Fisiopatología del receptor plaquetario de fibrinógeno

Tesis de la Universidad Complutense de Madrid, Facultad de Ciencias Biológicas, Departamento de Bioquímica y Biología Molecular, leída el 24-05-2001La tromboastenia de Glanzimann es una enfermedad hereditaria caracterizada por un defecto cuantitativo o funcional del receptor plaquetario de fibrinógeno (GPIIb/IIIa). Por ello, nos ha servido de modelo para ampliar los conocimientos acerca de este receptor. Hemos estudiado cinco familias de pacientes con diagnóstico clínico de tromboastenia de Glanzimann, en las cuales hemos hallado cuatro nuevas mutaciones, todas ellas localizadas en la subunidad GPIIb. El análisis cuantitativo del RNAm de GPIIb y GPIIIa en plaquetas y el estudio funcional de las mutaciones halladas demuestran que los mecanismos moleculares responsables de la falta de expresión y/o función de complejos GPIIb/IIIa. han sido: ausencia de RNAm; incapacidad de la subunidad mutada para formar heterodímetros; alteración en la velocidad de tránsito y/o ensamblaje enla membrana plasmática de complejos GPIIb/IIla; alteración de los mecanismos de señalización intracelular. Hemos demostrado que la disponibilidad de RNAm de GPIIb es limitante para la expresión superficial de receptores GPIIb/IIIa. También observamos que la disminución en la tasa de expresión de GPIIb/IIIa en mutaciones heterocigotas puede ser debida a un efecto "dominante negativo" de las formas mutadas de estas proteínas o el resultado de interacciones anómalas con chaperonas del retículo endoplasmático. Realizamos estudios de mutagénesis específica como método experimental para establecer una correlación precisa entre estructura y función de estas proteínas. Los resultados obtenidos nos permiten afirmar:(a) El puente disulfuro intracatenario 674- 687 de GPIIb es esencial para mantener una tasa de expresión superficial de GPIIb/IIIa normal. (b) La carga negativa del residuo 324 E de GPIIb es fundamental para la correcta formación de heterodímeros con GPIIIa. (c) Los segmentos transmembranar y citoplásmico de GPIIIa son imprescindibles para la expresión de GPIIb/IIIa. (d) La deleción de los residuos 616-690 del extremo carboxi-terminal extracelular de GPIIIa confiere activación constitutiva al receptor GPIIb/IIIa.Sección Deptal. de Bioquímica y Biología Molecular (Biológicas)Fac. de Ciencias BiológicasTRUEpu

Fisiopatología del receptor plaquetario de fibrinógeno

La tromboastenia de Glanzimann es una enfermedad hereditaria caracterizada por un defecto cuantitativo o funcional del receptor plaquetario de fibrinógeno (GPIIb/IIIa). Por ello, nos ha servido de modelo para ampliar los conocimientos acerca de este receptor. Hemos estudiado cinco familias de pacientes con diagnóstico clínico de tromboastenia de Glanzimann, en las cuales hemos hallado cuatro nuevas mutaciones, todas ellas localizadas en la subunidad GPIIb. El análisis cuantitativo del RNAm de GPIIb y GPIIIa en plaquetas y el estudio funcional de las mutaciones halladas demuestran que los mecanismos moleculares responsables de la falta de expresión y/o función de complejos GPIIb/IIIa. han sido: ausencia de RNAm; incapacidad de la subunidad mutada para formar heterodímetros; alteración en la velocidad de tránsito y/o ensamblaje enla membrana plasmática de complejos GPIIb/IIla; alteración de los mecanismos de señalización intracelular. Hemos demostrado que la disponibilidad de RNAm de GPIIb es limitante para la expresión superficial de receptores GPIIb/IIIa. También observamos que la disminución en la tasa de expresión de GPIIb/IIIa en mutaciones heterocigotas puede ser debida a un efecto "dominante negativo" de las formas mutadas de estas proteínas o el resultado de interacciones anómalas con chaperonas del retículo endoplasmático. Realizamos estudios de mutagénesis específica como método experimental para establecer una correlación precisa entre estructura y función de estas proteínas. Los resultados obtenidos nos permiten afirmar:(a) El puente disulfuro intracatenario 674- 687 de GPIIb es esencial para mantener una tasa de expresión superficial de GPIIb/IIIa normal. (b) La carga negativa del residuo 324 E de GPIIb es fundamental para la correcta formación de heterodímeros con GPIIIa. (c) Los segmentos transmembranar y citoplásmico de GPIIIa son imprescindibles para la expresión de GPIIb/IIIa. (d) La deleción de los residuos 616-690 del extremo carboxi-terminal extracelular de GPIIIa confiere activación constitutiva al receptor GPIIb/IIIa

Study of platelet kinetics in immune thrombocytopenia to predict splenectomy response

Despite the efficacy of splenectomy for chronic immune thrombocytopenia (ITP), its considerable failure rate and its possible related complications prove the need for further research into potential predictors of response. The platelet sequestration site determined by 111In-labelled autologous platelet scintigraphy has been proposed to predict splenectomy outcome, but without standardisation in clinical practice. Here, we conducted a single-centre study by analysing a cohort of splenectomised patients with ITP in whom 111In-scintigraphy was performed at La Paz University Hospital in Madrid to evaluate the predictive value of the platelet kinetic studies. We also studied other factors that could impact the splenectomy outcome, such as patient and platelet characteristics. A total of 51 patients were splenectomised, and 82.3% responded. The splenic sequestration pattern predicted a higher rate of complete response up to 12 months after splenectomy (p = 0.005), with 90% sensitivity and 77% specificity. Neither age, comorbidities, therapy lines nor previous response to them showed any association with response. Results from the platelet characteristics analysis revealed a significant loss of sialic acid in platelets from the non-responding patients compared with those who maintained a response (p = 0.0017). Our findings highlight the value of splenic sequestration as an independent predictor of splenectomy respons

The importance of platelet glycoside residues in the haemostasis of patients with immune thrombocytopaenia

Loss of sialic acid from the carbohydrate side chains of platelet glycoproteins can affect platelet clearance, a proposed mechanism involved in the etiopathogenesis of immune thrombocy-topaenia (ITP). We aimed to assess whether changes in platelet glycosylation in patients with ITP affected platelet counts, function, and apoptosis. This observational, prospective, and transversal study included 82 patients with chronic primary ITP and 115 healthy controls. We measured platelet activation markers and assayed platelet glycosylation and caspase activity, analysing samples using flow cytometry. Platelets from patients with ITP with a platelet count <30 × 103/µL presented less sialic acid. Levels of α1,6-fucose (a glycan residue that can directly regulate antibody-dependent cellular cytotoxicity) and α-mannose (which can be recognised by mannose-binding-lectin and acti-vate the complement pathway) were increased in the platelets from these patients. Platelet surface exposure of other glycoside residues due to sialic acid loss inversely correlated with platelet count and the ability to be activated. Moreover, loss of sialic acid induced the ingestion of platelets by human hepatome HepG2 cells. Changes in glycoside composition of glycoproteins on the platelets’ surface impaired their functional capacity and increased their apoptosis. These changes in platelet glycoside residues appeared to be related to ITP severity.This research was funded by FIS-Fondos FEDER PI19/00631, FIS-Fondos FEDER PI19/00772 and Platelet Disorder Support Associatio

Lung Transplant Improves Survival and Quality of Life Regardless of Telomere Dysfunction

Introduction: Fibrotic interstitial lung diseases (ILDs) are the first indication for lung transplantation (LT). Telomere dysfunction has been associated with poor post-transplant outcomes. The aim of the study was to evaluate the morbi-mortality and quality of life in fibrotic ILDs after lung transplant depending on telomere biology. Methods: Fibrotic ILD patients that underwent lung transplant were allocated to two arms; with or without telomere dysfunction at diagnosis based on the telomere length and telomerase related gene mutations revealed by whole-exome sequencing. Post-transplant evaluation included: (1) short and long-term mortality and complications and (2) quality of life. Results: Fifty-five percent of patients that underwent LT carried rare coding mutations in telomerase-related genes. Patients with telomere shortening more frequently needed extracorporeal circulation and presented a higher rate of early post-transplant hematological complications, longer stay in the intensive care unit (ICU), and a higher number of long-term hospital admissions. However, post-transplant 1-year survival was higher than 80% regardless of telomere dysfunction, with improvement in the quality of life and oxygen therapy withdrawal. Conclusions: Post-transplant morbidity is higher in patients with telomere dysfunction and differs according to elapsed time from transplantation. However, lung transplant improves survival and quality of life and the associated complications are manageable

GSE4‐loaded nanoparticles a potential therapy for lung fibrosis that enhances pneumocyte growth, reduces apoptosis and DNA damage

Idiopathic pulmonary fibrosis is a lethal lung fibrotic disease, associated with aging with a mean survival of 2-5 years and no curative treatment. The GSE4 peptide is able to rescue cells from senescence, DNA and oxidative damage, inflammation, and induces telomerase activity. Here, we investigated the protective effect of GSE4 expression in vitro in rat alveolar epithelial cells (AECs), and in vivo in a bleomycin model of lung fibrosis. Bleomycin-injured rat AECs, expressing GSE4 or treated with GSE4-PLGA/PEI nanoparticles showed an increase of telomerase activity, decreased DNA damage, and decreased expression of IL6 and cleaved-caspase 3. In addition, these cells showed an inhibition in expression of fibrotic markers induced by TGF-β such as collagen-I and III among others. Furthermore, treatment with GSE4-PLGA/PEI nanoparticles in a rat model of bleomycin-induced fibrosis, increased telomerase activity and decreased DNA damage in proSP-C cells. Both in preventive and therapeutic protocols GSE4-PLGA/PEI nanoparticles prevented and attenuated lung damage monitored by SPECT-CT and inhibited collagen deposition. Lungs of rats treated with bleomycin and GSE4-PLGA/PEI nanoparticles showed reduced expression of α-SMA and pro-inflammatory cytokines, increased number of pro-SPC-multicellular structures and increased DNA synthesis in proSP-C cells, indicating therapeutic efficacy of GSE4-nanoparticles in experimental lung fibrosis and a possible curative treatment for lung fibrotic patients

Multicentric study of cervical cancer screening with human papillomavirus testing and assessment of triage methods in Latin America : the ESTAMPA screening study protocol

Q1Q1Introduction Human papillomavirus (HPV) testing is replacing cytology in primary screening. Its limited specificity demands using a second (triage) test to better identify women at high-risk of cervical disease. Cytology represents the immediate triage but its low sensitivity might hamper HPV testing sensitivity, particularly in low-income and middle-income countries (LMICs), where cytology performance has been suboptimal. The ESTAMPA (EStudio multicéntrico de TAMizaje y triaje de cáncer de cuello uterino con pruebas del virus del PApiloma humano; Spanish acronym) study will: (1) evaluate the performance of different triage techniques to detect cervical precancer and (2) inform on how to implement HPV-based screening programmes in LMIC. Methods and analysis Women aged 30–64 years are screened with HPV testing and Pap across 12 study centres in Latin America. Screened positives have colposcopy with biopsy and treatment of lesions. Women with no evident disease are recalled 18 months later for another HPV test; those HPV-positive undergo colposcopy with biopsy and treatment as needed. Biological specimens are collected in different visits for triage testing, which is not used for clinical management. The study outcome is histological high-grade squamous intraepithelial or worse lesions (HSIL+) under the lower anogenital squamous terminology. About 50 000 women will be screened and 500 HSIL+ cases detected (at initial and 18 months screening). Performance measures (sensitivity, specificity and predictive values) of triage techniques to detect HSIL+ will be estimated and compared with adjustment by age and study centre. Ethics and dissemination The study protocol has been approved by the Ethics Committee of the International Agency for Research on Cancer (IARC), of the Pan American Health Organisation (PAHO) and by those in each participating centre. A Data and Safety Monitoring Board (DSMB) has been established to monitor progress of the study, assure participant safety, advice on scientific conduct and analysis and suggest protocol improvements. Study findings will be published in peer-reviewed journals and presented at scientific meetings. Trial registration number NCT01881659Revista Internacional - Indexad

Membrane dynamics in cell migration

Migration of cells is required in multiple tissue-level processes, such as in inflammation or cancer metastasis. Endocytosis is an extremely regulated cellular process by which cells uptake extracellular molecules or internalise cell surface receptors. While the role of endocytosis of focal adhesions (FA) and plasma membrane (PM) turnover at the leading edge of migratory cells is wide known, the contribution of endocytic proteins per se in migration has been frequently disregarded. In this review, we describe the novel functions of the most well-known endocytic proteins in cancer cell migration, focusing on clathrin, caveolin, flotillins and GRAF1. In addition, we highlight the relevance of the macropinocytic pathway in amoeboid-like cell migration

Protein-protein interactions in platelet alphaIIbbeta3 signaling

13 páginasThe major platelet integrin αIIbβ3 is the main receptor involved in platelet functions such as aggregation and spreading on extracellular matrix. Like all other integrins, αIIbβ3 is capable of transducing signals both from inside and outside of the cell. To mediate these functions, αIIbβ3 interacts with intracellular and transmembrane proteins. The identification of these proteins, as well as the study of their functions, has provided valuable insights into integrin-mediated function and signaling. This review summarizes the known proteins that directly interact with αIIbbβ3 and provides an overview of their roles in integrin functionPeer reviewe

Competition between normal [674C] and mutant [674R] subunits: role of the molecular chaperone BiP in the processing of GPIIb-IIIa complexes

8 páginas, 8 figuras -- PAGS nros. 2640-2647This work aimed at investigating the function of the [C674R] mutation in GPIIb that disrupts the intramolecular 674 to 687 disulfide bridge. Individuals heterozygous for this mutation show a platelet GPIIb-IIIa content approximately 30% of normal controls, which is less than expected from one normal functioning allele. Coexpression of normal [674C]GPIIb and mutant [674R]GPIIb with normal GPIIIa produced a [674R]GPIIb concentration-dependent inhibition of surface exposure of GPIIb-IIIa complexes in Chinese hamster ovary (CHO) cells, suggesting that [674R]GPIIb interferes with the association and/or intracellular trafficking of normal subunits. Mutation of either 674C or 687C had similar effects in reducing the surface exposure of GPIIb-IIIa. However, substitution of 674C for A produced a much lesser inhibition than R, suggesting that a positive-charged residue at that position renders a less efficient subunit conformation. The mutant [674R]GPIIb but not normal GPIIb was found associated with the endoplasmic reticulum chaperone BiP in transiently transfected CHO cells. BiP was also found associated with [674R]GPIIb-IIIa heterodimers, but not with normal GPIIIa or normal heterodimers. Overexpression of BiP did not increase the surface exposure of [674R]GPIIb-IIIa complexes, indicating that its availability was not a limiting step. Platelets from the thrombasthenic patient expressing [674R]GPIIb-IIIa were found to bind soluble fibrinogen in response to physiologic agonists or dithiothreitol treatment. Thus, the [674R]GPIIb mutation leads to a retardation of the secretory pathway, most likely related to its binding to the molecular chaperone BiP, with the result of a defective number of functional GPIIb-IIIa receptors in the cell surfaceSupported in part by grants from the Dirección General de Investigación Cientı́fica y Técnica (DGICYT PB97-1240 and DGICYT PM97-0016), Fondo de Investigaciones Sanitarias (96/2014), and Comunidad Autónoma de Madrid 08.4/0031/1998, and by a grant-in-aid from the Agencia Española de Cooperación Internacional (AECI, n/ref 99CN0009). E.G.A.-S. received a fellowship from the Fundación ArecesPeer reviewe