title: Application of 2-dimensional difference gel electrophoresis (2D-DIGE) to the study of thrombin-activated human platelet secretome.
abstract: Thrombin is an agonist inducing platelet activation. We combined two-dimensional difference gel electrophoresis (2D-DIGE) and mass spectrometry (MALDI-TOF MS) to analyse differentially expressed proteins secreted from thrombin-stimulated platelets. Human washed platelets, from healthy volunteers, were stimulated with thrombin 0.5 U/ml at 37 degrees C without stirring and the secreted proteins were resolved by 2D-DIGE. By image analysis, 1094 spots were detected in the 2D gel. The spots whose mean intensity showed at least a five-fold change intensity increase or decrease in the thrombin-activated platelet gel in comparison with unstimulated control were digested by trypsin and subjected to MALDI-TOF MS analysis. Peptides from mass spectra of in-gel digest samples were matched against available databases, using the Mascot search engine (Matrix Science) for peptide mass fingerprint. In the activated platelet secretome, transferrin, glutathione-transferase, WD repeat protein, ER-60, thrombospondin-1 precursor and thrombospondin were the most abundant. Also lamin A, a nuclear protein, not previously identified in platelets, appeared to be released. The novel strategy to combine 2D-DIGE with MALDI-TOF MS is a useful approach for a quantitative analysis of the effect of thrombin on the secretome profile of human platelets.
authors.affiliation: Research Laboratories, John Paul II Centre for High Technology Research and Education in Biomedical Sciences, Catholic University, Largo, 86100 Campobasso, Italy. anna.dellacorte@rm.unicatt.it
authors.forename: Anna
authors.initials: A
authors.lastname: Della Corte
authors.forename: Norma
authors.initials: N
authors.lastname: Maugeri
authors.forename: Agnieszka
authors.initials: A
authors.lastname: Pampuch
authors.forename: Chiara
authors.initials: C
authors.lastname: Cerletti
authors.forename: Giovanni
authors.initials: G
authors.lastname: de Gaetano
authors.forename: Domenico
authors.initials: D
authors.lastname: Rotilio
mesh_terms: D000328:Adult; D001792:Blood Platelets / Q000378:metabolism*; D015180:Electrophoresis, Gel, Two-Dimensional / Q000379:methods; D005260:Female; D006801:Humans; D008297:Male; D015539:Platelet Activation / Q000187:drug effects / Q000502:physiology*; D020543:Proteome / Q000378:metabolism*; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization / Q000379:methods; D013917:Thrombin / Q000494:pharmacology
chemical_list: D020543:Proteome; D013917:Thrombin