RESUMEN
The biomarkers for predicting venous thromboembolic events (VTEs) after oncologic surgery are still lacking. The current study aimed to analyze the relationships of CD62P and GP IIb/IIIa with hypercoagulation after oncologic surgery. A total of 76 patients with primary abdominopelvic tumors in our hospital were enrolled. The patients were divided into groups A (malignancy with no VTE group), B (malignancy with VTE group), and C (benign with no VTE group). Twenty healthy volunteers were selected as control. The plasma CD62P (4.69 ± 2.55 vs. 1.76 ± 0.48) and the GP IIb/IIIa (9.28 ± 3.79 vs. 1.76 ± 0.48) levels in group A were significantly higher than those in the control group preoperatively. The CD62P (31.46 ± 17.13 vs. 13.51 ± 7.43, P < 0.05), GP IIb/IIIa (42.33 ± 21.82 vs. 13.51 ± 7.43, P < 0.05), and D-dimer (7.33 ± 2.34 vs. 2.03 ± 0.55, P < 0.05) levels in group B were markedly higher 7 days after operation compared with those in group A. The CD62P and the GP IIb/IIIa exhibited a positive correlation with the hypercoagulable state after oncologic surgery.
Asunto(s)
Neoplasias/sangre , Neoplasias/cirugía , Selectina-P/sangre , Complejo GPIIb-IIIa de Glicoproteína Plaquetaria/análisis , Trombofilia/sangre , Tromboembolia Venosa/sangre , Adulto , Anciano , Coagulación Sanguínea , Plaquetas/patología , Femenino , Productos de Degradación de Fibrina-Fibrinógeno/análisis , Humanos , Masculino , Persona de Mediana Edad , Neoplasias/complicaciones , Neoplasias/patología , Trombofilia/etiología , Trombofilia/patología , Tromboembolia Venosa/etiología , Tromboembolia Venosa/patologíaRESUMEN
BACKGROUND: A strong correlation exists between type 2 diabetes mellitus (T2DM) and cardiovascular disease (CVD), with CVD and the presence of atherosclerosis being the prevailing cause of morbidity and mortality in diabetic populations. T2DM is accompanied by various coagulopathies, including anomalous clot formation or amyloid fibrin(ogen), the presence of dysregulated inflammatory molecules. Platelets are intimately involved in thrombus formation and particularly vulnerable to inflammatory cytokines. METHODS: The aim of this current study was therefore to assess whole blood (hyper)coagulability, platelet ultrastructure and receptor expression, as well as the levels of IL-1ß, IL-6, IL-8 and sP-selectin in healthy and diabetic individuals. Platelet morphology was assessed through scanning electron microscopy (SEM), while assessment of GPIIb/IIIa receptor expression was performed with confocal microscopy and flow cytometry with the addition of FITC-PAC-1 and CD41-PE antibodies. IL-1ß, IL-6 and IL-8 and sP-selectin levels were assessed using a multiplex assay. RESULTS: In T2DM there is significant upregulation of circulating inflammatory markers, hypercoagulation and platelet activation, with increased GPIIb/IIIa receptor expression, as seen with flow cytometry and confocal microscopy. Analyses showed that these receptors were additionally shed onto microparticles, which was confirmed with SEM. CONCLUSIONS: Cumulatively, this provides mechanistic evidence that pathological states of platelets together with amyloid fibrin(ogen) in T2DM, might underpin an increased risk for cardiovascular events.
Asunto(s)
Coagulación Sanguínea , Plaquetas/metabolismo , Micropartículas Derivadas de Células/metabolismo , Diabetes Mellitus Tipo 2/sangre , Activación Plaquetaria , Trombofilia/sangre , Trombosis/sangre , Anciano , Biomarcadores/sangre , Factores de Coagulación Sanguínea/análisis , Plaquetas/ultraestructura , Estudios de Casos y Controles , Micropartículas Derivadas de Células/ultraestructura , Diabetes Mellitus Tipo 2/complicaciones , Diabetes Mellitus Tipo 2/diagnóstico , Femenino , Humanos , Mediadores de Inflamación/sangre , Interleucinas/sangre , Masculino , Persona de Mediana Edad , Selectina-P/sangre , Complejo GPIIb-IIIa de Glicoproteína Plaquetaria/análisis , Trombofilia/diagnóstico , Trombofilia/etiología , Trombosis/diagnóstico , Trombosis/etiologíaRESUMEN
PURPOSE: To investigate the correlation of inhaled nitric oxide (NO) on plasma levels of cardiac troponin I (cTnI) and von Willebrand factor (vWF), glycoprotein (GP) IIb/IIIa, granule membrane protein 140 (GMP-140) in rabbits with acute massive pulmonary embolism (PE). METHODS: Thirty apanese white rabbits were divided into 3 groups, thrombus were injected in model group (n = 10), NO were inhalated for 24 h after massive PE in NO group (n = 10), saline were injected in control group (n = 10). The concentrations of vWF, GP IIb/IIIa, GMP-140 and cTnI were tested at 4, 8, 12, 16, 20, and 24 h, Correlation analyses were conducted between cTnI and vWF, GP IIb/IIIa, and GMP-140 by Pearson's correlation. RESULTS: The concentration of cTnI and vWF, GP IIb/IIIa, and GMP-140 was increased in the model group, compared to control group. In the inhaled group, the concentrations of cTnI, vWF, GP IIb/IIIa, and GMP-140 were reduced compared to model group. There was a positive correlation between cTnI and vWF, GP IIb/IIIa, and GMP-140. CONCLUSION: Inhaled nitric oxide can lead to a decrease in levels of cardiac troponin I, von Willebrand factor, glycoprotein, and granule membrane protein 140, after an established myocardial damage, provoked by acute massive pulmonary embolism.
Asunto(s)
Óxido Nítrico/administración & dosificación , Selectina-P/sangre , Complejo GPIIb-IIIa de Glicoproteína Plaquetaria/análisis , Embolia Pulmonar/sangre , Embolia Pulmonar/tratamiento farmacológico , Troponina I/sangre , Factor de von Willebrand/análisis , Administración por Inhalación , Animales , Modelos Animales de Enfermedad , Ventrículos Cardíacos/patología , Miocardio/patología , Selectina-P/efectos de los fármacos , Complejo GPIIb-IIIa de Glicoproteína Plaquetaria/efectos de los fármacos , Embolia Pulmonar/patología , Conejos , Valores de Referencia , Reproducibilidad de los Resultados , Factores de Tiempo , Resultado del Tratamiento , Troponina I/efectos de los fármacos , Microtomografía por Rayos X , Factor de von Willebrand/efectos de los fármacosRESUMEN
Abstract Purpose: To investigate the correlation of inhaled nitric oxide (NO) on plasma levels of cardiac troponin I (cTnI) and von Willebrand factor (vWF), glycoprotein (GP) IIb/IIIa, granule membrane protein 140 (GMP-140) in rabbits with acute massive pulmonary embolism (PE). Methods: Thirty apanese white rabbits were divided into 3 groups, thrombus were injected in model group (n = 10), NO were inhalated for 24 h after massive PE in NO group (n = 10), saline were injected in control group (n = 10). The concentrations of vWF, GP IIb/IIIa, GMP-140 and cTnI were tested at 4, 8, 12, 16, 20, and 24 h, Correlation analyses were conducted between cTnI and vWF, GP IIb/IIIa, and GMP-140 by Pearson's correlation. Results: The concentration of cTnI and vWF, GP IIb/IIIa, and GMP-140 was increased in the model group, compared to control group. In the inhaled group, the concentrations of cTnI, vWF, GP IIb/IIIa, and GMP-140 were reduced compared to model group. There was a positive correlation between cTnI and vWF, GP IIb/IIIa, and GMP-140. Conclusion: Inhaled nitric oxide can lead to a decrease in levels of cardiac troponin I, von Willebrand factor, glycoprotein, and granule membrane protein 140, after an established myocardial damage, provoked by acute massive pulmonary embolism.
Asunto(s)
Animales , Conejos , Embolia Pulmonar/sangre , Factor de von Willebrand/análisis , Complejo GPIIb-IIIa de Glicoproteína Plaquetaria/análisis , Complejo GPIIb-IIIa de Glicoproteína Plaquetaria/efectos de los fármacos , Selectina-P/sangre , Troponina I/sangre , Óxido Nítrico/administración & dosificación , Embolia Pulmonar/patología , Embolia Pulmonar/tratamiento farmacológico , Valores de Referencia , Factores de Tiempo , Administración por Inhalación , Factor de von Willebrand/efectos de los fármacos , Reproducibilidad de los Resultados , Resultado del Tratamiento , Selectina-P/efectos de los fármacos , Troponina I/efectos de los fármacos , Modelos Animales de Enfermedad , Microtomografía por Rayos X , Ventrículos Cardíacos/patología , Miocardio/patologíaRESUMEN
AIM: To find novel platelet aggregation inhibitors, two new series of 1,4-benzodioxine derivatives were synthesized and screened for the ability to inhibit platelet aggregation. MATERIALS & METHODS: The synthesized compounds were evaluated for antiplatelet aggregation activity using human blood platelet and GPIIb/IIIa antagonistic activity. RESULTS: Compound 9-2p showed significant antiplatelet activity with the IC50 values of 41.7 and 22.2 µM induced by ADP and thrombin, respectively, more potent than that of LX2421. Compound 9-2p exhibited GPIIb/IIIa antagonistic activity with the IC50 value of 2.3 µM, as potent as RGDs. In vivo study showed that 9-2p displayed remarkable antithrombotic activity, more effective than LX2421, but less effective than tirofiban. CONCLUSION: Compound 9-2p showed moderate antiplatelet activity and antithrombotic activity, which could be further optimized based on the target of GPIIb/IIIa.
Asunto(s)
Diseño de Fármacos , Inhibidores Enzimáticos/farmacología , Inhibidores de Agregación Plaquetaria/farmacología , Agregación Plaquetaria/efectos de los fármacos , Complejo GPIIb-IIIa de Glicoproteína Plaquetaria/antagonistas & inhibidores , Propanolaminas/farmacología , Animales , Plaquetas/efectos de los fármacos , Relación Dosis-Respuesta a Droga , Inhibidores Enzimáticos/síntesis química , Inhibidores Enzimáticos/química , Ensayo de Inmunoadsorción Enzimática , Femenino , Humanos , Masculino , Simulación del Acoplamiento Molecular , Estructura Molecular , Inhibidores de Agregación Plaquetaria/síntesis química , Inhibidores de Agregación Plaquetaria/química , Complejo GPIIb-IIIa de Glicoproteína Plaquetaria/análisis , Complejo GPIIb-IIIa de Glicoproteína Plaquetaria/metabolismo , Propanolaminas/síntesis química , Propanolaminas/química , Ratas , Ratas Sprague-Dawley , Relación Estructura-Actividad , Trombina/antagonistas & inhibidores , Trombina/metabolismoRESUMEN
BACKGROUND: Enhanced platelet activation has been implicated in several pathophysiological processes. Here, we evaluated the biological reproducibility of circulating P-Selectin, Thrombomodulin (TM), Thrombopoietin (TPO), and Glycoprotein IIb/IIIa (GPIIb/IIIa) to assess whether these analytes can be used as reliable biomarkers of platelet activation in epidemiological studies. METHODS: We measured circulating P-Selectin, TM, TPO and GPIIb/IIIa by immunoassays in two blood samples of 78 participants of the EPIC Heidelberg study (47-80years, 50% female) that were collected one year apart. Biological reproducibility of biomarker levels over time and associations with routine biochemistry parameters were assessed by Spearman's correlation coefficients. RESULTS: Statistical analyses revealed good reproducibility over one year for two of the analyzed markers, with Spearman coefficients of ρ=0.80 (P-Selectin) and ρ=0.73 (TPO) and reasonable reproducibility for TM (ρ=0.63) and GPIIb/IIIa (ρ=0.51). Levels of P-Selectin, TM, TPO and GPIIb/IIIa were not significantly associated with routine biochemistry parameters, such as glucose, HbA1c, LDL, HDL, Triglycerides and CRP. CONCLUSIONS: Our findings suggest that a single assessment of P-Selectin, TM, TPO and GPIIb/IIIa at baseline in prospective epidemiological studies is appropriate to investigate associations between platelet activation and risks of chronic diseases.
Asunto(s)
Selectina-P/sangre , Activación Plaquetaria , Complejo GPIIb-IIIa de Glicoproteína Plaquetaria/análisis , Trombomodulina/sangre , Trombopoyetina/sangre , Anciano , Anciano de 80 o más Años , Biomarcadores/sangre , Femenino , Humanos , Masculino , Persona de Mediana Edad , Estudios Prospectivos , Reproducibilidad de los ResultadosRESUMEN
STUDY OBJECTIVE: Platelets play a pivotal role in metastasis of tumor cells. The aim of this study is to explore the effects of sevoflurane and isoflurane on platelets activation of patients undergoing lung cancer surgery, and the effects of sevoflurane and isoflurane on platelets-induced invasion of lung cancer cells. DESIGN: Prospective and randomized study, and in vitro experiment. SETTING: University-affiliated teaching hospital and laboratory. PATIENTS: Forty-six patients scheduled for lung cancer radical surgery. INTERVENTIONS: Patients were randomized to two groups of 23 patients each and were received sevoflurane (group SEV) or isoflurane (group ISO) during surgery, respectively. In vitro, lung cancer cells were treated with platelets in the presence or absence anesthetics. MEASUREMENTS: Platelets activation were determined by detecting glycoproteinIIb/IIIa (GPIIb/IIIa), CD62P, and platelets aggregation rate (PAR) pre-, intra-, and postoperatively. Invasion ability of lung cancer cells were evaluated by Transwell assay. RESULTS: The levels of GPIIb/IIIa, CD62P, and PAR were reduced markedly in group SEV during perioperative period compared with group ISO. In vitro, activated platelets contributed profoundly to the invasive ability of lung cancer cells. Sevoflurane, but not isoflurane, inhibited platelets-induced invasion of lung cancer cells. Furthermore, sevoflurane suppressed the platelets activity in vitro. CONCLUSION: Sevoflurane attenuates platelets activation of patients undergoing lung cancer surgery. In vitro, sevoflurane suppresses platelets-induced invasion of lung cancer cells via decreasing platelets activity.
Asunto(s)
Anestésicos por Inhalación/farmacología , Plaquetas/patología , Isoflurano/farmacología , Neoplasias Pulmonares/patología , Éteres Metílicos/farmacología , Activación Plaquetaria/efectos de los fármacos , Células A549 , Anciano , Anestesia General , Anestésicos por Inhalación/administración & dosificación , Humanos , Isoflurano/administración & dosificación , Neoplasias Pulmonares/sangre , Neoplasias Pulmonares/inmunología , Neoplasias Pulmonares/cirugía , Masculino , Éteres Metílicos/administración & dosificación , Persona de Mediana Edad , Invasividad Neoplásica/inmunología , Selectina-P/análisis , Agregación Plaquetaria/efectos de los fármacos , Complejo GPIIb-IIIa de Glicoproteína Plaquetaria/análisis , Neumonectomía/métodos , Estudios Prospectivos , Sevoflurano , ToracoscopíaRESUMEN
AIM: The aim of this study was to investigate the possibility of using 99mtechnetium (99mTc)-labeled tirofiban (a reversible antagonist of glycoprotein IIb/IIIa) for detection of deep venous thrombosis (DVT) in rats without causing an antiplatelet effect. METHODS: The ability of in vitro tirofiban to inhibit adenosine 5'-diphosphate (ADP)-induced platelet aggregation was evaluated using optical aggregometer. Binding of 99mTc-tirofiban to platelets was evaluated. Serum levels of unlabeled (a validated high performance liquid chromatography method) and 99mTc-tirofiban after single intravenous injection were evaluated in male Wistar rats with or without induced DVT (femoral vein ligation model), and the rats were also subjected to whole body scintigraphy. RESULTS: Tirofiban in vitro inhibits ADP-induced aggregation of human platelets in a dose- and concentration-dependent manner (10 nM to 2 µM), but only if it is added before ADP and not after ADP. 99mTc labeling did not affect the ability of tirofiban to bind to either human or rat platelets, nor did it affect tirofiban pharmacokinetics in intact rats or in animals with induced DVT. When 99mTc-tirofiban was injected to rats after induction of DVT, at a molar dose lower than the one showing only a weak antiaggregatory effect in vitro, whole body scintigraphy indicated localization of 99mTc-tirofiban around the place of the induced DVT. CONCLUSION: 99mTc labeling of tirofiban does not affect its ability to bind to glycoprotein IIb/IIIa or its in vivo pharmacokinetics in rats, either intact or with DVT. A low, nonantiaggregatory dose of 99mTc-tirofiban may be used to visualize DVT at an early stage.
Asunto(s)
Adenosina Difosfato/química , Inhibidores de Agregación Plaquetaria/farmacología , Agregación Plaquetaria/efectos de los fármacos , Complejo GPIIb-IIIa de Glicoproteína Plaquetaria/análisis , Tirosina/análogos & derivados , Trombosis de la Vena/diagnóstico por imagen , Animales , Humanos , Masculino , Inhibidores de Agregación Plaquetaria/química , Complejo GPIIb-IIIa de Glicoproteína Plaquetaria/antagonistas & inhibidores , Complejo GPIIb-IIIa de Glicoproteína Plaquetaria/química , Radiofármacos , Ratas , Ratas Wistar , Tirofibán , Tirosina/química , Tirosina/farmacologíaRESUMEN
In patients with thrombocytopenia, it can be difficult to predict a patient's bleeding risk based on platelet count alone. Platelet reactivity may provide additional information; however, current clinical assays cannot reliably assess platelet function in the setting of thrombocytopenia. New methods to study platelet reactivity in thrombocytopenic samples are needed. In this study, we sought to develop a laboratory model of thrombocytopenia using blood from healthy subjects that preserves the whole blood environment and reproducibly produces samples with a specific platelet count and hematocrit. We compared the activation state of unstimulated and agonist-stimulated platelets in thrombocytopenic samples derived from this method with normocytic controls. Whole blood was diluted with autologous red blood cell concentrate and platelet-poor plasma, which were obtained via centrifugation, in specific ratios to attain a final sample with a predetermined platelet count and hematocrit. P-selectin exposure and GPIIbIIIa activation in unstimulated platelets and platelets stimulated with collagen-related peptide (CRP) or adenosine diphosphate (ADP) in thrombocytopenic samples and the normocytic control from which they were derived were quantified by flow cytometry. Our methodology reliably produced thrombocytopenic samples with a platelet count ≤50,000/µL and an accurately and precisely controlled hematocrit. P-selectin exposure and GPIIbIIIa activation on unstimulated platelets or on ADP- or CRP-stimulated platelets did not differ in thrombocytopenic samples compared to normocytic controls. We describe a new method for creating thrombocytopenic blood that can be used to better understand the contributions of platelet number and function to hemostasis.
Asunto(s)
Citometría de Flujo/métodos , Hematócrito , Trastornos Hemorrágicos/etiología , Recuento de Plaquetas , Pruebas de Función Plaquetaria , Trombocitopenia/sangre , Adenosina Difosfato/farmacología , Adulto , Proteínas Portadoras/farmacología , Centrifugación , Trastornos Hemorrágicos/sangre , Humanos , Técnicas In Vitro , Selectina-P/análisis , Péptidos/farmacología , Activación Plaquetaria/efectos de los fármacos , Complejo GPIIb-IIIa de Glicoproteína Plaquetaria/análisis , Reproducibilidad de los Resultados , Trombocitopenia/complicacionesRESUMEN
We have designed a novel peptide-Au cluster probe to specifically bind to αIIbß3 integrin. As indicated by molecular dynamics (MD) simulations, the binding mode of the native ligand of αIIbß3 integrin, γC peptide, can be realized by the designed probe. More importantly, the peptide-Au probe can provide multiple coating peptides to form additional salt bridges with protein, and the binding stability of the probe is comparable to the native ligand. The designed probe was then successfully synthesized. The specific binding in a cellular environment was validated by colocalization analysis of confocal microscopy. In addition, the binding affinity was confirmed by atomic force microscopy (AFM) based single molecule force spectroscopy. Our results suggest the combination of computational design and experimental verification can be a useful strategy for the development of nanoprobes.
Asunto(s)
Sondas Moleculares/química , Péptidos/química , Complejo GPIIb-IIIa de Glicoproteína Plaquetaria/análisis , Plata/química , Secuencia de Aminoácidos , Sitios de Unión , Ligandos , Microscopía de Fuerza Atómica , Microscopía Confocal , Simulación de Dinámica Molecular , Sondas Moleculares/metabolismo , Nanoestructuras/química , Péptidos/metabolismo , Unión Proteica , Estructura Terciaria de ProteínaRESUMEN
BACKGROUND AND OBJECTIVES: Pathogen reduction technologies may affect platelet quality during storage. We studied functional characteristics and clinical effectiveness of platelet concentrates (PCs) treated with Mirasol in plasma and in platelet-additive solution SSP+. MATERIALS AND METHODS: Mirasol-treated, gamma-irradiated and untreated apheresis PCs were examined on days 0, 1, 3 and 5 of storage. Phosphatidylserine, P-selectin and active glycoprotein IIb/IIIa were analysed using flow cytometry before and after platelet stimulation. Platelet count increments, the numbers of inefficient transfusions and post-transfusion reactions were analysed to estimate clinical effectiveness. RESULTS: A significant increase in all platelet activation markers occurred during storage in all PC groups. Activation markers in Mirasol-treated samples were already significantly higher compared with the control ones on the day of harvesting, and continued to grow during the storage. Mirasol treatment increased the number of platelets with a mitochondrial membrane potential loss. On the 3rd day of storage, 50% of Mirasol-treated platelets did not respond to activation; on the 5th day, none did. This agreed well with a decrease (approximately twofold) in the effectiveness of Mirasol-treated PC transfusions. Transfusions of PCs stored in SSP+ were accompanied by fewer inefficient transfusions and post-transfusion reactions than of PCs stored in plasma. CONCLUSION: Treatment with Mirasol decreased platelet function, particularly profoundly on the 5th day of storage, and led to a decrease in the effectiveness of transfusions. SSP+ did not affect laboratory parameters significantly compared with plasma, but decreased the percentage of transfusion complications.
Asunto(s)
Plaquetas/citología , Plaquetas/efectos de los fármacos , Riboflavina/farmacología , Rayos Ultravioleta , Plaquetas/efectos de la radiación , Conservación de la Sangre , Citometría de Flujo , Humanos , Potencial de la Membrana Mitocondrial , Selectina-P/sangre , Fosfatidilserinas/sangre , Activación Plaquetaria/efectos de los fármacos , Activación Plaquetaria/efectos de la radiación , Recuento de Plaquetas , Complejo GPIIb-IIIa de Glicoproteína Plaquetaria/análisis , Transfusión de Plaquetas , PlaquetoferesisRESUMEN
A key prerequisite in an ideal supported lipid bilayer based cell membrane model is that the mobility of both the lipid matrix and its components are unhindered by the underlying support. This is not trivial and with the exception of liposomes, many of even the most advanced approaches, although accomplishing lipid mobility, fail to achieve complete mobility of incorporated membrane proteins. This is addressed in a novel platform comprising lipid bilayers assembled over buffer-filled, arrays of spherical cap microcavities formed from microsphere template polydimethoxysilane. Prior to bilayer assembly the PDMS is rendered hydrophilic by plasma treatment and the lipid bilayer prepared using Langmuir Blodgett assembly followed by liposome/proteoliposome fusion. Fluorescence Lifetime Correlation Spectroscopy confirmed the pore suspended lipid bilayer exhibits diffusion coefficients comparable to free-standing vesicles in solution. The bilayer modified arrays are highly reproducible and stable over days. As the bilayers are suspended over deep aqueous reservoirs, reconstituted membrane proteins experience an aqueous interface at both membrane interfaces and attain full lateral mobility. Their utility as membrane protein platforms was exemplified in two case studies with proteins of different dimensions in their extracellular and cytoplasmic domains reconstituted into DOPC lipid bilayers; Glycophorin A, and Integrin αIIbß3. In both cases, the proteins exhibited 100% mobility with high lateral diffusion coefficients.
Asunto(s)
Membrana Dobles de Lípidos/química , Proteínas de la Membrana/análisis , Fosfatidilcolinas/química , Difusión , Dimetilpolisiloxanos/química , Diseño de Equipo , Glicoforinas/análisis , Humanos , Interacciones Hidrofóbicas e Hidrofílicas , Fluidez de la Membrana , Modelos Moleculares , Complejo GPIIb-IIIa de Glicoproteína Plaquetaria/análisis , Espectrometría de FluorescenciaRESUMEN
We report for the first time seeing and counting integrin α(IIb)ß3 on a single-cell level. The proposed method is based on the using of the Au cluster probe. With the fluorescent property of Au24 cluster and the specific targeting ability of peptide, our probe can directly visualize integrin α(IIb)ß3 on the membrane of human erythroleukemia cells (HEL) via confocal microscopy. On the basis of the accurate formula of our probe (Au24Peptide8), the number of integrin α(IIb)ß3 can be precisely counted by quantifying the gold content on a single HEL cell via laser ablation inductively coupled plasma mass spectrometry. Our results reveal that the number of integrin α(IIb)ß3 on a single cell varies from 5.75 × 10(-17) to 9.11 × 10(-17) mol, because of the heteroexpression levels of α(IIb)ß3 on individual cells.
Asunto(s)
Membrana Celular/metabolismo , Oro/química , Leucemia Eritroblástica Aguda/metabolismo , Fragmentos de Péptidos/análisis , Complejo GPIIb-IIIa de Glicoproteína Plaquetaria/análisis , Análisis de la Célula Individual/métodos , Plaquetas/metabolismo , Membrana Celular/ultraestructura , Humanos , Microscopía Confocal , Células Tumorales CultivadasRESUMEN
Platelet signatures indicating differential dysfunction, hyperactivation, aggregation or adhesion are capable of expressing their characters during the journey of a disease process, and can be utilized as cost effective biomarkers with immense clinical value. Type 2 diabetes mellitus (T2DM) is a major lifestyle disease of contemporary world with progression to diabetes associated cardiovascular diseases (DM-CVD). We identified a few potential biomarkers in platelets of T2DM to analyze the thrombotic risk in diabetes subjects by utilizing flow cytometric quantification with different flurochrome conjugated monoclonal antibodies. Our study describes interesting correlations (p<0.0001) for different clinical parameters of concurrent threat for vessel occlusion and the status of indices like reactive oxygen species, von Willebrand factor and mitochondrial membrane potential using western blotting and fluorescence techniques. Our study involved 32 T2DM, and 31 T2DM-CVD subjects compared to 29 healthy controls without any history of T2DM or CVD. An altered expression of platelet surface markers P-selectin (CD62p) and GpIIb/IIIa (CD 41/61, PAC1) along with changes in the platelet size due to agonist induced activation contributed to the enhanced thrombotic potential in the patients. This work elucidates the prospect of platelet biomarkers as diagnostic tool to predict cardiovascular risk in DM subjects.
Asunto(s)
Plaquetas/patología , Enfermedades Cardiovasculares/complicaciones , Enfermedades Cardiovasculares/diagnóstico , Diabetes Mellitus Tipo 2/complicaciones , Diabetes Mellitus Tipo 2/diagnóstico , Biomarcadores/análisis , Femenino , Citometría de Flujo , Humanos , Masculino , Potencial de la Membrana Mitocondrial , Persona de Mediana Edad , Selectina-P/análisis , Complejo GPIIb-IIIa de Glicoproteína Plaquetaria/análisis , Especies Reactivas de Oxígeno/análisis , Factores de Riesgo , Factor de von Willebrand/análisisRESUMEN
High levels of low-density lipoprotein cholesterol (LDL-C) enhance platelet activation, whereas high levels of high-density lipoprotein cholesterol (HDL-C) exert a cardioprotective effect. However, the effects on platelet activation of high levels of LDL-C combined with low levels of HDL-C (HLC) have not yet been reported. We aimed to evaluate the platelet activation marker of HLC patients and investigate the antiplatelet effect of atorvastatin on this population. Forty-eight patients with high levels of LDL-C were enrolled. Among these, 23 had HLC and the other 25 had high levels of LDL-C combined with normal levels of HDL-C (HNC). A total of 35 normocholesterolemic (NOMC) volunteers were included as controls. Whole blood flow cytometry and platelet aggregation measurements were performed on all participants to detect the following platelet activation markers: CD62p (P-selectin), PAC-1 (GPIIb/IIIa), and maximal platelet aggregation (MPAG). A daily dose of 20 mg atorvastatin was administered to patients with high levels of LDL-C, and the above assessments were obtained at baseline and after 1 and 2 months of treatment. The expression of platelets CD62p and PAC-1 was increased in HNC patients compared to NOMC volunteers (P<0.01 and P<0.05). Furthermore, the surface expression of platelets CD62p and PAC-1 was greater among HLC patients than among HNC patients (P<0.01 and P<0.05). Although the expression of CD62p and PAC-1 decreased significantly after atorvastatin treatment, it remained higher in the HLC group than in the HNC group (P<0.05 and P=0.116). The reduction of HDL-C further increased platelet activation in patients with high levels of LDL-C. Platelet activation remained higher among HLC patients regardless of atorvastatin treatment.
Asunto(s)
Anticolesterolemiantes/uso terapéutico , HDL-Colesterol/sangre , LDL-Colesterol/sangre , Ácidos Heptanoicos/uso terapéutico , Hipercolesterolemia/sangre , Activación Plaquetaria , Pirroles/uso terapéutico , Anciano , Análisis de Varianza , Atorvastatina , Biomarcadores/análisis , HDL-Colesterol/metabolismo , LDL-Colesterol/metabolismo , Femenino , Citometría de Flujo , Humanos , Hipercolesterolemia/tratamiento farmacológico , Masculino , Persona de Mediana Edad , Selectina-P/sangre , Agregación Plaquetaria/efectos de los fármacos , Inhibidores de Agregación Plaquetaria/metabolismo , Complejo GPIIb-IIIa de Glicoproteína Plaquetaria/análisis , Estadísticas no ParamétricasRESUMEN
BACKGROUND: Platelet (PLT) concentrates can be cryopreserved in dimethyl sulfoxide (DMSO) and stored at -80°C for 2 years. These storage conditions improve availability in both rural and military environments. Previous phenotypic and in vitro studies of cryopreserved PLTs are limited by comparison to fresh liquid-stored PLTs, rather than PLTs stored over their clinically relevant shelf life. Further, nothing is known of the effect of reconstituting cryopreserved PLTs in plasma stored at a variety of clinically relevant temperatures. STUDY DESIGN AND METHODS: Apheresis PLTs were either stored at room temperature for 5 days or cryopreserved at -80°C with 5% DMSO. Cryopreserved PLTs were thawed at 37°C and reconstituted in plasma (stored at different temperatures) and compared to fresh and expired liquid-stored PLTs. In vitro assays were performed to assess glycoprotein expression, PLT activity, microparticle content, and function. RESULTS: Compared to liquid PLTs over storage, cryopreserved PLTs had reduced expression of the key glycoprotein receptors GPIbα and GPIIb. However, the proportion of PLTs expressing activation markers CD62P and CD63 was similar between cryopreserved and liquid-stored PLTs at expiry. Cryopreserved PLT components contained significantly higher numbers of phosphatidylserine- and tissue factor-positive microparticles than liquid-stored PLTs, and these microparticles reduced the time to clot formation and increased thrombin generation. CONCLUSION: There are distinct differences between cryopreserved and liquid-stored PLTs. Cryopreserved PLTs also have an enhanced hemostatic activity. Knowledge of these in vitro differences will be essential to understanding the outcomes of a clinical trial comparing cryopreserved PLTs and liquid PLTs stored for various durations.
Asunto(s)
Plaquetas/metabolismo , Conservación de la Sangre/métodos , Criopreservación , Complejo GPIIb-IIIa de Glicoproteína Plaquetaria/análisis , Plaquetas/efectos de los fármacos , Micropartículas Derivadas de Células , Crioprotectores/farmacología , Dimetilsulfóxido , Hemostasis , Humanos , Técnicas In Vitro , Selectina-P/sangre , Agregación Plaquetaria , Transfusión de Plaquetas , Plaquetoferesis , Tetraspanina 30/sangreRESUMEN
We report here design of a graphene oxide-based electrochemical biosensor for detection of platelet-derived microparticles (PMPs), a major risk factor for arterial pro-thrombotic pathologies like acute myocardial infarction and stroke. Electrodes were fabricated with immobilized layers of graphene oxide and a specific antibody targeted against active conformation of integrin αIIbß3 on PMP surface. Results showed progressive rise in impedance in Nyquist plots with increasing number of PMPs in analyte. The sensor was highly specific for PMPs and did not identify microparticles originating from other cells. Blood obtained from patients diagnosed with acute myocardial infarction exhibited significantly higher values of impedance, consistent with larger number of circulating PMPs in these patients, as compared to samples from healthy individuals, thus validating biosensor as a specific, sensitive, label-free and cost-effective tool for rapid point-of-care detection of PMPs at bedside. Our biosensor is most ideal for mass population screening programs at periphery-level healthcare units with limited resources. It is aimed at early detection of individuals having higher imminent cardiovascular risk, as well as for routine analysis, which in turn would contribute to better management and survival of screened 'high-risk' subjects.
Asunto(s)
Técnicas Biosensibles/métodos , Plaquetas/patología , Micropartículas Derivadas de Células/patología , Grafito/química , Infarto del Miocardio/sangre , Sistemas de Atención de Punto , Anticuerpos Inmovilizados/química , Plaquetas/química , Micropartículas Derivadas de Células/química , Impedancia Eléctrica , Técnicas Electroquímicas/métodos , Humanos , Complejo GPIIb-IIIa de Glicoproteína Plaquetaria/análisis , Sensibilidad y Especificidad , Trombosis/sangre , Trombosis/etiologíaRESUMEN
BACKGROUND: After percutaneous coronary intervention (PCI) for non-ST-segment elevation myocardial infarction (NSTEMI), treatment with a P2Y12 antagonist with aspirin is recommended for 1 year. OBJECTIVES: The oral P2Y12 antagonists ticagrelor and prasugrel have higher recommendations than clopidogrel, but it is unknown if administration before the start of PCI is beneficial. METHODS: In the randomized, double-blind ACCOAST (A Comparison of prasugrel at the time of percutaneous Coronary intervention Or as pre-treatment At the time of diagnosis in patients with non-ST-segment elevation myocardial infarction) trial, 4,033 patients were diagnosed with NSTEMI and 68.7% underwent PCI; 1,394 received pre-treatment with prasugrel (30-mg loading dose), and 1,376 received placebo. At the time of PCI, patients who received pre-treatment with prasugrel received an additional 30-mg dose of prasugrel, and those who received placebo received a 60-mg loading dose of prasugrel. Primary efficacy was a composite of cardiovascular death, myocardial infarction, stroke, urgent revascularization, or glycoprotein IIb/IIIa bailout through 7 days from randomization. Investigators captured the presence of thrombus on initial angiography and during PCI. RESULTS: The incidence of the primary endpoint through 7 days from randomization in the pre-treatment group versus the no pre-treatment group was 13.1% versus 13.1% (p = 0.93). Pre-treatment with prasugrel was not associated with decreases in any ischemic event, including total mortality. Patients with thrombus on angiography had a 3-fold higher incidence of the primary endpoint than patients without thrombus. There was no impact of pre-treatment with prasugrel on the presence of thrombus before PCI or on occurrence of stent thrombosis after PCI. There was a 3-fold increase in all non-coronary artery bypass graft Thrombolysis In Myocardial Infarction (TIMI) major bleeding and a 6-fold increase in non-coronary artery bypass graft life-threatening bleeding with pre-treatment with prasugrel; the same trends persisted in patients who had radial or femoral access even with use of a closure device. CONCLUSIONS: These findings support deferring treatment with prasugrel until a decision is made about revascularization in patients with NSTEMI undergoing angiography within 48 h of admission. (A Comparison of prasugrel at the time of percutaneous Coronary intervention Or as pre-treatment At the time of diagnosis in patients with non-ST-segment elevation myocardial infarction [ACCOAST]; NCT01015287).
Asunto(s)
Hemorragia , Complicaciones Intraoperatorias , Infarto del Miocardio , Intervención Coronaria Percutánea , Piperazinas , Tiofenos , Anciano , Angiografía/métodos , Relación Dosis-Respuesta a Droga , Método Doble Ciego , Electrocardiografía/métodos , Determinación de Punto Final , Femenino , Hemorragia/inducido químicamente , Hemorragia/prevención & control , Humanos , Complicaciones Intraoperatorias/inducido químicamente , Complicaciones Intraoperatorias/prevención & control , Masculino , Persona de Mediana Edad , Infarto del Miocardio/diagnóstico , Infarto del Miocardio/terapia , Intervención Coronaria Percutánea/efectos adversos , Intervención Coronaria Percutánea/métodos , Piperazinas/administración & dosificación , Piperazinas/efectos adversos , Inhibidores de Agregación Plaquetaria/administración & dosificación , Inhibidores de Agregación Plaquetaria/efectos adversos , Complejo GPIIb-IIIa de Glicoproteína Plaquetaria/análisis , Clorhidrato de Prasugrel , Cuidados Preoperatorios/métodos , Medición de Riesgo , Tiofenos/administración & dosificación , Tiofenos/efectos adversos , Resultado del TratamientoRESUMEN
BACKGROUND: The activation of pro-coagulation mechanisms associated with the vascular wall's immune and inflammatory responses wall to injury plays a crucial role in the mechanisms of the induction and progression of atherosclerosis. OBJECTIVES: The aim of this study was to determine the role of protease activated receptors (PAR-1) expressed on the surface of blood platelets in the pathogenesis of chronic peripheral arterial obliterative disease (PAOD) in patients with obliterative atherosclerosis (n = 24) and diabetic macroangiopathy (n = 16), as well as in the controls (n = 12). MATERIAL AND METHODS: In addition to the expression of PAR-1, serum/plasma concentrations of thrombin-antithrombin complex (TAT), the von Willebrand factor (vWF), the platelet-derived growth factor, monocyte chemotactic protein, the soluble form of the platelet endothelial cell adhesion molecule, thrombin activatable fibrinolysis inhibitor and interleukin 6 (IL-6) were determined. RESULTS: Compared to the controls, PAOD patients were characterized by significantly higher levels of PAR-1 expression, vWF, TAT and IL-6. Individuals with diabetic macroangiopathy did not differ significantly from individuals with obliterative atherosclerosis in terms of PAR-1 expression. Upon activation with thrombin receptor antagonist peptide (TRAP), the levels of PAR-1 were comparable in all analyzed groups. In patients with diabetic macroangiopathy, a significant association was observed between the expression of PAR-1 on the surface of the platelet and the serum TAT concentration, as well as between TAT and serum IL-6 concentration. CONCLUSIONS: Enhanced expression of PAR-1 on the thrombocyte surface in chronic PAOD patients occurs equally in cases of diabetic macroangiopathy and in individuals free from this endocrine pathology.
Asunto(s)
Plaquetas/química , Enfermedad Arterial Periférica/sangre , Receptor PAR-1/sangre , Antitrombina III , Enfermedad Crónica , Humanos , Persona de Mediana Edad , Péptido Hidrolasas/sangre , Complejo GPIIb-IIIa de Glicoproteína Plaquetaria/análisis , Factor de von Willebrand/análisisRESUMEN
BACKGROUND: The beneficial effect of fruit- and vegetable-rich diets on cardiovascular health is partly attributed to the effect of their bioactive compounds on platelet function. The aim of this study was to investigate the effects of bioactive-rich plant extracts and isolated bioactive metabolites on platelet function. Blood samples from healthy subjects (n = 4) and subjects with metabolic syndrome (n = 4) were treated with six extracts of bioactive-rich plants consumed as traditional foods in the Black Sea region, or with human metabolites of the bioactives quercetin and sulforaphane. Markers of arachidonic acid induced platelet activation and platelet-leucocyte aggregation were assessed using flow cytometry. RESULTS: In subjects with metabolic syndrome, kale extract significantly inhibited agonist induced P-selectin expression (P = 0.004). Sulforaphane-cysteine-glycine, a human plasma metabolite of the related glucosinolate, glucoraphanin, significantly inhibited P-selectin and GPIIb-IIIa expression (P = 0.020 and 0.024, respectively) and platelet-neutrophil aggregation (P = 0.027). Additionally, pomegranate extract significantly inhibited GPIIb-IIIa expression (P = 0.046) in subjects with metabolic syndrome. In healthy subjects only dill extract significantly inhibited agonist induced P-selectin expression (P = 0.025). CONCLUSION: These data show that bioactive-rich extracts of kale and pomegranate that are consumed as traditional plant foods of Black Sea area countries were effective in modulating platelet function.