Dataset Information

Platelet-derived microparticles from obese individuals: characterization of number, size, proteomics and crosstalk with cancer and endothelial cells

ABSTRACT: Rationale. Obesity is a risk factor for atherothrombosis and various cancers. However, the mechanisms are not completely uncovered. Objectives. We aimed to verify whether the microparticles (MPs) released from thrombin-activated platelets differed in obese and nonobese women for number, size, and proteomics cargo and the capacity to modulate in vitro the expression of genes related to the epithelial to mesenchymal transition (EMT) and the endothelial to mesenchymal transition (EndMT), and COX-2, a pro-angiogenic pathway. Methods and Results. MPs were obtained from thrombin activated platelets of four obese women and their matched, lean controls . MPs were analyzed by cytofluorimeter and protein content by liquid chromatography-mass spectrometry. Obese MPs were not different in number but were characterized by increased heterogeneity in size. In obese individuals, MPs containing mitochondria (mitoMPs) expressed lower CD41 levels and increased phosphatidylserine associated with enhanced Factor V representing a signature of a prothrombotic state. Proteomics analysis identified 44 proteins downregulated and 3 upregulated in obese versus nonobese MPs . A reduction in the proteins involved in mitophagy and antioxidant defenses, and of the -granular membrane was detected in the MPs of obese individuals. MPs released from platelets of obese individuals were more prone to induce the expression of marker genes of EMT and EndMT when incubated with HT29 cells and human cardiac microvascular endothelial cells(HCMEC), respectively. A protein, highly enhanced in obese MPs, was the pro-platelet basic protein with pro-inflammatory and tumorigenic actions. MPs from obese, but not nonobese, women induced COX-2 in HCMEC. Conclusions. Platelet-derived MPs of obese women showed higher heterogeneity in size and contained different levels of proteins relevant to thrombosis and tumorigenesis. Obese MPs presented enhanced capacity to induce changes in the expression of EMT and EndMT markers and COX-2. These effects might contribute to the increased risk for the development of thrombosis and multiple malignancies in obesity.

INSTRUMENT(S): Q Exactive

ORGANISM(S): Homo Sapiens (human)

TISSUE(S): Blood Platelet, Blood Cell

DISEASE(S): Cardiovascular System Disease

SUBMITTER: Simone Marcone

LAB HEAD: Professor Paola Patrignani

PROVIDER: PXD011563 | Pride | 2019-02-19

REPOSITORIES: Pride

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			Action	DRS
	26_64_99_B1711_R1.raw	Raw
	26_64_99_B1711_R11.raw	Raw
	26_64_99_B1711_R11_171122200629.raw	Raw
	26_64_99_B1711_R11_171122212256.raw	Raw
	26_64_99_B1711_R12.raw	Raw

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Publications

Platelet-Derived Microparticles From Obese Individuals: Characterization of Number, Size, Proteomics, and Crosstalk With Cancer and Endothelial Cells.

Grande Rosalia R Dovizio Melania M Marcone Simone S Szklanna Paulina B PB Bruno Annalisa A Ebhardt H Alexander HA Cassidy Hilary H Ní Áinle Fionnuala F Caprodossi Anna A Lanuti Paola P Marchisio Marco M Mingrone Geltrude G Maguire Patricia B PB Patrignani Paola P

Frontiers in pharmacology 20190122

Rationale: Obesity is a risk factor for atherothrombosis and various cancers. However, the mechanisms are not yet completely clarified. Objectives: We aimed to verify whether the microparticles (MPs) released from thrombin-activated platelets differed in obese and non-obese women for number, size, and proteomics cargo and the capacity to modulate in vitro the expression of (i) genes related to the epithelial to mesenchymal transition (EMT) and the endothelial to mesenchymal ...[more]

PMID: 30723407

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Project description:Phosphoproteomic analysis of platelets activated by pro-thrombotic oxidized phospholipids and thrombin Alejandro Zimman, Bjoern Titz, Evangelia Komisopoulou, Sudipta Biswas, Thomas G. Graeber, and Eugene A. Podrez ABSTRACT Specific oxidized phospholipids (oxPCCD36) promote platelet hyper-reactivity and thrombosis in hyperlipidemia via the scavenger receptor CD36, however the signaling pathway(s) induced in platelets by oxPCCD36 are not well defined. We have now employed mass spectrometry-based tyrosine, serine, and threonine global phosphoproteomics for the unbiased analysis of platelet signaling pathways induced by oxPCCD36 as well as by the strong physiological agonist thrombin. oxPCCD36 and thrombin induced differential phosphorylation of 115 proteins (162 phosphorylation sites) and 181 proteins (334 phosphorylation sites) respectively. Most of the phosphoproteome changes induced by either agonist have never been reported in platelets; thus they provide a new resource to study platelet signaling. Bioinformatic analyses of protein phosphorylation dependent responses were used to categorize preferential motifs for (de)phosphorylation, predict pathways and kinase activity, and construct a phosphoproteome network regulating integrin activation. A putative signaling pathway involving Src-family kinases, SYK, and PLCy2 was identified in platelets activated by oxPCCD36. Subsequent ex vivo studies in human platelets demonstrated that this pathway is downstream of the scavenger receptor CD36 and is critical for platelet activation by oxPCCD36. Our results provide multiple novel insights into the mechanism of platelet activation and specifically in platelet regulation by oxPCCD36. Methods Human platelets isolated by gel filtration (2.7 x 108 / mL) were incubated in Tyrode's buffer with 50 uM KODA-PC or PLPC (as control) for 30 min at 37 degrees C. Platelets were then centrifuged at 3,700 g (10 min, 35 degrees C) and lysed in 8 M urea, 50 mM Tris-HCl (pH 7.4), 1 mM Na3VO4, and 1 mM NaF with sonication. Phosphoproteome changes induced by the agonists were assessed based on trypsin digestion of the protein lysate, phosphopeptide enrichment, mass spectrometry analysis, chromatography alignment, and label-free quantitation as described in the Supplemental Materials and Methods of the publication. The same protocol was applied when studying platelet activation by thrombin, where gel-filtered platelets were incubated in Tyrode's buffer with 0.05 U/mL thrombin or in buffer alone (Resting) for 3 min at 37 degrees C.