**4. Conclusions**

The human plasma depletion methods used in this study provided heterogeneous samples with respect to the range of dynamic protein concentration, which were compatible with the peptidomic analysis, and generated complementary results for the elucidation of the HF3 degradome. The two bioinformatics approaches used for the analysis of the peptide fraction gave robustness to the set of obtained results, since most proteins identified as HF3 substrates were detected by both. The determination of the primary specificity of HF3 on protein substrates showed that Leu at P1 is a major determinant of HF3 primary specificity, which agrees with previous studies using peptides, and reinforces the importance of this residue at P1 , regardless of the substrate structure.

As a result of this study, knowledge about the HF3 substrate repertoire in human plasma has been expanded in terms of number, as well as protein classes and functions (Figure 9). Taken together, the results illustrate the proteolytic signature of human plasma in the context of HF3-induced hemorrhage. By acting on distinct substrates, which are part of a highly connected biological circuit, the proteolytic signaling triggered by HF3 may not be fully anticipated by the results of in vitro incubation with single substrates. Actually, the activated/impaired biological pathways involved in the hemorrhagic and pro-inflammatory effects of SVMPs are the result of complex signaling circuits, which are significantly affected by limited proteolysis and protein degradation. In this regard, it was interesting to note that the hydrolysis of some proteins by HF3 seems to lead to antagonistic results, such as the hydrolysis of fibrinogen and plasminogen, which play roles in different steps of blood coagulation and fibrinolysis. In general, the characterization of HF3 substrate degradome in the human plasma suggests that it acts in a dysregulated manner, refractory to plasma inhibitors, causing an imbalance in hemostasis.

**Figure 9.** Schematic representation of some of the new substrates of HF3 revealed in this study, according to biological function.

The hydrolysis of human plasma proteins by an uncontrolled, exogeneous metalloproteinase has a direct impact in the plasma proteome, and it can be hypothesized that some hydrolysis products could also play synergistic roles in the pro-inflammatory and hemorrhagic processes generated by HF3.

### **5. Materials and Methods**

#### *5.1. HF3*

HF3 (Uniprot entry Q98UF9) was purified as described previously (Oliveira et al., 2009) from *B. jararaca* venom provided by the Laboratory of Herpetology of Butantan Institute (São Paulo, Brazil), and identified by trypsin digestion and mass spectrometric (LC–MS/MS) analysis.

#### *5.2. Analytical Procedures*

Protein and peptide contents were quantified using, respectively, Bradford assay (Sigma-Aldrich, St. Louis, MO, USA) and micro-BCA assay (Pierce) kits according to the manufacturers' recommendations. SDS–PAGE was carried out according to Laemmli (1970) [106]. Silver staining was carried out according to Mortz et al., 2001 [107].
