Phosphoproteomic Analysis of Platelets in Severe Obesity Uncovers Platelet Reactivity and Signaling Pathways Alterations

OBJECTIVE: Obesity is associated with a proinflammatory and prothrombotic state that supports atherosclerosis progression. The goal of this study was to gain insights into the phosphorylation events related to platelet reactivity in obesity and identify platelet biomarkers and altered activation pathways in this clinical condition. APPROACH AND RESULTS: We performed a comparative phosphoproteomic analysis of resting platelets from obese patients and their age- A nd gender-matched lean controls. The phosphoproteomic data were validated by mechanistic, functional, and biochemical assays. We identified 220 differentially regulated phosphopeptides, from at least 175 proteins; interestingly, all were up-regulated in obesity. Most of the altered phosphoproteins are involved in SFKs (Src-family kinases)-related signaling pathways, cytoskeleton reorganization, and vesicle transport, some of them validated by targeted mass spectrometry. To confirm platelet dysfunction, flow cytometry assays were performed in whole blood indicating higher surface levels of GP (glycoprotein) VI and CLEC (C-type lectin-like receptor) 2 in platelets from obese patients correlating positively with body mass index. Receiver operator characteristics curves analysis suggested a much higher sensitivity for GPVI to discriminate between obese and lean individuals. Indeed, we also found that obese platelets displayed more adhesion to collagen-coated plates. In line with the above data, soluble GPVI levels-indicative of higher GPVI signaling activation-were almost double in plasma from obese patients. CONCLUSIONS: Our results provide novel information on platelet phosphorylation changes related to obesity, revealing the impact of this chronic pathology on platelet reactivity and pointing towards the main signaling pathways dysregulatedThis work was supported by the Spanish Ministry of Science and Innovation (grants No. SAF2016-79662-R, and PID2019-108727RB-I00), co-funded by the European Regional Development Fund (ERDF). Financial support from the Consellería de Cultura, Educación e Ordenación Universitaria, Xunta de Galicia (Centro Singular de investigación de Galicia accreditation 2019–2022, ED431G 2019/02; predoctoral grant 2018 Call) and the ERDF is also gratefully acknowledged. E.E. Gardiner and R.K. Andrews are supported by the National Health and Medical Research Council of Australia. The Proteomics Laboratory CSIC/UAB (Centro Superior de Investigaciones Científicas/Universidad Autónoma de Barcelona) is a member of Proteored, PRB3-ISCIII (Instituto de Salud Carlos III), and is supported by Grant PT17/0019/0008, funded by ISCIII and ERDF. L.A. Morán is supported by the European Union’s Horizon 2020 research and innovation programme under the Marie Skłodowska-Curie grant agreement No 766118. S.P. Watson is supported by a BHF (British Heart Foundation) Chair (CH03/003)

El modelo integrador y de innovación docente del Grado en Veterinaria de Zaragoza: integración en équidos

Desde hace cuatro años se ha comenzado a impartir en la Universidad de Zaragoza el nuevo Grado en Veterinaria, de 300 créditos ECTS distribuidos en 5 cursos. En el diseño de este nuevo Plan de Estudios se ha tratado de dotar a la Titulación de un enfoque basado, fundamentalmente, en las competencias que la profesión exige y la sociedad necesita de un t itulado (graduado) en veterinaria. Tras una formación inicial básica el alumno llega al CUARTO CURSO del grado, en el que las materias se han organizado en asignaturas siguiendo una estructura totalmente novedosa para los estudios de veterinaria en España. Como consecuencia de esta nueva estructura desaparecen como tales muchas de las asignaturas tradicionales que han estado presentes como asignaturas propias (con denominaciones idénticas o similares) en la práctica totalidad de los planes de estudio de veterinaria españoles desde la década de los 70 del siglo pasado. Al cursar estas asignaturas, los alumnos de esos planes, estudiábamos las particularidades de las diferentes especies animales de interés veterinario desde el enfoque, inevitablemente compartimentalizado, de esas asignaturas

GPVI surface expression and signalling pathway activation are increased in platelets from obese patients: Elucidating potential anti-atherothrombotic targets in obesity.

BACKGROUND AND AIMS: Platelets play a fundamental role in the increased atherothrombotic risk related to central obesity since they show hyperactivation and lower sensitivity to antiplatelet therapy in obese patients. The main goal of this study was to identify platelet biomarkers related to the risk of atherothrombosis in obese patients, confirm platelet activation levels in these patients, and identify altered activation pathways. METHODS: Platelets were obtained from cohorts of obese patients and age- and sex-matched lean controls. Biochemical and proteome analyses were done by two-dimensional differential in-gel electrophoresis (2D-DIGE), mass spectrometry, and immunoblotting. Functional and mechanistic studies were conducted with aggregation assays and flow cytometry. RESULTS: We confirmed an up-regulation of αIIb and fibrinogen isoforms in platelets from obese patients. A complementary platelet aggregation approach showed platelets from obese patients are hyper-reactive in response to collagen and collagen-related peptide (CRP), revealing the collagen receptor Glycoprotein VI (GPVI) signalling as one of the altered pathways. We also found the active form of Src (pTyr418) is up-regulated in platelets from obese individuals, which links proteomics to aggregation data. Moreover, we showed that CRP-activated platelets present higher levels of tyrosine phosphorylated PLCγ2 in obese patients, confirming alterations in GPVI signalling. In line with the above, flow cytometry studies show higher surface expression levels of total GPVI and GPVI-dimer in obese platelets, both correlating with BMI. CONCLUSIONS: Our results suggest a higher activation state of SFKs-mediated signalling pathways in platelets from obese patients, with a primary involvement of GPVI signalling.In addition to the British Heart Foundation, the following are the sources of funding: Spanish Ministry of Economy and Competitiveness (MINECO) [grant No. SAF2016-79662-R], co-funded by the European Regional Development Fund (ERDF). Consellería de Cultura, Educación e Ordenación Universitaria, Xunta de Galicia (Centro Singular de investigación de Galicia accreditation 2016-2019, ED431G/05) Regional Development Fund (ERDF

Data on hyper-activation of GPVI signalling in obese patients: Towards the identification of novel antiplatelet targets in obesity.

This data article is associated with the manuscript "GPVI surface expression and signalling pathway activation are increased in platelets from obese patients: elucidating potential anti-atherothrombotic targets in obesity" [1]. The study refers to a combination of different approaches in order to identify platelet-derived biomarkers in obesity. A total of 34 obese patients and their lean-matched controls were included in the study. We carried out a proteomic and functional (aggregation assays) analysis to find alterations in platelet-derived signalling pathways. After that, biochemical and mechanistic (flow cytometry assays) approaches were done in order to confirm a hyperactivation of the GPVI-related signalling pathway.Spanish Ministry of Economy and Competitiveness (MINECO) [grant No. SAF2016-79662-R] European Regional Development Fund (ERDF). Consellería de Cultura, Educación e Ordenación Universitaria, Xunta de Galicia (Centro Singular de investigación de Galicia accreditation 2016-2019, ED431G/05) European Regional Development Fund (ERDF) British Heart Foundation, grants SP/13/7/30575, PG/18/36/338, and RG/15/4/31268

Platelet Lipidome Fingerprint: New Assistance to Characterize Platelet Dysfunction in Obesity

Obesity is associated with a pro-inflammatory and pro-thrombotic state that supports atherosclerosis progression and platelet hyper-reactivity. During the last decade, the platelet lipidome has been considered a treasure trove, as it is a source of biomarkers for preventing and treating different pathologies. The goal of the present study was to determine the lipid profile of platelets from non-diabetic, severely obese patients compared with their age- and sex-matched lean controls. Lipids from washed platelets were isolated and major phospholipids, sphingolipids and neutral lipids were analyzed either by gas chromatography or by liquid chromatography coupled to mass spectrometry. Despite a significant increase in obese patient’s plasma triglycerides, there were no significant differences in the levels of triglycerides in platelets among the two groups. In contrast, total platelet cholesterol was significantly decreased in the obese group. The profiling of phospholipids showed that phosphatidylcholine and phosphatidylethanolamine contents were significantly reduced in platelets from obese patients. On the other hand, no significant differences were found in the sphingomyelin and ceramide levels, although there was also a tendency for reduced levels in the obese group. The outline of the glycerophospholipid and sphingolipid molecular species (fatty-acyl profiles) was similar in the two groups. In summary, these lipidomics data indicate that platelets from obese patients have a unique lipid fingerprint that may guide further studies and provide mechanistic-driven perspectives related to the hyperactivate state of platelets in obesity

A Combination of Proteomic Approaches Identifies A Panel of Circulating Extracellular Vesicle Proteins Related to the Risk of Suffering Cardiovascular Disease in Obese Patients

Plasma-derived extracellular vesicles (EVs) have been extensively described as putative biomarkers in different diseases. Interestingly, increased levels of EVs subpopulations are well known to associate with obesity. The goal of this study is to identify EVs-derived biomarkers in plasma from obese patients in order to predict the development of pathological events associated with obesity. Samples are obtained from 22 obese patients and their lean-matched controls are divided into two cohorts: one for a 2D fluorescence difference gel electrophoresis (2D-DIGE)-based study, and the other one for a label free LC–MS/MS-based approach. EVs are isolated following a serial ultracentrifugation protocol. Twenty-two and 23 differentially regulated features are detected from 2D-DIGE and label free LC–MS/MS, respectively; most of them involve in the coagulation and complement cascades. Remarkably, there is an upregulation of complement C4, complement C3, and fibrinogen in obese patients following both approaches, the latter two also validated by 2D-western-blotting in an independent cohort. These results correlate with a proinflammatory and prothrombotic state of those individuals. On the other hand, a downregulation of adiponectin leading to an increased risk of suffering cardiovascular diseases has been shown. The results suggest the relevance of plasma-derived-EVs proteins as a source of potential biomarkers for the development of atherothrombotic events in obesity.This work was supported by the Spanish Ministry of Economy and Competitiveness; grant No. SAF2016-79662-R), co-funded by the European Regional Development Fund. Financial support from the Conseller ́ıa de Cultura, Educaci ́on e Ordenaci ́on Universitaria, Xunta de Galicia (Centro Singular de investigaci ́on de Galicia accreditation 2016–2019,ED431G/05), and the European Regional Development Fund is also grate-fully acknowledged. The Proteomics Laboratory CSIC/UAB is a member ofProteored, PRB2-ISCIII and is supported by Grant PT13/0001, of the PEI+D+i 2013–2016, funded by ISCIII and FEDER

The PI3Kδ Inhibitor Idelalisib Diminishes Platelet Function and Shows Antithrombotic Potential

Background: Clinical management of ischemic events and prevention of vascular disease is based on antiplatelet drugs. Given the relevance of phosphatidylinositol-4,5-bisphosphate 3-kinase (PI3K) as a candidate target in thrombosis, the main goal of the present study was to identify novel antiplatelet agents within the existing inhibitors blocking PI3K isoforms. Methods: We performed a biological evaluation of the pharmacological activity of PI3K inhibitors in platelets. The effect of the inhibitors was evaluated in intracellular calcium release and platelet functional assays, the latter including aggregation, adhesion, and viability assays. The in vivo drug antithrombotic potential was assessed in mice undergoing chemically induced arterial occlusion, and the associated hemorrhagic risk evaluated by measuring the tail bleeding time. Results: We show that PI3K Class IA inhibitors potently block calcium mobilization in human platelets. The PI3K p110δ inhibitor Idelalisib inhibits platelet aggregation mediated by ITAM receptors GPVI and CLEC-2, preferentially by the former. Moreover, Idelalisib also inhibits platelet adhesion and aggregation under shear and adhesion to collagen. Interestingly, an antithrombotic effect was observed in mice treated with Idelalisib, with mild bleeding effects at high doses of the drug. Conclusion: Idelalisib may have antiplatelet effects with minor bleeding effects, which provides a rationale to evaluate its antithrombotic efficacy in humans

Katacine is a new ligand of CLEC-2 that acts as a platelet agonist

Background: CLEC-2 is a platelet receptor with an important role in thrombo-inflammation but a minor role in haemostasis. Two endogenous ligands of CLEC-2 have been identified, the transmembrane protein podoplanin, and iron-containing porphyrin hemin, which is formed following haemolysis from red blood cells. Other exogenous ligands such as rhodocytin have contributed to our understanding of the role of CLEC-2. Objectives: To identify novel CLEC-2 small molecule ligands to aid therapeutic targeting of CLEC-2. Methods: ALPHA Screen technology has been used for development of a high throughput screening (HTS) assay recapitulating the podoplanin-CLEC-2 interaction. Light transmission aggregometry was used to evaluate platelet aggregation. Immunoprecipitation and western blot were used to evaluate direct phosphorylation of CLEC-2 and downstream protein phosphorylation. Autodock vina software was used to predict the molecular binding site of katacine and mass spectrometry to determine the polymeric nature of the ligand. Results and conclusions: We developed a CLEC-2-podoplanin interaction assay in a HTS format and screened 5016 compounds from an EU-Open screen library. We identified katacine, a mixture of polymers of proanthocyanidins, as a novel ligand for CLEC-2 and showed that it induces platelet aggregation and CLEC-2 phosphorylation via Syk- and Src kinases. Platelet aggregation induced by katacine is inhibited by the anti-CLEC-2 monoclonal antibody fragment AYP1 F(ab)’2. Katacine is a novel non-protein ligand of CLEC-2 that could contribute to a better understanding of CLEC-2 activation in human platelets

Katacine is a new ligand of CLEC-2 that acts as a platelet agonist

Background  CLEC-2 is a platelet receptor with an important role in thromboinflammation but a minor role in hemostasis. Two endogenous ligands of CLEC-2 have been identified, the transmembrane protein podoplanin and iron-containing porphyrin hemin, which is formed following hemolysis from red blood cells. Other exogenous ligands such as rhodocytin have contributed to our understanding of the role of CLEC-2. Objectives  To identify novel CLEC-2 small-molecule ligands to aid therapeutic targeting of CLEC-2. Methods  ALPHA screen technology has been used for the development of a high-throughput screening (HTS) assay recapitulating the podoplanin–CLEC-2 interaction. Light transmission aggregometry was used to evaluate platelet aggregation. Immunoprecipitation and western blot were used to evaluate direct phosphorylation of CLEC-2 and downstream protein phosphorylation. Autodock vina software was used to predict the molecular binding site of katacine and mass spectrometry to determine the polymeric nature of the ligand. Results and Conclusion  We developed a CLEC-2–podoplanin interaction assay in a HTS format and screened 5,016 compounds from a European Union-open screen library. We identified katacine, a mixture of polymers of proanthocyanidins, as a novel ligand for CLEC-2 and showed that it induces platelet aggregation and CLEC-2 phosphorylation via Syk and Src kinases. Platelet aggregation induced by katacine is inhibited by the anti-CLEC-2 monoclonal antibody fragment AYP1 F(ab)′2. Katacine is a novel nonprotein ligand of CLEC-2 that could contribute to a better understanding of CLEC-2 activation in human platelets

Qualitative and Quantitative Comparison of Plasma Exosomes from Neonates and Adults

Exosomes are extracellular vesicles that contain nucleic acids, lipids and metabolites, and play a critical role in health and disease as mediators of intercellular communication. The majority of extracellular vesicles in the blood are platelet-derived. Compared to adults, neonatal platelets are hyporeactive and show impaired granule release, associated with defects in Soluble N-ethylmaleimide-sensitive fusion Attachment protein REceptor (SNARE) proteins. Since these proteins participate in biogenesis of exosomes, we investigated the potential differences between newborn and adult plasma-derived exosomes. Plasma-derived exosomes were isolated by ultracentrifugation of umbilical cord blood from full-term neonates or peripheral blood from adults. Exosome characterization included size determination by transmission electron microscopy and quantitative proteomic analysis. Plasma-derived exosomes from neonates were significantly smaller and contained 65% less protein than those from adults. Remarkably, 131 proteins were found to be differentially expressed, 83 overexpressed and 48 underexpressed in neonatal (vs. adult) exosomes. Whereas the upregulated proteins in plasma exosomes from neonates are associated with platelet activation, coagulation and granule secretion, most of the underexpressed proteins are immunoglobulins. This is the first study showing that exosome size and content change with age. Our findings may contribute to elucidating the potential “developmental hemostatic mismatch risk” associated with transfusions containing plasma exosomes from adults