. Administration of COR-1 resulted in markedly reduced anti-ß1EC2-expressing memory B lymphocytes in the spleen despite continued antigenic boosts, but did not significantly decrease overall peripheral anti-ß1EC2 titers. COR-1 did not induce any anti-ß1EC2 or other immune response in naïve rats (corresponding to findings in healthy human volunteers). It did not cause any toxic side effects in GLP studies in dogs, rats or mice, and the "no observed adverse effect level" (NOAEL) exceeded the therapeutic doses by 100-fold. CONCLUSION: The second generation immunomodulating epitope-mimicking cyclopeptide COR-1 (also termed JNJ-5442840) offers promise to treat immune-mediated cardiac diseases.
Asunto(s)
Insuficiencia Cardíaca/tratamiento farmacológico , Péptidos Cíclicos/uso terapéutico , Receptores Adrenérgicos beta 1/inmunología , Secuencia de Aminoácidos , Animales , Anticuerpos Monoclonales/administración & dosificación , Especificidad de Anticuerpos , Modelos Animales de Enfermedad , Femenino , Cobayas , Insuficiencia Cardíaca/etiología , Insuficiencia Cardíaca/fisiopatología , Humanos , Masculino , Imitación Molecular/inmunología , Miocardio/metabolismo , Fragmentos de Péptidos/química , Fragmentos de Péptidos/uso terapéutico , Péptidos Cíclicos/química , Péptidos Cíclicos/toxicidad , ARN Mensajero/genética , ARN Mensajero/metabolismo , Ratas , Ratas Endogámicas Lew , Ratas Wistar , Receptores Adrenérgicos beta 1/química , Receptores Adrenérgicos beta 1/genéticaRESUMEN
Platelet glycoprotein VI (GPVI) acts as a decisive collagen receptor in atherothrombosis. Besides collagen, injured atherosclerotic plaques expose tissue factor (TF) that triggers fibrin formation. Two recent studies reported that platelet GPVI also functions as fibrin receptor, which would importantly widen the mode of action of GPVI-targeted antithrombotic drugs. We studied the binding of two GPVI fusion proteins to fibrin under static and arterial flow conditions. Fibrin was prepared from purified fibrinogen or generated more physiologically from endogenous fibrinogen by coagulating plasma with thrombin. Fibrin formation was also triggered by exposing TF-coated surfaces or human atherosclerotic plaque slices to arterially flowing blood. By binding studies and advanced optical imaging, we found that recombinant dimeric GPVI-Fc fusion proteins with Fc from either IgG1 (GPVI-Fc1) or IgG2 (GPVI-Fc2) bound to collagen fibres, but neither to fibrin prepared from purified fibrinogen obtained from three suppliers, nor to physiological fibrin formed by thrombin in plasma or triggered by exposing TF or atherosclerotic plaque slices to arterially flowing blood. Our findings do not support a role of dimeric platelet GPVI as receptor for fibrin. This is important for the understanding of plaque-triggered platelet thrombus formation and is clinically relevant for future GPVI-targeting therapies with recombinant GPVI-Fc and anti-GPVI antibodies.
Asunto(s)
Colágeno/metabolismo , Fibrina/metabolismo , Fibrinógeno/metabolismo , Glicoproteínas de Membrana Plaquetaria/metabolismo , Trombina/metabolismo , Aterosclerosis/metabolismo , Coagulación Sanguínea , Plaquetas/metabolismo , Humanos , Microscopía Fluorescente , Placa Aterosclerótica/metabolismo , Activación Plaquetaria , Adhesividad Plaquetaria , Agregación Plaquetaria , Unión Proteica , Multimerización de Proteína , Proteínas RecombinantesRESUMEN
A hyperadrenergic state is one of the key features of human and experimental heart failure. Decreased densities and activities of the presynaptic neuronal norepinephrine (NE) transporter uptake-1 occur both in patients and animal models. It is currently unclear to what extent the reduction of uptake-1 contributes to the deterioration of heart failure. Therefore, we investigated the effects of myocardial overexpression of uptake-1 in both nonfailing rabbit hearts and in an animal model of heart failure. Heart failure was induced in rabbits by rapid ventricular pacing. Adenoviral gene transfer was used to overexpress uptake-1 in the myocardium. Uptake-1 overexpression led to increased NE uptake capacity into the myocardium. In contrast, systemic plasma NE levels in uptake-1-overexpressing failing rabbits (uptake-1-CHF) did not differ from controls. Downregulation of SERCA-2 and beta-adrenergic receptors in the failing myocardium was significantly reversed after uptake-1 overexpression. Uptake-1 overexpression significantly improved left ventricular (LV) diameters (LV end-diastolic diameter: in GCP-overexpressing failing rabbits (GFP-CHF), 17.4+/-0.4 mm; in uptake-1-CHF rabbits, 15.6+/-0.6 mm) and systolic contractility (fractional shortening: GFP-CHF, 20.7+/-0.6%; uptake-1-CHF, 27.3+/-0.7%), as assessed by echocardiography at the end of the heart failure protocol. Intraventricular tip catheter measurements revealed enhanced contractile reserve (dP/dt max with isoproterenol 1.0 microg/kg: GFP-CHF, 6964+/-230 mm Hg/sec; uptake-1-CHF, 7660+/-315 mm Hg/sec) and LV relaxation (dP/dt min with isoproterenol 1.0 microg/kg: GFP-CHF: -3960+/-260 mm Hg/sec; uptake-1-CHF, -4910+/-490 mm Hg/sec). End-diastolic filling pressures (GFP-CHF, 8.5+/-1.2 mm Hg; uptake-1-CHF, 5.6+/-0.7 mm Hg) tended to be lower in uptake-1 overexpressing animals. In summary, local overexpression of uptake-1 in the myocardium results in marked structural and functional improvement of heart failure, thus underlining the importance of uptake-1 as a key protein in heart failure.
Asunto(s)
Terapia Genética , Insuficiencia Cardíaca/terapia , Proteínas de Transporte de Noradrenalina a través de la Membrana Plasmática/genética , Adenoviridae/genética , Animales , Células HeLa , Insuficiencia Cardíaca/fisiopatología , Humanos , Contracción Miocárdica , Miocardio/metabolismo , Norepinefrina/metabolismo , Células PC12 , Conejos , Ratas , Transgenes , Disfunción Ventricular Izquierda/terapia , Aumento de PesoRESUMEN
Various approaches have been used to model human Graves' disease in mice, including transfected fibroblasts, and plasmid or adenoviral immunisations with the extracellular A subunit of the human thyrotropin receptor (TSHR). Some of these models were only observed for a short time period or were self-limiting. A long-term model for human Graves' disease was established in mice using continuing immunisations (4-weekly injections) with recombinant adenovirus expressing TSHR. Generation of TSHR binding cAMP-stimulatory antibodies, thyroid enlargement and alterations, elevated serum thyroxin levels, tachycardia and cardiac hypertrophy were maintained for at least 9 months in all Ad-TSHR-immunised mice. Here, we show that these mice suffer from orbitopathy, which was detected by serial orbital sectioning and histomorphometry. Attempts to treat established Graves' disease in preclinical mouse model studies have included small molecule allosteric antagonists and specific antagonist antibodies which were isolated from hypothyroid patients. In addition, novel peptides have been conceived which mimic the cylindrical loops of the TSHR leucine-rich repeat domain, in order to re-establish tolerance toward the antigen. Here, we show preliminary results that one set of these peptides improves or even cures all signs and symptoms of Graves' disease in mice after six consecutive monthly injections. First beneficial effects were observed 3-4 months after starting these therapies. In immunologically naïve mice, administration of the peptides did not induce any immune response.