Leucocytes-decontaminated platelet-rich plasma (PRP) from 15 healthy volunteers was collected and activated with collagen, or Thrombin Activator Receptor Peptide (TRAP). Total protein extracts (75 microg protein/sample), coming from two biological replicates, were precipitated using 2-D Clean Up Kit (Amersham Bioscience). Total protein extracts were precipitated and dissolved into 20 microl of iTRAQ dissolution buffer. Protein alkylation, trypsin digestion and labeling of the resulting peptides were performed according to manufacturer’s instructions (Applied Biosystems) followed by strong cation exchange fractionation and LC-MS/MS analysis of the peptides as previously described (Lietzén et al, PLoS Pathogens). Protein identification and relative quantitation were performed with Paragon search algorithm using ProteinPilot 4 interface (Applied Biosystems). Database searches were performed against human protein sequences in UniProt database (version 120813 with 20231 human entries). The search criteria were: cysteine alkylation with MMTS, trypsin digestion, biological modifications allowed, thorough search and detected protein threshold of 95% confidence (Paragon Unused ProtScore > 1.3). Additionally, automatic bias correction was used to correct for uneven protein loading.