Hemorrhagic Activity of HF3, a Snake Venom Metalloproteinase

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Hemorrhagic Activity of HF3, a Snake Venom Metalloproteinase: Insights from the Proteomic Analysis of Mouse Skin and Blood Plasma Adriana F. Paes Leme,†,‡ Nicholas E. Sherman,§ David M. Smalley,§ Letícia O. Sizukusa,† Ana K. Oliveira,† Milene C. Menezes,† Jay W. Fox,§ and Solange M. T. Serrano*,† †

Laboratorio Especial de Toxinologia Aplicada-CAT/cepid, Instituto Butantan, S~ao Paulo, Brazil Laboratorio Nacional de Biociências (LNBio), Campinas, Brazil § Department of Microbiology, University of Virginia, Charlottesville, Virginia, United States ‡

bS Supporting Information ABSTRACT: Hemorrhage induced by snake venom metalloproteinases (SVMPs) is a complex phenomenon resulting in capillary disruption and blood extravasation. The mechanism of action of SVMPs has been investigated using various methodologies however the precise molecular events associated with microvessel disruption remains not fully understood. To gain insight into the hemorrhagic process, we analyzed the global effects of HF3, an extremely hemorrhagic SVMP from Bothrops jararaca, in the mouse skin and plasma. We report that in the HF3-treated skin there was evidence of degradation of extracellular matrix (collagens and proteoglycans), cytosolic, cytoskeleton, and plasma proteins. Furthermore, the data suggest that direct and indirect effects promoted by HF3 contributed to tissue injury as the activation of collagenases was detected in the HF3-treated skin. In the plasma analysis after depletion of the 20 most abundant proteins, fibronectin appeared as degraded by HF3. In contrast, some plasma proteinase inhibitors showed higher abundance compared to control skin and plasma. This is the first study to assess the complex in vivo effects of HF3 using high-throughput proteomic approaches, and the results underscore a scenario characterized by the interplay between the hydrolysis of intracellular, extracellular, and plasma proteins and the increase of plasma inhibitors in the hemorrhagic process. KEYWORDS: hemorrhage, peptidome, proteome, metalloproteinase, snake venom, skin, plasma

’ INTRODUCTION Manifestations of local tissue damage, such as hemorrhage and myonecrosis, are among the most dramatic effects of envenomation by snake species of the Viperidae family. In cases of less severe envenomation, the hemorrhagic effect is usually localized at the site of the bite. However, hemorrhage can be found spread widely through a substantial area of the involved extremity. In cases where the envenomation is severe, bleeding in organs distant from the site of envenomation, such as heart, lungs, kidneys and brain, may also occur.1 3 Virtually all of the snake venom hemorrhagic toxins isolated and characterized thus far have been determined to be Zn2+dependent metalloproteinases. Other features of viperid envenoming that occur in part by the direct action of metalloproteinases are the prominent local tissue damage associated with a series of acute pathological alterations in the area of venom inoculation and a profound consumption coagulopathy.4,5 Snake venom metalloproteinases (SVMPs) are members of the Reprolysin subfamily of the M12 family of metalloproteinases.6 Of the SVMPs, the P III class is distinguished by being comprised of pro-proteinase, proteinase, disintegrin-like, and cysteine-rich domains.7 Similar structures are found in the ADAMs, which contain an epidermal growth factor-like domain, a transmembrane region and a cytoplasmic tail,8 and in the r 2011 American Chemical Society

ADAMTSs, which contain thrombospondin type repeats.9 The proteinase domain of all of the SVMP hemorrhagic toxins is believed to function to degrade capillary basement membranes, endothelial cell surfaces, and stromal matrix ultimately causing extravasation of capillary contents into the surround stroma.10,11 Interestingly, the P III class of SVMPs is typically much more potent in causing hemorrhage compared with the P I and P II classes that lack the disintegrinlike/cysteine-rich domains found in the P III class,7 suggesting a role for these domains in the pathophysiology of the P III hemorrhagic toxins. In fact, the disintegrin-like/cysteine-rich domains of certain hemorrhagic toxins have been shown to be potent inhibitors of collagen-induced platelet aggregation as a result of their interaction with the α2β1 integrin on platelets.12 15 Proteolytic degradation of capillary basement membrane structures and inhibition of platelet aggregation have been considered to be the key features underlying the hemorrhagic potency of P III SVMP hemorrhagic toxins.11,14,15 Furthermore, in conjunction with the hemorrhagic effect, SVMPs Special Issue: Microbial and Plant Proteomics Received: July 8, 2011 Published: September 23, 2011 279

dx.doi.org/10.1021/pr2006439 | J. Proteome Res. 2012, 11, 279–291

Journal of Proteome Research are also capable of affecting hemostasis by degrading plasma proteins such as fibrinogen and von Willebrand factor.14 18 Over the past several years, our laboratories have reported the effects of SVMPs on tissues and cultured cells using techniques such as in vitro hydrolysis of extracellular matrix and plasma proteins, surface plasmon resonance, solid-phase binding assays, and gene expression analysis.10,11,17,19 22 These studies have shown that some P III SVMPs specifically interact with and cleave collagens I, VI, XII and XIV, matrilins 1, 3 and 4, and von Willebrand factor, which could lead to destabilization of extracellular matrix, disruption of cell matrix interactions and interference with the adhesion of platelets at the site of envenoming, and in the case of capillaries, it could contribute to their disruption and hemorrhage.17,20 23 These and other studies have provided considerable insight into the mechanisms associated with SVMP-induced pathogenesis; however, they also indicated that tissues are affected by SVMPs in a complex and heterogeneous fashion and that these alterations cannot be completely assessed by studying the action of SVMPs in isolated systems. From this perspective, the analysis of the action of a SVMP in the tissue or in plasma provides a general view of the pathologic events occurring but clearly misses the more subtle aspects of the heterogeneous tissue/plasma response to toxins. Therefore, for a better understanding of the pathogenesis of toxin-induced local tissue and systemic damage, one should consider experimental designs aimed at visualizing the spatial and temporal complexity of the response of cells and extracellular matrix. The proteomic/peptidomic analysis of skin and plasma is challenging due to the high complexity of the samples. The plasma, in addition to the classical “plasma proteins”, contains variable amounts of all tissue leakage products plus very numerous distinct immunoglobulin sequences, and it has an extraordinary dynamic range in that more than 10 orders of magnitude in concentration separate albumin and other rarest proteins.24 The skin is considered the largest organ of the human body and provides a physical and immunological barrier to potentially harmful environmental agents. The complexity of the skin proteome is illustrated by the presence of different types of cells and various types of components of the extracellular matrix.25 Snake venom toxins cause a large array of local and systemic pathological effects, most of which are detected at the site of venom injection and in plasma. Therefore, the use of systemic approaches, although difficult and challenging, is necessary to better understand the complexity of the local and systemic effects of venom toxins. To further explore the role of SVMPs in the venom-induced pathological alterations, we have focused on the application of proteomic approaches to elucidate the in vivo effects of HF3, an extremely hemorrhagic metalloproteinase from the venom of Bothrops jararaca.17,26,27 HF3 is a highly glycosylated P III SVMP that shows a minimum hemorrhagic dose of 240 fmol on the rabbit skin or 2.6 pmol on the mouse skin.26,28 Moreover, it hydrolyses fibrinogen, fibronectin, vitronectin, von Willebrand factor, collagens IV and VI, laminin and Matrigel in vitro17 and promotes leukocyte rolling in the microcirculation at a concentration of 1.6 nM.29 In this study, we used for the first time proteomic/peptidomic technologies to analyze the effects of HF3 on the mouse skin and plasma, and our results revealed new extracellular, intracellular and plasma target proteins for HF3 as well as the increase of plasma inhibitors. The data suggest that direct and indirect effects promoted by the proteolytic activity of HF3 result in tissue injury and disruption of hemostasis.

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’ EXPERIMENTAL SECTION HF3

HF326,27 was obtained as described elsewhere.28 Analysis of the in vivo Effects of HF3 on the Mouse Skin

Swiss mice were housed in temperature-controlled rooms and received water and food ad libitum. These studies were approved by the Experimental Animals Committee of Butantan Institute according to procedures laid down by the Universities Federation for Animal Welfare. Male swiss mice (n = 3) weighing 18 22 g were injected intradermally on the dorsal region with 100 μL of a control solution (0.15 M NaCl) or with 100 μL of a solution containing 1 μg of HF3 in 0.15 M NaCl. After 2 h, the dorsal skin was sectioned and homogenized with liquid nitrogen using mortar and pestle. Skin proteins (∼100 mg) from each of the three mice were separately resuspended with 1 mL of extraction buffer (7 M urea, 2 M thiourea, 4% (w/v) CHAPS, 30 mM Tris-HCl, pH 8.5) and incubated at room temperature for 30 min. After centrifugation at 12 000 g for 10 min at 4 °C, the solubilized protein solution was quantified using the Bradford reagent (BioRad) and bovine serum albumin (Sigma) as a standard, resulting in 7 9 mg/mL protein depending on the sample. Then the protein samples from the three mice were pooled for the next analyses. The experiment was carried out three times (three biological replicates) using three mice (injected with 0.15 M NaCl or with HF3) per experiment and each sample (composed of the pooled skin proteins from three mice) was submitted to 2-DE or direct digestion with trypsin. 2-DE and Image Analysis

Prior to the first dimension, precast IPG strips (18 cm, linear pH 3 10 gradient, GE Healthcare) were rehydrated with 400 μL of IEF solution (8 M urea, 4% (w/v) CHAPS, 70 mM DTT, 0.8% (v/v) pH 3 10 ampholytes (GE Healthcare) and 0.006% bromophenol blue) containing 400 μg of protein for 12 h at 25 °C. The first dimension was carried out in an Ettan IPGphor Isoeletric Focusing System (GE Healthcare) at 20 °C using the following program: 30 V for 6 h, 150 V for 2 h, 350 V for 1 h, 500 V for 1 h, 1000 V for 1 h, 3000 V for 1 h and 5000 V for 13 h. Prior to running the second dimension, the IPG strips were placed in the rehydration tray and the proteins in the strip were reduced and alkylated by sequential incubation in the following solutions: 0.05 M Tris-HCl, pH 8.4, 2% SDS; 30% glycerol, 6 M urea, 0.006% bromophenol blue (equilibration buffer-EB), 20 mg/mL DTT in EB; and then a solution of 30 mg/mL iodoacetamide in EB. Then the strips were directly applied on 8 18% SDSpolyacrylamide gels for second-dimension electrophoresis at 200 V in an Ettan DALTsix electrophoresis unit. The gels were fixed and then stained with silver.30 Twelve 2-DE runs were carried out including control samples and HF3-treated samples (duplicate runs for each sample of three in vivo experiments). The pairs of gels (control skin and HF3-treated skin) were stained in parallel and under the same conditions to avoid differential silver staining signal for a given protein. Gels were imaged using ImageScanner (GE Healthcare) and analyzed using the ImageMaster 2-D platinum software version 6.0 (GE Healthcare). The spots were quantified using the % of spot volume criterion, which is automatically calculated by the ImageMaster software. The match analysis was performed in an automatic mode, and further manual editing was performed to correct the mismatched and unmatched spots. Spots present in all gels with a fold change of 1.5 were considered with differential abundance and a criterion of

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Journal of Proteome Research

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centrifuged at 12 000 g for 10 min at 4 °C. The supernatant was submitted to protein quantification using the Bradford reagent (BioRad) and bovine serum albumin (Sigma) as a standard. One milligram of protein of control skin and HF3-treated skin was precipitated with acetonitrile (1:3, v/v) for 1 h at 20 °C32 and after centrifugation at 12 000 g for 10 min at 4 °C the supernatant was lyophilized, resuspended in 40 μL of 0.1% formic acid and analyzed by LC MS/MS using an ESI Q-Tof mass spectrometer. An aliquot (4.5 μL) of the peptide mixture was separated by C18 (75 μm 100 mm) RP-nanoUPLC (nanoAcquity, Waters) coupled with a Q-Tof Ultima mass spectrometer (Micromass) with nanoelectrospray source at a flow rate of 600 nL/min. The gradient was 2 90% acetonitrile in 0.1% formic acid over 45 min. The instrument was operated in the “top three” mode, in which one MS spectrum is acquired followed by MS/MS of the top three most-intense peaks detected. The spectra were acquired using software MassLynx v.4.1 and the raw data files were converted to a peak list format (mgf) by the software Mascot Distiller v.2.2.1.0, 2008 (Matrix Science Ltd.) and searched against International Protein Index mouse protein database (release date April 10, 2007) database restricted to Mus musculus (53 847 entries) using Mascot search engine (Matrix Science Ltd.). The search parameters were: no enzyme restriction, oxidation of methionine as variable modification and a tolerance of 0.1 Da for both precursor and fragment ions. Only peptides with a minimum of five amino acid residues which showed significant threshold (p < 0.05) in Mascot-based score were considered as a product of peptide cleavage. The peptidomic analysis was carried out with a mixture of peptides obtained from three separate experiments (biological replicates) using three mice (injected with 0.15 M NaCl or with HF3) per experiment.

p < 0.05 was used to define the significant difference when analyzing the paired spots between the control group and treated group (n = 6) according to Student’s t test. Tryptic Digestion and Mass Spectrometric Analysis

2-DE Spots. Protein spots were excised and in-gel trypsin digestion was performed according to Hanna et al.31 An aliquot (7.5 μL) of the resulting peptide mixture was separated by RPHPLC using a C18 (8 cm 75 μm) (Phenomenex) column coupled with nanoelectrospray tandem mass spectrometry on a Thermo Electron LTQXL ion-trap mass spectrometer at a flow rate of 500 nL/min. The gradient was 0 80% acetonitrile in 0.1 M acetic acid over 30 min. The instrument was operated in the “top ten” mode, in which one MS spectrum is acquired followed by MS/MS of the top ten most-intense peaks detected. Full dynamic exclusion was used to enhance dynamic range one spectrum before exclusion for 120 s. The resulting fragment spectra were searched using SEQUEST (Bioworks v3.3.1) against the International Protein Index (release date April 10, 2007) database restricted to Mus musculus (53 847 entries), with a parent tolerance of 2.0 Da and fragment tolerance of 1.0 Da and one trypsin missed cleavage. Iodoacetamide derivative of cysteine and oxidation of methionine were specified as fixed and variable modifications, respectively. The output of the search was loaded into Scaffold (v1_17_00) and initially filtered using xcorr cutoffs (+1 > 1.8, +2 > 2.5 and +3 > 3.5). A peptide was considered as unique when it differed in at least 1 amino acid residue; modified peptides, including N- or C-terminal elongation (i.e., missed cleavages), were also considered as unique while different charge states of the same peptide and modifications were not counted as unique. Whole Extract of Skin Proteins. Skin proteins (50 μg) were directly reduced, with DTT (final concentration 10 mM) and alkylated with iodoacetamide (final concentration 100 mM) for 30 min at room temperature and digested with trypsin (20 ng/μL). The resulting peptides were desalted by loading onto a capillary column (5 cm 150 μm) packed with Oligo R3 packing material (PerSeptive Byosystems) and rinsing with 1% acetic acid. Peptides were eluted with 80% acetonitrile/1% acetic acid into tubes and subsequently dried using a SpeedVac concentrator. For detergent removal, the dried peptide mixture was loaded into a capillary column (5 cm 150 μm) packed with Partisil 10 SCX (Whatman) packing material. After rinsing using 1% acetic acid, one step elution with 1 M ammonium acetate, pH 9.0, was performed and the eluted peptides were dried in a SpeedVac concentrator and resuspended in 1% acetic acid. An aliquot (5 μg) of the resulting peptide mixture was separated by C18 (8 cm 75 μm) (Phenomenex) RP-HPLC coupled with nanoelectrospray tandem mass spectrometry on a Thermo Electron LTQXL ion-trap mass spectrometer at a flow rate of 500 nL/ min. The gradient was 0 80% acetonitrile in 0.1 M acetic acid over 120 min. Samples from two biological replicates (control skin proteins and HF3-treated skin proteins) were submitted to one LC MS/MS analysis. The resulting fragment spectra were searched as described above for 2-DE spots.

Zymography of Mouse Skin Proteins

Proteins from the control skin and the HF3-treated skin (30 μg), extracted as described above, were solubilized in 50 mM TrisHCl, pH 8.0, and zymography was performed as described before33 using 12% SDS-polyacrylamide gels copolymerized with 1 mg/mL gelatin (Sigma), 1 mg/mL collagen I (B-D Biosciences), 100 μg/mL collagen IV (B-D Biosciences) or 250 μg/mL collagen VI (Sigma). The gels were stained with Coomassie blue and destained. Gelatin digestion was identified as clear zones of lysis against a blue background. Analysis of the in vivo Effects of HF3 on the Mouse Plasma

The experiment was carried out once using Swiss mice (n = 3) weighing 18 22 g which were injected in the thigh muscle with 100 μL of a control solution (0.15 M NaCl) or with 100 μL of a solution containing 5 μg of HF3 in 0.15 M NaCl. After 4 h, blood was collected from the mice by cardiac puncture and the plasma was submitted to depletion of the 20 most proteins using the Prot20 ProteoPrep 20 plasma immunodepletion kit (Sigma) according to manufacturers instructions. Briefly, samples of 8 μL of control or HF3-treated plasma (n = 3) were separately loaded to the column that had been previously equilibrated with phosphate-buffered saline, pH 7.2. The nonbound protein fraction was recovered and submitted to precipitation with acetone (1:3, v/v) for 1 h at 20 °C. After centrifugation at 12 000 g for 10 min at 4 °C proteins were resuspended with 100 mM ammonium bicarbonate, submitted to trypsin digestion as described above and desalinized using the column OASIS HLB (Waters). Tryptic fragments were fractionated by cation exchange (SCX) chromatography using a PolySulfethyl A column

Analysis of the Mouse Skin Peptide Fraction

Male swiss mice (n = 3) weighing 18 22 g were injected intradermally on the dorsal region as described above for the analysis of the in vivo effects of HF3 on the mouse skin. After 2 h, the dorsal skin was sectioned and homogenized with liquid nitrogen using mortar and pestle. The pooled skin mixture from the three mice was solubilized with 50 mM Tris-HCl, pH 8.0, and 281



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Figure 1. Experimental design for the analysis of the effects of HF3 on the mouse skin and plasma. (Left) One-hundred microliters of 0.15 M NaCl (control) or 1 μg of HF3 in 100 μL of 0.15 M NaCl was injected intradermically in the dorsal region of Swiss mice; the skin was removed after 2 h and processed for proteomic/peptidomic analysis and zymography. (Right) One-hundred microliters of 0.15 M NaCl (control) or 5 μg of HF3 in 100 μL of 0.15 M NaCl was injected in the thigh muscle of Swiss mice; the blood was collected after 2 h and processed for plasma proteomics analysis.

(PolyLC Inc.) coupled to a HPLC Waters 2796 Bioseparations Module and Waters 2996 Photodiode Array Detector. The following gradient was employed for peptide elution at 0.5 mL/min: buffer A (5 mM NH4Cl in 25% acetonitrile, pH 3.0) for 30 min, 0 60% buffer B (800 mM NH4Cl in 25% acetonitrile, pH 3.0) in 60 min, 5 min at 60% buffer B, 60 100% buffer B in 20 min, 100% buffer for 30 min. Eluting peptides were monitored at 214 nm. Forty fractions of 3.0 mL were collected from the SCX separation, and lyophilized. Fractions were combined in 10 pools and desalted using Sep-Pak (Sep-Pak Light tC18) cartridges (Waters). The samples were resuspended in 50 μL of 0.1% formic acid and an aliquot of 4.5 μL was analyzed by LC MS/MS using an ESI Q-Tof mass spectrometer as described above.

after 2 h the hemorrhagic skin was removed and processed for proteomic/peptidomic analysis and zymography. 2-DE and LC MS/MS Analysis. Skin proteins were initially submitted to 2-DE using 18 cm IPG strips (pH 3 10) and 8 18% SDS-polyacrylamide gels. A total of 12 2-DE runs were carried out including duplicate runs for each control and HF3treated sample of three in vivo experiments. Figure 2 shows the 2-DE profiles of the control and HF3-treated skin proteins. The image analysis using Image Master 2D Platinum detected 687.0 ( 95.4 spots in the control skin and 654.8 ( 57.7 spots in the HF3-treated skin reference gels from which 481 spots were paired. Spots present in all gels with a fold change >1.5 were considered with differential abundance. Twenty-six protein spots were statistically different among the twelve gels (p < 0.05) and were submitted to in gel trypsin digestion and LC-MS/MS analysis. From the mass spectral analysis of the 2-DE spots of the HF3-treated skin, 26 proteins were identified with XCorr scores greater than 1.8, 2.25, and 2.50 for +1, +2, and +3 ions, respectively, and protein identification probability above 95%. The identified proteins were sorted into three categories: cellular, extracellular and plasma proteins (Table 1). Spots containing proteins of the three categories were found as significantly less abundant or absent in the hemorrhagic skin. The proteins identified in these spots were: actin, annexin A5, apobec2, chloride intra cellular channel protein 1, creatine kinase, galactose binding lectin, myosin, mouse protein 40 kDa, thioredoxin, collagens I, III and VI, and alpha-1-antitrypsin. Most of the identified proteins showed experimental values of molecular mass on the 2D gels similar to the theoretical ones, indicating that these proteins were in part degraded and their abundance decreased in the hemorrhagic skin, however, the hydrolysis products were likely small fragments that were not detected on the gels. Interestingly, the proteins actin, creatine kinase and

Validation of in vivo Protein Targets of HF3 by their Incubation with HF3 in vitro

Annexin V, actin, vimentin, albumin and decorin (Sigma) were incubated at a 1:10 (w/w) enzyme-to-substrate ratio with HF3 in 0.020 M Tris-HCl, pH 8.0, 0.5 mM CaCl2 for 2 h at 37 °C. Decorin was incubated with HF3 as described above except that the enzyme-to-substrate ratio was 1:20 (w/w). A sample of each protein was incubated without enzyme under identical conditions. Reactions were stopped by adding Laemmli sample buffer prior to incubation for 5 min at 95 °C and electrophoresis on 12% or 4 15% SDS-polyacrylamide gels.34 Gels were silver stained.

’ RESULTS Analysis of the in vivo Effects of HF3 on the Mouse Skin

To explore the in vivo effects of HF3 on the mouse skin we used the experimental design depicted in Figure 1. Mice were injected in the dorsal area with 1 μg of HF3 (or 16 pmoles) and 282



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more spots, and as a consequence, changes in spot volume could be below the selected threshold of 1.5-fold. The 2-DE approach should therefore be used in combination with other approaches to address some of these concerns. In this study, the complex mixture of skin proteins was also analyzed using a label-free quantitative proteomics approach. The presence of several hundred proteins in the skin samples gives rise to thousands of peptides after digestion with trypsin. We evaluated the major skin proteins affected in the hemorrhagic process by examining the difference in protein abundance between control and HF3treated samples taking into account the number of unique peptides and the number of spectral counts for each protein (Table 2). The proteins of the HF3-treated skin that showed a lower number of unique peptides and spectral counts compared to the control skin were considered as decreased in abundance due to the in vivo effects of HF3. Interestingly, these results confirmed the 2-DE/LC MS/MS findings, among which, many proteins were found decreased by the effect of HF3, such as actin, creatine kinase, tropomyosin, vimentin, collagen I, decorin, lumican and mimecan. The plasma proteins alpha-2-macroglobulin, hemoglogin, hemopexin and serotransferrin were notably more abundant in the HF3-treated skin while albumin remained unchanged. Peptidomic Analysis of the Skin. To gain further insight into the molecular changes induced by HF3 in the mouse skin we next examined the skin peptide fraction by LC-MS/MS in order to find hydrolysis products resulting from the proteolysis occurring in the hemorrhagic process. Supplemental Tables 1 and 2 (Supporting Information) show the complete lists of peptides detected in the control and HF3-treated skin samples, respectively. The peptide lists contain a similar number of total queries identified in the control skin (1366) and HF3-treated skin (1236), and the individual analysis of proteins from which these peptides were derived showed that a significantly higher number of peptides from hemoglobin, fibrinogen and apolipoprotein A-II were detected in the hemorrhagic skin, indicating that these proteins were cleaved in vivo (Table 3). Although in a low number, peptides from calcium-binding protein and eukaryotic translation initiation factor 4H were also detected in the HF3treated skin peptide fraction. Interestingly, a high number of peptides from AHNAK nucleoprotein isoform 1, desmoyokin, elongation factor 1-alpha 1, filaggrin and high mobility group protein B1 were detected in the control skin but were absent in the HF3-treated skin, indicating that these peptides were further degraded in the hemorrhagic process caused by HF3 (Table 3). Validation of HF3 Targets in the Mouse Skin. The distinct differences observed in the proteome composition between samples are consistent with the different macroscopic aspect of the mouse control and hemorrhagic skins; thus, we further investigated the newly identified proteins as candidates for in vivo substrates of HF3. For this analysis we selected some proteins that are commercially available and that were detected as decreased in the hemorrhagic skin. The proteins were incubated at a 1:10 (w/w) enzyme-to-substrate ratio with HF3 for 2 h and submitted to SDS-PAGE. Under these conditions, actin, vimentin and decorin were degraded by HF3, while annexin V remained intact (Figure 4). The albumin level did not decrease in the hemorrhagic skin (Table 2) and, likewise, was not cleaved in vitro by HF3. Evaluation of the Activity of HF3-Treated Skin Proteinases by Zymography. We have previously shown that despite its extremely high hemorrhagic activity HF3 did not

Figure 2. Analysis of the effects of HF3 on the mouse skin by 2-DE. Skin proteins (400 μg) from (A) control and (B) HF3-treated mice were submitted to isoelectric focusing on 3 10 IPG strips (18 cm) followed by electrophoresis on 8 18% SDS-PAGE. Gels were silver-stained. Numbers on the right indicate molecular mass marker mobility. Spots indicated with arrows were identified by LC MS/MS as described in the Experimental Section.

myosin were identified in spots showing two or three different molecular masses; however, these masses were the same on both control and HF3-treated skin gels. These data suggest that these proteins may have undergone some degradation process upon sample preparation for 2-DE. On the other hand, elongation factor 2, muscle form of glycogen phosphorylase, T-complex protein 1, alpha-2-macroglobulin and Mu-Crystallin homologue appeared as significantly more abundant in the HF3-treated skin. Figure 3 summarizes the changes in abundance detected among the skin proteins as examined by 2-DE and LC MS/MS analysis. In Solution Trypsin Digestion and LC MS/MS Analysis of Whole Extract of Skin Proteins. It is important to point out that 2-DE is not an absolute separation technology in the sense that not all proteins are fully resolved from each other on the gel and that not all proteins are present on the gel. As a consequence, this is also likely to have led to an underestimation of the number of proteins affected by HF3 in the hemorrhagic skin. Specific concerns include the following: (1) nondetection of a relevant protein due to its pI being outside the experimentally chosen analytical pH window; (2) a differentially regulated protein not being detected because of comigration with proteins that are present at a much higher level; (3) failure of a protein to enter the gel; and (4) the protein may be represented by an array of two or 283



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Table 1. Identification of Differentially Expressed Proteins Indicated in Figure 2 by LC MS/MS number of unique

molecular mass

average of spot

peptides/spectral counts

observed on the 2D gel

volume ratio hemorrhagic skin/control skina

IPI00110827 IPI00110827 IPI00125150 IPI00125150 IPI00317309 IPI00130344 IPI00127596 IPI00127596.1 IPI00466069 gi|12805209 IPI00312700 IPI00224549

30/362 6/15 11/20 10/22 4/4 15/46 28/153 23/81 16/20 7/11 10/85 18/128

45250 12667 32166 32166 31916 31583 43000 35700 92090 12545 13500 18250

0.63 0.49 Only in the control skin Only in the control skin 0.32 0.46 0.64 0.33 1.51 0.43 0.40 0.46

muscle isoform 2481/1422: Myosin light chain 1, skeletal muscle isoform 521/593: Myosin A1 catalytic light chain, skeletal

IPI00312700 IPI00312700

14/60 8/11

24583 24666

0.54 0.50

muscle - mouse -/426: Mu-Crystallin homologue 283/339: Protein 40 kDa

IPI00120030 gi|226005

14/46 17/19

35667 44583

Only in the hemorrhagic skin 0.61

797/932: T-complex protein 1 subunit alpha A 622/-: Thioredoxin

prf||1405340A IPI00118678 IPI00226993

19/32 8/27

60272 10500

3.41 Only in the control skin

385/455: Col3a1 Collagen alpha-1(III) chain precursor 373/449: Col1a1 Isoform 1 of Collagen alpha-1(I)

IPI00129571 IPI00329872

11/32 12/38

33416 34000

0.39 0.52

chain precursor 238/261: Col6a3 Type VI collagen alpha 3 subunit

IPI00131114

4/5

53500

0.55

245/855: Alpha-1-antitrypsin 1 6 precursor 248/-: Spi1 6 Alpha-1-antitrypsin 1 6 precursor 346/410: Alpha-2-macroglobulin precursor 613/675: Transthyretin

IPI00117857 IPI00117857 IPI00624663 IPI00127560

11/15 9/15 11/38 10/12

50250 50833 37333 12750

0.10 Only in the control skin 2.62 2.38

spot number control skin/ hemorrhagic skin: protein name

accession number

257/337: Actin alpha skeletal muscle 2617/1518: Actin alpha skeletal muscle 414/-: Apobec2 Probable CfU-editing enzyme APOBEC-2 416/-: Apobec2 Probable CfU-editing enzyme APOBEC-2 2334/1305: Annexin A5 418/499: Chloride intracellular channel protein 1 299/386: Creatine kinase 337/413: Creatine kinase 81/100: Elongation factor 2 616/683: Lectin, galactose binding, soluble 1 609/672: Myosin light chain 1, skeletal muscle isoform 2562/1466: Myosin regulatory light chain 2, skeletal

Cellular proteins

Extracellular proteins

Plasma proteins

a

n = 6; the significance level was set at 5%.

degrade collagen I while it only discretely cleaved laminin and Matrigel in vitro.17 Therefore, the fact that collagen I appeared as a protein that decreased in the hemorrhagic skin suggests that collagen I might have been cleaved by HF3 or by a tissue collagenase. To evaluate this hypothesis we tested the effects of skin proteins upon gelatin, and three types of collagen. Interestingly, the total extract of proteins from the hemorrhagic skin showed significantly higher proteolytic activity upon gelatin and collagens I and VI at the molecular mass range above 80 kDa than the control skin. Although at a lower extent, proteins from the hemorrhagic skin also cleaved collagen IV (Figure 5). These data indicate that collagenases were generated in the HF3-treated skin and these may have a role in the hemorrhagic process.

for HF3 among the less abundant plasma proteins. For this purpose, mice were injected in the thigh muscle with 5 μg (or 80 pmoles) of HF3 and after 4 h the blood was collected and the plasma of control and HF3-treated mice were submitted to depletion of the 20 most abundant proteins (acid-1-glycoprotein, albumin, alpha-1-antitrypsin, alpha-2-macroglobulin, apolipoprotein A1, apolipoprotein A2, apolipoprotein B, ceruloplasmin, complement C1q, complement C3, complement C4, fibrinogen, haptoglobin, IgAs, IgDs, IgGs, IgMs, plasminogen, prealbumin and transferrin) by affinity chromatography followed by trypsin digestion of the nonbound plasma fraction and LC MS/MS analysis. Table 4 shows a list of selected proteins that were detected as differentially abundant in the plasma of control and HF3-treated mice as judged by the number of unique peptides and spectral counts. Fibronectin was detected as less abundant in the plasma of HF3-treated mice suggesting that it was cleaved by HF3. On the other hand, the proteins afamin, alpha1-B glycoprotein, coagulation factor II, complement factor B, hemopexin and pregnancy zone protein were found as more abundant in the plasma of HF3-treated mice (Table 4).

Analysis of the in vivo Effects of HF3 on the Mouse Plasma

We have previously shown that HF3 cleaves fibrinogen and von Willebrand factor in vitro, either in their isolated forms or in human plasma.17 Here we evaluated the ability of HF3 to cleave plasma proteins in vivo as an attempt to underscore new substrates 284



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Figure 3. Most differentially expressed proteins between the control and the HF3-treated mouse skin, as indicated in Table 1. Numbers on the left side indicate spot number control skin/hemorrhagic skin, as shown in Figure 2.

’ DISCUSSION The complexity of the in vivo effects of P III SVMPs is high due combination of their proteolytic activity promoted by the metalloproteinase domain, and the effects of the noncatalytic disintegrin-like and cysteine-rich domains, which play a critical role in the interaction of the metalloproteinase with specific cellular and extracellular targets.20 23 Therefore, the analysis of the effects of the hemorrhagic metalloproteinase HF3 in the mouse skin revealed a complex scenario of dramatic changes in abundance of various plasma, extracellular and cellular proteins as a result of its proteolytic activity along with signaling events promoted by its noncatalytic domains. In this study, we provide evidence that the hemorrhagic process promoted by HF3 in the skin involves multiple mechanisms and is characterized by the degradation of a number of extracellular matrix, plasma and cellular proteins with the concomitant increase of plasma proteinase inhibitors, carriers and chaperonins. Thus, due to the complexity of the events of the hemorrhagic process, we shall discuss only the main changes detected by the proteomic/ peptidomic analysis of the hemorrhagic skin which were summarized in Figure 6. Knowledge of SVMP effects was generally derived from in vitro biochemical analyses, with only a few studies assessing proteolytic cleavage in vivo.35,36 One of these studies included immunohistochemical analyses of tissues injected with SVMPs and showed the degradation of laminin, nidogen and type IV collagen, as well as of the endothelial cell marker VEGFR-2 in the hemorrhagic areas in the muscle.35 Gallagher and colleagues assessed the indirect effects of jararhagin, a hemorrhagic P III SVMP from B. jararaca, on host tissue local necrosis by the analysis of gene expression profiles of human fibroblasts in vitro and mouse tissue in vivo and found proteolysis as the primary mechanism affecting gene expression of cells and tissues resulting in a pro-inflammatory, pro-apoptotic host response.19 In the only

extensive analysis investigating the effects of snake toxins in vivo by proteomic approaches, wound exudates from muscular wounds in mice caused by BaP1, a P I metalloproteinase and Mtx-I, a phospholipase A2, were considered a “window” on the cellular processes associated with specific wound types.37 In the case of BaP1, the composition of the wound exudate showed a complex picture and there was clear evidence of degradation of nonfibrillar collagens (collagens VI, XIX, XII, XIV, XV, and XVI) and laminin.37 Our proteomic and peptidomic analyses of the mouse skin injected with HF3 revealed an extensive proteolysis process illustrated by various proteins that were detected as less abundant in the hemorrhagic skin. Among the extracellular proteins, collagens I, III and VI and the proteoglycans decorin, lumican and mimecan were clearly less abundant in the hemorrhagic skin. Collagens, in particular types I, III and VI, are important platelet activators in the vascular subendothelium and vessel wall. Moreover, collagen III is a fibrilar collagen often found in association with collagen I in the skin while collagen VI is a major structural component of microfibrils where it appears associated with collagen I. Since collagen I plays a major role in plateletaggregation and extracellular matrix stability, its degradation together with the associated collagens III and VI would lead to the weakening of the capillaries and eventually to hemorrhage. However, we recently showed that HF3 was able to cleave collagen VI but not collagen I in vitro.17 To check whether the cleavage of collagen I in the hemorrhagic process was due to the direct activity of HF3 we carried out zymography experiments that showed that the proteins extracted from the hemorrhagic skin have a clearly higher activity upon gelatin and collagens I, IV and VI than the control skin, indicating that the cleavage of collagen I in the HF3-induced hemorrhagic process may be the due to the action of HF3 or of the newly activated collagenases or both (Figure 5). Capillary vessels are known to be stabilized by 285



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Table 2. Identification of Proteins from the Skin Protein Extract by LC MS/MS number of unique peptides/spectral counts experiment 1 accession number

protein

control skin

experiment 2

hemorrhagic skin

control skin

hemorrhagic skin

Cellular proteins IPI00110827

Actin, alpha skeletal muscle

16/92

13/61

19/100

IPI00468481

ATP synthase subunit beta, mitochondrial

12/20

9/16

13/67

19/83 9/49

IPI00221890

Carbonic anhydrase 3

10/32

6/23

7/53

5/38

IPI00127596 IPI00123316

Creatine kinase M-type Tropomyosin-1 alpha chain

10/21 10/24

7/8 5/13

12/74 8/23

10/61 7/14

IPI00227299

Vimentin

10/32

8/23

9/36

11/26

IPI00329872

Collagen alpha-1(I) chain

7/50

4/35

12/79

10/55

IPI00330632

Collagen alpha-1(XIV) chain

5/18

5/13

6/30

3/10

IPI00222188

Collagen alpha-2(I) chain

12/35

6/19

11/28

12/25

IPI00123196

Decorin precursor

9/35

6/27

7/53

6/47

IPI00313900 IPI00120848

Lumican precursor Mimecan precursor

6/27 5/14

6/23 3/11

4/22 4/23

4/11 4/18

Extracellular proteins

Plasma proteins IPI00624663

Alpha-2-macroglobulin

0/0

14/27

3/8

13/55

IPI00316491

Hemoglobin subunit beta-2

6/27

6/43

6/28

11/71

IPI00128484

Hemopexin

7/25

10/49

5/14

5/20

IPI00139788

Serotransferrin

11/28

21/56

10/38

18/77

IPI00131830

Serpina3k Serine protease inhibitor A3K

11/97

IPI00131695

Serum albumin

5/12

5/15

9/59

25/148

26/196

44/758

48/1156

Table 3. Identification of Peptides with Differential Abundance in Skin by LC MS/MS skin number of unique peptides/spectral counts accession number

protein

control

HF3-treated mice

43/44 0

0 19/28

IPI00553798 IPI00869381

AHNAK nucleoprotein isoform 1 Apolipoprotein A-II

IPI00831055

Beta-globin

0

27/29

IPI00458924

Calcium-binding protein

0

3/4

IPI00605894

Desmoyokin

18/22

0

IPI00307837

Elongation factor 1-alpha 1

16/17

0

IPI00885793

Fibrinogen, alpha polypeptide isoform 1

0

8/16

IPI00467138

Filaggrin

33/47

0

IPI00621440 IPI00553333

Filaggrin Hemoglobin subunit beta-1

35/48 0

0 36/39

IPI00316491

Hemoglobin subunit beta-2

0

24/28

IPI00845802

Hemoglobin alpha, adult chain 2

0

114/165

IPI00420261

High mobility group protein B1

22/35

0

IPI00124742

Isoform long of eukaryotic translation initiation factor 4H

0

6/7

binding of the basal surface of vascular endothelial cells to the basement membrane. Therefore, specific degradation of collagen I, III and IV cause destruction of the basement membrane, breakdown of capillary vessels, and leakage of blood components. Although disorganization of the vascular integrity is likely to be the most important mechanism, HF3 induces hemorrhage through

several cooperative mechanisms. The cleavage of decorin, lumican and mimecan is an interesting new finding as the proteolysis of proteoglycans by SVMPs has not been reported in vivo. The only evidence of the cleavage of a proteoglycan by a SVMP was reported by Tortorella et al.,38 who showed that atrolysin C, a P I SVMP from the venom of Crotalus atrox, was able to cleave the core protein 286



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Figure 4. Proteolytic activity of HF3 upon isolated proteins. Annexin V, actin, vimentin and albumin were incubated at a 1:10 (w/w) enzyme-tosubstrate ratio with HF3 for 2 h. Decorin was incubated with HF3 at a 1:20 (w/w) enzyme-to-substrate ratio. (1) Protein incubated for 2 h. (2) Protein incubated for 2 h with HF3. The arrow indicates the mobility of HF3. Numbers on the left indicate molecular mass marker mobility. Gels were silver stained.

Figure 5. Zymography of skin proteins. Protein samples (30 μg) from control skin proteins (CSP) or hemorrhagic skin proteins (HSP), prepared under nonreducing conditions, were submitted to electrophoresis on 10 or 12% SDS-PAGE copolymerized with gelatin, collagen I, collagen IV and collagen VI. Gels were stained with Coomassie blue. Proteins with activity were identified as clear zones of lysis against a dark background. Numbers on the right indicate molecular mass marker mobility. White boxes indicate areas of higher proteolytic activity by the hemorrhagic skin proteins.

of aggrecan at the aggrecanase site in vitro.38 Proteoglycans are key interfibrillary components of the animal extracellular matrix. Their major biological function derives from the physicochemical characteristics of the glycosaminoglycan component of the molecule, which provides hydration and swelling pressure to the tissue enabling it to withstand compressional forces.39 Decorin, lumican and mimecan are members of the small leucine-rich proteoglycan (SLRP) family involved in collagen fibrilogenesis. In these bifunctional molecules, the protein moiety binds collagen fibrils and the charged hydrophilic glycosaminoglycans regulate interfibrillar spacings. As decorin can be degraded by MMP2 we tested the ability of HF3 to cleave decorin in vitro. The clear degradation of decorin by HF3 suggests that the lower abundance of this proteoglycan detected by the proteomic analysis is likely the result of the direct activity of HF3 in vivo (Figure 4). The hydrolysis of decorin, lumican, and mimecan in conjunction with the degradation of collagens I, III and VI in the hemorrhagic process seems to be a synergistic phenomenon that plays a role in the overall destabilization of extracellular matrix and cell-matrix interactions and in the case of capillaries it could contribute to their disruption and ultimately to hemorrhage.

Among the intracellular proteins that were detected as less abundant in the hemorrhagic skin is actin, one of the three major components of cytoskeleton, which is involved in various cellular functions such as muscle contraction, motility, cell division, cell signaling, establishment and maintenance of cell junctions and cell shape.40 Interestingly, a recent study that investigated the effect of another SVMP, jararhagin, on the gene expression profile of human fibroblasts in vitro and mouse tissue in vivo showed that the expression of actin was significantly downregulated by the presence of jararhagin.19 Myosins compose a large family of motor proteins that move along actin filaments, while hydrolyzing ATP. Various forms of myosin were detected as clearly degraded in the hemorrhagic process suggesting that skeletal muscle fibers have been affected by the proteolytic activity of HF3. Moreover, creatine kinase, another protein normally found in the cytosol of muscle fibers was found as less abundant in the hemorrhagic skin. Hemorrhagic SVMPs are in general not related to the disruption of the integrity of muscle cell plasma membrane, a pathological effect that is usually attributed to the venom myotoxins.41 In this study, HF3 probably did not affect actin, myosin and creatine kinase expression since the skin was evaluated only 2 h after the metalloproteinase injection. 287



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Table 4. Identification of Plasma Proteins after Depletion of the 20 Most Abundant Proteins by LC MS/MS

hemorrhagic SVMP atrolysin A, but remained intact and was found in the extracellular milieu.47 Taken together, these data suggest that annexin A5 has been released by HF3 from the cell and further cleaved by tissue collagenases. The only class of proteins that showed differential abundance in both directions in the hemorrhagic skin was the plasma proteins, of which some were detected as increased and others decreased. Interestingly, alpha-1-antitrypsin, a general serine proteinase inhibitor was found as nearly depleted in the HF3treated skin. Since alpha-1-antitrypsin shows antiapoptosis effects and can act as a negative inflammatory regulator,48 it can be hypothesized that its absence in the hemorrhagic skin would facilitate inflammation and apoptosis. Apoliprotein A-II, the second main constitutive component of HDL, was also found as clearly degraded in the hemorrhagic skin. Although the significance of this finding in the context of the hemorrhagic process is unclear, it could be speculated that the decrease of apoliprotein A-II in the skin microcirculation could play a role in the imbalance of the hemostatic function as it has been shown that apoliprotein A-II appears to inhibit factor X activation by preventing the appropriate association of tissue factor with factor VIIa.49 The presence of a high number of peptides from hemoglobin in the hemorrhagic skin suggests that an extensive process of hemolysis occurred exposing hemoglobin to proteolysis. SVMPs have not been described as capable of causing direct lysis of erythrocytes, therefore, the degradation of hemoglobin probably occurred by the indirect effect of HF3 upon released hemoglobin. Hemoglobin has been suggested to stimulate the secretion of uPA, MMP-2 and MMP-9 by synovial tissues, and to play a role in joint damage after intra-articular bleeding.50 In the hemorrhagic skin, the increase of hemoglobin peptides could play a role in the increased collagenolytic activity observed (Figure 5). Moreover, the broad spectrum of biological effects exhibited by endogenous hemoglobin-derived peptides suggests that the proteolytic degradation of hemoglobin may affect the peptidergic regulation of tissue homeostasis. The cleavage of fibrinogen in vivo likely occurred as a direct effect of HF3. Fibrinogen is degraded by various SVMPs in vitro.7 and we recently showed that HF3 can cleave isolated fibrinogen and fibrinogen in the human plasma,17 suggesting that the proteolytic activity of HF3 upon fibrinogen is not abolished by plasma inhibitors. However, the presence of fibrinogen peptides in the hemorrhagic skin could also be due to the activity of HF3 upon fibrin, which would accumulate at the damaged microvessel wall. Whichever the source of the high number of fibrinogen peptides observed in the hemorrhagic skin, the degradation of this crucial coagulation protein would have a direct impact in the hemostasis by causing delay in plasma coagulation and increase the escape of erythrocytes from damaged capillary vessels. As a counterbalance to the extensive degradation of proteins observed in the hemorrhagic skin, the levels of the proteinase inhibitor alpha-2-macroglobulin, serotransferrin, and the T-complex protein 1 clearly increased. The increase of alpha-2-macroglobulin level is particularly interesting in the sense that it shows a response from the skin to damage by proteolytic enzymes. Considering that alpha-2-macroglobulin is a poor inhibitor of P III SVMPs,51 the increase of this protein likely did not affect the activity of HF3. The higher abundance of iron carrier serotransferrin and of the chaperonin T-complex protein 1 in plasma does not offer an obvious interpretation; however, they could be the result of the inflammatory process induced by SVMPs.

number of unique peptides/ spectral counts accession

HF3-treated

number

protein

control plasma

plasma

IPI00130654 Afamin

1/1

11/20

IPI00129965 Alpha-1-B glycoprotein

5/6

10/15

IPI00114206 Coagulation factor II IPI00928163 Complement factor

2/2 6/7

4/5 8/12

9/10

6/6

IPI00877345 Hemopexin

13/46

16/56

IPI00624663 Pregnancy zone protein

23/57

27/81

B, isoform CRA_b IPI00113539 Fibronectin 1, isoform CRA_b

Instead, HF3 likely promoted cell lysis and subsequently cleaved these proteins as suggested by the fact that it cleaved actin in vitro (Figure 4). Other cytoskeleton proteins were affected in vivo by the presence of HF3, such as vimentin. Vimentin is the most widely distributed of all intermediate filament proteins and can be found in fibroblasts, leukocytes, and endothelial cells.42 The effect of HF3 on intermediate filaments might promote disruption of cell integrity and activate tissue metalloproteinases. Although it is reported that vimentin is secreted by macrophages in response to pro-inflammatory signaling pathways,43 it decreased in vivo in the hemorrhagic process caused by HF3 whereas it was cleaved in vitro by HF3 (Figure 4). Collectively, cytoskeleton proteins are involved in essential functions such as the maintenance of the cell membranes and some organelles in a fixed place within the cytoplasm, and their degradation could lead to a variety of cell responses such as loss of cell anchorage and apoptosis as previously reported in vitro using jararhagin.44 A great number of peptides of various lengths from AHNAK/ desmoyokin, filaggrin and high mobility group protein B1 were detected in the control skin which were absent in the hemorrhagic skin. AHNAK/desmoyokin is a ubiquitous protein expressed in a variety of cell types. In epithelial cells AHNAK/ desmoyokin is distributed mainly on the cell membranes, suggesting its role in cell cell adhesion. At the plasma membrane, AHNAK/desmoyokin interacts with the annexin 2/S100A10 complex and regulates cortical actin cytoskeleton organization and cell membrane cytoarchitecture.45 Filaggrins are filamentassociated proteins which bind to keratin fibers in epithelial cells, and high mobility group protein B1 is a multifunctional protein with roles in chromatin structure, transcriptional regulation, V(D)J recombination, and inflammation. The reason for the presence of peptides from these proteins in the control skin is unknown; however, a possible hypothesis is that they were in the cytosol of skin cells and were further degraded by HF3 or by the tissue collagenases. Among the altered proteins, annexin A5 appeared decreased in the hemorrhagic skin. This protein is proposed as a potent anticoagulant and exerts antithrombotic activity by binding to phosphatidylserine, inhibiting activation of serine proteases important in blood coagulation.46 However, annexin A5 was not cleaved in vitro by HF3 (Figure 4). This finding is in agreement with a previous study that showed that annexin A5 was released from fibroblasts in culture treated with the 288



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Figure 6. Selected proteins detected as differentially abundant by the proteomic/peptidomic analysis of the effects of HF3 in the mouse skin and plasma.

The analysis of the effects on the plasma proteins caused by the injection of HF3 in the mouse thigh showed rather less alterations compared to the effects of the injection in the skin; nevertheless, interestingly, the analysis of plasma depleted of the 20 most abundant proteins showed the degradation of fibronectin by HF3. Fibronectin was previously shown to be cleaved in vitro by HF317 and in the coagulation process, the incorporation of fibronectin into fibrin clots is thought to be important for the formation of a provisional matrix that promotes cell adhesion and migration during wound healing.52 Therefore, the degradation of plasma fibronectin contributes to the aggravation of the hemorrhagic process. Hemopexin was detected as more abundant in the HF3 treated plasma analysis confirming the finding of this protein being also more abundant in the analysis of skin proteins (Table 2). Hemopexin is the plasma protein with the highest binding affinity to heme and is induced during inflammation.53 Hence, its higher abundance in the plasma of HF3-treated mice is

likely a sign of the organism reaction to a pro-inflammatory agent. The increase of coagulation factor II (prothrombin) is interesting and can be interpreted as an attempt to overcome the hemorrhagic process by providing more prothrombin for the coagulation cascade. The complement factor B is part of the alternative pathway of complement activation and its increase in plasma can be seen as a compensation for the cleavage of complement component 3 also detected in plasma. Afamin is a member of the albumin gene family that was recently shown to be involved in binding/transport of proteins contributing to α-tocopherol homeostasis at the blood brain barrier.54 The significance of its higher abundance in the plasma of HF3-treated mice is not obvious unless we hypothesize that this protein is a more general plasma carrier. Alpha-1-B glycoprotein is an immunoglobulin superfamily member that has homologous forms in the opossum serum, which were shown to bind to and neutralize SVMPs.55 Therefore, the increase of this 289



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glycoprotein in plasma of mice treated with HF3 could likely be correlated to its ability of neutralizing metalloproteinases; however, this is a hypothesis that remains to be confirmed by other experiments. As an interesting outcome of this study, the fact that various plasma components considered as acute phase proteins have been detected as increased or decreased by the proteomic analyses (alpha-2-macroglobulin, prothrombin, fibrinogen, alpha-1-antitrypsin, complement factor B, hemopexin, serotransferrin and transthyretin) may be directly related to the overall proteolytic and pro-inflammatory properties of HF3. In summary, this study shows that the overall effects of HF3 in the mouse skin and in plasma likely promote a cascade of proteolytic events, possibly including shedding effects, specific substrate cleavage, activation of tissue collagenases and increase of plasma inhibitors. These events create a panel of several tissue networks triggering physiological and pathological responses and ultimately, disruption of hemostasis and generation of hemorrhage by destabilization of the microvasculature via disruption of cell cell and cell-extracellular matrix interactions. This is the first study to assess the complex in vivo effects of HF3 using highthroughput approaches, which was conducted as an attempt to understand the complex pattern of alterations caused by a potent hemorrhagic toxin from a systems biology perspective.

lethal toxicity, proteolytic activities and other pathological activities. Br. J. Exp. Pathol. 1960, 41, 478–486. (3) Bjarnason, J. B.; Fox, J. W. Hemorrhagic metalloproteinases from snake venoms. Pharmacol. Ther. 1994, 62, 325–372. (4) White, J. Snake venoms and coagulopathy. Toxicon 2005, 45, 951–967. (5) Gutierrez, J. M.; Rucavado, A.; Escalante, T.; Díaz, C. Hemorrhage induced by snake venom metalloproteinases: biochemical and biophysical mechanisms involved in microvessel damage. Toxicon 2005, 45, 997–1011. (6) Bjarnason, J. B.; Fox, J. W. Snake venom metalloendopeptidases: reprolysins. Methods Enzymol. 1995, 248, 345–368. (7) Fox, J. W.; Serrano, S. M. Insights into and speculations about snake venom metalloproteinase (SVMP) synthesis, folding and disulfide bond formation and their contribution to venom complexity. FEBS J. 2008, 275, 3016–3030. (8) White, J. M. ADAMs: modulators of cell-cell and cell-matrix interactions. Curr. Opin. Cell Biol. 2003, 15, 598–606. (9) Tang, B. L.; Hong, W. ADAMTS: a novel family of proteases with an ADAM protease domain and thrombospondin 1 repeats. FEBS Lett. 1999, 445, 223–225. (10) Baramova, E. N.; Shannon, J. D.; Bjarnason, J. B.; Fox, J. W. Degradation of extracellular matrix proteins by hemorrhagic metalloproteinases. Arch. Biochem. Biophys. 1989, 275, 63–71. (11) Shannon, J. D.; Baramova, E. N.; Bjarnason, J. B.; Fox, J. W. Amino acid sequence of a Crotalus atrox venom metalloproteinase which cleaves type IV collagen and gelatin. J. Biol. Chem. 1989, 264, 11575–11583. (12) Jia, L. G.; Wang, X. M.; Shannon, J. D.; Bjarnason, J. B.; Fox, J. W. Inhibition of platelet aggregation by the recombinant cysteine-rich domain of the hemorrhagic snake venom metalloproteinase, atrolysin A. Arch. Biochem. Biophys. 2000, 373, 281–286. (13) Zigrino, P.; Kamiguti, A. S.; Eble, J.; Drescher, C.; Nischt, R.; Fox, J. W.; Mauch, C. The reprolysin jararhagin, a snake venom metalloproteinase, functions as a fibrillar collagen agonist involved in fibroblast cell adhesion and signaling. J. Biol. Chem. 2002, 277, 40528–40535. (14) Kamiguti, A. S.; Gallagher, P.; Marcinkiewicz, C.; Theakston, R. D.; Zuzel, M.; Fox, J. W. Identification of sites in the cysteine-rich domain of the class P-III snake venom metalloproteinases responsible for inhibition of platelet function. FEBS Lett. 2003, 549, 129–134. (15) Kamiguti, A. S.; Hay, C. R.; Zuzel, M. Inhibition of collageninduced platelet aggregation as the result of cleavage of alpha 2 beta 1-integrin by the snake venom metalloproteinase jararhagin. Biochem. J. 1996, 320, 635–641. (16) Lu, Q.; Clemetson, J. M.; Clemetson, K. J. Snake venoms and hemostasis. J. Thromb. Haemost. 2005, 3, 1791–1799. (17) Oliveira, A. K.; Paes Leme, A. F.; Asega, A. F.; Camargo, A. C. M.; Fox, J. W.; Serrano, S. M. T. New insights into the structural elements involved in the skin hemorrhage induced by snake venom metalloproteinases. Thromb. Haemost. 2010, 104, 485–497. (18) Kamiguti, A. S.; Slupsky, J. R.; Zuzel, M.; Hay, C. R. Properties of fibrinogen cleaved by Jararhagin, a metalloproteinase from the venom of Bothrops jararaca. Thromb. Haemost. 1994, 72, 244–249. (19) Gallagher, P.; Bao, Y.; Serrano, S. M.; Laing, G. D.; Theakston, R. D.; Gutierrez, J. M.; Escalante, T.; Zigrino, P.; Moura-da-Silva, A. M.; Nischt, R.; Mauch, C.; Moskaluk, C.; Fox, J. W. Role of the snake venom toxin jararhagin in proinflammatory pathogenesis: in vitro and in vivo gene expression analysis of the effects of the toxin. Arch. Biochem. Biophys. 2005, 441, 1–15. (20) Serrano, S. M.; Jia, L. G.; Wang, D.; Shannon, J. D.; Fox, J. W. Function of the cysteine-rich domain of the haemorrhagic metalloproteinase atrolysin A: targeting adhesion proteins collagen I and von Willebrand factor. Biochem. J. 2005, 391, 69–76. (21) Serrano, S. M.; Kim, J.; Wang, D.; Dragulev, B.; Shannon, J. D.; Mann, H. H.; Veit, G.; Wagener, R.; Koch, M.; Fox, J. W. The cysteinerich domain of snake venom metalloproteinases is a ligand for von Willebrand factor A domains: role in substrate targeting. J. Biol. Chem. 2006, 281, 39746–39756. (22) Serrano, S. M.; Wang, D.; Shannon, J. D.; Pinto, A. F.; PolanowskaGrabowska, R. K.; Fox, J. W. Interaction of the cysteine-rich domain of

’ ASSOCIATED CONTENT

bS

Supporting Information Supplementary tables. This material is available free of charge via the Internet at http://pubs.acs.org.

’ AUTHOR INFORMATION Corresponding Author

*Solange M. T. Serrano, Laboratorio Especial de Toxinologia Aplicada, Instituto Butantan, Av. Vital Brasil, 1500, 05503-900, S~ao Paulo, Brazil. Tel./Fax: +55 11 3726-1024. E-mail: solangeserrano@ butantan.gov.br.

’ ACKNOWLEDGMENT This work was supported by grants from Fundac-~ao de Amparo a Pesquisa do Estado de S~ao Paulo (04/15974-1; 06/50059-8; 98/14307-9), and Rede de Proteoma de S~ao Paulo (FAPESP 2004/14846-0/FINEP 01.07.0290.00). ’ ABBREVIATIONS: ADAM, A disintegrin and metalloproteinase; ADAMTS, A disintegrin and metalloproteinase with thrombospondin motifs; EB, equilibration buffer; HDL, high-density lipoprotein; LTQ, linear quadrupole ion Trap; MMP, Matrix Metalloproteinase; RPUPLC, reverse phase-ultra performance liquid chromatography; SCX, strong cation exchange; SVMPs, snake venom metalloproteinases; VEGFR, vascular endothelial growth factor receptor. ’ REFERENCES (1) Ownby, C. L.; Bjarnason, J.; Tu, A. T. Hemorrhagic toxins from rattlesnake (Crotalus atrox) venom. Pathogenesis of hemorrhage induced by three purified toxins. Am. J. Pathol. 1978, 93, 201–218. (2) Ohsaka, A.; Ikezawa, H.; Kondo, H.; Kondo, S.; Uchida, N. Haemorrhagic activities of habu snake venom, and their relations to 290



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Hemorrhagic Activity of HF3, a Snake Venom Metalloproteinase

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