*2.4. Mapping the Primary Specificity of HF3 on Plasma Proteins*

A large number of peptides derived from the activity of HF3 on plasma proteins were identified in the experiments performed with P(W), P(Alb-D), P(20-MAP-D), and P(LAP-E), and these were used for the evaluation of the proteins that were hydrolyzed by HF3, as well as to carry out the mapping of amino acid sequences adjacent to the cleavage sites, which are preferential for the proteinase (primary specificity). For this purpose, we used the amino acid sequences of the peptides, corresponding to the positions P1'–P6' [26], identified in the samples treated with HF3, and originating from the proteins considered as substrate, whereas the complementary amino acid sequences (P6-P1) adjacent to the cleavage sites of the respective peptides were obtained using a tool available online at http://clipserve.clip.ubc.ca/pics; accessed on 1 April 2014 [27], which also generated the graphical representation (heat map) of the proteinase cleavage consensus sequence. The heat maps representing the occurrence of amino acids at positions P6-P6' are described in Figure 7, corresponding to the identification results obtained by the Mascot/TPP and

PEAKS approaches. It was interesting to verify that, although the number of cleavage sites identified by the PEAKS approach was higher than that of Mascot/TPP, the heat maps generated with data from both approaches from data from all plasma samples were very similar and evidenced a clear preference of HF3 for Leu at the P1' position.

The analysis of hydrolysis products of plasma proteins incubated with HF3 showed that fibrinogen was cleaved in the alpha, beta, and gamma chains (Supplementary Material Tables S1–S8). These data corroborate previous studies that described extensive cleavage of the alpha chain, followed by more limited beta chain cleavage [9] by HF3, however, proteolysis of the fibrinogen gamma chain by HF3 was unknown. Here, peptides from the C-terminal region of the fibrinogen gamma chain were identified as a product of HF3 activity in P(20-MAP-D) and P(LAP-E). Studies have shown that the C-terminal region of the fibrinogen gamma chain plays important roles in platelet interaction [28] and fibrin stabilization [29], and that the C-terminal peptide also plays a role in the inhibition of platelet aggregation [30]. Thus, for the validation of peptides generated by cleavage of fibrinogen by HF3 in plasma, it was incubated with isolated fibrinogen, followed by analysis of the resulting peptide fraction by LC-MS/MS. This analysis revealed the peptides generated by the enzymatic activity of HF3, and indirectly, the cleavage points of HF3 in the protein (Supplementary Material Table S9). Figure 8 shows the location of these peptides in the fibrinogen sequence, corresponding to 73 peptides of the alpha chain and 15 peptides of the beta chain. Eight fibrinogen gamma chain peptides were also identified, including two located in the C-terminal region, thus confirming the data obtained by the incubation of HF3 with plasma.

**Figure 7.** *Cont*.

**Figure 7.** Substrate specificity assessed by the identification of peptides generated by the proteolytic activity of HF3 on human plasma proteins. (**A**) Heat maps showing the relative occurrence (in %) of each amino acid residue and the fold-change over the natural abundance of amino acids, identified using Mascot/TPP. (**B**) Heat maps showing the relative occurrence (in %) of each amino acid residue and the fold-change over the natural abundance of amino acids, identified using PEAKS. Only peptides derived from proteins identified as HF3 substrates were considered, and peptides identified both in control and treated samples were excluded. Heat maps were created at http://clipserve.clip.ubc.ca/pics (accessed on 1 April 2014).

**Figure 8.** Amino acid sequence of human fibrinogen alpha (**A**), beta (**B**), and gamma (**C**) chains, indicating the identified cleavage products (blue bars) generated by the incubation with HF3. Graphical view generated with PEAKS Studio 7.
