*5.9. Mass Spectrometry Data Analysis*

Two strategies were employed for the identification of peptides originated from HF3 cleavage of plasma proteins. In the first, we used the Mascot program, and validated the results using the PeptideProphet and ProteinProphet tools of the Trans-Proteomic Pipeline (TPP) platform (Mascot/TPP). In the second, de novo sequencing, searching the database, and validating the results were carried out using the Peaks Studio 7 program (Peaks).

Mascot/TPP: Acquired MS/MS raw data were converted to the mgf and mzXML format using MS-Convert. Database searches were performed against the human UniProtKB/ Swiss-Prot protein database (available at http://www.uniprot.org; accessed on 3 February 2014) using Mascot 2.4.1 (Matrix Science) with the following parameters: no enzyme specificity indicated; 10 ppm precursor tolerance; 20 mmu fragment ion tolerance; variable Met oxidation (+15.9949 Da); variable *N* terminal acetylation (+42.0105 Da); and variable Asn and Gln deamidation (+0.9840 Da). Mascot search results were further processed using the Trans-Proteomic Pipeline (TPP, version 4.6) (Keller and Shteynberg, 2011). Peptides were included in the analysis if they were identified at a false discovery rate (FDR) of ≤1% at peptide level (PeptideProphet), and (ii) proteins at an FDR of ≤1% at protein level (ProteinProphet).

Peaks: Acquired MS/MS raw data were imported into Peaks Studio 7 software (Ma et al., 2003). De novo analysis was performed with the following parameters: no enzyme specificity indicated; 10 ppm precursor tolerance; 0.02 Da fragment ion tolerance; variable Met oxidation (+15.9949 Da); variable *N* terminal acetylation (+42.0105 Da); and variable Asn and Gln deamidation (+0.9840 Da). After de novo sequencing, a database search (Peaks DB) was performed against the human UniProtKB/Swiss-Prot protein database (available at http://www.uniprot.org; accessed on 10 February 2014), using the same parameters. Peptides were included in the analysis if they were identified at a Peaks DB FDR of ≤1%.

#### *5.10. Identification of Peptides Generated the Incubation of Fibrinogen with HF3*

Fibrinogen (50 μg) was incubated with HF3 (0.5 μg) in 0.025 M Tris-HCl, pH 7.5, 5 mM CaCl2 for 2 h at 37 ◦C. The reaction was stopped by adding eight volumes of cold acetone and one volume of cold methanol, and stored for 12 h at −20 ◦C. After centrifugation at 14,000× *g* for 10 min at 4 ◦C, the supernatant, corresponding to the peptide fraction, was dried in a SpeedVac concentrator, subjected to desalination procedures using Sep-Pak Light C18 cartridges, vacuum dried, and resuspended in 20 μL of 0.1% formic acid for analysis by LC-MS/MS, as described above, and a database search using the Peaks Studio 7 program.
