RESUMEN
There is currently a demand for new highly efficient and specific drugs to treat osteoporosis, a chronic bone disease affecting millions of people worldwide. We have developed a combinatorial strategy for engineering bispecific inhibitors that simultaneously target the unique combination of c-FMS and αvß3 integrin, which act in concert to facilitate bone resorption by osteoclasts. Using functional fluorescence-activated cell sorting (FACS)-based screening assays of random mutagenesis macrophage colony-stimulating factor (M-CSF) libraries against c-FMS and αvß3 integrin, we engineered dual-specific M-CSF mutants with high affinity to both receptors. These bispecific mutants act as functional antagonists of c-FMS and αvß3 integrin activation and hence of osteoclast differentiation in vitro and osteoclast activity in vivo. This study thus introduces a versatile platform for the creation of new-generation therapeutics with high efficacy and specificity for osteoporosis and other bone diseases. It also provides new tools for studying molecular mechanisms and the cell signaling pathways that mediate osteoclast differentiation and function.
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Conservadores de la Densidad Ósea/farmacología , Resorción Ósea/prevención & control , Integrina alfaVbeta3/antagonistas & inhibidores , Factor Estimulante de Colonias de Macrófagos/farmacología , Osteoclastos/efectos de los fármacos , Osteoporosis/tratamiento farmacológico , Receptor de Factor Estimulante de Colonias de Macrófagos/antagonistas & inhibidores , Animales , Sitios de Unión , Conservadores de la Densidad Ósea/química , Conservadores de la Densidad Ósea/metabolismo , Células de la Médula Ósea/efectos de los fármacos , Células de la Médula Ósea/metabolismo , Células de la Médula Ósea/patología , Resorción Ósea/genética , Resorción Ósea/metabolismo , Resorción Ósea/patología , Diferenciación Celular , Clonación Molecular , Escherichia coli/genética , Escherichia coli/metabolismo , Expresión Génica , Regulación de la Expresión Génica , Humanos , Integrina alfaVbeta3/química , Integrina alfaVbeta3/genética , Integrina alfaVbeta3/metabolismo , Factor Estimulante de Colonias de Macrófagos/química , Factor Estimulante de Colonias de Macrófagos/genética , Factor Estimulante de Colonias de Macrófagos/metabolismo , Macrófagos/efectos de los fármacos , Macrófagos/metabolismo , Macrófagos/patología , Ratones , Simulación del Acoplamiento Molecular , Mutación , Osteoclastos/metabolismo , Osteoclastos/patología , Osteoporosis/genética , Osteoporosis/metabolismo , Osteoporosis/patología , Unión Proteica , Conformación Proteica en Hélice alfa , Conformación Proteica en Lámina beta , Ingeniería de Proteínas , Dominios y Motivos de Interacción de Proteínas , Receptor de Factor Estimulante de Colonias de Macrófagos/química , Receptor de Factor Estimulante de Colonias de Macrófagos/genética , Receptor de Factor Estimulante de Colonias de Macrófagos/metabolismo , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Transducción de SeñalRESUMEN
BACKGROUND: Increased activity of the receptor tyrosine kinase Tie2 has been implicated in the promotion of pathological angiogenesis. This activity is mainly mediated through angiopoietin (Ang)1- and Ang2-dependent activation of integrins by Tie2, rendering the Ang/Tie2/integrin axis an attractive putative target for cancer therapeutics. RESULTS: To target this axis, we developed single domain, non-immunoglobulin high-affinity bi-specific protein inhibitors against both Tie2 and αvß3 integrin. We have previously engineered the Ang2-binding domain of Tie2 (Ang2-BD) as a Tie2 inhibitor. Here, we engineered an exposed loop in Ang2-BD to generate variants that include an integrin-binding Arg-Gly-Asp (RGD) motif and used flow cytometry screening of a yeast-displayed Ang2-BD RGD loop library to identify the integrin antagonists. The bi-specific antagonists targeting both Tie2 and αvß3 integrin inhibited adhesion and proliferation of endothelial cells cultured together with the αvß3 integrin ligand vitronectin, as well as endothelial cell invasion and tube formation. The bi-specific reagents inhibited downstream signaling by Tie2 intracellularly in response to its agonist Ang1 more effectively than the wild-type Ang2 BD that binds Tie2 alone. CONCLUSIONS: Collectively, this study-the first to describe inhibitors targeting all the known functions resulting from Tie2/integrin αvß3 cross-talk-has created new tools for studying Tie2- and integrin αvß3-dependent molecular pathways and provides the basis for the rational and combinatorial engineering of ligand-Tie2 and ligand-integrin αvß3 receptor interactions. Given the roles of these pathways in cancer angiogenesis and metastasis, this proof of principle study paves the route to create novel Tie2/integrin αvß3-targeting proteins for clinical use as imaging and therapeutic agents.
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Inhibidores de la Angiogénesis/farmacología , Neovascularización Fisiológica/genética , Receptor TIE-2/antagonistas & inhibidores , Receptores de Vitronectina/genética , Ribonucleasa Pancreática/antagonistas & inhibidores , Inhibidores de la Angiogénesis/química , Animales , Ratones , Receptor TIE-2/química , Receptor TIE-2/genética , Receptor TIE-2/metabolismo , Receptores de Vitronectina/química , Receptores de Vitronectina/metabolismo , Ribonucleasa Pancreática/química , Ribonucleasa Pancreática/genética , Ribonucleasa Pancreática/metabolismoRESUMEN
Thrombocytopenia (TCP) and Glanzmann thrombasthenia (GT) are typical platelet disorders characterized by mild to severe bleeding. This study aims to create in vitro models of TCP and GT and to correct the impaired clot formation by fibrinogen and coagulation factor XIII. The TCP model (mean platelet count, 16â¯×â¯109 L-1) was produced by differential centrifugation of normal blood followed by mixing plasma with packed cells. The GT model was created by treating normal blood with 50⯵g/mL eptifibatide, an inhibitor of platelet integrin αIIbß3. Clot formation was evaluated in whole blood by rotation thromboelastometry. In both models, the extent of clot strength was two-three times lower compared to normal blood. Fibrinogen and, to a lesser extent, factor XIII stimulated the propagation phase of clot formation both in TCP and GT models. Clot strength in TCP was increased by both agents, while in GT by fibrinogen only. Similar results were obtained in blood from patients with primary immune thrombocytopenia and Glanzmann thrombasthenia. In conclusion, the created models may be useful in the development of new ways to correct the impaired coagulation potential in TCP and GT.
RESUMEN
The ß3 subunit of αIIbß3 and αvß3 integrins contains four epidermal growth factor (EGF)-like domains. Each domain harbors four disulfide bonds of which one is unique for integrins. We previously discerned a regulatory role of the EGF-4 Cys-560-Cys-583 unique bond for αIIbß3 activation. In this study we further investigated the role of all four integrin unique bonds in both αIIbß3 and αvß3. We created ß3 mutants harboring serine substitutions of each or both cysteines that disrupt the four unique bonds (Cys-437-Cys-457 in EGF-1, Cys-473-Cys-503 in EGF-2, Cys-523-Cys-544 in EGF-3, and Cys-560-Cys-583 in EGF-4) and transfected them into baby hamster kidney cells together with normal αv or αIIb. Flow cytometry was used to measure surface expression of αIIbß3 and αvß3 and their activity state by soluble fibrinogen binding. Most cysteine substitutions caused similarly reduced surface expression of both receptors. Disrupting all four unique disulfide bonds by single cysteine substitutions resulted in variable constitutive activation of αIIbß3 and αvß3. In contrast, whereas double C437S/C457S and C473S/C503S mutations yielded constitutively active αIIbß3 and αvß3, the C560S/C583S mutation did not, and the C523S/C544S mutation only yielded constitutively active αIIbß3. Activation of C523S/C544S αvß3 mutant by activating antibody and dithiothreitol was also impaired. Molecular dynamics of C523S/C544S ß3 in αIIbß3 but not in αvß3 displayed an altered stable conformation. Our findings indicate that unique disulfide bonds in ß3 differently affect the function of αIIbß3 and αvß3 and suggest a free sulfhydryl-dependent regulatory role for Cys-560-Cys-583 in both αIIbß3 and αvß3 and for Cys-523-Cys-544 only in αvß3.
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Disulfuros/química , Factor de Crecimiento Epidérmico/metabolismo , Integrina alfaVbeta3/química , Integrina alfaVbeta3/metabolismo , Complejo GPIIb-IIIa de Glicoproteína Plaquetaria/química , Complejo GPIIb-IIIa de Glicoproteína Plaquetaria/metabolismo , Secuencia de Aminoácidos , Animales , Línea Celular , Cricetinae , Disulfuros/metabolismo , Factor de Crecimiento Epidérmico/genética , Humanos , Integrina alfaVbeta3/genética , Datos de Secuencia Molecular , Complejo GPIIb-IIIa de Glicoproteína Plaquetaria/genética , Unión Proteica , Estructura Terciaria de ProteínaRESUMEN
BACKGROUND: Current methods used to diagnose the thalassemia minor (TM) patients require high-cost assays, while broader screening based on routine blood count has limited specificity and sensitivity. This study developed a new screening technique for TM patients' diagnosis. METHODS: The study enrolled 526 patients database that included 185 verified α and ß TM cases, and control group consisted of iron-deficiency anemia (IDA), myelodysplastic syndrome (MDS), and healthy patients. More than 1,500 artificial neural networks (ANNs) models were created and the networks that gave high accuracy were selected for the study. TM patients were identified from the general database using the best-optimized ANNs. RESULTS: Comparison between three or six routine blood count parameters determined a slightly higher accuracy of the model with the three-parameter scheme, including mean corpuscular volume, red blood cell distribution width, and red blood cell. Based on these parameters, we were able to separate TM patients from the control group and MDS group, with specificity of 0.967 and sensitivity of 1. Including IDA patients into comparison gave lower but, still, very good values of specificity of 0.968 and sensitivity of 0.9. CONCLUSION: ANN-based TM diagnostics should be used for broad automatic screening of general population prior diagnosis with high-cost tests.
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Simulación por Computador , Diagnóstico por Computador/métodos , Redes Neurales de la Computación , Talasemia beta/diagnóstico , Bases de Datos Factuales , Diagnóstico Diferencial , Humanos , Talasemia beta/sangre , Talasemia beta/epidemiologíaRESUMEN
Glanzmann's thrombasthenia (GT) is characterized by increased bleeding risk. The treatment options in GT are limited. The aim of this study was to test the effect of GT blood supplementation with fibrinogen and factor XIII on thrombin generation, blood clotting, and fibrinolysis. Whole blood samples of GT patients and normal donors treated with eptifibatide (GT model) were subjected to clotting by CaCl(2) and tissue factor. Thrombin generation was measured in platelet-rich plasma. Clot formation and tPA-induced fibrinolysis were evaluated in whole blood by rotation thromboelastometry (ROTEM). Blood was supplemented with fibrinogen (3 g/L) and/or FXIII (2 IU/mL). Thrombin generation analysis of blood derived from GT model and GT patients revealed decreased endogenous thrombin potential and peak height and extended lag time compared to control. However, this method was not sensitive to blood spiking with fibrinogen and FXIII. ROTEM revealed lower maximum clot firmness (MCF) and area under curve (AUC) in the blood of GT model and GT patients. In the absence of exogenous tPA, blood spiking with fibrinogen markedly enhanced clot quality while FXIII had no effect. Combination of fibrinogen and FXIII did not add to the effect of fibrinogen. In contrast, by the addition of tPA, both fibrinogen and FXIII separately and, to more extent, in combination enhanced clot quality as well as resistance against tPA-induced fibrinolysis (increasing MCF, AUC, and lysis onset time). In conclusion, fibrinogen and FXIII exerted stimulation of blood clotting and inhibition of fibrinolysis. Treating normal blood with eptifibatide mimics the changes of coagulopathy in GT blood.
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Coagulación Sanguínea/efectos de los fármacos , Factor XIII/farmacología , Fibrinógeno/farmacología , Trombastenia/sangre , Trombina/metabolismo , Área Bajo la Curva , Cloruro de Calcio/farmacología , Estudios de Casos y Controles , Eptifibatida , Humanos , Péptidos/farmacología , Inhibidores de Agregación Plaquetaria/farmacología , Plasma Rico en Plaquetas/química , Tromboelastografía , Trombina/agonistas , Tiempo de Trombina , Tromboplastina/farmacología , Activador de Tejido Plasminógeno/farmacologíaRESUMEN
BACKGROUND: Bleeding tendencies in immune thrombocytopenia (ITP) do not always correlate with the number of platelets, suggesting platelet function variation. We used a model of normal whole blood thrombocytopenia to compare platelet function and other hemostatic variables with ITP patients. We further investigated the effect of in vitro spiking with von Willebrand factor (vWF) and fibrinogen on platelet function and hemostatic variables. METHODS: The Cone and Plate(let) Analyzer was used to measure platelet adhesion (surface coverage [SC], %) and aggregation (average size, µm(2)) under defined shear rate (1200 s(-1)). Rotational thromboelastometry was used to determine variables of clot formation triggered by CaCl(2) and tissue factor. RESULTS: In both the model of thrombocytopenia as well as in ITP, the SC and to some extent the average size were correlated to the platelet number over a range of 5 to 80 × 10(6)/mL. The results obtained for most ITP samples were within the boundaries of the lower and upper limits set by the whole blood model of thrombocytopenia. The addition of 2 U/mL vWF (Haemate-P) to whole blood (calculated to plasma volume) results in an increase in the SC and average size without affecting clot formation. Spiking with fibrinogen (100 and 300 mg/dL) did not affect platelet deposition but improved clot formation. CONCLUSIONS: Using a model of whole blood thrombocytopenia enables us to establish reference variables for the Cone and Plate(let) Analyzer and rotational thromboelastometry and to assess platelet function and clot formation in the presence of severe thrombocytopenia. We demonstrated that in most cases of ITP, platelet function is comparable to normal platelets. This work also suggests that vWF and fibrinogen differentially affect primary and secondary hemostasis and therefore both may perform a function in the bleeding phenotype and possibly may be considered for treatment in patients with ITP.
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Fibrinógeno/metabolismo , Hemorragia/etiología , Hemostasis , Adhesividad Plaquetaria , Púrpura Trombocitopénica Idiopática/complicaciones , Trombocitopenia/complicaciones , Trombosis/etiología , Factor de von Willebrand/metabolismo , Adulto , Anciano , Anciano de 80 o más Años , Femenino , Hemorragia/sangre , Hemorragia/inmunología , Humanos , Masculino , Persona de Mediana Edad , Fenotipo , Agregación Plaquetaria , Recuento de Plaquetas , Púrpura Trombocitopénica Idiopática/sangre , Púrpura Trombocitopénica Idiopática/inmunología , Tromboelastografía , Trombocitopenia/sangre , Trombosis/sangre , Trombosis/inmunología , Adulto JovenRESUMEN
PURPOSE: Coagulation abnormalities are common following major trauma. The aim of this study was to assess the improvement of trauma-induced coagulopathy (TIC) in an in vitro model. METHODS: TIC was created on blood taken from healthy individuals by inducing hemodilution, acidosis, hypothermia and fibrinolysis. Next, blood samples were subjected to rotational thromboelastometry to assess the effect of hemostasis modulators on blood coagulation and fibrinolysis. RESULTS: Introducing to blood fibrinogen at 0.75 mg/mL, prothrombin complex concentrate at 0.66 IU/mL or tranexamic acid at 95 µg/mL increased clot strength. Higher effect was observed by combination of fibrinogen with tranexamic acid and prothrombin complex with tranexamic acid, whereas the maximal effect was achieved using all agents together. Fibrinolysis was inhibited by tranexamic acid and stronger by triple combination of the agents. Selective treating the TIC blood with fibrinogen, prothrombin complex or tranexamic acid at two time lower concentrations did not affect clot strength. Combining fibrinogen with prothrombin complex or with tranexamic acid stimulated clot strength but at lower extent compared to higher concentrations. Lysis onset time was prolonged by tranexamic acid. Maximal effect on both clot formation and fibrinolysis was achieved using all three agents together. CONCLUSIONS: Blood clotting stimulation and fibrinolysis inhibition in the TIC model was enough combining subthreshold concentrations of fibrinogen, prothrombin complex and tranexamic acid. Further experiments are warranted in both in vitro and in vivo conditions with minimally effective concentrations of both pro-coagulant and anti-fibrinolytic drugs assuming that this combinatorial approach may not only improve coagulopathy but also minimize the risk of thrombotic complications.
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Ácido Tranexámico , Factores de Coagulación Sanguínea , Fibrinógeno , Fibrinólisis , Humanos , Tromboelastografía , Ácido Tranexámico/farmacología , Ácido Tranexámico/uso terapéuticoRESUMEN
Background The cardiac ryanodine receptor type 2 (RyR2) is a large homotetramer, located in the sarcoplasmic reticulum (SR), which releases Ca2+ from the SR during systole. The molecular mechanism underlying Ca2+ sensing and gating of the RyR2 channel in health and disease is only partially elucidated. Catecholaminergic Polymorphic Ventricular Tachycardia (CPVT1) is the most prevalent syndrome caused by RyR2 mutations. Methods and Results This study involves investigation of a family with 4 cases of ventricular fibrillation and sudden death and physiological tests in HEK 293 cells and normal mode analysis (NMA) computation. We found 4 clinically affected members who were homozygous for a novel RyR2 mutation, G3118R, whereas their heterozygous relatives are asymptomatic. G3118R is located in the periphery of the protein, far from the mutation hotspot regions. HEK293 cells harboring G3118R mutation inhibited Ca2+ release in response to increasing doses of caffeine, but decreased the termination threshold for store-overload-induced Ca2+ release, thus increasing the fractional Ca2+ release in response to increasing extracellular Ca2+. NMA showed that G3118 affects RyR2 tetramer in a dose-dependent manner, whereas in the model of homozygous mutant RyR2, the highest entropic values are assigned to the pore and the central regions of the protein. Conclusions RyR2 G3118R is related to ventricular fibrillation and sudden death in recessive mode of inheritance and has an effect of gain of function on the protein. Despite a peripheral location, it has an allosteric effect on the stability of central and pore regions in a dose-effect manner.
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ADN/genética , Muerte Súbita Cardíaca/epidemiología , Ventrículos Cardíacos/fisiopatología , Mutación , Canal Liberador de Calcio Receptor de Rianodina/genética , Taquicardia Ventricular/genética , Función Ventricular/fisiología , Adolescente , Análisis Mutacional de ADN , Muerte Súbita Cardíaca/etiología , Ecocardiografía , Electrocardiografía Ambulatoria , Femenino , Ventrículos Cardíacos/diagnóstico por imagen , Heterocigoto , Humanos , Incidencia , Israel/epidemiología , Masculino , Canal Liberador de Calcio Receptor de Rianodina/metabolismo , Tasa de Supervivencia/tendencias , Taquicardia Ventricular/complicaciones , Taquicardia Ventricular/epidemiologíaRESUMEN
: Trauma-induced coagulopathy (TIC) occurs commonly as a second event following severe injury. We evaluated the effects of fibrinogen, recombinant factor VIIa and factor XIII on blood clotting and fibrinolysis in an in-vitro TIC model. The TIC model included hemodilution, hyperfibrinolysis, acidosis and hypothermia. The extent of clot formation and fibrinolysis was evaluated using rotational thromboelastometry. Clot strength was increased following spiking the TIC blood with either 1.0âmg/ml fibrinogen, 3.0âµg/ml recombinant factor VIIa or 2.0 IU/ml factor XIII. Maximal effect was achieved by all agents in combination approximating the extent of clot formation to those in normal blood. Fibrinolysis was inhibited by factor XIII, while the reduction was stronger using all agents together. When each of the agents used in two times lower concentrations, clot strength was near to threshold. Fibrinogen and factor XIII but not factor VIIa exerted stimulation of clot strength, whereas synergistic effect of fibrinogen and factor XIII was observed. Maximal effect was achieved combining all agents. The antifibrinolytic effect was observed only by co-administration of fibrinogen, factor XIII and factor VIIa. On the basis of our study, we suggest that stimulation of clot formation and inhibition of fibrinolysis may be achieved by combination of FG, rFVIIa an FXIII using each of them at minimal effective concentration. Taken into consideration, multifactorial TIC pathogenesis, this approach may be preferable for improving coagulopathy than separate blood spiking with the essayed factors at high concentrations.
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Trastornos de la Coagulación Sanguínea/tratamiento farmacológico , Factor XIII/uso terapéutico , Fibrinógeno/uso terapéutico , Adulto , Factor XIII/farmacología , Femenino , Fibrinógeno/farmacología , Voluntarios Sanos , Humanos , MasculinoRESUMEN
PURPOSE: To investigate the urinary levels of TGF-ß1, VEGF, and MCP-1 as potential biomarkers of latent inflammation and fibrosis in the kidney before and 6 months after correction of vesicoureteral reflux (VUR) in children. METHODS: A total of 88 patients (mean age 26 months) with VUR were divided into three groups: group A-patients with grades II-III VUR, conservative treatment; group B-patients with grades III-V VUR, endoscopic correction of VUR; group C-patients with grades III-V VUR, ureteral reimplantation after failed endoscopic correction. Control group included 20 healthy children. Biomarker levels were measured by ELISA. 99mTc-DMSA scintigraphy and renal histology were performed if possible. RESULTS: At admission, TGF-ß1 was close to control in all study groups, VEGF increased with severity of the disease, and MCP-1 increased in group C. Six months after correction of VUR, despite clinical and laboratory improvement, TGF-ß1 and MCP-1 increased while VEGF decreased compared to the admission values in all groups; no amelioration of renal scarring was detected either by 99mTc-DMSA scintigraphy or renal histology. CONCLUSION: The results support our hypothesis that successful correction of VUR is not sufficient to stop or reduce the latent inflammatory and fibrotic processes that have already started in the kidney regardless of the reflux grade and treatment option. Measuring the urinary levels of TGF-ß1, VEGF, and MCP-1 may aid in the development of non-invasive, pathophysiologically relevant approach to diagnosis and monitoring of kidney injury and fibrosis in children with VUR.
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Quimiocina CCL2/orina , Inflamación/orina , Riñón/patología , Factor de Crecimiento Transformador beta1/orina , Factor A de Crecimiento Endotelial Vascular/orina , Reflujo Vesicoureteral/complicaciones , Biomarcadores/orina , Estudios de Casos y Controles , Niño , Preescolar , Tratamiento Conservador , Endoscopía , Femenino , Fibrosis , Estudios de Seguimiento , Humanos , Lactante , Inflamación/etiología , Riñón/diagnóstico por imagen , Masculino , Cintigrafía , Reflujo Vesicoureteral/patología , Reflujo Vesicoureteral/terapiaRESUMEN
Unresponsiveness to clopidogrel or aspirin has been reported in patients with acute coronary syndrome (ACS). Platelet aggregometry (PA) and the Impact-R [Cone and Plate(let) Analyzer (CPA) technology, measuring whole blood platelet adhesion under flow conditions] were compared in detecting laboratory unresponsiveness to clopidogrel and aspirin among ACS patients. Platelet-rich plasma (PRP) samples were evaluated in 404 patients by PA using adenosine diphosphate (ADP) and arachidonic acid (AA) and whole blood samples by the Impact-R ADP- and AA-response tests. The first cohort (n=114) was assayed by PA on days 1 and 4 of the onset of ACS. A patient with relative decrease of =10% in ADP-induced maximal platelet aggregation after clopidogrel treatment was defined as laboratory non-responding (NR) patient to clopidogrel. This relative value correlated well with an absolute value of ADP-induced aggregation >/=70%. A patient with an absolute value of AA-induced maximal aggregation >/=60% was defined as laboratory NR patient to aspirin. The second cohort (n=290) was tested on day 4 by both systems and results analyzed by receiver operating characteristic curve. The following cut-off values of the Impact-R surface coverage were obtained: =2.8% and =3.4% for clopidogrel and aspirin NR patients, respectively. The incidence of NR patients to clopidogrel and aspirin, according to the two methods was 27% and 22%, respectively. Impact-R compared to PA in detecting clopidogrel and aspirin NR patients revealed: 79% and 82% agreement, 71% and 73% sensitivity, 83% and 86% specificity, respectively. In conclusion, the Impact-R and PA results demonstrated high degree of similarity.
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Síndrome Coronario Agudo/tratamiento farmacológico , Aspirina/administración & dosificación , Inhibidores de Agregación Plaquetaria/administración & dosificación , Agregación Plaquetaria/efectos de los fármacos , Pruebas de Función Plaquetaria/métodos , Ticlopidina/análogos & derivados , Síndrome Coronario Agudo/sangre , Adenosina Difosfato/análogos & derivados , Adenosina Difosfato/farmacología , Adulto , Anciano , Ácido Araquidónico/farmacología , Clopidogrel , Femenino , Humanos , Masculino , Persona de Mediana Edad , Nefelometría y Turbidimetría/instrumentación , Nefelometría y Turbidimetría/métodos , Pruebas de Función Plaquetaria/instrumentación , Plasma Rico en Plaquetas/efectos de los fármacos , Curva ROC , Sensibilidad y Especificidad , Ticlopidina/administración & dosificaciónRESUMEN
BACKGROUND: Trauma-induced coagulopathy (TIC) is commonly seen among patients with severe injury. The dynamic process of TIC is characterized by variability of the features of the disease. METHODS: A model of TIC was created. Hemodilution was produced by mixing the blood with 40% Tris/saline solution, fibrinolysis by treating the blood with 160 ng/mL tPA, acidosis by adding 1.2 mg/mL lactic acid achieving pH 7.0 to 7.1, and hypothermia by running the assay at 31°C. Intact blood tested at 37°C served as control. Clot formation was evaluated using rotation thromboelastometry. Platelet adhesion and aggregation were assayed at a shear rate of 1800 s(-) using Impact-R device. RESULTS: Clotting time was not affected by any of the TIC constituents used. Clotting initiation was reduced by hemodilution and further reduced by additive hypothermia. The propagation phase of blood clotting was reduced by hemodilution, further reduced by additive hypothermia, and maximally reduced if additionally combined with fibrinolysis. No effect of fibrinolysis on clot propagation was observed at 37°C. Maximum clot firmness was reduced by hemodilution, further reduced by additive fibrinolysis, and maximally reduced if additionally combined with hypothermia. No effect of hypothermia on clot strength was observed in the absence of fibrinolysis. Platelet adhesion (percentage of surface coverage) and aggregation (aggregate size) under flow condition were reduced by hemodilution and further reduced by additive acidosis. Introduction of tPA to diluted blood had no effect on platelet function. CONCLUSION: The study revealed a differential effect of TIC constituents-hemodilution, hypothermia, fibrinolysis, and acidosis-on clot formation and platelet function. The effect of one factor may influence that of another factor. These data may be helpful to better understand the pathogenesis of TIC and to elaborate an individually tailored treatment strategy. LEVEL OF EVIDENCE: A new model of TIC is created. Contribution of various constituents to pathogenesis of TIC and their interactions are evaluated.
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Acidosis/fisiopatología , Trastornos de la Coagulación Sanguínea/etiología , Trastornos de la Coagulación Sanguínea/fisiopatología , Fibrinólisis/fisiología , Hemodilución/métodos , Hipotermia/fisiopatología , Heridas y Lesiones/complicaciones , Heridas y Lesiones/fisiopatología , Adulto , Femenino , Humanos , Masculino , Persona de Mediana Edad , Pruebas de Función Plaquetaria , TromboelastografíaRESUMEN
Previously, we demonstrated the establishment of synthetic lethality screening in cultured somatic human cells, or mouse embryo fibroblasts (MEFs), for chemicals or mutant genes synergistically lethal with a mutated gene of interest. Here, we show in MEFs that the usage of RNA interference-based genetic suppressor elements encoding short hairpin RNAs (shRNAs) enables for genetic synthetic lethality screening at a frequency much higher than that achieved before with short truncated sense and antisense RNAs. These findings open up the possibility of using in mammalian cells genome-wide shRNA libraries for genetic synthetic lethality screening at the multi-gene level.
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Genes Letales/genética , Genómica/métodos , Interferencia de ARN , ARN Interferente Pequeño/genética , Animales , Ligasas de Carbono-Nitrógeno/genética , Embrión de Mamíferos/citología , Fibroblastos/metabolismo , Vectores Genéticos/genética , Ratones , Plásmidos/genética , Purinas/biosíntesis , ARN Mensajero/genética , ARN Mensajero/metabolismoRESUMEN
Recently, we demonstrated the establishment of chemical and genetic synthetic lethality screens in cultured human cells. Here, we report the establishment of this method in mouse embryonal fibroblasts (MEF). The method employs an immortalized mammalian cell line, deficient in a gene of interest, which is complemented by an episomal survival plasmid expressing the wild-type cDNA for the gene of interest and the use of a novel green fluorescent protein (GFP)-based double-label fluorescence system. The crucial part in this endeavor has been the identification of a DNA replicon that could stably replicate in MEFs while under selection for survival and gets spontaneously lost relatively fast in the absence of such a pressure. Here, we show for the first time that EBV-based replicons but not polyoma virus-based ones can replicate and be stably maintained in MEFs. In the chemical screen, selective pressure imposed by synthetic lethal drugs prevented the spontaneous loss of the GFP-marked episome, enabling drug identification. Retention or spontaneous loss over time of the episomal survival plasmid could be sensitively detected in a large-scale blind test in the presence or absence of synthetic lethal chemicals, respectively. Establishing the synthetic lethality screen should thus permit high throughput screening for chemicals, which are synthetically lethal with any mouse mutant/knockout gene of interest. Moreover, it forms the basis for a genetic synthetic lethality screen in MEFs, an important new tool for mouse functional genomics.
Asunto(s)
Replicación del ADN/genética , ADN Viral/biosíntesis , Prueba de Complementación Genética/métodos , Vectores Genéticos/genética , Genómica/métodos , Herpesvirus Humano 4/genética , Plásmidos/genética , Animales , Muerte Celular/efectos de los fármacos , Línea Celular , Supervivencia Celular/efectos de los fármacos , Metilación de ADN , ADN Complementario/genética , ADN Viral/genética , Fibroblastos , Fluorescencia , Eliminación de Gen , Humanos , Ratones , Poliomavirus/genética , Selección GenéticaRESUMEN
Thrombotic microangiopathies (TMAs) include several diseases, most prominently are thrombotic thrombocytopenic purpura (TTP) and hemolytic-uremic syndrome (HUS). TMAs are characterized by profound thrombocytopenia, microangiopathic hemolytic anemia and organ ischemia. In most cases TTP results from deficiency of ADAMTS13, the von Willebrand factor-cleaving protease leading to increase of ultra-large von Willebrand factor (ULVWF) multimers. Congenital TTP is due to mutations in the gene of ADAMTS13 whereas acquired TTP is due to production of autoantibodies against ADAMTS13. In both cases severe deficiency of ADAMTS13 exists. However, the presence of ADAMTS13 activity does not rule out TTP. Diagnostic criteria of TTP are based on clinical features of neurologic and renal disfunction along with anemia and thrombocytopenia, low ADAMTS13 activity, and the presence of ULVWF. The standard treatment of TTP includes plasma exchange, protein A immunoabsobtion, immunosuppressive drugs, CD20 antibodies against B cells, and splenectomy. HUS is commonly caused by infection with Shiga-toxin produced by Escherichia coli. HUS is characterized by thrombocytopenia, anemia, renal impairment and diarrhea. Rarely, atypical HUS appears as a consequence of mutations related to the alternative pathway for the compliment system. Plasmapheresis in HUS is not efficient. Alternatively, plasma therapy and in some cases dialysis are used. TMA diseases may be associated with other infections, bone marrow transplantation, pregnancy, systemic vasculitis, and certain drugs.
Asunto(s)
Anemia Hemolítica/diagnóstico , Púrpura Trombocitopénica Trombótica/diagnóstico , Proteínas ADAM/inmunología , Proteína ADAMTS13 , Anemia Hemolítica/epidemiología , Anemia Hemolítica/inmunología , Animales , Autoanticuerpos/sangre , Humanos , Mutación , Pronóstico , Púrpura Trombocitopénica Trombótica/epidemiología , Púrpura Trombocitopénica Trombótica/inmunología , Factor de von Willebrand/metabolismoRESUMEN
The platelet integrin αIIbß3 is widely accepted as a structural and a functional model of the broad integrin protein family. The four calcium-binding sites in the αIIb subunit contribute to biogenesis and stability of the protein. Mansour et al. (J Thromb Haemost 9:192-200, 2011) showed that the natural Asn2Asp mutation causing Glanzmann thrombasthenia, prevented surface expression of αIIbß3, whereas the artificial Asn2Gln mutation only decreased its level. Molecular dynamics simulations and EDTA chelation assay were used here to explore the mechanism of these structural deformations. We show a considerable expansion of the calcium-binding site 3 in Asn2Asp mutation, whereas the Asn2Gln toggles between normal and expanded conformations. The αIIbß3 surface expression level correlates to the relative spending time in the expanded conformation. By a comparison to other calcium-binding sites of αIIb and of other α integrins we show that the size of a calcium-binding loop is conserved. EDTA chelation assay shows a sensitivity to calcium removal, which correlates with the reduction in αIIbß3 surface expression and with the calcium binding site expansion, thus verifying the simulation data. Here we indicate that Asn2 mutation affects a calcium-binding site 3 of αIIb, which structural deformation is proposed to deprive calcium binding and interfere with an integrin intracellular trafficking and its surface expression.
Asunto(s)
Sitios de Unión/genética , Calcio/metabolismo , Mutación , Glicoproteína IIb de Membrana Plaquetaria/química , Glicoproteína IIb de Membrana Plaquetaria/genética , Animales , Calcio/química , Línea Celular , Cricetinae , Ácido Edético , Enlace de Hidrógeno , Simulación de Dinámica Molecular , Mutación/genética , Mutación/fisiología , Glicoproteína IIb de Membrana Plaquetaria/metabolismoRESUMEN
BACKGROUND: The treatment options in severe thrombocytopenia (platelet count ≤20×10(9)/L) are limited. The aim of this study was to investigate ways of improving blood clotting and stability in reconstituted thrombocytopenia. MATERIALS AND METHODS: Thrombocytopenia (platelets [16±4]×10(9)/L) was created by differential centrifugation of normal blood followed by reconstitution of whole blood which was subjected to clotting in a rotation thromboelastometer by CaCl2 and tissue factor, and to fibrinolysis by tissue plasminogen activator (tPA). In separate experiments, blood was diluted by 40% with TRIS/saline solution. Blood was treated with fibrinogen (fib), factor XIII (FXIII), and thrombin-activatable fibrinolysis inhibitor (TAFI). RESULTS: The maximum clot firmness of thrombocytopenic blood was approximately 2-fold less than that of intact blood. Supplementation of blood with fib and FXIII improved clot formation. In the presence of tPA, among fib, FXIII and TAFI, only fib stimulated clot propagation whereas each of these agents increased clot strength. There was a synergistic effect when fib was added together with FXIII or TAFI. Fibrinolysis was inhibited by TAFI and to a greater extent by TAFI + FXIII. Fourty percent dilution of blood reduced clot strength and increased susceptibility to tPA. Clot strength was increased by the treatments in the following order: fib/FXIII/TAFI > fib/TAFI > fib > TAFI > FXIII. In the presence of tPA, TAFI and FXIII lysed the clots significantly more slowly. This effect was stronger when blood was treated with the combination of fib/FXIII/TAFI. Doubling the fib concentration, alone or together with other agents, did not improve clot strength or stability. DISCUSSION: Augmentation of clot formation and anti-fibrinolysis by combining fib, FXIII and TAFI may be beneficial for the treatment of patients with severe thrombocytopenia especially when complicated by haemodilution following introduction of fluids to compensate for massive blood loss.
Asunto(s)
Coagulación Sanguínea , Carboxipeptidasa B2/metabolismo , Factor XIII/metabolismo , Fibrinógeno/metabolismo , Modelos Biológicos , Trombocitopenia/metabolismo , Femenino , Humanos , Masculino , Tromboelastografía/instrumentación , Tromboelastografía/métodos , Trombocitopenia/terapia , Activador de Tejido Plasminógeno/metabolismoRESUMEN
The integrins are a family of membrane receptors that attach a cell to its surrounding and play a crucial function in cell signaling. The combination of internal and external stimuli alters a folded non-active state of these proteins to an extended active configuration. The ß3 subunit of the platelet αIIbß3 integrin is made of well-structured domains rich in disulfide bonds. During the activation process some of the disulfides are re-shuffled by a mechanism requiring partial reduction of some of these bonds; any disruption in this mechanism can lead to inherent blood clotting diseases. In the present study we employed Molecular Dynamics simulations for tracing the sequence of structural fluctuations initiated by a single cysteine mutation in the ß3 subunit of the receptor. These simulations showed that in-silico protein mutants exhibit major conformational deformations leading to possible disulfide exchange reactions. We suggest that any mutation that prevents Cys560 from reacting with one of the Cys(567)-Cys(581) bonded pair, thus disrupting its ability to participate in a disulfide exchange reaction, will damage the activation mechanism of the integrin. This suggestion is in full agreement with previously published experiments. Furthermore, we suggest that rearrangement of disulfide bonds could be a part of a natural cascade of thiol/disulfide exchange reactions in the αIIbß3 integrin, which are essential for the native activation process.
Asunto(s)
Comunicación Celular/fisiología , Disulfuros/química , Integrina beta3/química , Compuestos de Sulfhidrilo/química , Análisis por Conglomerados , Biología Computacional , Humanos , Integrina beta3/genética , Simulación de Dinámica Molecular , Mutación/genética , Subunidades de Proteína/químicaRESUMEN
Patients suffering major traumatic or surgical bleeding are often exposed to hemodilution resulting in dilutional coagulopathy. The aim of this study was to evaluate in vitro the effects of fibrinogen, factor XIII and thrombin-activatable fibrinolysis inhibitor (TAFI) on clot formation and resistance to fibrinolysis in hemodilution conditions. Citrated whole blood from 36 healthy volunteers was diluted to 30 and 60% with lactated Ringer's solution. Blood samples were subsequently supplemented with fibrinogen, FXIII, TAFI or their combinations. Rotation thromboelastometry (ROTEM) in whole blood and thrombin generation in plasma were performed in the presence of CaCl2 and tissue factor/EXTEM reagent, and fibrinolysis was induced by tissue plasminogen activator (tPA). Hemodilution was expressed by decrease of peak height in thrombin generation and α-angle and maximum clot firmness (MCF) in ROTEM. Fibrinogen, FXIII or TAFI did not correct the decrease in thrombin generation peak height. In ROTEM, spiking of diluted blood with fibrinogen stimulated clot propagation. In tPA-treated blood fibrinogen, FXIII and TAFI increased clot firmness and inhibited fibrinolysis. Stronger protection against fibrinolysis was achieved combining FXIII with TAFI. Hemodilution was associated with inhibition of thrombin generation; however, this effect was not sensitive to blood spiking with fibrinogen, FXIII and TAFI. In ROTEM, these hemostasis agents improved clot strength and decreased clot susceptibility to tPA in nondiluted and to more extent in diluted blood. The maximal protection against fibrinolysis was caused by TAFI. Combining FXIII with TAFI exerted synergistic inhibitory effect on fibrinolysis.