RESUMEN
Lipids participate in many important biological functions through energy storage, membrane structure stabilization, signal transduction, and molecular recognition. Previous studies have shown that patients with esophageal squamous cell carcinoma (ESCC) have abnormal lipid metabolism. However, studies characterizing lipid metabolism in ESCC patients through lipidomics are limited. Plasma lipid profiles of 65 ESCC patients and 42 healthy controls (HC) were characterized by lipidomics-based ultraperformance liquid chromatography quadrupole time-of-flight mass spectrometry (UPLC-Q-TOF-MS). Single-factor and multi-factor statistical analysis were used to screen the differences in blood lipids between groups, and combined with component ratio analysis and receiver operating characteristic (ROC) curve diagnostic efficiency assessment, to reveal the potential mechanisms and biomarkers of ESCC. There were significant differences in lipid profiles between the ESCC and HC groups. Thirty-six differential lipids (11 up-regulated and 25 down-regulated) were selected based on the criteria of p < .05 and fold change > 1.3 or < 0.77. Glycerophospholipids were the major differential lipids, suggesting that these lipid metabolic pathways exhibit a significant imbalance that may contribute to the development of esophageal squamous cell carcinoma. Among them, the seven candidate biomarkers for esophageal squamous cell carcinoma with the highest diagnostic value are three phosphatidylserine (PS), three fatty acids (FA) and one phosphatidylcholine (PC).
Asunto(s)
Biomarcadores de Tumor , Neoplasias Esofágicas , Carcinoma de Células Escamosas de Esófago , Lipidómica , Humanos , Carcinoma de Células Escamosas de Esófago/metabolismo , Carcinoma de Células Escamosas de Esófago/sangre , Masculino , Neoplasias Esofágicas/sangre , Neoplasias Esofágicas/metabolismo , Lipidómica/métodos , Femenino , Persona de Mediana Edad , Biomarcadores de Tumor/sangre , Biomarcadores de Tumor/metabolismo , Carcinoma de Células Escamosas/sangre , Estudios de Casos y Controles , Anciano , Metabolismo de los Lípidos , Lípidos/sangre , Curva ROC , Glicerofosfolípidos/sangre , Fosfatidilserinas/metabolismo , Fosfatidilserinas/sangre , Ácidos Grasos/sangreRESUMEN
Chronic lymphocytic leukemia (CLL) is a chronic lymphoproliferative disorder characterized by monoclonal B cell proliferation. Studies carried out in recent years suggest that extracellular vesicles (EVs) may be a potential biomarker in cancer. Tyro3-Axl-Mertk (TAM) Receptor Tyrosine Kinases (RTKs) and Phosphatidylserine (PS) have crucial roles in macrophage-mediated immune response under normal conditions. In the tumor microenvironment, these molecules contribute to immunosuppressive signals and prevent the formation of local and systemic antitumor immune responses. Based on this, we aimed to evaluate the amount of PS and TAM RTK in plasma and on the surface of EVs in CLL patients and healthy volunteers in this study. In this study, 25 CLL (11 F/14 M) patients in the Rai (O-I) stage, newly diagnosed or followed up without treatment, and 15 healthy volunteers (11 F/4 M) as a control group were included. For all samples, PS and TAM RTK levels were examined first in the plasma and then in the EVs obtained from the plasma. We detected a significant decrease in plasma PS, and TAM RTK levels in CLL patients compared to the control. Besides, we determined a significant increase in TAM RTK levels on the EV surface in CLL, except for PS. In conclusion, these receptor levels measured by ELISA in plasma may not be effective for the preliminary detection of CLL. However, especially TAM RTKs on the surface of EVs may be good biomarkers and potential targets for CLL therapies.
Asunto(s)
Vesículas Extracelulares , Leucemia Linfocítica Crónica de Células B , Fosfatidilserinas , Proteínas Tirosina Quinasas Receptoras , Humanos , Leucemia Linfocítica Crónica de Células B/sangre , Leucemia Linfocítica Crónica de Células B/diagnóstico , Leucemia Linfocítica Crónica de Células B/metabolismo , Vesículas Extracelulares/metabolismo , Vesículas Extracelulares/química , Femenino , Fosfatidilserinas/metabolismo , Fosfatidilserinas/sangre , Proteínas Tirosina Quinasas Receptoras/metabolismo , Proteínas Tirosina Quinasas Receptoras/sangre , Masculino , Persona de Mediana Edad , Anciano , Tirosina Quinasa del Receptor Axl , Proteínas Proto-Oncogénicas/sangre , Proteínas Proto-Oncogénicas/metabolismo , Adulto , Tirosina Quinasa c-Mer/metabolismo , Anciano de 80 o más AñosRESUMEN
BACKGROUND: As a method with insignificant adverse effects on in vitro quality of platelet concentrates (PCs), gamma irradiation is applied to abrogate the risk of transfusion-associated graft-vs-host disease in vulnerable recipients. However, there is some evidence of lower posttransfusion responses and proteomic alterations in gamma-irradiated platelets (PLTs), which raises some questions about their quality, safety, and efficacy. Since reactive oxygen species (ROS) are considered as markers of PLT storage lesion (PSL), the study presented here investigated oxidant state in gamma-irradiated PCs. STUDY DESIGN AND METHODS: PLT-rich plasma PC was split into two bags, one kept as control while other was subjected to gamma irradiation. Within 7 days of storage, the levels of intra-PLT superoxide, H2 O2 , mitochondrial ROS, P-selectin expression, and phosphatidylserine (PS) exposure were detected by flow cytometry while intracellular reduced glutathione (GSH), glucose concentration, and lactate dehydrogenase (LDH) activity were measured by enzymocolorimetric method. RESULTS: GSH decreased, while ROS generation and LDH activity increased, during storage. Gamma irradiation significantly attenuated GSH whereas increased ROS generation in earlier and later stages of storage associated with either P-selectin or PS exposure increments. CONCLUSION: Gamma irradiation can significantly increase cytosolic ROS generation in two distinct phases, one upon irradiation and another later in longer-stored PCs. While earlier ROS influx seems to be governed by direct effect of irradiation, the second phase of oxidant stress is presumably due to the storage-dependent PLT activation. Intriguingly, these observations were also in line with early P-selectin increments and increased PS exposure in longer-stored PLTs. Given the mutual link between ROS generation and PLT activation, further investigation is required to explore the effect of gamma irradiation on the induction of PSL.
Asunto(s)
Plaquetas/efectos de la radiación , Conservación de la Sangre , Rayos gamma , Glucemia/análisis , Plaquetas/metabolismo , Glutatión/sangre , Humanos , Peróxido de Hidrógeno/sangre , Concentración de Iones de Hidrógeno , L-Lactato Deshidrogenasa/sangre , Mitocondrias/metabolismo , Mitocondrias/efectos de la radiación , Oxidación-Reducción , Selectina-P/sangre , Fosfatidilserinas/sangre , Activación Plaquetaria , Plasma Rico en Plaquetas , Especies Reactivas de Oxígeno/sangre , Superóxidos/sangre , Factores de TiempoRESUMEN
BACKGROUND AND AIM: Lipids play important roles in inflammation and may be involved in the pathophysiology of inflammatory bowel disease (IBD). Here, we evaluated the characteristics of the plasma lipid profile in patients with IBD. METHODS: Plasma samples were collected from 20 patients with Crohn's disease (CD), 20 patients with ulcerative colitis (UC), and 10 healthy volunteers (HVs) after overnight fasting. The subjects were men between 20 and 49 years of age with no history of hyperlipidemia. A total of 698 molecular species in 22 lipid classes were analyzed by ultra-performance liquid chromatography-electrospray ionization-tandem mass spectrometry. RESULTS: Lipid classes of lysophosphatidic acid, lysophosphatidylserine (LPS), phosphatidylserine (PS), and shingosine-1-phosphate (S1P) were significantly increased in UC patients compared with the HV. The LPS, PS, and S1P levels were significantly increased, while those of lysophosphatidylinositol and phosphatidylcholine were significantly decreased in CD patients compared with HV. Among PS species, the levels of PSacyl (PSa) 40:3, PSa 38:3, and PSa 42:4 were significantly higher in CD patients, both active and remissive stage, than in HV. The LPS 18:0 level was significantly higher in CD and UC patients compared with HV. PSa 40:3 and PSa 38:3 levels positively correlated with the Crohn's Disease Activity Index, erythrocyte sedimentation rate, and platelet count and negatively correlated with hemoglobin, hematocrit, and albumin levels in CD patients. CONCLUSION: The lipid profile in IBD patients exhibits significant alterations, and PS levels are associated with clinical disease activity in CD patients.
Asunto(s)
Cromatografía Líquida de Alta Presión/métodos , Enfermedades Inflamatorias del Intestino/diagnóstico , Lipidómica/métodos , Fosfatidilserinas/sangre , Espectrometría de Masa por Ionización de Electrospray/métodos , Adulto , Biomarcadores/sangre , Colitis Ulcerosa/sangre , Colitis Ulcerosa/diagnóstico , Enfermedad de Crohn/sangre , Enfermedad de Crohn/diagnóstico , Femenino , Humanos , Enfermedades Inflamatorias del Intestino/sangre , Lisofosfolípidos/sangre , Masculino , Persona de Mediana Edad , Adulto JovenRESUMEN
Extracellular vesicles (EV) act as a cellular communication tool by carrying lipids, proteins and micro RNA (miR) between cells, thereby playing a pivotal role in thromboembolic processes. The effect of P2Y12 inhibitors on pro-coagulatory, phosphatidylserine (PS)-expressing EV has been investigated previously, but only in vitro or during confounding clinical conditions, such as acute coronary syndrome. Hence, we enrolled 62 consecutive patients 12 month after percutaneous coronary intervention and stent implantation and consequent treatment with dual-antiplatelet therapy consisting of low-dose aspirin and P2Y12 inhibitors. Blood for platelet function testing and EV and miR measurements was taken on the last day of P2Y12 inhibitor intake (baseline, on-treatment) and 10, 30 and 180 days thereafter (off-treatment). We did not observe any influence of P2Y12 inhibitors on the levels of PS-EV or EV sub-population from platelets, erythrocytes, monocytes or endothelial cells, respectively. There was no relationship between platelet function and EV levels in plasma. However, the association of miR-21 and miR-150 with platelet EVs was significantly different between on- and off-treatment measurements. Hence, our study suggests no influence of P2Y12 inhibition on the count of EVs in plasma, but on the potential cargo of platelet-derived EV.
Asunto(s)
Enfermedad de la Arteria Coronaria/tratamiento farmacológico , Enfermedad de la Arteria Coronaria/genética , Vesículas Extracelulares/efectos de los fármacos , Vesículas Extracelulares/metabolismo , MicroARNs/sangre , Antagonistas del Receptor Purinérgico P2Y/uso terapéutico , Anciano , Aspirina/farmacología , Clopidogrel/uso terapéutico , Enfermedad de la Arteria Coronaria/sangre , Enfermedad de la Arteria Coronaria/cirugía , Femenino , Humanos , Masculino , MicroARNs/genética , Persona de Mediana Edad , Fosfatidilserinas/sangre , Fosfatidilserinas/metabolismo , Clorhidrato de Prasugrel/uso terapéutico , Ticagrelor/uso terapéuticoRESUMEN
Programmed cell death of non-nucleated blood cells - platelets - could be associated with pathophysiology of oncologic and oncohematologic diseases. It contributes to both bleedings (caused by the thrombocytopenia, which is induced by elimination of the platelets) and thrombosis (caused by the processes of blood coagulation on the surface of phosphatidylserine exposing platelets). Here we characterized functional responses of platelets from the patients with various oncological disorders undergoing chemotherapy and compared them to the platelets from the healthy donors and platelets pre-incubated with apoptosis inducer ABT-737. Some patients exhibited diminished capability of platelets to aggregate. Immunophenotyping of these platelets revealed their pre-activation in comparison to the platelets from the healthy donors. Calcium signaling analysis revealed that in the patient-derived platelets, as well as in the apoptotic platelets, intracellular calcium levels were increased in resting cells. However, moderate level of this increase together with weak expression of phosphatidylserine allows us to assume that apoptotic processes in the circulating platelets from the patients are limited.
Asunto(s)
Antineoplásicos/farmacología , Apoptosis/efectos de los fármacos , Plaquetas/efectos de los fármacos , Neoplasias Hematológicas , Adolescente , Compuestos de Bifenilo/farmacología , Coagulación Sanguínea/efectos de los fármacos , Calcio/metabolismo , Niño , Preescolar , Femenino , Neoplasias Hematológicas/sangre , Neoplasias Hematológicas/tratamiento farmacológico , Humanos , Masculino , Nitrofenoles/farmacología , Fosfatidilserinas/sangre , Piperazinas/farmacología , Sulfonamidas/farmacologíaRESUMEN
N,N-Diethyl-3-methylbenzamide (DEET) is the most widely used insect repellent in the world. Adverse effects following DEET exposure are well documented. Moreover, DEET has been shown to possess cytotoxic and apoptotic properties in nucleated cells. Although red blood cells (RBCs) lack intracellular organelles, they nevertheless undergo programmed cell death termed eryptosis. Compromised RBC health contributes to the development of anemia; a condition affecting 25% of the global population. This study investigated the interaction between DEET and human RBCs, and explored accompanying biochemical and molecular alterations. RBCs at 5% hematocrit were incubated in presence and absence of 1-5â¯mM (0.02%-0.1%) of DEET for 6â¯h at 37⯰C. Hemolysis was spectrophotometrically determined by hemoglobin release, while major eryptotic events were analyzed by flow cytometer. Phosphatidylserine (PS) exposure was detected with Annexin-V-FITC, cell volume by forward scatter (FSC) of light, intracellular calcium with Fluo-3/AM, and reactive oxygen species with 2',7'-dichlorodihydrofluorescein diacetate (H2DCFDA). DEET caused slight hemolysis at 4 and 5â¯mM, and significantly increased Annexin-V-FITC and Fluo3 fluorescence, with reduced FSC at 5â¯mM. Removal of extracellular Ca2+ abolished DEET-induced Fluo3 fluorescence but had no effect on Annexin-V binding. Importantly, blockade of eryptotic signaling mediators p38 MAPK, caspases, protein kinase C, casein kinase 1, or necroptotic kinases receptor-interacting protein 1 and mixed lineage kinase domain-like protein, with small molecule inhibitors, did not ameliorate DEET-mediated PS externalization. In conclusion, DEET elicits suicidal erythrocyte death; an event characterized by loss of membrane asymmetry, cell shrinkage, and elevations in intracellular Ca2+ mainly through dysregulated Ca2+ influx.
Asunto(s)
DEET/toxicidad , Eriptosis/efectos de los fármacos , Eritrocitos/efectos de los fármacos , Repelentes de Insectos/toxicidad , Compuestos de Anilina , Anexina A5 , Calcio/sangre , Muerte Celular/fisiología , Índices de Eritrocitos/efectos de los fármacos , Eritrocitos/fisiología , Citometría de Flujo , Fluoresceína-5-Isotiocianato/análogos & derivados , Hemólisis/efectos de los fármacos , Humanos , Fosfatidilserinas/sangre , Especies Reactivas de Oxígeno/sangre , Transducción de Señal/efectos de los fármacos , Transducción de Señal/fisiología , XantenosRESUMEN
BACKGROUND: Preterm newborns are at thrombohemorrhagic risk during the early neonatal period. Taking into account the lack of informative tools for the laboratory diagnosis of hemostasis disorders in newborns, our goal was to determine the baseline values of thrombodynamics and platelet functional activity in healthy term and moderately preterm newborns during the early neonatal period future potential clinical use of these tests. METHODS: Coagulation was assessed using an integral assay of thrombodynamics and standard coagulation assays, and platelet functional activity was estimated by flow cytometry. RESULTS: Hypercoagulation of newborns, represented by a significantly higher clot growth velocity and the presence of spontaneous clots in the thrombodynamics, was combined with platelet hypoactivity. Granule release, phosphatidylserine exposure, and the ability to change shape upon activation were decreased in the platelets of moderately preterm newborns. The platelet function remained at the same level over the first four days of life, whereas the hypercoagulation became less pronounced. CONCLUSIONS: The hemostasis of newborns is characterized by hypercoagulation combined with reduced platelet functional activity. Moderately preterm and term newborns do not differ in the parameters of coagulation, while some of the functional responses of platelets are lower in moderately preterm newborns than in term.
Asunto(s)
Coagulación Sanguínea , Plaquetas/metabolismo , Recien Nacido Prematuro/sangre , Activación Plaquetaria , Nacimiento Prematuro , Trombofilia/sangre , Biomarcadores/sangre , Femenino , Edad Gestacional , Humanos , Recién Nacido , Masculino , Selectina-P/sangre , Fosfatidilserinas/sangre , Nacimiento a Término , Trombofilia/diagnósticoRESUMEN
OBJECTIVE: The objective of this study was to measure the role of platelets and red blood cells on thrombus propagation in an in vitro model of venous valvular stasis. APPROACH AND RESULTS: A microfluidic model with dimensional similarity to human venous valves consists of a sinus distal to a sudden expansion, where for sufficiently high Reynolds numbers, 2 countercurrent vortices arise because of flow separation. The primary vortex is defined by the points of flow separation and reattachment. A secondary vortex forms in the deepest recess of the valve pocket characterized by low shear rates. An initial fibrin gel formed within the secondary vortex of a tissue factor-coated valve sinus. Platelets accumulated at the interface of the fibrin gel and the primary vortex. Red blood cells at physiological hematocrits were necessary to provide an adequate flux of platelets to support thrombus growth out of the valve sinus. A subpopulation of platelets that adhered to fibrin expose phosphatidylserine. Platelet-dependent thrombus growth was attenuated by inhibition of glycoprotein VI with a blocking Fab fragment or D-dimer. CONCLUSIONS: A 3-step process regulated by hemodynamics was necessary for robust thrombus propagation: First, immobilized tissue factor initiates coagulation and fibrin deposition within a low flow niche defined by a secondary vortex in the pocket of a model venous valve. Second, a primary vortex delivers platelets to the fibrin interface in a red blood cell-dependent manner. Third, platelets adhere to fibrin, activate through glycoprotein VI, express phosphatidylserine, and subsequently promote thrombus growth beyond the valve sinus and into the bulk flow.
Asunto(s)
Coagulación Sanguínea , Plaquetas/metabolismo , Eritrocitos/metabolismo , Glicoproteínas de Membrana Plaquetaria/metabolismo , Trombosis de la Vena/sangre , Válvulas Venosas/metabolismo , Velocidad del Flujo Sanguíneo , Fibrina/metabolismo , Hematócrito , Hemodinámica , Humanos , Dispositivos Laboratorio en un Chip , Técnicas Analíticas Microfluídicas/instrumentación , Fosfatidilserinas/sangre , Transducción de Señal , Estrés Mecánico , Tromboplastina/metabolismo , Trombosis de la Vena/patología , Trombosis de la Vena/fisiopatología , Válvulas Venosas/patología , Válvulas Venosas/fisiopatologíaRESUMEN
BACKGROUND: Obesity plays crucial roles in the pathogenesis of metabolic diseases such as hyperlipidemia, nonalcoholic fatty liver disease (NAFLD), and type 2 diabetes (T2D). The underlying mechanisms linking obesity to metabolic diseases are still less understandable. METHODS: Previously, we screened a group of spontaneously obese rhesus monkeys. Here, we performed a plasma lipidomic analysis of normal and obese monkeys using gas chromatography/mass spectroscopy (GC/MS) and ultra-high performance liquid chromatography/mass spectroscopy (UPLC/MS). RESULTS: In total, 143 lipid species were identified, quantified, and classified into free fatty acids (FFA), phosphatidylcholine (PC), phosphatidylethanolamine (PE), phosphatidylinositol (PI), phosphatidylserine (PS), phosphatidylglycerol (PG), lysophosphatidylcholine (LPC), lysophosphatidic acid (LPA), and sphingomyelin (SM). Data analysis showed that the obese monkeys had increased levels of fatty acids palmitoleic acid (C16:1) and arachidonic acid (C20:4), FFA especially palmitic acid (C16:0), as well as certain PC species and SM species. Surprisingly, the plasma level of LPA-C16:0 was approximately four-fold greater in the obese monkeys. Conversely, the levels of most PE species were obviously reduced in the obese monkeys. CONCLUSION: Collectively, our work suggests that lipids such as FFA C16:0 and 16:0-LPA may be potential candidates for the diagnosis and study of obesity-related diseases.
Asunto(s)
Ácidos Grasos no Esterificados/sangre , Metabolismo de los Lípidos , Metaboloma , Obesidad/sangre , Obesidad/veterinaria , Animales , Estudios de Casos y Controles , Ácidos Grasos no Esterificados/clasificación , Femenino , Cromatografía de Gases y Espectrometría de Masas , Lisofosfatidilcolinas/sangre , Lisofosfolípidos/sangre , Macaca mulatta , Obesidad/fisiopatología , Fosfatidilcolinas/sangre , Fosfatidiletanolaminas/sangre , Fosfatidilgliceroles/sangre , Fosfatidilinositoles/sangre , Fosfatidilserinas/sangre , Esfingomielinas/sangreRESUMEN
Sepsis is associated with dysfunctional coagulation. Recent data suggest that platelets play a role in sepsis by promoting neutrophil accumulation. Herein, we show that cecal ligation and puncture (CLP) triggered systemic inflammation, which is characterized by formation of IL-6 and CXC chemokines as well as neutrophil accumulation in the lung. Platelet depletion decreased neutrophil accumulation, IL-6, and CXC chemokines formation in septic lungs. Depletion of platelets increased peak thrombin formation and total thrombin generation (TG) in plasma from septic animals. CLP elevated circulating levels of platelet-derived microparticles (PMPs). In vitro generated PMPs were a potent inducer of TG. Interestingly, in vitro wild-type recombinant annexin V abolished PMP-induced thrombin formation whereas a mutant annexin V protein, which does not bind to phosphatidylserine (PS), had no effect. Administration of wild-type, but not mutant annexin V, significantly inhibited thrombin formation in septic animals. Moreover, CLP-induced formation of thrombin-antithrombin complexes were reduced in platelet-depleted mice and in animals pretreated with annexin V. PMP-induced TG attenuated in FXII- and FVII-deficient plasma. These findings suggest that sepsis-induced TG is dependent on platelets. Moreover, PMPs formed in sepsis are a potent inducer of TG via PS exposure, and activation of both the intrinsic and extrinsic pathway of coagulation. In conclusion, these observations suggest that PMPs and PS play an important role in dysfunctional coagulation in abdominal sepsis.
Asunto(s)
Coagulación Sanguínea , Plaquetas/metabolismo , Micropartículas Derivadas de Células/metabolismo , Fosfatidilserinas/sangre , Sepsis/sangre , Trombina/metabolismo , Animales , Anexina A5/sangre , Antitrombina III , Plaquetas/inmunología , Plaquetas/microbiología , Plaquetas/ultraestructura , Micropartículas Derivadas de Células/inmunología , Micropartículas Derivadas de Células/microbiología , Micropartículas Derivadas de Células/ultraestructura , Quimiocinas CXC/metabolismo , Modelos Animales de Enfermedad , Inflamación/sangre , Inflamación/inmunología , Inflamación/microbiología , Interleucina-6/metabolismo , Pulmón/inmunología , Pulmón/metabolismo , Pulmón/microbiología , Masculino , Ratones Endogámicos C57BL , Infiltración Neutrófila , Péptido Hidrolasas/sangre , Sepsis/inmunología , Sepsis/microbiología , Sepsis/patología , Transducción de Señal , Factores de TiempoRESUMEN
Binding of coagulation factors to phosphatidylserine (PS)-exposing procoagulant-activated platelets followed by formation of the membrane-dependent enzyme complexes is critical for blood coagulation. Procoagulant platelets formed upon strong platelet stimulation, usually with thrombin plus collagen, are large "balloons" with a small (â¼1 µm radius) "cap"-like convex region that is enriched with adhesive proteins. Spatial distribution of blood coagulation factors on the surface of procoagulant platelets was investigated using confocal microscopy. All of them, including factors IXa (FIXa), FXa/FX, FVa, FVIII, prothrombin, and PS-sensitive marker Annexin V were distributed nonhomogeneously: they were primarily localized in the "cap," where their mean concentration was by at least an order of magnitude, higher than on the "balloon." Assembly of intrinsic tenase on liposomes with various PS densities while keeping the PS content constant demonstrated that such enrichment can accelerate this reaction by 2 orders of magnitude. The mechanisms of such acceleration were investigated using a 3-dimensional computer simulation model of intrinsic tenase based on these data. Transmission electron microscopy and focal ion beam-scanning electron microscopy with Annexin V immunogold-labeling revealed a complex organization of the "caps." In platelet thrombi formed in whole blood on collagen under arterial shear conditions, ubiquitous "caps" with increased Annexin V, FX, and FXa binding were observed, indicating relevance of this mechanism for surface-attached platelets under physiological flow. These results reveal an essential heterogeneity in the surface distribution of major coagulation factors on the surface of procoagulant platelets and suggest its importance in promoting membrane-dependent coagulation reactions.
Asunto(s)
Factores de Coagulación Sanguínea/metabolismo , Coagulación Sanguínea/fisiología , Plaquetas/metabolismo , Adulto , Anexina A5/metabolismo , Plaquetas/ultraestructura , Membrana Celular/metabolismo , Membrana Celular/ultraestructura , Simulación por Computador , Humanos , Imagenología Tridimensional , Técnicas In Vitro , Microscopía Confocal , Microscopía Inmunoelectrónica , Fosfatidilserinas/sangre , Activación Plaquetaria/fisiología , Unión Proteica , Trombina/metabolismo , Trombosis/metabolismo , Trombosis/patologíaRESUMEN
BACKGROUND: Several circumstances require the accurate measurement of red blood cell (RBC) survival and clearance, such as determination of posttransfusion recovery of stored RBCs to investigate the effect of new additive solutions. To this end, biotin as a marker of RBCs to track donor RBCs in the blood of the recipient has been used in many studies. However, so far only experimental, nonvalidated, biotin-labeled red cell concentrates (RCCs) are transfused. The goal of this study was to produce a standardized biotin-labeled RCC product in a fast, simple, and sterile manner that can be used for clinical research and for the evaluation of new blood products according to Good Practice Guidelines (GPG) for blood establishments. STUDY DESIGN AND METHODS: RCC fractions were labeled with two different concentrations of biotinylation reagent in a closed system, to prevent bacterial contamination of the end product. Using flow cytometry, the reproducibility and robustness of the biotin labeling was assessed, as well as the stability of the biotin label on the (un-)irradiated RCC fraction. Additionally, parameters such as phosphatidylserine (PS) exposure, sodium (Na), potassium (K), free hemoglobin, adenosine triphosphate (ATP), pH, and morphology were determined prior to and after biotin labeling to rule out detrimental effects of the labeling procedure on the RCC. RESULTS: Our data show that RCCs can be labeled under sterile conditions in a closed system with two different biotinylation reagent concentrations, without affecting the biological activity. CONCLUSION: An easy, rapid (<2 hr), and robust method was developed to manufacture biotin-labeled RCCs for clinical research compliant to GPG.
Asunto(s)
Biotinilación/métodos , Rastreo Celular/métodos , Transfusión de Eritrocitos , Eritrocitos , Adenina , Adenosina Trifosfato/sangre , Anexina A5/sangre , Bacteriemia/prevención & control , Biotina/farmacología , Conservación de la Sangre , Forma de la Célula , Supervivencia Celular , Eritrocitos/química , Eritrocitos/efectos de los fármacos , Glucosa , Hemoglobinas/análisis , Humanos , Concentración de Iones de Hidrógeno , Indicadores y Reactivos , Manitol , Fosfatidilserinas/sangre , Potasio/sangre , Sodio/sangre , Cloruro de SodioRESUMEN
OBJECTIVES: The liver X receptors (LXRs) and farnesoid X receptor (FXR) have been identified in human platelets. Ligands of these receptors have been shown to have nongenomic inhibitory effects on platelet activation by platelet agonists. This, however, seems contradictory with the platelet hyper-reactivity that is associated with several pathological conditions that are associated with increased circulating levels of molecules that are LXR and FXR ligands, such as hyperlipidemia, type 2 diabetes mellitus, and obesity. APPROACH AND RESULTS: We, therefore, investigated whether ligands for the LXR and FXR receptors were capable of priming platelets to the activated state without stimulation by platelet agonists. Treatment of platelets with ligands for LXR and FXR converted platelets to the procoagulant state, with increases in phosphatidylserine exposure, platelet swelling, reduced membrane integrity, depolarization of the mitochondrial membrane, and microparticle release observed. Additionally, platelets also displayed features associated with coated platelets such as P-selectin exposure, fibrinogen binding, fibrin generation that is supported by increased serine protease activity, and inhibition of integrin αIIbß3. LXR and FXR ligand-induced formation of coated platelets was found to be dependent on both reactive oxygen species and intracellular calcium mobilization, and for FXR ligands, this process was found to be dependent on cyclophilin D. CONCLUSIONS: We conclude that treatment with LXR and FXR ligands initiates coated platelet formation, which is thought to support coagulation but results in desensitization to platelet stimuli through inhibition of αIIbß3 consistent with their ability to inhibit platelet function and stable thrombus formation in vivo.
Asunto(s)
Benzoatos/farmacología , Bencilaminas/farmacología , Coagulación Sanguínea/efectos de los fármacos , Plaquetas/efectos de los fármacos , Isoxazoles/farmacología , Receptores X del Hígado/agonistas , Activación Plaquetaria/efectos de los fármacos , Inhibidores de Agregación Plaquetaria/farmacología , Receptores Citoplasmáticos y Nucleares/agonistas , Plaquetas/metabolismo , Señalización del Calcio/efectos de los fármacos , Micropartículas Derivadas de Células/efectos de los fármacos , Micropartículas Derivadas de Células/metabolismo , Ciclofilinas/sangre , Relación Dosis-Respuesta a Droga , Fibrina/metabolismo , Fibrinógeno/metabolismo , Humanos , Ligandos , Receptores X del Hígado/sangre , Potencial de la Membrana Mitocondrial/efectos de los fármacos , Selectina-P/sangre , Fosfatidilserinas/sangre , Complejo GPIIb-IIIa de Glicoproteína Plaquetaria/antagonistas & inhibidores , Complejo GPIIb-IIIa de Glicoproteína Plaquetaria/metabolismo , Especies Reactivas de Oxígeno/sangre , Receptores Citoplasmáticos y Nucleares/sangreRESUMEN
OBJECTIVE: Phosphatidylserine exposure mediates platelet procoagulant function and regulates platelet life span. Apoptotic, necrotic, and integrin-mediated mechanisms have been implicated as intracellular determinants of platelet phosphatidylserine exposure. Here, we investigate (1) the role of mitochondrial events in platelet phosphatidylserine exposure initiated by these distinct stimuli and (2) the cellular interactions of the procoagulant platelet in vitro and in vivo. APPROACH AND RESULTS: Key mitochondrial events were examined, including cytochrome c release and inner mitochondrial membrane (IMM) disruption. In both ABT-737 (apoptotic) and agonist (necrotic)-treated platelets, phosphatidylserine externalization was temporally correlated with IMM disruption. Agonist stimulation resulted in rapid cyclophilin D-dependent IMM disruption that coincided with phosphatidylserine exposure. ABT-737 treatment caused rapid cytochrome c release, eventually followed by caspase-dependent IMM disruption that again closely coincided with phosphatidylserine exposure. A nonmitochondrial and integrin-mediated mechanism has been implicated in the formation of a novel phosphatidylserine-externalizing platelet subpopulation. Using image cytometry, this subpopulation is demonstrated to be the result of the interaction of an aggregatory platelet and a procoagulant platelet rather than indicative of a novel intracellular mechanism regulating platelet phosphatidylserine externalization. Using electron microscopy, similar interactions between aggregatory and procoagulant platelets are demonstrated in vitro and in vivo within a mesenteric vein hemostatic thrombus. CONCLUSIONS: Platelet phosphatidylserine externalization is closely associated with the mitochondrial event of IMM disruption identifying a common pathway in phosphatidylserine-externalizing platelets. The limited interaction of procoagulant platelets and integrin-active aggregatory platelets identifies a potential mechanism for procoagulant platelet retention within the hemostatic thrombus.
Asunto(s)
Apoptosis , Plaquetas/metabolismo , Mitocondrias/metabolismo , Membranas Mitocondriales/metabolismo , Fosfatidilserinas/sangre , Agregación Plaquetaria , Trombosis de la Vena/sangre , Animales , Apoptosis/efectos de los fármacos , Compuestos de Bifenilo/farmacología , Coagulación Sanguínea/efectos de los fármacos , Plaquetas/efectos de los fármacos , Plaquetas/ultraestructura , Caspasas/sangre , Venenos de Crotálidos/farmacología , Peptidil-Prolil Isomerasa F , Ciclofilinas/sangre , Ciclofilinas/genética , Citocromos c/sangre , Modelos Animales de Enfermedad , Genotipo , Integrinas/sangre , Cinética , Lectinas Tipo C , Ratones Noqueados , Mitocondrias/efectos de los fármacos , Mitocondrias/ultraestructura , Membranas Mitocondriales/efectos de los fármacos , Necrosis , Nitrofenoles/farmacología , Fenotipo , Piperazinas/farmacología , Agregación Plaquetaria/efectos de los fármacos , Transducción de Señal , Sulfonamidas/farmacología , Trombina/farmacología , Trombosis de la Vena/genética , Trombosis de la Vena/patología , Proteína Destructora del Antagonista Homólogo bcl-2/sangre , Proteína Destructora del Antagonista Homólogo bcl-2/genética , Proteína X Asociada a bcl-2/sangre , Proteína X Asociada a bcl-2/genéticaRESUMEN
We compared the effects of cycling and running exercise on hemorheological and hematological properties, as well as eryptosis markers. Seven endurance-trained subjects randomly performed a progressive and maximal exercise test on a cycle ergometer and a treadmill. Blood was sampled at rest and at the end of the exercise to analyze hematological and blood rheological parameters including hematocrit (Hct), red blood cell (RBC) deformability, aggregation, and blood viscosity. Hemoglobin saturation (SpO2), blood lactate, and glucose levels were also monitored. Red blood cell oxidative stress, calcium content, and phosphatidylserine exposure were determined by flow cytometry to assess eryptosis level. Cycling exercise increased blood viscosity and RBC aggregation whereas it had no significant effect on RBC deformability. In contrast, blood viscosity remained unchanged and RBC deformability increased with running. The increase in Hct, lactate, and glucose concentrations and the loss of weight at the end of exercise were not different between running and cycling. Eryptosis markers were not affected by exercise. A significant drop in SpO2 was noted during running but not during cycling. Our study showed that a progressive and maximal exercise test conducted on a cycle ergometer increased blood viscosity while the same test conducted on a treadmill did not change this parameter because of different RBC rheological behavior between the 2 tests. We also demonstrated that a short maximal exercise does not alter RBC physiology in trained athletes. We suspect that exercise-induced hypoxemia occurring during running could be at the origin of the RBC rheological behavior differences with cycling.
Asunto(s)
Ciclismo/psicología , Eriptosis , Deformación Eritrocítica , Carrera/fisiología , Adulto , Glucemia , Viscosidad Sanguínea , Calcio/sangre , Femenino , Hematócrito , Humanos , Hipoxia , Ácido Láctico/sangre , Masculino , Consumo de Oxígeno , Fosfatidilserinas/sangre , Especies Reactivas de Oxígeno/sangreRESUMEN
BACKGROUND: There has been increasing interest in the detection of tumour exosomes in blood for cancer diagnostics. Most studies have focussed on miRNA and protein signatures that are surrogate markers for specific tumour types. Because tumour cells and tumour-derived exosomes display phosphatidylserine (PS) in their outer membrane leaflet, we developed a highly sensitive ELISA-based system that detects picogram amounts of exosomal phospholipid in plasma as a cancer biomarker. METHODS: This report describes the development of a highly specific and sensitive ELISA for the capture of PS-expressing tumour exosomes in the blood of tumour-bearing mice. To monitor the relationship between tumour burden and tumour exosome plasma concentrations, plasma from one transplantable breast cancer model (MDA-MB-231) and three genetic mouse models (MMTV-PyMT; breast and KIC and KPC; pancreatic) were screened for captured exosomal phospholipid. RESULTS: We show that quantitative assessment of PS-expressing tumour exosomes detected very early-stage malignancies before clinical evidence of disease in all four model systems. Tumour exosome levels showed significant increases by day 7 after tumour implantation in the MDA-MB-231 model while palpable tumours appeared only after day 27. For the MMTV-PyMT and KIC models, tumour exosome levels increased significantly by day 49 (P⩽0.0002) and day 21 (P⩽0.001) while tumours developed only after days 60 and 40, respectively. For the KPC model, a significant increase in blood exosome levels was detected by day 70 (P=0.023) when only preinvasive lesions are microscopically detectable. CONCLUSIONS: These data indicate that blood PS exosome levels is a specific indicator of cancer and suggest that blood PS is a biomarker for early-stage malignancies.
Asunto(s)
Biomarcadores de Tumor/sangre , Neoplasias de la Mama/sangre , Exosomas/química , Neoplasias Ováricas/sangre , Neoplasias Pancreáticas/sangre , Fosfatidilserinas/sangre , Animales , Anticuerpos/inmunología , Neoplasias de la Mama/diagnóstico , Neoplasias de la Mama/patología , Estudios de Casos y Controles , Línea Celular Tumoral , Ensayo de Inmunoadsorción Enzimática/métodos , Femenino , Humanos , Ratones , Estadificación de Neoplasias , Trasplante de Neoplasias , Neoplasias Ováricas/diagnóstico , Neoplasias Pancreáticas/diagnóstico , Neoplasias Pancreáticas/patología , Fosfatidilserinas/inmunología , Factores de Tiempo , Carga Tumoral , beta 2 Glicoproteína I/inmunologíaRESUMEN
Bone marrow transplantation (BMT) serves as the only curative treatment for patients with ß-thalassemia major; however, hemostatic changes have been observed in these BMT patients. Aggregability of thalassemic red blood cells (RBCs) and increased red blood cell-derived microparticles (RMPs) expressing phosphatidylserine (PS) are thought to participate in thromboembolic events by initially triggering platelet activation. To our knowledge, there has been no report providing quantitation of these circulating PS-expressing RBCs and RMPs in young ß-thalassemia patients after BMT. Whole blood from each subject was fluorescently labeled to detect RBC markers (CD235a) and annexin-V together with the known number TruCount™ beads. PS-expressing RBCs, RMPs, and activated platelets were identified by flow cytometry. In our randomized study, we found the decreased levels of three aforementioned factors compared to levels in patients receiving regular blood transfusion (RT). This study showed that BMT in ß-thalassemia patients decreases the levels of circulating PS-expressing RBCs, their MPs, and procoagulant platelets when compared to patients who received RT. Normalized levels of these coagulation markers may provide the supportive evidence of the effectiveness of BMT for curing thalassemia.
Asunto(s)
Plaquetas/metabolismo , Trasplante de Médula Ósea , Micropartículas Derivadas de Células/metabolismo , Eritrocitos/metabolismo , Fosfatidilserinas/sangre , Activación Plaquetaria , Talasemia beta , Adolescente , Aloinjertos , Anexina A5/sangre , Niño , Femenino , Humanos , Masculino , Talasemia beta/sangre , Talasemia beta/terapiaRESUMEN
OBJECTIVE: It is known that both platelets and coagulation strongly influence infarct progression after ischemic stroke, but the mechanisms and their interplay are unknown. Our aim was to assess the contribution of the procoagulant platelet surface, and thus platelet-driven thrombin generation, to the progression of thromboinflammation in the ischemic brain. APPROACH AND RESULTS: We present the characterization of a novel platelet and megakaryocyte-specific TMEM16F (anoctamin 6) knockout mouse. Reflecting Scott syndrome, platelets from the knockout mouse had a significant reduction in procoagulant characteristics that altered thrombin and fibrin generation kinetics. In addition, knockout mice showed significant defects in hemostasis and arterial thrombus formation. However, infarct volumes in a model of ischemic stroke were comparable with wild-type mice. CONCLUSIONS: Platelet TMEM16F activity contributes significantly to hemostasis and thrombosis but not cerebral thromboinflammation. These results highlight another key difference between the roles of platelets and coagulation in these processes.
Asunto(s)
Plaquetas/metabolismo , Enfermedades de las Arterias Carótidas/sangre , Encefalitis/sangre , Encefalitis/genética , Hemostasis , Infarto de la Arteria Cerebral Media/sangre , Fosfatidilserinas/sangre , Proteínas de Transferencia de Fosfolípidos/sangre , Trombina/metabolismo , Trombosis/sangre , Animales , Anoctaminas , Coagulación Sanguínea , Enfermedades de las Arterias Carótidas/genética , Enfermedades de las Arterias Carótidas/patología , Modelos Animales de Enfermedad , Encefalitis/patología , Fibrina/metabolismo , Infarto de la Arteria Cerebral Media/genética , Infarto de la Arteria Cerebral Media/patología , Cinética , Megacariocitos/metabolismo , Ratones Endogámicos C57BL , Ratones Noqueados , Proteínas de Transferencia de Fosfolípidos/deficiencia , Proteínas de Transferencia de Fosfolípidos/genética , Activación Plaquetaria , Transducción de Señal , Trombosis/genética , Trombosis/patologíaRESUMEN
BACKGROUND/AIMS: Arteritis is an inflammatory disease of the vascular wall leading to ischemia and vascular occlusion. Complications of arteritis include anemia, which could, at least in theory, result from suicidal erythrocyte death or eryptosis, which is characterized by erythrocyte shrinkage and phosphatidylserine (PS) exposure at the erythrocyte surface. Cellular mechanisms involved in the stimulation of eryptosis include increased cytosolic Ca2+-concentration ([Ca2+]i), oxidative stress and ceramide formation. The present study explored whether and how arteritis influences eryptosis. METHODS: Blood was drawn from patients suffering from arteritis (n=17) and from healthy volunteers (n=21). PS exposure was estimated from annexin V-binding, erythrocyte volume from forward scatter, [Ca2+]i from Fluo3-fluorescence, reactive oxygen species (ROS) from DCFDA fluorescence and ceramide abundance from FITC-conjugated antibody binding in flow cytometry. The patients suffered from anemia despite 2.8±0.4% reticulocytes. RESULTS: The percentage of PS-exposing erythrocytes was significantly higher in patients (1.1±0.1%) than in healthy volunteers (0.3±0.1%). The increase in PS exposure was paralleled by increase in oxidative stress and [Ca2+]i but not by significant changes of ceramide abundance. Erythrocyte PS exposure and ROS production were significantly enhanced in erythrocytes exposed to patient plasma as compared to exposure to plasma from healthy volunteers. CONCLUSION: Arteritis is associated with enhanced eryptosis due to increased [Ca2+]i and oxidative stress. The eryptosis contributes to or even accounts for the anemia in those patients. As eryptotic erythrocytes adhere to endothelial cells of the vascular wall, they could impede microcirculation and thus contribute to vascular occlusion.