**1. Introduction**

Among the drastic consequences of viperid snakebite envenomation, manifestations of local tissue damage, such as hemorrhage and myonecrosis, may result in permanent tissue damage and sequelae [1,2]. In cases of severe envenomation, bleeding in organs distant from the site of bite, such as the heart, lungs, kidneys, and brain, may also occur [3–5]. Coagulopathy, including procoagulant, blood-clotting, fibrinolytic, and anticoagulant effects, is

**Citation:** Bertholim, L.; Chaves, A.F.A.; Oliveira, A.K.; Menezes, M.C.; Asega, A.F.; Tashima, A.K.; Zelanis, A.; Serrano, S.M.T. Systemic Effects of Hemorrhagic Snake Venom Metalloproteinases: Untargeted Peptidomics to Explore the Pathodegradome of Plasma Proteins. *Toxins* **2021**, *13*, 764. https:// doi.org/10.3390/toxins13110764

Received: 19 August 2021 Accepted: 6 October 2021 Published: 28 October 2021

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another cause of morbidity and mortality upon viperid snakebite accidents [6,7]. Local and systemic effects of viperid envenomation involve the synergistic effects of snake venom metalloproteinases (SVMPs) on plasma proteins, connective tissue, platelets, and blood vessels. SVMPs are zinc-dependent enzymes classified in the M12B subfamily of metallopeptidases, in which the P-III class protein precursors are comprised pro-, catalytic, disintegrin-like, and cysteine-rich domains [8]. SVMPs target specific capillary basement components, cell surface proteins, and extracellular matrix and plasma proteins, thereby promoting capillary rupture and content extravasation, and resulting in hemostasis disturbance and hemorrhage [8–11].

HF3 is a very potent P-III class SVMP of *Bothrops jararaca* venom that induces local hemorrhage with minimum hemorrhagic doses of 15 ng on rabbit skin, and 160 ng on mouse skin [12,13]. The precursor of HF3 is composed of 606 amino acid residues, including five putative *N*-glycosylation sites. The calculated molecular mass of the mature form of HF3 is 46 kDa, whereas on SDS-PAGE, it shows a mobility corresponding to a protein of ~70 kDa, indicating that it is heavily glycosylated [13,14]. HF3 was shown to degrade proteins of the plasma and extracellular matrix, including fibrinogen, fibronectin, vitronectin, von Willebrand factor, collagens IV and VI, laminin, matrigel, antithrombin III, complement components C3 and C4, prothrombin, and plasminogen in vitro [9,15]. Moreover, HF3 showed degradation or limited proteolysis of the proteoglycans aggrecan, brevican, biglycan, decorin, glypican-1, lumican, mimecan, and syndecan-1 [15]. Proteins extracted from the hemorrhagic dorsal skin of mice injected with HF3 were submitted to SDS-PAGE and immunostained with specific anti-proteoglycan antibodies, resulting in the demonstration of in vivo cleavage of biglycan, decorin, glypican-1, lumican, and syndecan-1 [15]. Interestingly, HF3 cleaved the platelet derived growth factor receptor (PDGFR; alpha and beta), and PDGF, in vitro, and both receptor forms were also detected as degraded in vivo in the hemorrhagic process generated by HF3 in the mouse skin [15]. Moreover, the proteolytic activity of HF3 is not affected by plasma proteinase inhibitors, including α2-macroglobulin, which is cleaved by HF3 [16]. The disintegrin-like and cysteine-rich domains of HF3 play a role in its activities upon cells, as reported on their ability to inhibit collagen-induced platelet aggregation, to activate macrophage phagocytosis mediated by αMβ2 integrin, and to induce inflammation by increasing leukocyte rolling in the microcirculation [14,17,18].

The potent hemorrhage generated by HF3 on the mouse skin was analyzed using proteomic approaches, which corroborated the hydrolysis of intracellular, extracellular, and plasma proteins, including some proteoglycans [19]. Indeed, the cleavage of proteoglycans suggested a critical role of the destabilization of the mouse skin integrity in the hemorrhagic process generated by HF3, along with the release of pro-inflammatory fragments acting in the imbalance of tissue homeostasis [15,19]. Despite the strong evidence of the role of proteolysis in the local hemorrhage promoted by SVMPs, the full substrate repertoire of HF3 is unknown. Recently, we reported positional proteomic studies of HF3 cleavage sites in mouse embryonic fibroblast secreted proteins using terminal amine isotopic labeling of substrates (TAILS), which revealed a number of substrates, including proteins of the extracellular matrix and focal adhesions, and the cysteine protease inhibitor cystatin-C [20]. Proteomic identification of cleavage site specificity (PICS) was also used for identifying cleavage sites and sequence preferences in peptides upon incubation with HF3. Two studies using tryptic libraries of proteins from human plasma [21] and from THP-1 monocytic cells [20] revealed a clear preference for leucine at P1 position and the influence of amino acid sequences adjacent to the scissile bond in the substrate specificity of HF3, similarly to other metalloproteinases from viperid venoms [22].

The aim of this study was to gain new insights into the mechanisms of hemorrhage production by HF3 by expanding the analysis of the substrate repertoire of this SVMP on plasma proteins. To this end, approaches for the depletion of the most abundant proteins and for the enrichment of low abundant proteins of the human plasma were used to minimize the dynamic range of protein concentration. In order to assess the proteolytic activity of HF3 on a wide spectrum of proteins, we used untargeted peptidomics to detect the degradation products by mass spectrometry.

#### **2. Results**

The aim of this study was to evaluate the proteolytic activity of HF3 on human plasma proteins. In order to overcome the large protein dynamic range and complexity of human plasma [23,24], four different types of human plasma preparations were compared in in vitro incubations with HF3: whole plasma [P(W)]; plasma depleted of albumin [P(Alb-D)]; plasma depleted of 20 most abundant proteins [P(20-MAP-D)]; and plasma enriched of low-abundance proteins [P(LAP-E)]. Further, the resulting degradation products present in the peptide fraction were analyzed by LC-MS/MS and a database search using tools of Mascot in conjunction with the Trans-Proteomic Pipeline (TPP) and PEAKS Studio. An overview of the study is provided in Figure 1, as well as the nomenclature used for the different plasma fractionation methods.

**Figure 1.** Experimental workflow for the analysis of the proteolytic activity of HF3 on human plasma proteins. Human plasma was depleted of (i) albumin, or (ii) 20 most abundant proteins, or (iii) enriched of low abundant proteins. Whole plasma [P(W)], albumin-depleted plasma [P(Alb-D)], plasma depleted of the 20 most abundant proteins [P(20-MAP-D)], and plasma enriched of low abundant proteins [P(LAP-E)] were individually incubated with HF3, or vehicle, for 2 h at 37 ◦C. Peptide fractions from control (C) or HF3-treated (T) samples were obtained and analyzed by LC-MS/MS, and peptides were identified using Mascot in conjunction with the TPP and PEAKS Studio.

Three individual experiments of incubation of plasma proteins with HF3 were carried out with each of the four types of plasma samples, P(W), P(Alb-D), P(20-MAP-D), and P(LAP-E), and the following criteria were used in the untargeted peptidomic analysis to accept a protein as being cleaved by HF3: (i) peptides identified in the control samples were disregarded in the analysis, therefore only proteins that showed peptides identified exclusively in the sample of plasma treated with HF3 were considered; (ii) only proteins identified in at least two experiments were considered.

#### *2.1. Electrophoretic Profiles of Plasma Samples Incubated with HF3*

As shown in Figure 2, the three methods used for the decomplexation of the plasma proteome to achieve adequate sampling of proteins resulted in distinct electrophoretic profiles, containing proteins of 15−250 kDa. The incubation of these plasma samples with HF3 resulted in protein bands that decreased in intensity in the treated plasma in comparison to the control plasma, indicating proteins that may have been degraded by HF3. The bands that increased in intensity in the treated plasma indicated HF3 proteolysis products derived from proteins of higher molecular mass. In the case of P(W) incubated with HF3, it was possible to observe an increase in the intensity of bands in the region above 225 kDa, and a slight variation in the mobility of an intense band in the region of 52 kDa. P(Alb-D) treated with HF3 showed a decrease in the intensity of bands of ~140 kDa and above, and also a small alteration in the mobility of the ~52 kDa band. On the other hand, the electrophoretic profile of P(20-MAP-D) treated with HF3 showed only a slight decrease in the intensity of the bands in the region between 80 kDa and 120 kDa. However, upon incubation with HF3, P(LAP-E) showed a clear decrease in the intensity of the bands in of ~55 kDa, 40 kDa, and 25 kDa, and an increase in the intensity of the bands of 34 kDa and 22 kDa.

**Figure 2.** SDS-PAGE profile (12% SDS-polyacrylamide gel) of plasma samples incubated with HF3. M: molecular mass markers; C: control plasma; +HF3: plasma incubated with HF3. Red rectangles indicate regions showing different staining intensities between the control and HF3-treated plasma. Proteins were stained with silver.

Overall, all electrophoretic profiles indicated differences between control and treated plasma samples, evidencing the proteolytic effect of HF3 on plasma proteins in vitro. Interestingly, the electrophoretic profile of P(LAP-E) showed the most significant differences between control and treated samples, possibly due to the fact that the method applied for the enrichment of low abundant proteins of the plasma proteome was more efficient in minimizing the dynamic range of protein concentration, thus enabling a better visualization of the degraded proteins.
