Aplicacion Farmacologica Venenos

Minor snake venom proteins: structure, function and potential applications
Johara Boldrini-França, Camila Takeno Cologna, Manuela Berto Pucca,

Karla de Castro Figueiredo Bordon, Fernanda Gobbi Amorim, Fernando
Antonio Pino Anjolette, Francielle Almeida Cordeiro, Gisele Adriano Wiezel,
Felipe Augusto Cerni, Ernesto Lopes Pinheiro-Junior, Priscila Yumi Tanaka
Shibao, Isabela Gobbo Ferreira, Isadora Sousa de Oliveira, Iara Aimê Cardoso,
Eliane Candiani Arantes
PII: S0304-4165(16)30516-5
DOI: doi:10.1016/j.bbagen.2016.12.022
Reference: BBAGEN 28722
To appear in: BBA - General Subjects
Received date: 20 August 2016

Revised date: 12 December 2016
Accepted date: 20 December 2016
Please cite this article as: Johara Boldrini-França, Camila Takeno Cologna, Manuela
Berto Pucca, Karla de Castro Figueiredo Bordon, Fernanda Gobbi Amorim, Fernando
Antonio Pino Anjolette, Francielle Almeida Cordeiro, Gisele Adriano Wiezel, Felipe Au-
gusto Cerni, Ernesto Lopes Pinheiro-Junior, Priscila Yumi Tanaka Shibao, Isabela Gobbo
Ferreira, Isadora Sousa de Oliveira, Iara Aimê Cardoso, Eliane Candiani Arantes, Mi-
nor snake venom proteins: structure, function and potential applications, BBA - General
Subjects (2016), doi:10.1016/j.bbagen.2016.12.022
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Minor snake venom proteins: structure, function and potential applications
Johara Boldrini-Françaa; Camila Takeno Colognaa; Manuela Berto Puccab; Karla de

Castro Figueiredo Bordona; Fernanda Gobbi Amorima; Fernando Antonio Pino
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Anjolettea; Francielle Almeida Cordeiroa; Gisele Adriano Wiezela; Felipe Augusto
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Cernia; Ernesto Lopes Pinheiro-Juniora; Priscila Yumi Tanaka Shibaoa; Isabela Gobbo
Ferreiraa; Isadora Sousa de Oliveiraa; Iara Aimê Cardosoa; Eliane Candiani Arantesa
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a
School of Pharmaceutical Sciences of Ribeirão preto, University of São Paulo, Ribeirão
Preto, Brazil;
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Medical School of Roraima, Federal University of Roraima, Boa Vista, Brazil.
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Address correspondence to: Eliane Candiani Arantes, Universidade de São Paulo,
Faculdade de Ciências Farmacêuticas de Ribeirão Preto, Departamento de Física e
Química. Av. do Café s/nº, Monte Alegre, 14040-903 - Ribeirão Preto, SP – Brazil,
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Phone: +55 16 33154275, Fax: +55 16 33154880, E-mail: ecabraga@fcfrp.usp.br

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E-mail adresses:
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Camila Takeno Cologna: camilatcbio@gmail.com;

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Eliane Candiani Arantes: ecabraga@fcfrp.usp.br;

Ernesto Lopes Pinheiro Júnior: ernesto.pinheiro@usp.br;
Felipe Augusto Cerni: felipe_cerni@hotmail.com;
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Fernanda Gobbi Amorim: fernandagamorim@gmail.com;

Fernando Antonio Pino Anjolette: fanjolette@yahoo.com.br;
Francielle Almeida Cordeiro: fran_acordeiro@hotmail.com;
Gisele Adriano Wiezel: gisele.wiezel@gmail.com;
Iara Aimê Cardoso: iara_cardoso@hotmail.com;
Isabela Gobbo Ferreira: igobboferreira@yahoo.com.br;
Isadora Sousa de Oliveira: isadora_so@yahoo.com;
Johara Boldrini França: joharafran@gmail.com;
Karla de Castro Figueiredo Bordon: karla@fcfrp.usp.br;
Manuela Berto Pucca: manupucca@hotmail.com;
Priscila Yumi Tanaka Shibao: priscila.shibao@gmail.com;
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Abstract
Snake venoms present a great diversity of pharmacologically active compounds

that may be applied as research and biotechnological tools, as well as in drug
development and diagnostic tests for certain diseases. The most abundant toxins have
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been extensively studied in the last decades and some of them have already been used
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for different purposes. Nevertheless, most of the minor snake venom protein classes
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remain poorly explored, even presenting potential application in diverse areas. The main
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difficulty in studying these proteins lies on the impossibility of obtaining sufficient
amounts of them for a comprehensive investigation. The advent of more sensitive
techniques in the last few years allowed the discovery of new venom components and
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the in-depth study of some already known minor proteins. This review summarizes
information regarding some structural and functional aspects of low abundant snake
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venom proteins classes, such as growth factors, hyaluronidases, cysteine-rich secretory
proteins, nucleases and nucleotidases, cobra venom factors, vespryns, protease
inhibitors, antimicrobial peptides, among others. Some potential applications of these
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molecules are discussed herein in order to encourage researchers to explore the full
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venom repertoire and to discover new molecules or applications for the already known
venom components.
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Keywords: snake venoms; toxins; drug discovery.

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1. Introduction
Snake venoms are complex mixtures of organic and inorganic compounds that
act on a variety of specific metabolic and physiologic targets of preys or victims,
assisting feeding and defense [1]. Protein composition of snake venoms may undergo
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pronounced qualitative and quantitative variation in all levels of taxa, as well as in
populations and individuals of the same species [2, 3]. Variation in protein expression
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of venom components may also be observed in the same specimen in different
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ontogenetic stages [4]. Such great diversification in proteins that compose the venom
directly reflects its toxicity and pathophysiologic effects, and may represent an
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evolutionary arms race, in which the venom mixture is adapted to improve the
predator’s ability of subduing different preys and to overcome the resistance of some
prey species to the venom [5, 6].
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Elapidae and Viperidae venom proteins are produced and secreted by oral
exocrine glands that present a basal-central lumen, where the produced venom is stored
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until the moment of its delivery [7]. The production of toxins is activated by
morphological and biochemical changes in secretory epithelial cells after venom
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injection or extraction [8-10]. However, synthesis and secretion of different protein

classes are not synchronized and may result in variation in venom composition in
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different stages of the venom production cycle [11].

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Many regulatory mechanisms are supposed to impact protein composition in

snake venoms, such as mutations affecting gene expression [12], duplication and loss of
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toxin-related genes [13], post-transcriptional microRNAs regulation [14] and proteolytic

processing [13]. Although presenting variable expression levels in different snake
venoms, the pathologically important toxins, such as phospholipases A2,
metalloproteases, kallikrein toxin families and three-finger toxins are usually the most
abundant proteins found in these biological samples. Some of these toxins compose up
to 80% of the total venom proteins, such as phospholipase A2 from Crotalus durissus
venom, which forms the crotoxin complex that is a neurotoxin responsible for the major
clinical symptoms of crotalic envenoming in South America [15]. The major snake
toxins are encoded by large multilocus gene families that exhibit substantial gene
duplication and directional selection and consequently the greatest variation [13].
On the other hand, the minor snake venom proteins are supposed to have
ancillary functions, less variability and experienced little to no gene duplication and
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positive selection. This contrast may be a result of the likely conservative functions and
the low relative abundance of ancillary proteins in the venoms, which precluded their
participation in the pray-predator arm’s race, resulting in a lack of selective pressures
[13, 16]. Nevertheless, although some minor snake venom protein classes are supposed
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to present secondary effects, their ecological relevance to the snake and their
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physiologic effects should be better explored. It is also worth to mention that most of
these proteins interact with specific targets in some physiological systems, which will
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be discussed thereafter. Accordingly, these features have aroused an interest in
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investigating possible medical and biotechnological applications for these families of
venom proteins.
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Another issue to be considered is that venom production has a substantial
metabolic cost [17] and a long regeneration cycle [9, 18], which indicates that the
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venom composition is optimized in order to maximize efficacy while metabolic
expenditure is minimized [19, 20]. Thus, the synthesis and secretion of this variety of
protein classes presenting merely ancillary and unessential functions seems
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contradictory considering that the maintenance of the venom repertoire is considerably

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metabolic expensive. Therefore, researchers should look into minor proteins classes in
the venom aiming to unveil possible functions they may play in the envenoming or in
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the physiology of the venom gland. Moreover, the expression of these proteins in
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nonvenomous tissues, the conservation degree of physiologic activities comparing with

their related orthologs and the structure and rate of duplication, mutations and
rearrangement of their encoding genes may provide relevant information regarding
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snake venom evolution and the generation of toxins diversity.

In spite of their possible great scientific value, some of these classes are poorly
explored mainly due to difficulties in purifying them from the venom for an in-depth
investigation [21]. In this context, this review will focus on some classes of proteins that
are known to be expressed in relatively low amounts in most snake venoms (less than
20% of the whole venom proteins, as showed in Table1), such as growth factors,
hyaluronidases, cysteine-rich secretory proteins, nucleases and nucleotidases, cobra
venom factors, vespryns, protease inhibitors, antimicrobial peptides, among others.
Moreover, this review also aims to discuss the importance of minor proteins to
toxinology and how the advent of innovative technological resources may enable the
assessment of their structural and functional properties and their role in the evolutionary
history of snake venoms. We also highlight that this review represents the first
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compilation of snake venom minor proteins and provides useful information of those
still neglected molecule that may hide outstanding potential applications.
2. Omic approaches in the identification of minor venom proteins
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Until a few years ago, comprehensive investigation of snake venoms
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composition was considered a time-costing and laborious work. Mainly due to the low
sensitivity of the applied methods, usually only the major components, such as
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proteases, neurotoxins and phospholipases A2, were studied in detail [22, 23]. The
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advances of proteomics, transcriptomic and genomic techniques, as well as the
improvement of their related instrumentation, have led to progress in knowledge
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regarding the complexity of snake venom composition and the evolutionary history of
those sophisticated lethal cocktails [24, 25]. Not long ago, the term “venomics” was
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used for the first time to describe the proteomic study of snake venom composition [26,
27]. However, the term has evolved for a more comprehensive meaning and nowadays
is also used to describe the studies which combine strategies such as genomics,
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transcriptomics and proteomics to unveil the full picture of venom components [25, 28].
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A genomic analysis of the genes encoding venom proteins may answer a number of
fundamental questions, especially regarding the evolutionary origin of those remarkable
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compounds and the genetic basis of generating toxin function and diversity [28, 29]. In
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defiance of this great value, there is still a lack of snakes’ full genomes in the literature
[29, 30]. The big size of snakes’ genomes which could turn the assembling of novel
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genomes an extreme difficult task could explain this deficiency [28]. In a recent study
regarding the loss of venom toxins genes in rattlesnakes, Dowell and colleagues [29]
stated that most investigations of snake venom diversity have been conducted without
genome sequences; besides their article, only other two groups reported full genomes
from snakes [30].
Following a different direction, transcriptomics, essentially the next generation
sequencing (NGS) transcriptomic approach, have allowed the investigation of the
venom repertoire in a broader way, revealing transcripts that are rarely expressed or
present in very low abundance and therefore poorly studied hitherto [24, 31].
Nevertheless, such strategy cannot always provide accurate quantitative data nor predict
the post translational modifications (PTM), which include the cleavage of the mature
region, disulfide bonds formation, modifications of the side chains and/or N- and C-
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terminal [25, 32, 33]. Glycosylation, as an example, is one of the most frequent
modifications found in snake venoms and play notable role in protein folding,
conformation, stability, pharmacodynamics, and biological activity. As for other types
of PTMs, glycosylation increase the complexity of venom proteomes and expand
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functions of its components [34], highlighting the relevance to elucidate those protein
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adornments. [33, 35]. In parallel and to fulfill those gaps, proteomics, supported by
advances of mass spectrometry, has been widely employed to explore animal venoms.
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This strategy can elucidate not only the molecular mass of the toxin, but also fragments
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of its sequence which can be used as tags for database searches, the identification of
post translational modifications and their localization within the sequence [36].
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Individually, proteomics and transcriptomics strategies have already contributed
massively to the investigation of snake venom diversity (Table 1). Notwithstanding, the
“venomics” strategy, which is the integration of the data coming from different cutting-
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edge technologies, will replace the classical approach, giving toxinologists the access
beyond the traditional and paving the way to explore bona fide novel compounds and
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minor venom proteins [37-39].

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Table 1 – Main minor protein classes in snake venoms.
Approaches Relative
Main biological Other relevant
Venom protein class used in the abundance in References
activities characteristics
identification snake venoms (%)*
Act in synergism with [40-42]

Hydrolysis of 5'- ADPases,
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nucleotides to phospholipases, and
5’-Nucleotidases T, P and T.A. <0,1 - 4,8 nucleosides, leading to disintegrins,
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platelet aggregation potentiating the
inhibition anticoagulant effect in
the envenoming
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Cathelicidin-derived and
cathelicidin-related T -- Antimicrobial activity -- [43-54]
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peptides
Cobra venom factors Mainly found in [55-59]

P, T And T.A. 0.1 – 2.8 Complement depletion
(CVFs) Elapidae snakes
Inhibition of tumor
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Cystatins T, P and T.A. 1,7 Protease inhibition [60-62]
cells metastasis
Presence of sixteen
cysteine residues
Cysteine-rich secretory
P, T and TA 0,1 - 15,9 Blockage of ion channels highly conserved [63-67]
proteins (CRISPs)
which form eight
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disulfide bonds
Act as a “spreading
Degradation of factor” in the
Hyaluronidases P, T and TA 0.1 – 1.9 hyaluronan in envenoming and [68-70]
extracellular matrix potentiate the effects
of other toxins
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Kazal-type inhibitors P, T 8,3 Protease inhibition -- [71, 72]

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Diversity of biological
effects, including
P, T and blockage of ion
Kunitz-type inhibitors 0,3 - 12,6** Protease inhibition channels, disturbances [73-82]
TA
P
in blood coagulation,
fibrinolysis and
inflammation
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Increases the vascular

Neuronal differentiation, permeability and
synaptic plasticity, and facilitates the diffusion
Nerve growth factors
P, T and TA 0.1 – 5 neuroprotection in the of other toxins in [25, 83, 84]
(NGFs)
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peripheral and central envenoming. May act

nervous systems. as a proapoptotic
factor
Hypotension, locomotor
Phosphodiesterases (PDEs) P, T and T.A. 0.1 – 3,2 depression and platelet -- [85-88]
aggregation
Cleavage of membrane Hydrolysis of

Phospholipases B (PLB) P, T and TA <0.1 – 1,4 phospholipids and phospholipids at sn-1 [89-91]
hemolytic activity and sn-2 positions
Proliferation of vascular
Vascular endothelial
P, T and TA <0.1 – 3,2 endothelial cells and -- [92-94]
growth factors (VEGFs)
hypotension
Hypolocomotion, Ohanin presents a

Vespryns T, P and TA 0.2 –14,4 [95]
hypernociception single cysteine residue
Structural similarity to
Waprins T and TA -- Antimicrobial activity whey acidic protein [96-98]
(WAP)
T=transcriptome; P = Proteome; T.A. = traditional approach (activity-guided assays)

*Relative abundance determined by proteomic approach
**Exception of Dendroaspis polylepis venom that is composed by 61.1% of Kunitz-
type inhibitor.
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3. Minor venom protein classes
3.1 Growth factors
The designation "growth factor" was historically associated with growth and cell
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proliferation. Later, other cellular responses were attributed to these neurotophins, such
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as cell differentiation, transformation, synthesis, secretions, death and motility [99]. The
first identified growth factor was a nerve growth factor (NGF) from mouse sarcoma 180
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in the 1950s [100, 101]. Snake venom NGF (sv-NGF) was firstly isolated in 1956 from
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the venom of Agkistrodon piscivorus [102]. Some years later, epidermal growth factor
(EGF) from the submaxillary gland of mouse and platelet-derived growth factor
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(PDGF) from monkey blood were discovered [103, 104]. Since then, over 200 growth
factors have been isolated and studied [99].
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Among the several known families of growth factors, the following have been
identified in snakes and are deposited in UniProt (Universal Protein Resource
Knowledgebase) databank: latent-transforming growth factor beta-binding protein
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(LTBP), transforming growth factor beta-2 (TGF-β), hepatocyte growth factor-regulated

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tyrosine kinase (HGF), hepatoma-derived growth factor (HDGF), delta/notch-like

epidermal growth factor, connective tissue growth factor, insulin-like growth factor-
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binding protein 5 (IGF), epidermal growth factor (EGF-like protein), brain-derived

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neurotrophic factor (BDNF), interferon (IF), platelet-derived growth factor (PDGF),

tumor necrosis factor (TNF), snake venom vascular endothelial growth factor (sv-
VEGF) and nerve growth factor (NGF). However, this review will focus on VEGF and
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NGF, which are the most studied snake venom growth factors.
3.1.1 Nerve growth factors (NGFs)
NGFs participate in neuronal differentiation, synaptic plasticity, and

neuroprotection in the peripheral and central nervous systems [83]. NGF acts also on no
neuronal cells, especially on hematopoietic stem cells. However, most of these effects
were reported for murine NGF [105].
The physiological responses to sv-NGF have not been elucidated yet [105].
However, the effects of mammalian NGF on non-neuronal cells might assist the
comprehension of some effects exerted by sv-NGFs on mammalian systems [106]. It
has been described that part of the sv-NGF injected at the bite site may reach the
circulation, leading to some physiological activities on non-neuronal cells or tissues. Sv-
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NGF from Naja atra (formerly N. naja atra) exerts important systemic effects in the
envenoming, such as plasma extravasation and histamine release, which result in tissue
vulnerability and facilitate toxin diffusion in the prey organism [105]. In contrast, the
sv-NGF isolated from Naja naja has shown no toxic effects [107].
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In regards to the presence of PTMs, some sv-NGFs were confirmed to have an
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Asn23 glyscosilation site (NXS/T). After treatment with PNGase F, the sv-NGFs from
Acanthophis antarcticus, Anilios nigrescens (formerly Ramphotyphlops nigrescens) and
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Oxyuranus scutellatus were confirmed to be N-glycosylated proteins [108]. Notably,
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this glycosilation site is missing in Notechis scutatus, Pseudechis australis and
Pseudechis porphyriacus [108]. Although the sv-NGFs from the snakes Hoplocephalus
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stephensii [108] and Naja sputatrix [109] have shown a N-linked glycosylation site, it
migrate as a non-glycosylated protein. The functional relevance of these differences in
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PTMs for sv-NGF demands further assays.
Sv-NGF has low abundance in snake venoms, corresponding to 0.1%-0.5%
(w/w) of crude venoms of Daboia russelii (formerly Vipera russelli) [111] and of
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snakes from Oxyuranus [106, 108], Naja [84, 112]and Sistrurus [113] genus. The
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relative abundance of ESTs coding sv-NGF comprised only 0.1% of the total transcripts
from Lachesis muta venom gland [92].
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The cDNA encoding sv-NGF exhibits a signal peptide, a pre-prodomain and the
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mature protein [109]. The precursor contains a presumptive 18 amino acid signal
sequence, which is followed by a propetide (proNGF) of approximately 109 amino acid
residues [114]. The prodomain is implicated to the correct folding of mature NGF
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[106]. The first cDNA of a sv-NGF was reported in 2002 [116] from Bothrops
jararacussu venomous gland. The molecular model revealed that this mature sv-NGF
(containing 118 amino acid residues) is formed by a pair of β-sheets, three β-hairpin
loops, a reverse turn and a short α- helix.
Concerning the functional properties and potential applications of sv-NGFs, the
injection of cobra venom NGF (cvNGF) was capable of slow down the growth of
Ehrlich ascites carcinoma (EAC) cells in mice, probably via an indirect mechanism in
which tyrosine kinase A (TrkA) receptors are involved. On the other hand, cvNGF
showed proliferative activity and had no cytotoxic effect on breast cancer cell line
MCF-7 [110].
Sv-NGF from N. sputatrix upregulates endogenous expression of NGF in PC12
cells, pro-survival cell surface receptors and ion channels [109]. The neurite
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differentiation activity evidenced with sv-NGF from N. sputatrix [109] and O.

scutellatus [108] was similar to that of mammalian homolog. These results may lead to
further studies on sv-NGFs as potential therapeutic agents against neuronal injury.
3.1.2 Vascular endothelial growth factor (VEGF)
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Vascular endothelial growth factor (VEGF), formerly designated as vascular
permeability factor, stimulates vasculogenesis, angiogenesis or lymphangiogenesis. The
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VEGF family is divided into seven groups, designated as VEGF-A to VEGF-F and
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placental growth factor (PGF) [93]. Snake venom glands present at least three different
VEGF-Fs with unique features and with distinct receptor-selectivity, designated as
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VEGF-F1 to VEGF-F3. Additionally, VEGF-A-like transcripts have also been
identified in some snake venoms [117]. VEGF-A is a cytokine secreted by tumor cells
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that play important role in normal and tumor-related angiogenesis.
Up to now, 26 expressed sequence tags (ESTs) for sv-VEGF were identified by
transcriptomic approach in the venom glands of Bothropoides insularis (formerly
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Bothrops insularis) (19), Bothropoides pauloensis (former Bothrops pauloensis) (1),

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Bothrops atrox (former Bothrops colombiensis) (3), Crotalus durissus collilineatus (2)
and L. muta (1) snakes [92, 118-120]. The sv-VEGFs corresponded to 0.1% of the total
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ESTs from L. muta venom gland [92].The VEGF gene is comprised of eight exons
which yields the splicing isoforms VEGF-An, where “n” may be 121, 145, 165, 183,
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189 or 206 and represents the number of amino acid residues in the protein [93]. VEGF-
A165 is the major isoform and a biological indicator of the invasiveness of
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hepatocellular carcinoma [121]. In 1999, a VEGF-like protein, named HF, was firstly
isolated from Vipera aspis aspis venom [117, 122]. A cDNA encoding for sv-VEGF
was identified in B. insularis venom gland in 2001 [123]. Two sv-VEGFs (vammin and
VR-1 from Vipera ammodytes ammodytes and Daboia russelli russelli venoms,
respectively) are known to lack the C-terminal heparin-binding region found in other
heparin-binding VEGF subtypes. On the other hand, their C-terminal recognizes similar
heparin/heparan sulfate molecules and shows high selectivity for the kinase insert
domain-containing receptor (KDR) when compared with VEGF-A165. Besides that,
their C-terminal specifically blocks the VEGF-A165 activity [93, 124].
VEGF-F induces hypotension and proliferation of vascular endothelial cells by
binding to the KDR receptor [94]. VEGF-A165 and vammin induced the synthesis and
secretion of perlecan via VEGF receptor-2 (VEGFR-2) in cultured human brain
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microvascular endothelial cells. That proteoglycan may help to regulate the

angiogenesis, maintain the endothelial barrier function and contribute to a synergistic
effect on angiogenesis [125]. The activation of VEGFR-2 depends on the affinity and
concentration of the ligands and the angiogenic activity is potentiated when the ligand
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binds to the co-receptor Neuropilin (Nrp). Hence, drugs with the VEGFR-2/Nrp binding
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domains may be successfully designed as potential proangiogenic therapies [126, 127].
However, further studies are necessary to elucidate the biological activities, mechanism
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of action and possible applications of these neurothophins from snake venoms.
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3.2 Hyaluronidases
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Venom hyaluronidases are enzymes that hydrolyze preferentially the
hyaluronan, the major glycosaminoglycan found in the extracellular matrix [68]. These
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enzymes belong to EC 3.2.1.35 class, which includes the hyaluronidases found in
mammals’ sperm cells, lysosomes and animals venoms. They are capable of
hydrolyzing glycosidic linkages β→1-4 of the residues N-acetyl-β-D-glucosamine and
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D-glucuronate from hyaluronan producing tetra and hexasaccharides [69]. Therefore,

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during the envenoming, hyaluronidases facilitate the venom diffusion in the victim’s
tissue due to their hydrolytic characteristics, acting as a “spreading factor” and
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enhancing the toxins’ effects [70, 135, 136]. This class of enzyme has been found in
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several organisms, being ubiquitous in snake venoms [137, 138].

Hyaluronidases from different sources have been used in different applications
in medicine. Examples include the use as a diffusion promoter for active substances
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(drugs, for instance), the treatment of hyaluronan-induced diseases (such as some types
of cancers), and in the aesthetic medicine [139]. Despite their great therapeutic
potential, these enzymes are found in small proportions in snake venoms and they have
an extremely unstable catalytic activity. These issues hamper their isolation from
venoms, as well as their in-depth functional and structural characterization [140, 141].
This is reinforced by the few hyaluronidase amino acids sequences deposited in
databases, which are currently fourteen for Uniprot and seventeen for NCBI.
The first report indicating the occurrence of hyaluronidase in snake venoms was
in 1939 by Duran-Reynalds, who analyzed the hyaluronidase activity in the venom of
nine different snake species [135]. However, up to date, limited studies have
successfully isolated these enzymes from snake venoms. Reports include the
hyaluronidases from Deinagkistrodon acutus (formerly Agkistrodon acutus) [142],
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Agkistrodon contortrix [143], N. naja [137], D. russelli [144], Cerastes cerastes [136],
Crotalus durissus terrificus [138] and Lachesis muta rhombeata [141].
Snake venom hyaluronidases are glycoproteins with molecular mass ranging
from 28 to 70 kDa, with optimum enzyme activity detected around pH 5.5, 37°C and
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0.15-0.2 M NaCl. They hold specificity for hyaluronan and show no significant activity
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upon chondroitin sulphate [136, 137, 141-144]. Moreover, hyaluronidase activity can
vary according to the snake’s age [145], species [146, 147] and habitat [148, 149]. The
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enzyme activity is inhibited by divalent cations, high temperatures and salt
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concentrations, extreme pH and by EDTA, heparin, denaturing agents and herbal
extracts [136, 138, 143, 144].
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As previously mentioned, in vivo experiments have proven that snake venom
hyaluronidase acts as a spreading factor, enhancing the effect of venom toxins [136-138,
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144]. Therefore, many recent studies put a spotlight on the production of antibodies and
on the search for synthetic and natural inhibitors of these enzymes in order to improve
antivenom therapy, since the importance of hyaluronidases in the envenoming process
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is outstanding [136, 137, 150-156].

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3.3 Cysteine-rich secretory proteins

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Cysteine-rich secretory proteins (CRISPs) are non-enzymatic components

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present in various organisms [63]. They are also found in snake venoms, but their
function in envenoming has not been fully understood thus far [157]. Snake venom
CRISPs are single chain proteins with molecular masses ranging from 20 to 30 kDa.
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They display sixteen highly conserved cysteine residues that form eight disulfide bonds
[63].
CRISPs are distributed among Viperidae and Elapidae families from different
continents. There are reports of the isolation and cloning of three snake venom CRISPs
from Agkistrodon piscivorus piscivous, Ophiophagus hannah and Crotalus atrox,
named piscivorin, ophanin and catrin, respectively [158]. Other CRISPs such as triflin,
ablomin, latisemin and tigrin were isolated from the venoms of Protobothrops
flavoviridis, Agkistrodon blomhoffi, Laticauda semifasciata and Rhabdophis tigrinus
tigrinus, respectively. Ablomin, triflin and latisemin block the caffeine-induced
depolarization, but they have no effects on the contraction of the smooth muscle,
indicating their L-type Ca2+ channel blocker action. On the other hand, tigrin does not
inhibit depolarization and contraction induced by caffeine [64]. Pseudechetoxin (PsTx)
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and pseudecin, from P. australis and P. porphyriacus venoms, respectively, block the
cyclic nucleotide-gated (CNG) ion channel [64, 65].
Natrin, from N. a. atra venom, was reported to be a BKCa channel blocker [66].
Its three-dimensional structure, determined by X-ray crystallography, revealed three
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functional sites: a pathogenesis-related protein from the group 1 (PR-1) like domain
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localized at the N-terminal region, a highly flexible cysteine-rich domain (CRD) at the
C-terminal extremity of the protein, and a hinge between these domains. The CRD
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domain plays an important role in blocking the ion channel activity since it probably
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interacts with the pore of Kv1.3 channels by hydrogen bonds formation [67].
Besides blocking the ion channel activity, CRISPs may act as inflammatory
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modulators once they can regulate the expression of adhesion molecules in endothelial
cells. Lecth and colleagues [159] reported that a CRISP from Echis carinatus sochureki
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negatively regulates angiogenesis, thus disturbing the wound healing process of
envenomed victims [160]. Furthermore, crovirin, a CRISP purified from Crotalus
viridis viridis [161] was tested in vitro for its antiparasitic activity. This CRISP
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demonstrated a dose-dependent antiparasitic activity on Leishmania amazonensis and

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Trypanosoma cruzi amastigote forms and Trypanosoma brucei rhodesiense metacyclic

and blood trypomastigotes form.
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In the light of the above, CRISPs are a promising class of snake venom
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component that can be employed as a model for future cancer drugs due to the negative
regulation of angiogenesis. In addition, this class of components may assist the
development of new drugs against leishmaniosis and Chagas disease.
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3.4 Nucleases and nucleotidases
Nucleases and nucleotidases are hydrolytic enzymes ubiquitously distributed in

snake venoms. Their main function is related to the generation of adenosine through
their catalytic activity, as shown in Figure 1, which schematically illustrates adenosine
production by these enzymes. Adenosine may support toxin biodistribution and also
contributes to prey immobilization, whereas it increases vascular permeability, inhibits
neurotransmitter release and promotes hypotension, sedation, locomotor depression and
bradycardia. Although nucleases and nucleotidases activities are broadly detected in
snake venoms, the biological effects of these enzymes are not completely clear [162-
166].
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3.4.1 Nucleases
Nucleases are hydrolytic enzymes that cleave nucleic acids and their derivatives
[85]. They are classified as endonucleases and exonucleases. Endonucleases [167]
comprise DNAses [168], and RNAses [169] which hydrolyze DNA and RNA,
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respectively. Exonucleases comprise phosphodiesterases (PDEs) [88] that catalyze the
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hydrolysis of phosphodiester bonds, releasing 5’-mononucleotides [85].
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Endonucleases show optimum activity on acidic pH and do not require divalent
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cations for nucleic acids hydrolysis. On the other hand, PDEs have optimum activity on
basic pH and divalent metal ions are required for PDE’s hydrolytic activity [170-172].
Endonucleases and PDEs have been found in snake venoms from Elapidae
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family and crotaline and viperine snakes from Viperidae family [164]. The biological
activities of endonucleases have not been elucidated yet [85] Nevertheless, PDEs are
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known to lead to hypotension, locomotor depression [86] and inhibition of platelet
aggregation [87].
In general, nucleases can lead to the production of purine and pyrimidine
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nucleotides that might act synergistically with nucleotidases and others snake venom
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toxins (phospholipases A2, cardiotoxins, myotoxins, cytolytic peptides and

proteases/hemorrhagins) [85, 162, 164, 173-176], contributing to the symptomatology
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and overall pathology caused by the snake bite envenoming [37].

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3.4.2 Nucleotidases
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Nucleotidases are enzymes involved in the cleavage of nucleic acid derivatives

and nucleic acid-related substrates (ATP, ADP and AMP). However, specific
identification of snake venom nucleotidases is impaired by the presence of other toxins
that share similar substrate specificity [85].
Nucleotidases have been found in a great number of snake venoms [171], but
there are only few nucleotidases isolated and characterized so far due to their minor
amounts in venoms [85]. The biological activities of nucleotidases are known to be
involved in the digestive process and in endogenous purines release, potentiating the
venom-induced hypotension and paralysis via purine receptors. However, nucleotidases
activities need to be deeper investigated [162, 164]. Furthermore, some reports in the
literature show that nucleotidases can act independently or in synergism with other
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toxins, like phosphodiesterase, phosphomonoesterase, endonuclease, phospholipases A2,

cytotoxins, myotoxins, and heparinases [42, 162, 164, 177-179].
Snake venom nucleotidases comprise 5'-nucleotidase, ATPase and ADPase. 5'-
nucleotidase are metalloenzymes of high molecular mass [42, 171, 180, 181], ranging
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from 73 to 100 kDa [42, 178, 182, 183]. Gulland and Jackson [184] firstly identified the
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5'-nucleotidase activity in snake venoms. These enzymes selectively hydrolyze 5'-
nucleotides to nucleosides [181] and inhibit platelet aggregation by adenosine release
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[40-42]. Thus, 5'-nucleotidase increases the anticoagulant effect of ADPases,
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phospholipases A2 and disintegrins by acting synergistically with these toxins [185].
ATPases were firstly described by Zeller in 1950 and are known to hydrolyze
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ATP, releasing adenosine and pyrophosphate. Depending on the reaction conditions,
ATPases can cleave ATP into AMP and pyrophosphate or into ADP and phosphate.
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ATPases have not been isolated from snake venoms yet, and their biological activity
remains unclear. However, they may be involved in shock symptoms by depletion of
ATP [186], since ATP released from skeletal muscles cells (due to activity of
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myotoxins) is quickly hydrolyzed by ATPase, and act as a danger signal molecule

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stimulating purinergic receptors, contributing to the pathophysiology of envenomation

[187-189].
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ADPase hydrolyzes ADP, producing adenosine and orthophosphate [171, 190].

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The only ADPase from snake venom isolated so far has 94 kDa and it was purified from
D. acutus. This toxin inhibits platelet aggregation in platelet rich plasma induced by
different molecules (ADP, collagen and sodium arachidonate), but it does not inhibit
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thrombin-induced aggregation in platelet poor plasma [178]. The inhibition of platelet

aggregation is also attributed to adenosine release due to ADPase activity [85].
3.5 Protease inhibitors
Snake venoms are interesting sources of protease inhibitors, although these

molecules represent, in most cases, only a small proportion of snake venoms. The
control of most body’s chemical reactions is sustained by the antagonism of: (i)
proteases, which play key functions in different systems and biochemical pathways, and
(ii) the correspondent protease inhibitors, responsible for controlling protease activity
[191]. In this context, the possible function of protease inhibitors in snake venoms is to
promote an imbalance in prey homeostasis, since these proteins can inhibit some classes
of enzymes, like serine proteases [192, 193], acetylcholinesterase (AchE) [194] as well
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as interact with ion channels [195, 196], thus interfering in the physiological systems
mediated by these components. On the other hand, it was suggested that these protease
inhibitors may also assist the proteolysis control while the venom is stored at the venom
gland [74, 197].
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The first protease inhibitor found in snake venom was reported in 1972 when
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Takahashi and colleagues [198] isolated a potent kunitz-type protease inhibitor from the
venom of D. russelli. The occurrence of this family of protease inhibitors is reported in
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Elapidae and Viperidae snake venoms [74, 199-201].
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This class of protease inhibitors presents a conserved motif found in kunitz
bovine pancreatic trypsin inhibitor, which is the active domain responsible for the
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inhibition of proteases such as trypsin, chymotrypsin, elastase, thrombin, and activated
factor X [202, 203]. Structurally, they display approximately sixty amino acid residues,
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with six conserved cysteine residues involved in three disulfide bonds, supporting the
stable and compact structure of the folded peptide [204-206].
These venom components represent from 0.3% to 4.6% of total proteins in snake
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venoms [199, 207-209]. The exceptions identified thus far are the species Dendroaspis
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polylepis and D. angusticeps, whose venom proteome presented larger amounts of

kunitz type protease inhibitors [166]. Although these inhibitors may exhibit several
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structural similarities, they show an extensive range of biological functions, including

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blockage of ion channels, disturbances in blood coagulation, inflammation and

fibrinolysis [73-76].
A kunitz-type protease inhibitor isolated from Macrovipera lebetina venom
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displayed a remarkable anti-tumor activity by the interaction with an integrin receptor

[77]. In a further study, this molecule inhibited angiogenesis and endothelial cell
adhesion and migration, as well as increased microtubule dynamics. Therefore, this
toxin is able to interfere in crucial mechanisms related to the process of tumorigenesis
[78]. Some studies also proposed an antibleeding function for these toxins, since some
kunitz-type snake venom protease inhibitors are capable of inhibiting enzymes involved
in the blood coagulation [79-82]. Thus, due to their extensive biological roles, these
venom proteins may present promising biotechnological and pharmaceutical
applications.
Other minor classes of protease inhibitors found in snake venoms are the
cysteine protease inhibitors (cystatins), the metalloproteases inhibitors and the kazal-
type protease inhibitors. The latter was only reported in Bothriechis ssp. venom, with a
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relative abundance of 8.3 and 9% in B. schlegelii and B. supraciliaris species,

respectively [71, 72]. Recently, a kazal-type inhibitor-like protein that is able to inhibit
trypsin activity at pH 5.4 was purified from B. schlegelii venom. This finding suggests
that such protein may act controlling venom proteolysis, considering that the venom
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gland has an acidic pH [197].
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Metalloprotease inhibitors were found by proteomic approaches in snake
venoms from Echis ocellatus, C. c. cerastes [210] and V. anatolica [207]. Recently, a
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new group of metalloprotease peptide inhibitors (Poly-Gly- and poly-His-poly-Gly-
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peptides and tripeptides) was detected in snake venoms. Tripeptides were identified in
the venom of Bothrops ayerbei [211], Bothriechis supraciliaris [72] and Bothrocophias
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campbellii [212]. Interestingly, these inhibitors are encoded by the same precursor of
bradykinin potentiating and C-natriuretic peptides. Furthermore, metalloprotease
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inhibitors such as Poly-His-poly-Gly-peptides (pHpG peptides) were identified in the
venoms of Bothrops snakes [213, 214] and E. ocellatus [210], whereas poly-Gly-
peptides (pG peptides) were found in the venom of Bothriechis supraciliaris [72].
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Regarding snake venom cystatins, the first toxin of this class was isolated from
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Vipera ammodytes by Kregar and colleagues, in 1981 [215]. The only proteomic study
that determined the percentage of this class of toxin in snake venom revealed that it
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represents 1.8% and 9.8% of total venom proteins of the species Bitis gabonica
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gabonica and Bitis arietans, respectively [199]. Moreover, three other cystatins were
purified from snake venom, all of them belonging to type-2 cystatins, which present
about 120 amino acid residues and two characteristic disulfide loops [216-218].
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Nonetheless, no studies have elucidated their role in the envenoming process yet.
Recently, the pharmaceutical application of a recombinant snake venom cystatin
(sv-cystatin) in cancer therapy was investigated. The recombinant cystatin is capable of
inhibiting the invasion and metastasis of tumor cells, both in in vitro and in vivo assays
[61]. Furthermore, it was reported that this molecule is capable of inhibiting tumor
angiogenesis and tumor-endothelial cell adhesion [60, 62]. Additionally, a recombinant
adenovirus transfected with the sv-cystatin gene has demonstrated the ability to inhibit
the invasion and metastasis of several types of tumors, such as mouse melanoma cells,
human gastric carcinoma cells, and MHCC97H cells. This recombinant adenovirus
approach demonstrated an increased antitumor activity compared to the recombinant
protein itself [60].
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Protease inhibitors are becoming molecules of interest to biotechnological and

pharmaceutical areas since proteases are one of the main potential drug targets. There
are about 553 human gene products incorporating protease domains. In addition, these
enzymes are also found in bacteria, virus and parasites [219, 220]. In this context, the
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search for new protease inhibitors plays a key role to warrant the discovery of more
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effective drugs for human therapeutics, especially for cancer treatment.
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3.6 Antimicrobial peptides
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Antimicrobial peptides are found in several organisms and are part of their
innate-immune system, participating of host defense and contributing to the survival of
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these organisms in microbe-rich environments [221, 222]. Snake venoms contain
biologically active peptides, which are a rich source of possible novel antimicrobials
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agents [223]. Furthermore, several well-characterized snake toxins, such as
phospholipases A2, metalloproteases, L-amino acid oxidases and crotamine have shown
effects against many microorganisms [224, 225].
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Most of the studies regarding antimicrobial peptides from snake venoms report
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only the screening of crude venom antimicrobial activity [226-229]. These peptides are
mainly identified by mass spectrometry following chromatographic isolation, although
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genomic sequencing and functional characterization by different antimicrobial assays

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are also employed [43, 44, 98, 221]. Among the previously described snake venom
antimicrobial peptides, it is possible to highlight cathelicidins, viperidins, waprins, β-
defensins, beyond other not yet classified molecules.
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One of the major AMP families in mammals is composed by cathelicidin

peptides that are cationic peptides with highly heterogeneous structures [230, 231].
These peptides are composed of 12 to 80 amino acid residues which are mainly folded
in α-helical structures [231].
The cathelicidin-BF, a cathelicidin-derived antimicrobial peptide, was the first
cathelicidin peptide identified in reptiles (Bungarus fasciatus snake) [45, 46] and
presents in vitro antimicrobial activity on bacterial and fungal species [45]. Other
cathelicidin-derived peptides were identified in O. hannah (OH-CATH and its
derivatives OH-CATH30 and OH-CM6) [43, 44, 47, 48], B. fasciatus (BF-CATH and
its derivatives BF-30 and BF-15) [45, 49-51], Hydrophis cyanocinctus (Hc-CATH) [52]
and N. atra (NA-CATH) [43, 53]. These peptides exhibit antimicrobial activity against
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gram-positive and gram-negative bacterial strains and fungi. Furthermore, it presents

antitumor activity, as demonstrated against BF-CATH cells [45].
Another family of snake venom AMPs is the vipericidins (cathelicidin-related
peptides). This family comprises toxins from South American pit vipers [54], such as
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crotalicidin (C. d. terrificus), lachesicidin (L. m. rhombeata), batroxicidin (Bothops
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atrox) and lutzicidin (Bothrops lutzi). These venom-derived peptides showed in vitro
antimicrobial activity against several bacterial strains, including Streptococcus
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pyogenes, Staphylococcus aureus, Escherichia coli, Klebsiella pneumonia,
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Enterococcus faecalis, Acinotobacter baumannii and Pseudomonas aeruginosa [54].
Omwaprin, a cationic antimicrobial peptide belonging to the waprin family, was
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isolated from O. microlepidotus snake venom [98]. This antimicrobial peptide is
composed of 50 amino acid residues and contains a whey acid protein (WAP) domain in
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its structure (PDB ID. 3NGG). Omwaprin features in vitro antimicrobial activity against
Bacillus megaterium and Staphylococcus warnei gram-positive bacterial strains [98].
On the other hand, the occurrence of waprins in the venoms from Thrasops jacksonii,
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Liophis poecilogyrus, Philodryas olfersii, Pseudoferania polylepis (formerly Enhydris

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polylepis and Rabdophis tigrinus was reported only through transcriptomic approaches
[96]. Corrêa-Netto et al. [97] reported that waprin-like peptides correspond to 0.2% of
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the total toxins in the cDNA library of Micrurus altirostris venom gland.
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The peptide Pep5Bj (1.37 kDa), isolated from Bothropoides jararaca venom,
was able to inhibit the growth of yeast (Candida albicans) and phytopathogenic fungi
(Fusarium oxysprorum and Colletotrichum lindemuthianum) cell cultures [232].
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Another peptide of 2.49 kDa, known as NAP (Naja Antibacterial Peptide), was isolated
from Indian cobra (N. naja) venom and showed in vitro potent antibacterial activity
against gram-negative and gram-positive bacterial strains [233].
Phylogenetic analysis enabled the identification of 13 β-defensin-like sequences
from Bothrops spp. and Lachesis spp. snakes by PCR approach [234]. β-defensins-like
peptides have also been described in other snake venoms [235, 236] and display distinct
biological effects, including antimicrobial activity [237].
These snake venom antimicrobial peptides can be valuable to the development
of novel antimicrobial drugs [44, 45, 223, 238], besides their potential as biomarkers as
well as diagnostic tools [239, 240].
3.7 Phospholipases B
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Phospholipases are enzymes that catalyze the cleavage of phospholipids,

releasing fatty acids and lysophospholipids. Depending on the class of phospholipase,
this reaction may take place in four different sites in membrane phospholipids. When
catalyzed by phospholipase B (PLB), the cleavage occurs at sn-1 and sn-2 positions
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[89]. PLBs were poorly reported so far since they are rarely found in snake venoms, in
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contrast to phospholipases A2 (PLA2s) that are amongst the major venom components.
There are several snake species that have PLB activity in their venom, such as
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Pseudechis phorphyriacus, P. australis, N. naja, D. russelii , Cerastes vipera and
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Crotalus adamanteus [241-243]. PLBs were firstly reported in Pseudechis colletti
venom as a dimeric protein of 16.5 kDa that presents high hemolytic activity in
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erythrocytes and cytotoxicity to rhabdomyosarcoma cells [90].
The omics era not only facilitated but also fastened the discovery of novel PLBs.
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The proteomic analysis of the Australian snake Pseudechis guttatus revealed two
peptide sequences that shared identity to PLB. They were identified in one basic spot of
2D-PAGE and these two peptides covered 5% of the PLB sequence. The authors
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suggest that this basic toxin may contribute to the hemolysis observed in P. guttatus
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envenoming [91].
Recently, proteins with PLB sequence identity were found in Micrurus dumerilii
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snake venom proteome, although in small relative abundance (less than 0.1%). This
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venom is mainly composed of PLA2 and three-finger toxins [244]. Interestingly, one
PLB was identified by mass spectrometry analysis in the venom of the Brazilian snake
L. m. rhombeata. It was the first report of this class of protein in Lachesis genus [141].
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Although PLBs have been reported in some snake venom proteomes, the advent
of transcriptome techniques has allowed the identification of this class in different snake
genus. One EST that encodes a PLB from a new lipase family was identified in the
cDNA library of Drysdalia coronoides venom gland. This EST comprises a signal
peptide of 36 amino acid residues and a mature protein of around 60 kDa. Moreover, the
authors also found some peptides in D. coronoides venom proteome that correspond to
this protein [245].
Rokyta and colleagues [246] analyzed the transcriptome from C. adamanteus
venom gland and found one transcript that encodes a PLB precursor presenting a signal
peptide of 36 amino acid residues and the mature PLB of 526 amino acid residues. It
represents just 0.06% of the whole transcriptome.
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Furthermore, the P. flavoviridis venom gland transcriptome revealed that PLB

comprises 0.14% of the transcripts, while in Ovophis okinavensis the percentage of
sequences that encodes this protein was just 0.15%. Some peptide sequences
corresponding to PLB were also found in the proteome of these two snake venoms and
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they cover from 26.1 to 61.6% of the PLB sequence [163].
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The venom-gland transcriptome of the coral snake Micrurus fulvius revealed one
PLB sequence, representing 0.154% of total reads. This venom is mostly composed of
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PLA2 (64.9% of toxins reads), which is responsible for the neurotoxic, cardiotoxic,
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myotoxic, anticoagulant and hemolytic effects of the envenoming [248].
Vonk and colleagues [16] analyzed the genome from the venom and accessory
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glands of king cobra snake (O. hannah) and observed that they are mainly composed of
three-finger toxins (venom) and lectins (accessory gland). In addition to these findings,
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they also reported PLB transcripts in these two glands. In contrast, PLB could not be
found in the venom proteome of this snake.
Although some PLBs have been identified in snake venoms by genomic,
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transcriptomic and proteomic approaches, they are poorly studied by traditional

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approach, mainly due to their low abundance in crude venoms [243]. Therefore the
importance of omics approaches to the disclosure of minor components such as PLB is
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reinforced and and further structural and functional studies regarding this neglected
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class of venom protein are required.
3.8 Cobra venom factor

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Cobra venom factor (CVF) is a family of proteins that activates the complement
system. The CVF function was reported by [55]. However, before that, Flexner and
Noguchi [249] had already demonstrated that Naja genus venom produces hemolysis,
which is dependent of the complement system, when incubated with mammalian
erythrocytes and serum [250]. In 1971, it was shown that the venom of N. haje
(Egyptian cobra) selectively consumes the complement components C3 → C9 [251].
Lately, several studies focused on CVF purification and complete functional
characterization [252-255]. Thereby, CVF has been studied for more than a century.
CVF presents approximately 149 kDa and shares high identity degree with
human complement component C3 (50% of identity, 69% of similarity) [59]. The C3 is
the most important protein of the complement system, being activated by all the three
pathways: classical, lectin and alternative, besides their possible cross-reactivity [56].
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The C3 activation and the generation of its products lead to all biological functions of
the complement system [256]. Since CVF is a C3-like protein, after the cleavage of
factor B (by factor D), it is turned into CVFBb, which acts as a C3-convertase. The
continuous action of CVFBb is responsible for complement depletion and production of
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components of the terminal phase, including the cleavage of C5 and the generation of
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C5a. The C5a component is a powerful pro-inflammatory peptide. Along with C3a, C5a
could be an important product for the snake since they increase the vascular
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permeability and blood flow in the bite site, allowing the rapid diffusion of the toxic
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venom compounds into the blood stream. Excellent detailed reviews can be found
elsewhere [59, 257].
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So far, CVF is mainly present in Elapidae snake family and it has been described
in four different genera: Hemachatus, Naja, Ophiophagus and Austrelaps [57-59, 251].
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Nevertheless, complement depletion caused by CVF is most studied using the venoms
of N. naja, Naja kaouthia and N. atra, even being found in low amounts in these
venoms. Laustsen et al. [165] demonstrated, by proteomic analysis, that CVF accounts
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for 0.1 % of N. kaouthia whole venom, which is a very low percentage in comparison to
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other components, such as PLA2 (13.5%), neurotoxins (53.2%) and LAAO (0.4%).
However, recently, CVF was also found in the Viperidae snake (Bothrops jararaca)
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using transcriptomic [258] and proteopeptidomic [259] analysis.

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Based on the low relative abundance of CVF in snake venoms and its important
anti-complement effect, molecular biology emerges as a convenient approach to CVF
production for therapeutic purposes. In this way, recombinant [59, 260] and humanized
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[261] CVF are available nowadays.
3.9 Vespryn
Vespryn is a new family of venom proteins comprising ohanin from O. hannah

[95], Thai cobrin (P82885) from N. kaouthia [262] and ohanin-like proteins from N. n.
atra [263], L. muta [92] and Tropidechis carinatus [264]. However, only ohanin (the
first family member) has been well characterized so far.
Ohanin was firstly purified in 2005 from the crude venom of O. hannah (king
cobra). It presents a molecular mass of approximately 12 kDa, with 107 amino acid
residues and a single cysteine residue, which is a unique feature of this protein class.
The amino acid sequence of ohanin shares 93% of sequence identity with Thai cobrin
isolated from N. kaouthia [95].
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Ohanin induces hypolocomotion and hypernociception in mice after

intraperitoneal injection in a dose-dependent manner. However, it is considered non-
toxic since the analysis of gross pathology, after 24 h of its injection in mice, showed no
signs of hemorrhage or necrosis in the brain, heart, lungs, kidneys, spleen and liver.
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Thus, the ohanin effects are possibly important to slow down the mobility of the prey,
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assisting in its capture [95].
The complete cDNA and genomic organization of ohanin was already reported.
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The ohanin-encoding cDNA (1558 bp) show that this toxin is synthesized as a prepro-
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protein in the venom gland [265]. In this context, due to ohanin low recovery from the
venom (0.1%), heterologous expression of this protein has already been accomplished
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and the resulting recombinant form functionally resembles the native toxin.
Recombinant ohanin may be helpful in the investigation of the structure-function
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relationship of this class of toxins [95].
3.10 Dipeptidylpeptidase IV
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Dipeptidylpeptidase IV (DPP IV) presents a wide distribution among snake

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venoms. It has been identified by proteome and transcriptome approaches, for example,
in Rhinocerophis alternatus (formerly Bothrops alternatus), Gloydius brevicaudus, P.
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flavoviridis and O. okinavensis venoms and its activity have been detected in a great
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variety of snake venoms [266, 267]. The DPP IV G. brevicaudus was partially isolated
from this snake venom and the cDNA sequence was cloned from the venom gland
cDNA library. Two DPP IV, named DPP IVa and DPP IVb, containing 751 amino acid
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residues were obtained. They contain a consensus sequence similar to serine proteases
around the Ser616 and a putative catalytic triad (Ser616, Asp694 and His76) is located
at the C-terminus region. The mature protein presented 116 kDa estimated by SDS-
PAGE, and about 25% of their molecular mass is due to glycosylation. In fact, the
sequences present 10 potential N-glycosylation sites [266] .It is interesting that DPP IV
is secreted into the venom gland through exosome-like vesicles not through the
common pathway by secretory granules and exocytosis [268].
The role of DPP IV during envenoming is still not clear, but these enzymes
might contribute to alterations in the cardiovascular system leading to hypotension by
cleaving endogenous hypertensive factors. Additionally, DPP IV may also present
important roles in the immune and neuroendocrine systems and in the glucose
homeostasis.
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3.11 Aminopeptidases
Aminopeptidases have been identified by “omics” techniques in the venoms of

B. jararaca [271], O. okinavensis [163], P. flavoviridis [163] and G. brevicaudus [270].
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Aminopeptidase activity was detected in the mice envenoming by C. d. terrificus venom
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[272]. Furthermore, aminopeptidase A (APA) is release to the venom gland lumen
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through exosome-like vesicles, the same say as DPP IV [268].
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The rhiminopeptidase A is an APA isolated from Bitis rhinoceros which is an
exo-metallopeptidase that removes acidic terminal residues from proteins and peptides
at presence of calcium. No specificity for acidic, basic or neutral pH was detected in the
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absence of calcium. Its molecular mass was estimated by SDS-PAGE being150 kDa,
and its sequence is composed of 951 amino acid residues [273].
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The aminopeptidases mammalian A and N act by different ways in the renin-
angiotensin system. Aminopeptidase N (APN) catalyzes the conversion of Angiotensin
III to angiotensin IV through the hydrolysis of the N-terminal arginine from angiotensin
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III thus preventing hypertension. In contrast, APA induces hypotension by degrading

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angiotensin II to angiotensin III through the hydrolysis of the N-terminal aspartic acid
since angiotensin III is less hypertensive that angiotensin II at peripheral sites [163].
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Both angiotensins (II and III) also take part in the brain renin-angiotensin system,
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involved in the blood pressure control and both present hypertensive action with affinity
to the AT1 and AT2 receptors [274].
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Besides that, a leucine aminopeptidase was also identified and it might be linked
to the action of hemorrhagic snake venom metalloproteases, other venom peptidases and
the L-amino acid oxidase activity [163].
3.12 Other minor snake venom toxins
Table 2 summarizes the main minor snake venom toxins identified only by
“omic” approaches.
3.12.1 Cytokine-like molecules
Transcriptomic analyses have enabled the identification of cytokine-like

molecules in the venoms of D. acutus, R. alternatus and B. gabonica. Their role in the
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envenoming remains unknown, but they possibly act in the vascular system,
contributing to the local inflammatory process induced by the venom [269, 275, 276].
3.12.2 Veficolins
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Ficolins are a group of proteins similar to complement-activating lectins present
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in different tissues. They show a collagen-like domain in the N-terminal and a
fibrinogen-like domain in the C-terminal region and are related to the innate defense
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system [277-279]. Veficolins (venom ficolins) were identified for the first time as a
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venom toxin in the venom gland transcriptome of the colubrid snake Cerberus rynchops
[279], although Ching et al. [280] reported a veficolin from P. olfersii as a putative
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protein in its venom. The collagen-like domain of ryncolins (the C. rynchops ficolins)
may induce platelet aggregation while the dimerization of two C-terminal fibrinogen-
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like domains might interfere in fibrin formation by mimicking the C-terminal globular
domain of fibrinogen. Veficolins were also identified in the transcriptome of Micrurus
corallinus [97, 279, 280].
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Table 2 – Snake venom proteins identified by “omic” techniques.
Approach used in the

Snake venom protein classes Snake venoms Possible function in the venom References
identification
Bungarus fasciatus, Naja atra,

Cathelicidins Transcriptome Antimicrobial activity [43, 45]
PT
Ophiophagus hannah
Deinagkistrodon acutus,
Local inflammation and alterations in the
Cytokine-like molecules Rhinocerophis alternatus, Bitis Transcriptome [269, 275, 276]
RI
vascular system
gabonica
Crotalus adamanteus, Bothropoides Transcriptome and
C
EF-hand protein Unknown [246, 281, 282]
insularis Proteome
US
Transcriptome and
Insulin-like growth factor Ophiophagus hannah Unknown [16, 58, 209]
Proteome
Transcriptome and
N
Lysosomal acid lipase (LAL) Echis coloratus Unknown [97, 283]
Proteome
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Bothropoides jararaca, Bothrops
Metalloprotease inhibitors (poly-Gly- fonsecai, Bothrops cotiara, Bothrops
and poly-His-poly-Gly-peptides and atrox, Bothrops barnetti, Bothrops Proteome and Peptidome Inhibition of metalloprotease activity [72, 210-214]
tripeptides) ayerbei, Bothriechis supraciliaris,
Bothrocophias campbelli
ED
Neuroprotective protein Deinagkistrodon acutus Transcriptome Unknown [276]
Increased vascular permeability, hypotension,

Nucleosides Dendroaspis polylepis
PT Proteome
inhibition of neurotransmitter release, sedation,
locomotor depression, bradycardia, influence in
the toxin distribution
[162-166]
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Transforming growth factor Deinagkistrodon acutus Transcriptome Unknown [276]
Sistrurus catenatus edwardsii, Echis Transcriptome and

Renin-like aspartic protease Local hypertension [284, 285]
ocellatus Proteome
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Transferrin-like proteins (TLP) Pseudechis australis Proteome Antimicrobial activity [286]
Deinagkistrodon acutus,
Tumor necrosis factor Transcriptome Local inflammation [269, 276]
Rhinocerophis alternatus
Cerberus rynchops, Micrurus Platelet aggregation induction and interference

Veficolins Transcriptome [97, 279, 280]
corallinus, Philodryas olfersii in the fibrin formation
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3.12.3 EF-hand protein
EF-hand proteins present a conserved calcium-binding domain, composed of

two alpha helixes linked by a loop (helix-loop-helix) usually formed by 12 amino acid
residues. This helix-loop-helix motif that coordinates calcium-binding resembles the
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human hand thumb and forefinger, which provided the name EF-hand for this motif.
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This domain is important to calcium-dependent processes, such as membrane fusion and
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vesicular transport [281]. Calglandulin, an EF-hand protein identified in the B insularis
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venom gland, has been implicated in the secretion of toxins from the venom gland cells
[246, 281, 287]. On the other hand, reticulocalbin 2 EF-hand calcium-binding domain
precursor is a highly expressed nontoxin in the venom gland C. adamanteus [246, 282].
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However, it is not yet clear if this molecule present toxic properties and potential role in
the envenoming or if it presence in the venom proteome is just a result of the leakage of
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a housekeeping protein.
4. Conclusions and Perspectives

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Snake venoms are rich combinations of bioactive compounds that act on specific
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biochemical and physiological targets of the prey or victim, causing an imbalance on

their homeostasis. Thus, snake venoms are considered a source of harmful toxins,
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possessing a diversity of molecules that mimic some natural regulatory components [1,
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288, 289]. On the other hand, snake venoms toxins are also considered promising
molecules for the treatment and diagnosis of certain pathologies, as well as for research
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and biotechnological purpose [289-292]. Figure 2 summarizes the main potential

application of the minor venom protein classes discussed herein. The most abundant
toxins are easily identified and purified from snake venoms by classical approaches.
They have been extensively studied in the last decades and some of them have already
been directly applied in the treatment of some disorders or used as models for drug
designs, such as serine proteases, disintegrins and bradykinin-potentiating peptides
[292]. A classic example is Captopril, which is an orally available peptidomimetic of a
bradykinin potentiating peptide from B. jararaca venom. This molecule competitively
inhibits the angiotensin-converting enzyme (ACE) and is extensively used worldwide in
the treatment of human hypertension [293]. Currently, nine snake venom protein
derivatives are commercially available for therapeutic application or under clinical trials
[292], but none of them are related to the protein classes approached in this review.
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Most of the minor snake venom proteins remained unidentified or poorly

explored for many years, mainly due to the difficulties in obtaining them in sufficient
amounts for a comprehensive study [21]. Only with the advent of new highly sensitive
methods of molecular identification, such as the “omic” approaches, this hidden world
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of snake toxins could be brought to light. However, an in-depth investigation of the
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biological effects and the potential for therapeutic and biotechnological application of
these toxins are still necessary.
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Heterologous protein expression and peptide synthesis represent alternatives to
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produce toxins in both laboratory and industrial scale, which may enable the study of
minor snake toxin classes and generate perspectives for their further investigation and
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application [21]. Furthermore, we highlight the importance of continuing the search for
novel toxin scaffolds, classes and isoforms, in order to enrich our knowledge on these
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natural libraries of pharmacologically active molecules. Improving the understanding in
snake proteins diversity is indispensable not only to drug discovery, but also to better
comprehend the pathophysiology of snake bites and, thereby, improving the still
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outdated antivenom therapy.

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Minor snake venom protein classes may also provide important information
regarding the evolutionary processes that generated snake venom repertories. Snake
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toxins are supposed to have been originated by genes that were expressed both on
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venom gland and other tissues, followed by tissue-specific enhanced expression in the
venom gland and reduction of expression or gene loss in nonvenomous-related tissues
[258, 288, 294]. Evidences that support this hypothesis is the expression of various
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venom homolog genes in nonvenom gland-related tissues of venomous and

nonvenomous snakes [294]. Subsequently, the main toxic and abundant snake venom
proteins probably suffered selective pressures and underwent diversification by gene
duplication that occurs via “birth and death” model [295], followed by mutations in key
residues or domains in the molecular surface [16, 296] and/or gene recombination [29].
This process typically results in multi-gene families, with conserved few disulfide-rich
molecular scaffolds, but encoding a variety of proteins exhibiting diverse biological and
toxic activities [6, 297, 298]. The reason for generating toxin diversification in the
venom is still not completely clarified. Nevertheless, it may represent a selective
advantage to these animals since different toxins may act on synergism, enhancing toxic
effects, and may extend molecular targets in preys [298, 299].
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On the other hand, genes encoding many low abundant snake venom proteins
were presumably devoid of selective pressures, mainly due to their small contribution in
the prey capture [13]. Thus, the minor protein families discussed herein are expected to
present low degree of diversification and neofunctionalization, retaining most of the
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biological effects of their homologous from nonvenomous tissues. However, studies
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related to the origin of these protein classes in the venom are recent and still very scarce
and inconclusive. The analysis of the differences in the expression rates of these
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proteins in the venom gland and in other tissues and the comparative characterization of
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venom proteins and their homologs may indicate whether protein may have evolved to
display toxic effects or another role in these complexes biological. Moreover,
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investigations related to the gene structure and expression of venom proteins will assist
the understanding of the evolutionary processes that originated venom components and
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enlightening of the selective forces that have been driven adaptation of snake venoms to
different ecological niches and preys.
5. Acknowledgments
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The authors acknowledge the financial support from the Fundação de Amparo à
Pesquisa do Estado de São Paulo (FAPESP, São Paulo Research Foundation, grant n.
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2011/23236-4; scholarships to: GAW, n. 2014/06170-8; KCFB, n. 2013/26619-7; JBF,

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n. 2015/16714-8; PYTS n. 2014/15644-3; CTC, n. 2013/26200-6; FAC, n. 2012/13590-

8; MBP, n. 2012/12954-6 and FGA, n. 2011/12317-3), Conselho Nacional de
Desenvolvimento Científico e Tecnológico (CNPq, The National Council for Scientific
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and Technological Development, 303689/2013-7; scholarship to FAC n. 140949/2015-

1), Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES,
Coordination for the Improvement of Higher Education Personnel, scholarship to
GAW) (scholarship to ELPJ, ISO and IGF) and the Support Nucleus for Research on
Animal Toxins (NAP-TOXAN-USP, grant n. 12-125432.1.3).
6. References
1. Fry, B.G., et al., The Toxicogenomic Multiverse: Convergent Recruitment of

Proteins Into Animal Venoms. Annual Review of Genomics and Human
Genetics, 2009. 10: p. 483-511, DOI: 10.1146/annurev.genom.9.081307.164356.
2. Casewell, N.R., Venom Evolution: Gene Loss Shapes Phenotypic Adaptation.
Current Biology, 2016. 26(18): p. R849-R851, DOI: 10.1016/j.cub.2016.07.082.
3. Chippaux, J.P., V. Williams, and J. White, Snake venom variability: methods of
study, results and interpretation. Toxicon, 1991. 29(11): p. 1279-303.
ACCEPTED MANUSCRIPT
30
4. Calvete, J.J., et al., Snake Venomics of the Central American Rattlesnake

Crotalus simus and the South American Crotalus durissus Complex Points to
Neurotoxicity as an Adaptive Paedomorphic Trend along Crotalus Dispersal in
South America. Journal of Proteome Research, 2010. 9(1): p. 528-544, DOI:
10.1021/pr9008749.
5. Calvete, J.J., et al., Venoms, venomics, antivenomics. FEBS Lett, 2009. 583(11):
T
p. 1736-43, DOI: 10.1016/j.febslet.2009.03.029.
6. Kordis, D. and F. Gubensek, Molecular evolution of Bov-B LINEs in
IP
vertebrates. Gene, 1999. 238(1): p. 171-8.
7. Rosenberg, H.I., Histology, histochemistry, and emptying mechanism of the
R
venom glands of some elapid snakes. J Morphol, 1967. 123(2): p. 133-55, DOI:
10.1002/jmor.1051230204.
SC
8. Carneiro, S.M., et al., Morphometric studies on venom secretory cells from
Bothrops jararacussu (Jararacucu) before and after venom extraction. Toxicon,
1991. 29(6): p. 569-80.
NU
9. Oron, U. and A. Bdolah, Regulation of protein synthesis in the venom gland of
viperid snakes. J Cell Biol, 1973. 56(1): p. 177-90.
10. Kochva, E., Oral glands of the Reptilia. Biology of the Reptilia, 1978. 8: p. 43-
MA
161.
11. Luna, M.S., et al., Activation of Bothrops jararaca snake venom gland and
venom production: A proteomic approach. Journal of Proteomics, 2013. 94: p.
460-472, DOI: 10.1016/j.jprot.2013.10.026.
12. Rokyta, D.R., et al., The transcriptomic and proteomic basis for the evolution of
D
a novel venom phenotype within the Timber Rattlesnake (Crotalus horridus).

TE
Toxicon, 2015. 98: p. 34-48, DOI: 10.1016/j.toxicon.2015.02.015.

13. Casewell, N.R., et al., Medically important differences in snake venom
composition are dictated by distinct postgenomic mechanisms. Proceedings of
P
the National Academy of Sciences of the United States of America, 2014.

111(25): p. 9205-9210, DOI: 10.1073/pnas.1405484111.
CE
14. Durban, J., et al., Integrated "omics" profiling indicates that miRNAs are
modulators of the ontogenetic venom composition shift in the Central American
rattlesnake, Crotalus simus simus. Bmc Genomics, 2013. 14, DOI:
AC
10.1186/1471-2164-14-234.
15. Boldrini-Franca, J., et al., Snake venomics and antivenomics of Crotalus
durissus subspecies from Brazil: Assessment of geographic variation and its
implication on snakebite management. Journal of Proteomics, 2010. 73(9): p.
1758-1776, DOI: 10.1016/j.jprot.2010.06.001.
16. Vonk, F.J., et al., The king cobra genome reveals dynamic gene evolution and
adaptation in the snake venom system. Proc Natl Acad Sci U S A, 2013.
110(51): p. 20651-6, DOI: 10.1073/pnas.1314702110.
17. McCue, M.D., Cost of producing venom in three North American pitviper
species. Copeia, 2006(4): p. 818-825, DOI: Doi 10.1643/0045-
8511(2006)6[818:Copvit]2.0.Co;2.
18. Rotenberg, D., E.S. Bamberger, and E. Kochva, Studies on ribonucleic acid
synthesis in the venom glands of Vipera palaestinae (Ophidia, Reptilia).
Biochem J, 1971. 121(4): p. 609-12.
19. Hayes, W., The snake venom-metering controversy: levels of analysis,
assumptions, and evidence. The biology of rattlesnakes, 2008: p. 191-220.
20. Morgenstern, D. and G.F. King, The venom optimization hypothesis revisited.
ACCEPTED MANUSCRIPT
31
21. Boldrini-Franca, J., et al., Expression of a new serine protease from Crotalus
durissus collilineatus venom in Pichia pastoris and functional comparison with
the native enzyme. Appl Microbiol Biotechnol, 2015. 99(23): p. 9971-86, DOI:
10.1007/s00253-015-6836-2.
22. Calvete, J.J., Venomics, what else? Toxicon, 2012. 60(4): p. 427-33, DOI:
10.1016/j.toxicon.2012.05.012.
T
23. Kaas, Q. and D.J. Craik, Bioinformatics-Aided Venomics. Toxins (Basel), 2015.
7(6): p. 2159-87, DOI: 10.3390/toxins7062159.
IP
24. Brahma, R.K., et al., Venom gland transcriptomics for identifying, cataloging,
and characterizing venom proteins in snakes. Toxicon, 2015. 93: p. 1-10, DOI:
R
10.1016/j.toxicon.2014.10.022.
25. Sunagar, K., et al., Molecular evolution of vertebrate neurotrophins: co-option
SC
of the highly conserved nerve growth factor gene into the advanced snake venom
arsenalf. PLoS One, 2013. 8(11): p. e81827, DOI:
10.1371/journal.pone.0081827.
NU
26. Bazaa, A., et al., Snake venomics: comparative analysis of the venom proteomes
of the Tunisian snakes Cerastes cerastes, Cerastes vipera and Macrovipera
lebetina. Proteomics, 2005. 5(16): p. 4223-35, DOI: 10.1002/pmic.200402024.
MA
27. Juarez, P., L. Sanz, and J.J. Calvete, Snake venomics: characterization of protein
families in Sistrurus barbouri venom by cysteine mapping, N-terminal
sequencing, and tandem mass spectrometry analysis. Proteomics, 2004. 4(2): p.
327-38, DOI: 10.1002/pmic.200300628.
28. Menez, A., R. Stocklin, and D. Mebs, 'Venomics' or : The venomous systems
D
genome project. Toxicon, 2006. 47(3): p. 255-9, DOI:

TE
10.1016/j.toxicon.2005.12.010.
29. Dowell, N.L., et al., The Deep Origin and Recent Loss of Venom Toxin Genes in
Rattlesnakes. Curr Biol, 2016. 26(18): p. 2434-45, DOI:
P
10.1016/j.cub.2016.07.038.
30. Yin, W., et al., Evolutionary trajectories of snake genes and genomes revealed
CE
by comparative analyses of five-pacer viper. Nat Commun, 2016. 7: p. 13107,

DOI: 10.1038/ncomms13107.
31. Zelanis, A. and A.K. Tashima, Unraveling snake venom complexity with 'omics'
AC
approaches: challenges and perspectives. Toxicon, 2014. 87: p. 131-4, DOI:

10.1016/j.toxicon.2014.05.011.
32. Conticello, S.G., et al., Mechanisms for evolving hypervariability: the case of
conopeptides. Mol Biol Evol, 2001. 18(2): p. 120-31.
33. Dutertre, S., et al., Deep venomics reveals the mechanism for expanded peptide
diversity in cone snail venom. Mol Cell Proteomics, 2013. 12(2): p. 312-29,
DOI: 10.1074/mcp.M112.021469.
34. Andrade-Silva, D., et al., Proteomic and Glycoproteomic Profilings Reveal That
Post- translational Modifications of Toxins Contribute to Venom Phenotype in
Snakes. Journal of Proteome Research, 2016. 15(8): p. 2658-2675, DOI:
10.1021/acs.jproteome.6b00217.
35. Prashanth, J.R., R.J. Lewis, and S. Dutertre, Towards an integrated venomics
approach for accelerated conopeptide discovery. Toxicon, 2012. 60(4): p. 470-
7, DOI: 10.1016/j.toxicon.2012.04.340.
36. Favreau, P., et al., Mass spectrometry strategies for venom mapping and peptide
sequencing from crude venoms: case applications with single arthropod
specimen. Toxicon, 2006. 47(6): p. 676-87, DOI:
10.1016/j.toxicon.2006.01.020.
ACCEPTED MANUSCRIPT
32
37. Fox, J.W., A brief review of the scientific history of several lesser-known snake
venom proteins: l-amino acid oxidases, hyaluronidases and phosphodiesterases.
38. Calvete, J.J. and B. Lomonte, A bright future for integrative venomics. Toxicon,
2015. 107(Pt B): p. 159-62, DOI: 10.1016/j.toxicon.2015.10.024.
39. Campos, P.F., et al., Trends in the evolution of snake toxins underscored by an
T
integrative omics approach to profile the venom of the colubrid Phalotris
mertensi. Genome Biol Evol, 2016, DOI: 10.1093/gbe/evw149.
IP
40. CHEN, X., et al., Purification and Characterization of 5'-nucleotidase from
Trimeresurus albolabris Venom. Zoological Research, 2008. 29(4): p. 399-404.
R
41. Hart, M.L., et al., Direct treatment of mouse or human blood with soluble 5'-
nucleotidase inhibits platelet aggregation. Arterioscler Thromb Vasc Biol, 2008.
SC
28(8): p. 1477-83, DOI: 10.1161/ATVBAHA.108.169219.
42. Ouyang, C. and T.F. Huang, Inhibition of platelet aggregation by 5′-
nucleotidase purified from Trimeresurus gramineus snake venom. Toxicon,
NU
1983. 21(4): p. 491-501.
43. Zhao, H., et al., Identification and characterization of novel reptile cathelicidins
from elapid snakes. Peptides, 2008. 29(10): p. 1685-91, DOI:
MA
10.1016/j.peptides.2008.06.008.
44. Li, S.A., W.H. Lee, and Y. Zhang, Efficacy of OH-CATH30 and its analogs
against drug-resistant bacteria in vitro and in mouse models. Antimicrob
Agents Chemother, 2012. 56(6): p. 3309-17, DOI: 10.1128/AAC.06304-11.
45. Wang, Y., et al., Snake cathelicidin from Bungarus fasciatus is a potent peptide
D
antibiotics. PLoS One, 2008. 3(9): p. e3217, DOI:

TE
10.1371/journal.pone.0003217.
46. Wang, Y., et al., Cathelicidin-BF, a Snake Cathelicidin-Derived Antimicrobial
Peptide, Could Be an Excellent Therapeutic Agent for Acne Vulgaris. PLoS
P
ONE, 2011. 6(7): p. e22120, DOI: 10.1371/journal.pone.0022120.

47. Zhang, Y., et al., Structure-function relationship of king cobra cathelicidin.
CE
Peptides, 2010. 31(8): p. 1488-93, DOI: 10.1016/j.peptides.2010.05.005.

48. Zhang, B.Y., et al., Protective effects of snake venom antimicrobial peptide OH-
CATH on E. coli induced rabbit urinary tract infection models. Dongwuxue
AC
Yanjiu, 2013. 34(1): p. 27-32, DOI: 10.3724/SP.J.1141.2013.01027.

49. Chen, W., et al., Structure-activity relationships of a snake cathelicidin-related
peptide, BF-15. Peptides, 2011. 32(12): p. 2497-503, DOI:
10.1016/j.peptides.2011.10.005.
50. Wang, H., et al., BF-30 selectively inhibits melanoma cell proliferation via
cytoplasmic membrane permeabilization and DNA-binding in vitro and in
B16F10-bearing mice. Eur J Pharmacol, 2013. 707(1-3): p. 1-10, DOI:
10.1016/j.ejphar.2013.03.028.
51. Zhou, H., et al., The antibacterial activity of BF-30 in vitro and in infected
burned rats is through interference with cytoplasmic membrane integrity.
Peptides, 2011. 32(6): p. 1131-8, DOI: 10.1016/j.peptides.2011.04.002.
52. Wei, L., et al., Identification and Characterization of the First Cathelicidin from
Sea Snakes with Potent Antimicrobial and Anti-inflammatory Activity and
Special Mechanism. J Biol Chem, 2015. 290(27): p. 16633-52, DOI:
10.1074/jbc.M115.642645.
53. Blower, R.J., S.M. Barksdale, and M.L. van Hoek, Snake Cathelicidin NA-
CATH and Smaller Helical Antimicrobial Peptides Are Effective against
ACCEPTED MANUSCRIPT
33
Burkholderia thailandensis. PLoS Negl Trop Dis, 2015. 9(7): p. e0003862, DOI:
10.1371/journal.pntd.0003862.
54. Falcao, C.B., et al., Vipericidins: a novel family of cathelicidin-related peptides
from the venom gland of South American pit vipers. Amino Acids, 2014. 46(11):
p. 2561-71, DOI: 10.1007/s00726-014-1801-4.
55. Ewing, C.B., The action of rattlesnake venom upon the bactericidal power of the
T
blood serum. The Boston Medical and Surgical Journal, 1894. 130(20): p. 487-
490.
IP
56. Laich, A. and R.B. Sim, Complement C4bC2 complex formation: an
investigation by surface plasmon resonance. Biochim Biophys Acta, 2001.
R
1544(1-2): p. 96-112.
57. Rehana, S. and R. Manjunatha Kini, Molecular isoforms of cobra venom factor-
SC
like proteins in the venom of Austrelaps superbus. Toxicon, 2007. 50(1): p. 32-
52, DOI: 10.1016/j.toxicon.2007.02.016.
58. Tan, C.H., et al., Venom-gland transcriptome and venom proteome of the
NU
Malaysian king cobra (Ophiophagus hannah). BMC Genomics, 2015. 16(1): p.
687, DOI: 10.1186/s12864-015-1828-2.
59. Vogel, C.W. and D.C. Fritzinger, Cobra venom factor: Structure, function, and
MA
humanization for therapeutic complement depletion. Toxicon, 2010. 56(7): p.
1198-222, DOI: 10.1016/j.toxicon.2010.04.007.
60. Xie, Q., et al., Recombinant adenovirus snake venom cystatin inhibits the
growth, invasion, and metastasis of B16F10 cells in vitro and in vivo. Melanoma
Res, 2013. 23(6): p. 444-51, DOI: 10.1097/CMR.0000000000000031.
D
61. Xie, Q., et al., Recombinant snake venom cystatin inhibits the growth, invasion
TE
and metastasis of B16F10 cells and MHCC97H cells in vitro and in vivo.
Toxicon, 2011. 57(5): p. 704-11, DOI: 10.1016/j.toxicon.2011.02.002.
62. Xie, Q., et al., Recombinant snake venom cystatin inhibits tumor angiogenesis in
P
vitro and in vivo associated with downregulation of VEGF-A165, Flt-1 and

bFGF. Anticancer Agents Med Chem, 2013. 13(4): p. 663-71.
CE
63. Yamazaki, Y. and T. Morita, Structure and function of snake venom cysteine-
rich secretory proteins. Toxicon, 2004. 44(3): p. 227-31, DOI:
10.1016/j.toxicon.2004.05.023.
AC
64. Yamazaki, Y., et al., Cloning and characterization of novel snake venom
proteins that block smooth muscle contraction. Eur J Biochem, 2002. 269(11): p.
2708-15.
65. Brown, R.L., et al., Pseudechetoxin binds to the pore turret of cyclic nucleotide-
gated ion channels. J Gen Physiol, 2003. 122(6): p. 749-60, DOI:
10.1085/jgp.200308823.
66. Wang, J., et al., Blocking effect and crystal structure of natrin toxin, a cysteine-
rich secretory protein from Naja atra venom that targets the BKCa channel.
Biochemistry, 2005. 44(30): p. 10145-52, DOI: 10.1021/bi050614m.
67. Wang, F., et al., Structural and functional analysis of natrin, a venom protein
that targets various ion channels. Biochem Biophys Res Commun, 2006.
351(2): p. 443-8, DOI: 10.1016/j.bbrc.2006.10.067.
68. Bordon, K.C.F., et al., Arthropod venom Hyaluronidases: biochemical
properties and potential applications in medicine and biotechnology. Journal of
Venomous Animals and Toxins Including Tropical Diseases, 2015. 21, DOI:
10.1186/s40409-015-0042-7.
69. Mackessy, S.P., Handbook of venoms and toxins of reptiles. 2010, Boca Raton:
Taylor & Francis. xvi, 521 p., 8 p. of plates.
ACCEPTED MANUSCRIPT
34
70. Pukrittayakamee, S., et al., The Hyaluronidase Activities of Some Southeast

Asian Snake-Venoms. Toxicon, 1988. 26(7): p. 629-637, DOI: Doi
10.1016/0041-0101(88)90245-0.
71. Lomonte, B., et al., Snake venomics and antivenomics of the arboreal
neotropical pitvipers Bothriechis lateralis and Bothriechis schlegelii. J
Proteome Res, 2008. 7(6): p. 2445-57, DOI: 10.1021/pr8000139.
T
72. Lomonte, B., et al., Snake venomics and toxicological profiling of the arboreal
pitviper Bothriechis supraciliaris from Costa Rica. Toxicon, 2012. 59(5): p.
IP
592-9, DOI: 10.1016/j.toxicon.2012.01.005.
73. Chen, W., et al., Fasxiator, a novel factor XIa inhibitor from snake venom, and
R
its site-specific mutagenesis to improve potency and selectivity. J Thromb
Haemost, 2015. 13(2): p. 248-61, DOI: 10.1111/jth.12797.
SC
74. Earl, S.T.H., et al., Identification and characterisation of Kunitz-type plasma
kallikrein inhibitors unique to Oxyuranus sp snake venoms. Biochimie, 2012.
94(2): p. 365-373, DOI: 10.1016/j.biochi.2011.08.003.
NU
75. Guo, C.T., et al., Trypsin and chymotrypsin inhibitor peptides from the venom of
Chinese Daboia russellii siamensis. Toxicon, 2013. 63: p. 154-64, DOI:
10.1016/j.toxicon.2012.12.013.
MA
76. Mukherjee, A.K., S.P. Mackessy, and S. Dutta, Characterization of a Kunitz-
type protease inhibitor peptide (Rusvikunin) purified from Daboia russelii
russelii venom. Int J Biol Macromol, 2014. 67: p. 154-62, DOI:
10.1016/j.ijbiomac.2014.02.058.
77. Morjen, M., et al., PIVL, a new serine protease inhibitor from Macrovipera
D
lebetina transmediterranea venom, impairs motility of human glioblastoma

TE
cells. Matrix Biol, 2013. 32(1): p. 52-62, DOI: 10.1016/j.matbio.2012.11.015.

78. Morjen, M., et al., PIVL, a snake venom Kunitz-type serine protease inhibitor,
inhibits in vitro and in vivo angiogenesis. Microvasc Res, 2014. 95: p. 149-56,
P
DOI: 10.1016/j.mvr.2014.08.006.
79. Flight, S.M., et al., Textilinin-1, an alternative anti-bleeding agent to aprotinin:
CE
Importance of plasmin inhibition in controlling blood loss. Br J Haematol, 2009.

145(2): p. 207-11, DOI: 10.1111/j.1365-2141.2009.07605.x.
80. Masci, P.P., et al., Textilinins from Pseudonaja textilis textilis. Characterization
AC
of two plasmin inhibitors that reduce bleeding in an animal model. Blood

Coagul Fibrinolysis, 2000. 11(4): p. 385-93.
81. Millers, E.K., et al., Crystal structure of textilinin-1, a Kunitz-type serine
protease inhibitor from the venom of the Australian common brown snake
(Pseudonaja textilis). FEBS J, 2009. 276(11): p. 3163-75, DOI: 10.1111/j.1742-
4658.2009.07034.x.
82. Perez, J.C. and E.E. Sanchez, Natural protease inhibitors to hemorrhagins in
snake venoms and their potential use in medicine. Toxicon, 1999. 37(5): p. 703-
28.
83. Mannion, R.J., et al., Neurotrophins: peripherally and centrally acting
modulators of tactile stimulus-induced inflammatory pain hypersensitivity. Proc
Natl Acad Sci U S A, 1999. 96(16): p. 9385-90.
84. Kostiza, T., et al., Nerve growth factor from the venom of the Chinese cobra
Naja naja atra: purification and description of non-neuronal activities. Toxicon,
1995. 33(10): p. 1249-61.
85. Dhananjaya, B.L. and D.S. CJ, An overview on nucleases (DNase, RNase, and
phosphodiesterase) in snake venoms. Biochemistry (Mosc), 2010. 75(1): p. 1-6.
ACCEPTED MANUSCRIPT
35
86. Russell, F.E., F.W. Buess, and Strassbe.J, Zootoxicological Properties of Venom
Phosphodiesterase. Federation Proceedings, 1962. 21(2): p. 242-&.
87. Santoro, M.L., et al., NPP-BJ, a nucleotide pyrophosphatase/phosphodiesterase
from Bothrops jararaca snake venom, inhibits platelet aggregation. Toxicon,
2009. 54(4): p. 499-512, DOI: 10.1016/j.toxicon.2009.05.016.
88. UZAWA, S., Über die phosphomonoesterase und die phosphodiesterase.
T
Journal of Biochemistry, 1932. 15(1): p. 1-10.
89. Aloulou, A., et al., Phospholipases: an overview. Methods Mol Biol, 2012. 861:
IP
p. 63-85, DOI: 10.1007/978-1-61779-600-5_4.
90. Bernheimer, A.W., et al., Isolation and characterization of a phospholipase B
R
from venom of Collett's snake, Pseudechis colletti. Toxicon, 1987. 25(5): p. 547-
54.
SC
91. Viala, V.L., et al., Pseudechis guttatus venom proteome: Insights into evolution
and toxin clustering. Journal of Proteomics, 2014. 110: p. 32-44, DOI:
10.1016/j.jprot.2014.07.030.
NU
92. Junqueira-de-Azevedo, I.L., et al., Lachesis muta (Viperidae) cDNAs reveal
diverging pit viper molecules and scaffolds typical of cobra (Elapidae) venoms:
implications for snake toxin repertoire evolution. Genetics, 2006. 173(2): p.
MA
877-89, DOI: 10.1534/genetics.106.056515.
93. Otrock, Z.K., J.A. Makarem, and A.I. Shamseddine, Vascular endothelial
growth factor family of ligands and receptors: review. Blood Cells Mol Dis,
2007. 38(3): p. 258-68, DOI: 10.1016/j.bcmd.2006.12.003.
94. Yamazaki, Y., et al., Snake venom vascular endothelial growth factors (VEGFs)
D
exhibit potent activity through their specific recognition of KDR (VEGF

TE
receptor 2). J Biol Chem, 2003. 278(52): p. 51985-8, DOI:

10.1074/jbc.C300454200.
95. Pung, Y.F., et al., Ohanin, a novel protein from king cobra venom, induces
P
hypolocomotion and hyperalgesia in mice. J Biol Chem, 2005. 280(13): p.

13137-47, DOI: 10.1074/jbc.M414137200.
CE
96. Fry, B.G., et al., Evolution of an arsenal: structural and functional

diversification of the venom system in the advanced snakes (Caenophidia). Mol
Cell Proteomics, 2008. 7(2): p. 215-46, DOI: 10.1074/mcp.M700094-MCP200.
AC
97. Corrêa-Netto, C., et al., Snake venomics and venom gland transcriptomic
analysis of Brazilian coral snakes, Micrurus altirostris and M. corallinus. J
Proteomics, 2011. 74(9): p. 1795-809, DOI: 10.1016/j.jprot.2011.04.003.
98. Nair, D.G., et al., Antimicrobial activity of omwaprin, a new member of the
waprin family of snake venom proteins. Biochem J, 2007. 402(1): p. 93-104,
DOI: 10.1042/BJ20060318.
99. Robert J. Wordinger, A.F.C., Growth Factors and Neurotrophic Factors as
Targets, in Ocular Therapeutics: Eye on New Discoveries, A.F.C.a.M.B.W.
Thomas Yorio, Editor. 2008, Academic Press: Amsterdam; Boston; London. p.
87-116, DOI: http://dx.doi.org/10.1016/B978-012370585-3.50007-8.
100. Aloe, L., Rita Levi-Montalcini: the discovery of nerve growth factor and modern
neurobiology. Trends Cell Biol, 2004. 14(7): p. 395-9, DOI:
10.1016/j.tcb.2004.05.011.
101. Cohen, S. and R. Levi-Montalcini, Purification and properties of a nerve
growth-promoting factor isolated from mouse sarcoma 180. Cancer Res, 1957.
17(1): p. 15-20.
102. Cohen, S. and R. Levi-Montalcini, A Nerve Growth-Stimulating Factor Isolated
from Snake Venom. Proc Natl Acad Sci U S A, 1956. 42(9): p. 571-4.
ACCEPTED MANUSCRIPT
36
103. Cohen, S., The stimulation of epidermal proliferation by a specific protein

(EGF). Dev Biol, 1965. 12(3): p. 394-407.
104. Ross, R., et al., A platelet-dependent serum factor that stimulates the
proliferation of arterial smooth muscle cells in vitro. Proc Natl Acad Sci U S A,
1974. 71(4): p. 1207-10.
105. Kostiza, T. and J. Meier, Nerve growth factors from snake venoms: chemical
T
properties, mode of action and biological significance. Toxicon, 1996. 34(7): p.
787-806.
IP
106. Lavin, M.F., et al., Snake Venom Nerve Growth Factors, in Handbook of venoms
and toxins of reptiles, S.P. Mackessy, Editor. 2010, CRC Press/Taylor &
R
Francis: Boca Raton. p. 377-391.
107. Angeletti, R.H., Nerve growth factor from cobra venom. Proc Natl Acad Sci U S
SC
A, 1970. 65(3): p. 668-74.
108. Earl, S.T., et al., Post-translational modification accounts for the presence of
varied forms of nerve growth factor in Australian elapid snake venoms.
NU
Proteomics, 2006. 6(24): p. 6554-65, DOI: 10.1002/pmic.200600263.
109. Koh, D., A. Armugam, and K. Jeyaseelan, Sputa nerve growth factor forms a
preferable substitute to mouse 7S-beta nerve growth factor. Biochem J, 2004.
MA
383(Pt 1): p. 149-58, DOI: 10.1042/BJ20040569.
110. Osipov, A.V., et al., Nerve growth factor from cobra venom inhibits the growth
of Ehrlich tumor in mice. Toxins (Basel), 2014. 6(3): p. 784-95, DOI:
10.3390/toxins6030784.
111. Pearce, F.L., et al., The isolation and characterization of nerve-growth factor
D
from the venom of Vipera russelli. Eur J Biochem, 1972. 29(3): p. 417-25.
TE
112. Hogue-Angeletti, R.A., et al., Purification, characterization, and partial amino

acid sequence of nerve growth factor from cobra venom. Biochemistry, 1976.
15(1): p. 26-34.
P
113. Sanz, L., et al., Venom proteomes of closely related Sistrurus rattlesnakes with
divergent diets. J Proteome Res, 2006. 5(9): p. 2098-112, DOI:
CE
10.1021/pr0602500.
114. Guo, L.Y., et al., Cloning of a cDNA encoding a nerve growth factor precursor
from the Agkistrodon halys Pallas. Toxicon, 1999. 37(3): p. 465-70.
AC
115. Suter, U., J.V. Heymach, Jr., and E.M. Shooter, Two conserved domains in the
NGF propeptide are necessary and sufficient for the biosynthesis of correctly
processed and biologically active NGF. EMBO J, 1991. 10(9): p. 2395-400.
116. Kashima, S., et al., cDNA sequence and molecular modeling of a nerve growth
factor from Bothrops jararacussu venomous gland. Biochimie, 2002. 84(7): p.
675-80.
117. Yamazaki, Y. and T. Morita, Molecular and functional diversity of vascular
endothelial growth factors. Mol Divers, 2006. 10(4): p. 515-27, DOI:
10.1007/s11030-006-9027-3.
118. Boldrini-Franca, J., et al., Crotalus durissus collilineatus venom gland
transcriptome: analysis of gene expression profile. Biochimie, 2009. 91(5): p.
586-95, DOI: 10.1016/j.biochi.2009.02.001.
119. Junqueira-de-Azevedo Ide, L. and P.L. Ho, A survey of gene expression and
diversity in the venom glands of the pitviper snake Bothrops insularis through
the generation of expressed sequence tags (ESTs). Gene, 2002. 299(1-2): p. 279-
91.
ACCEPTED MANUSCRIPT
37
120. Rodrigues, R.S., et al., Combined snake venomics and venom gland
transcriptomic analysis of Bothropoides pauloensis. J Proteomics, 2012. 75(9):
p. 2707-20, DOI: 10.1016/j.jprot.2012.03.028.
121. Sheen, I.S., et al., Clinical significance of the expression of isoform 165 vascular
endothelial growth factor mRNA in noncancerous liver remnants of patients
with hepatocellular carcinoma. World J Gastroenterol, 2005. 11(2): p. 187-92.
T
122. Komori, Y., et al., Vascular endothelial growth factor VEGF-like heparin-
binding protein from the venom of Vipera aspis aspis (Aspic viper).
IP
Biochemistry, 1999. 38(36): p. 11796-803.
123. Junqueira de Azevedo, I.L., et al., Molecular cloning and expression of a
R
functional snake venom vascular endothelium growth factor (VEGF) from the
Bothrops insularis pit viper. A new member of the VEGF family of proteins. J
SC
Biol Chem, 2001. 276(43): p. 39836-42, DOI: 10.1074/jbc.M106531200.
124. Yamazaki, Y., et al., Identification of the heparin-binding region of snake venom
vascular endothelial growth factor (VEGF-F) and its blocking of VEGF-A165.
NU
Biochemistry, 2005. 44(24): p. 8858-64, DOI: 10.1021/bi050197d.
125. Kaji, T., et al., The vascular endothelial growth factor VEGF165 induces
perlecan synthesis via VEGF receptor-2 in cultured human brain microvascular
MA
endothelial cells. Biochim Biophys Acta, 2006. 1760(9): p. 1465-74, DOI:
10.1016/j.bbagen.2006.06.010.
126. Nieminen, T., et al., The impact of the receptor binding profiles of the vascular
endothelial growth factors on their angiogenic features. Biochim Biophys Acta,
2014. 1840(1): p. 454-63, DOI: 10.1016/j.bbagen.2013.10.005.
D
127. Simons, M., E. Gordon, and L. Claesson-Welsh, Mechanisms and regulation of

TE
endothelial VEGF receptor signalling. Nat Rev Mol Cell Biol, 2016, DOI:
10.1038/nrm.2016.87.
128. Tuszynski, M.H. and A. Blesch, Nerve growth factor: from animal models of
P
cholinergic neuronal degeneration to gene therapy in Alzheimer's disease. Prog

Brain Res, 2004. 146: p. 441-9.
CE
129. Sun, W., et al., The effect of collagen-binding NGF-beta on the promotion of
sciatic nerve regeneration in a rat sciatic nerve crush injury model.
Biomaterials, 2009. 30(27): p. 4649-56, DOI:
AC
10.1016/j.biomaterials.2009.05.037.
130. Allen, S.J. and D. Dawbarn, Clinical relevance of the neurotrophins and their
receptors. Clin Sci (Lond), 2006. 110(2): p. 175-91, DOI: 10.1042/CS20050161.
131. Crafts, T.D., et al., Vascular endothelial growth factor: therapeutic possibilities
and challenges for the treatment of ischemia. Cytokine, 2015. 71(2): p. 385-93,
DOI: 10.1016/j.cyto.2014.08.005.
132. Yla-Herttuala, S., et al., Vascular endothelial growth factors: biology and
current status of clinical applications in cardiovascular medicine. J Am Coll
Cardiol, 2007. 49(10): p. 1015-26, DOI: 10.1016/j.jacc.2006.09.053.
133. Dalal, S., et al., Vascular endothelial growth factor: a therapeutic target for
tumors of the Ewing's sarcoma family. Clin Cancer Res, 2005. 11(6): p. 2364-
78, DOI: 10.1158/1078-0432.CCR-04-1201.
134. Ferrara, N., Vascular endothelial growth factor as a target for anticancer
therapy. Oncologist, 2004. 9 Suppl 1: p. 2-10.
135. Duran-Reynals, F., A Spreading Factor in Certain Snake Venoms and Its
Relation to Their Mode of Action. J Exp Med, 1939. 69(1): p. 69-81.
136. Wahby, A.F., et al., Egyptian horned viper Cerastes cerastes venom
hyaluronidase: purification, partial characterization and evidence for its action
ACCEPTED MANUSCRIPT
38
as a spreading factor. Toxicon, 2012. 60(8): p. 1380-9, DOI:

10.1016/j.toxicon.2012.08.016.
137. Girish, K.S., et al., Hyaluronidase and protease activities from Indian snake
venoms: neutralization by Mimosa pudica root extract. Fitoterapia, 2004. 75(3-
4): p. 378-80, DOI: 10.1016/j.fitote.2004.01.006.
138. Bordon, K.C., et al., Isolation, enzymatic characterization and antiedematogenic
T
activity of the first reported rattlesnake hyaluronidase from Crotalus durissus
terrificus venom. Biochimie, 2012. 94(12): p. 2740-8, DOI:
IP
10.1016/j.biochi.2012.08.014.
139. Wohlrab, J., et al., Use of hyaluronidase for pharmacokinetic increase in
R
bioavailability of intracutaneously applied substances. Skin Pharmacol Physiol,
2014. 27(5): p. 276-82, DOI: 10.1159/000360545.
SC
140. Tan, C.H., et al., Unveiling the elusive and exotic: Venomics of the Malayan
blue coral snake (Calliophis bivirgata flaviceps). J Proteomics, 2016. 132: p. 1-
12, DOI: 10.1016/j.jprot.2015.11.014.
NU
141. Wiezel, G.A., et al., Identification of hyaluronidase and phospholipase B in
Lachesis muta rhombeata venom. Toxicon, 2015. 107(Pt B): p. 359-68, DOI:
10.1016/j.toxicon.2015.08.029.
MA
142. Xu, X., et al., Purification and partial characterization of hyaluronidase from
five pace snake (Agkistrodon acutus) venom. Toxicon, 1982. 20(6): p. 973-81.
143. Kudo, K. and A.T. Tu, Characterization of hyaluronidase isolated from
Agkistrodon contortrix contortrix (Southern Copperhead) venom. Arch Biochem
Biophys, 2001. 386(2): p. 154-62, DOI: 10.1006/abbi.2000.2204.
D
144. Mahadeswaraswamy, Y.H., et al., Daboia russelli venom hyaluronidase:

TE
purification, characterization and inhibition by beta-3-(3-hydroxy-4-oxopyridyl)

alpha-amino-propionic Acid. Curr Top Med Chem, 2011. 11(20): p. 2556-65.
145. Antunes, T.C., et al., Comparative analysis of newborn and adult Bothrops
P
jararaca snake venoms. Toxicon, 2010. 56(8): p. 1443-58, DOI:

10.1016/j.toxicon.2010.08.011.
CE
146. Queiroz, G.P., et al., Interspecific variation in venom composition and toxicity of
Brazilian snakes from Bothrops genus. Toxicon, 2008. 52(8): p. 842-51, DOI:
10.1016/j.toxicon.2008.10.002.
AC
147. Guerra-Duarte, C., et al., Partial in vitro analysis of toxic and antigenic
activities of eleven Peruvian pitviper snake venoms. Toxicon, 2015. 108: p. 84-
96, DOI: 10.1016/j.toxicon.2015.09.007.
148. Shashidharamurthy, R., et al., Variations in biochemical and pharmacological
properties of Indian cobra (Naja naja naja) venom due to geographical
distribution. Mol Cell Biochem, 2002. 229(1-2): p. 93-101.
149. Shashidharamurthy, R. and K. Kemparaju, Region-specific neutralization of
Indian cobra (Naja naja) venom by polyclonal antibody raised against the
eastern regional venom: A comparative study of the venoms from three different
geographical distributions. Int Immunopharmacol, 2007. 7(1): p. 61-9, DOI:
10.1016/j.intimp.2006.08.014.
150. Pukrittayakamee, S., et al., Characterization of a monoclonal antibody that
neutralizes the hyaluronidase activity of Russell's viper venom. Southeast Asian
J Trop Med Public Health, 1990. 21(2): p. 231-7.
151. Girish, K.S. and K. Kemparaju, Inhibition of Naja naja venom hyaluronidase by
plant-derived bioactive components and polysaccharides. Biochemistry (Mosc),
2005. 70(8): p. 948-52.
ACCEPTED MANUSCRIPT
39
152. Khanum, S.A., et al., Synthesis of benzoyl phenyl benzoates as effective

inhibitors for phospholipase A2 and hyaluronidase enzymes. Bioorg Med Chem
Lett, 2005. 15(18): p. 4100-4, DOI: 10.1016/j.bmcl.2005.06.012.
153. Sunitha, K., et al., Inhibition of hyaluronidase by N-acetyl cysteine and
glutathione: role of thiol group in hyaluronan protection. Int J Biol Macromol,
2013. 55: p. 39-46, DOI: 10.1016/j.ijbiomac.2012.12.047.
T
154. Molander, M., et al., Hyaluronidase, phospholipase A2 and protease inhibitory
activity of plants used in traditional treatment of snakebite-induced tissue
IP
necrosis in Mali, DR Congo and South Africa. J Ethnopharmacol, 2014. 157: p.
171-80, DOI: 10.1016/j.jep.2014.09.027.
R
155. Liu, Y., et al., High-resolution hyaluronidase inhibition profiling combined with
HPLC-HRMS-SPE-NMR for identification of anti-necrosis constituents in
SC
Chinese plants used to treat snakebite. Phytochemistry, 2015. 119: p. 62-9, DOI:
10.1016/j.phytochem.2015.09.005.
156. Nanjaraj Urs, A.N., et al., Local and systemic toxicity of Echis carinatus venom:
NU
neutralization by Cassia auriculata L. leaf methanol extract. J Nat Med, 2015.
69(1): p. 111-22, DOI: 10.1007/s11418-014-0875-3.
157. Utkin, Y.N. and A.V. Osipov, Non-lethal polypeptide components in cobra
MA
venom. Curr Pharm Des, 2007. 13(28): p. 2906-15.
158. Yamazaki, Y., F. Hyodo, and T. Morita, Wide distribution of cysteine-rich
secretory proteins in snake venoms: isolation and cloning of novel snake venom
cysteine-rich secretory proteins. Arch Biochem Biophys, 2003. 412(1): p. 133-
41.
D
159. Lecht, S., et al., Anti-angiogenic activities of snake venom CRISP isolated from
TE
Echis carinatus sochureki. Biochim Biophys Acta, 2015. 1850(6): p. 1169-79,

DOI: 10.1016/j.bbagen.2015.02.002.
160. Wang, Y.L., et al., Cobra CRISP functions as an inflammatory modulator via a
P
novel Zn2+- and heparan sulfate-dependent transcriptional regulation of

endothelial cell adhesion molecules. J Biol Chem, 2010. 285(48): p. 37872-83,
CE
DOI: 10.1074/jbc.M110.146290.
161. Adade, C.M., et al., Crovirin, a snake venom cysteine-rich secretory protein
(CRISP) with promising activity against Trypanosomes and Leishmania. PLoS
AC
Negl Trop Dis, 2014. 8(10): p. e3252, DOI: 10.1371/journal.pntd.0003252.

162. Aird, S.D., Taxonomic distribution and quantitative analysis of free purine and
pyrimidine nucleosides in snake venoms. Comp Biochem Physiol B Biochem
Mol Biol, 2005. 140(1): p. 109-26, DOI: 10.1016/j.cbpc.2004.09.020.
163. Aird, S.D., et al., Quantitative high-throughput profiling of snake venom gland
transcriptomes and proteomes (Ovophis okinavensis and Protobothrops
flavoviridis). BMC Genomics, 2013. 14: p. 790, DOI: 10.1186/1471-2164-14-
790.
164. Aird, S.D., Ophidian envenomation strategies and the role of purines. Toxicon,
2002. 40(4): p. 335-93.
165. Laustsen, A.H., et al., Snake venomics of monocled cobra (Naja kaouthia) and
investigation of human IgG response against venom toxins. Toxicon, 2015. 99:
p. 23-35, DOI: 10.1016/j.toxicon.2015.03.001.
166. Laustsen, A.H., et al., Unveiling the nature of black mamba (Dendroaspis
polylepis) venom through venomics and antivenom immunoprofiling:
Identification of key toxin targets for antivenom development. J Proteomics,
2015. 119: p. 126-42, DOI: 10.1016/j.jprot.2015.02.002.
ACCEPTED MANUSCRIPT
40
167. Delezenne, C. and H. Morel, Action catalytique des venins des serpents sur les
acids nucleiques. CR Acad. Sci, 1919. 168: p. 244-246.
168. Taborda, A.R., et al., A study of the desoxyribonuclease activity of snake
venoms. J Biol Chem, 1952. 195(1): p. 207-13.
169. Taborda, A.R., et al., A study of the ribonuclease activity of snake venoms. J Biol
Chem, 1952. 194(1): p. 227-33.
T
170. Georgatsos, J.G. and M. Laskowski, Sr., Purification of an endonuclease from
the venom of Bothrops atrox. Biochemistry, 1962. 1: p. 288-95.
IP
171. Iwanaga, S. and T. Suzuki, Enzymes in snake venom, in Snake venoms. 1979,
Springer. p. 61-158.
R
172. Mackessy, S.P., Phosphodiesterases, ribonucleases and deoxyribonucleases.
Enzymes from Snake Venoms, 1998: p. 361-404.
SC
173. Ownby, C.L., J. Bjarnason, and A.T. Tu, Hemorrhagic toxins from rattlesnake
(Crotalus atrox) venom. Pathogenesis of hemorrhage induced by three purified
toxins. Am J Pathol, 1978. 93(1): p. 201-18.
NU
174. Bernheimer, A.W. and B. Rudy, Interactions between membranes and cytolytic
peptides. Biochim Biophys Acta, 1986. 864(1): p. 123-41.
175. Nunez, C.E., Y. Angulo, and B. Lomonte, Identification of the myotoxic site of
MA
the Lys49 phospholipase A(2) from Agkistrodon piscivorus piscivorus snake
venom: synthetic C-terminal peptides from Lys49, but not from Asp49
myotoxins, exert membrane-damaging activities. Toxicon, 2001. 39(10): p.
1587-94.
176. Ma, D., A. Armugam, and K. Jeyaseelan, Cytotoxic potency of cardiotoxin from
D
Naja sputatrix: development of a new cytolytic assay. Biochem. J, 2002. 366: p.

TE
35-43.
177. Boffa, M.C. and G.A. Boffa, Correlations between the enzymatic activities and
the factors active on blood coagulation and platelet aggregation from the venom
P
of Vipera aspis. Biochimica et Biophysica Acta (BBA)-General Subjects, 1974.

354(2): p. 275-290.
CE
178. Ouyang, C. and T.F. Huang, Platelet aggregation inhibitors from Agkistrodon
acutus snake venom. Toxicon, 1986. 24(11): p. 1099-1106.
179. Dhananjaya, B.L., et al., Anticoagulant effect of Naja naja venom
AC
5'nucleotidase: demonstration through the use of novel specific inhibitor,

vanillic acid. Toxicon, 2006. 48(4): p. 411-21, DOI:
10.1016/j.toxicon.2006.06.017.
180. Francis, B., C. Seebart, and I.I. Kaiser, Citrate is an endogenous inhibitor of
snake venom enzymes by metal-ion chelation. Toxicon, 1992. 30(10): p. 1239-
1246.
181. Rael, E.D., Venom phosphatases and 5’-nucleotidases. Enzymes from Snake
Venoms, 1998: p. 405-423.
182. Chen, Y.H. and T.B. Lo, Chemical Studies of Formosan Cobra (Naja Naja Atra)
Venom. Part V. Properties of 5′‐ Nucleotidase. Journal of the Chinese Chemical
Society, 1968. 15(3): p. 84-96.
183. DIECKHOFF, J., et al., An improved procedure for purifying 5′‐ nucleotidase
from various sources. European Journal of Biochemistry, 1985. 151(2): p. 377-
383.
184. Gulland, J.M. and E.M. Jackson, 5-Nucleotidase. Biochemical Journal, 1938.
32(3): p. 597.
ACCEPTED MANUSCRIPT
41
185. da Silva, N.J. and S.D. Aird, Prey specificity, comparative lethality and
compositional differences of coral snake venoms. Comparative Biochemistry
and Physiology Part C: Toxicology & Pharmacology, 2001. 128(3): p. 425-456.
186. Zeller, E.A., The formation of pyrophosphate from adenosine triphosphate in the
presence of a snake venom. Archives of biochemistry, 1950. 28(1): p. 138-139.
187. Caccin, P., et al., Why myotoxin-containing snake venoms possess powerful
T
nucleotidases? Biochem Biophys Res Commun, 2013. 430(4): p. 1289-93, DOI:
10.1016/j.bbrc.2012.11.129.
IP
188. Cintra-Francischinelli, M., et al., Bothrops snake myotoxins induce a large efflux
of ATP and potassium with spreading of cell damage and pain. Proc Natl Acad
R
Sci U S A, 2010. 107(32): p. 14140-5, DOI: 10.1073/pnas.1009128107.
189. Gay, C., et al., Snake Venomics and Antivenomics of Bothrops diporus, a
SC
Medically Important Pitviper in Northeastern Argentina. Toxins (Basel), 2015.
8(1), DOI: 10.3390/toxins8010009.
190. Johnson, M., et al., Enzymic hydrolysis of adenosine phosphates by cobra
NU
venom. Biochemical Journal, 1953. 54(4): p. 625.
191. Shamsi, T.N., R. Parveen, and S. Fatima, Characterization, biomedical and
agricultural applications of protease inhibitors: A review. Int J Biol Macromol,
MA
2016. 91: p. 1120-1133, DOI: 10.1016/j.ijbiomac.2016.02.069.
192. Possani, L.D., et al., Isolation and physiological characterization of taicatoxin, a
complex toxin with specific effects on calcium channels. Toxicon, 1992. 30(11):
p. 1343-64.
193. Willmott, N., et al., Novel Serine-Protease Inhibitor from the Australian Brown
D
Snake, Pseudonaja-Textilis Textilis - Inhibition-Kinetics. Fibrinolysis, 1995.

TE
9(1): p. 1-8, DOI: Doi 10.1016/S0268-9499(08)80040-9.

194. Rodriguez-Ithurralde, D., et al., Fasciculin, a powerful anticholinesterase
polypeptide from Dendroaspis angusticeps venom. Neurochem Int, 1983. 5(3):
P
p. 267-74.
195. Harvey, A.L., Twenty years of dendrotoxins. Toxicon, 2001. 39(1): p. 15-26,
CE
DOI: Doi 10.1016/S0041-0101(00)00162-8.

196. Yang, W.S., et al., BF9, the First Functionally Characterized Snake Toxin
Peptide with Kunitz-Type Protease and Potassium Channel Inhibiting
AC
Properties. Journal of Biochemical and Molecular Toxicology, 2014. 28(2): p.

76-83, DOI: 10.1002/jbt.21538.
197. Fernandez, J., et al., Characterization of a novel snake venom component:
Kazal-type inhibitor-like protein from the arboreal pitviper Bothriechis
schlegelii. Biochimie, 2016. 125: p. 83-90, DOI: 10.1016/j.biochi.2016.03.004.
198. Takahash.H, T. Suzuki, and S. Iwanaga, Isolation of a Novel Inhibitor of
Kallikrein, Plasmin and Trypsin from Venom of Russells Viper (Vipera Russelli).
Febs Letters, 1972. 27(2): p. 207-&, DOI: Doi 10.1016/0014-5793(72)80621-5.
199. Calvete, J.J., C. Marcinkiewicz, and L. Sanz, Snake venomics of Bitis gabonica
gabonica. Protein family composition, subunit organization of venom toxins,
and characterization of dimeric disintegrins bitisgabonin-1 and bitisgabonin-2.
J Proteome Res, 2007. 6(1): p. 326-36, DOI: 10.1021/pr060494k.
200. Chang, L., et al., Purification and characterization of a chymotrypsin inhibitor
from the venom of Ophiophagus hannah (King Cobra). Biochem Biophys Res
Commun, 2001. 283(4): p. 862-7, DOI: 10.1006/bbrc.2001.4878.
201. Shafqat, J., Z.H. Zaidi, and H. Jornvall, Purification and characterization of a
chymotrypsin Kunitz inhibitor type of polypeptide from the venom of cobra
(Naja naja naja). FEBS Lett, 1990. 275(1-2): p. 6-8.
ACCEPTED MANUSCRIPT
42
202. Mukherjee, A.K. and S.P. Mackessy, Pharmacological properties and

pathophysiological significance of a Kunitz-type protease inhibitor (Rusvikunin-
II) and its protein complex (Rusvikunin complex) purified from Daboia russelii
russelii venom. Toxicon, 2014. 89: p. 55-66, DOI:
10.1016/j.toxicon.2014.06.016.
203. Zupunski, V., D. Kordis, and F. Gubensek, Adaptive evolution in the snake
T
venom Kunitz/BPTI protein family. FEBS Lett, 2003. 547(1-3): p. 131-6.
204. Chou, W.M., et al., Structure-function studies on inhibitory activity of Bungarus
IP
multicinctus protease inhibitor-like protein on matrix metalloprotease-2, and
invasion and migration of human neuroblastoma SK-N-SH cells. Toxicon, 2010.
R
55(2-3): p. 353-60, DOI: 10.1016/j.toxicon.2009.08.012.
205. Meta, A., et al., High-yield production and characterization of biologically
SC
active recombinant aprotinin expressed in Saccharomyces cerevisiae. Protein
Expr Purif, 2009. 66(1): p. 22-7, DOI: 10.1016/j.pep.2009.02.005.
206. Ritonja, A., B. Meloun, and F. Gubensek, The primary structure of Vipera
NU
ammodytes venom trypsin inhibitor I. Biochim Biophys Acta, 1983. 748(3): p.
429-35.
207. Gocmen, B., et al., Mass spectrometry guided venom profiling and bioactivity
MA
screening of the Anatolian Meadow Viper, Vipera anatolica. Toxicon, 2015.
107(Pt B): p. 163-74, DOI: 10.1016/j.toxicon.2015.09.013.
208. Tan, N.H., et al., Functional venomics of the Sri Lankan Russell's viper (Daboia
russelii) and its toxinological correlations. J Proteomics, 2015. 128: p. 403-23,
DOI: 10.1016/j.jprot.2015.08.017.
D
209. Petras, D., et al., Venom Proteomics of Indonesian King Cobra, Ophiophagus
TE
hannah: Integrating Top-Down and Bottom-Up Approaches. J Proteome Res,

2015. 14(6): p. 2539-56, DOI: 10.1021/acs.jproteome.5b00305.
210. Wagstaff, S.C., et al., Molecular characterisation of endogenous snake venom
P
metalloproteinase inhibitors. Biochem Biophys Res Commun, 2008. 365(4): p.

650-6, DOI: 10.1016/j.bbrc.2007.11.027.
CE
211. Mora-Obando, D., et al., Proteomic and functional profiling of the venom of
Bothrops ayerbei from Cauca, Colombia, reveals striking interspecific variation
with Bothrops asper venom. J Proteomics, 2014. 96: p. 159-72, DOI:
AC
10.1016/j.jprot.2013.11.005.
212. Salazar-Valenzuela, D., et al., Proteomic and toxicological profiling of the
venom of Bothrocophias campbelli, a pitviper species from Ecuador and
Colombia. Toxicon, 2014. 90: p. 15-25, DOI: 10.1016/j.toxicon.2014.07.012.
213. Kohlhoff, M., et al., Exploring the proteomes of the venoms of the Peruvian pit
vipers Bothrops atrox, B. barnetti and B. pictus. J Proteomics, 2012. 75(7): p.
2181-95, DOI: 10.1016/j.jprot.2012.01.020.
214. Tashima, A.K., et al., Peptidomics of three Bothrops snake venoms: insights into
the molecular diversification of proteomes and peptidomes. Mol Cell
Proteomics, 2012. 11(11): p. 1245-62, DOI: 10.1074/mcp.M112.019331.
215. Kregar, I., et al., Bovine intracellular cysteine proteinases. Acta Biol Med Ger,
1981. 40(10-11): p. 1433-8.
216. Brillard-Bourdet, M., et al., Purification and characterization of a new cystatin
inhibitor from Taiwan cobra (Naja naja atra) venom. Biochem J, 1998. 331 ( Pt
1): p. 239-44.
217. Evans, H.J. and A.J. Barrett, A cystatin-like cysteine proteinase inhibitor from
venom of the African puff adder (Bitis arietans). Biochem J, 1987. 246(3): p.
795-7.
ACCEPTED MANUSCRIPT
43
218. Ritonja, A., et al., Amino acid sequence of a cystatin from venom of the African
puff adder (Bitis arietans). Biochem J, 1987. 246(3): p. 799-802.
219. Hamada, Y. and Y. Kiso, New directions for protease inhibitors directed drug
discoverydagger. Biopolymers, 2015, DOI: 10.1002/bip.22780.
220. Puente, X.S., et al., Human and mouse proteases: a comparative genomic
approach. Nat Rev Genet, 2003. 4(7): p. 544-58, DOI: 10.1038/nrg1111.
T
221. van Hoek, M.L., Antimicrobial peptides in reptiles. Pharmaceuticals (Basel),
2014. 7(6): p. 723-53, DOI: 10.3390/ph7060723.
IP
222. Ganz, T., Defensins: antimicrobial peptides of innate immunity. Nat Rev
Immunol, 2003. 3(9): p. 710-20, DOI: 10.1038/nri1180.
R
223. Samy, R.P., et al., Snake Venom Proteins and Peptides as Novel Antibiotics
Against Microbial Infections. Current Proteomics, 2013. 10(1): p. 10-28, DOI:
SC
http://dx.doi.org/10.2174/1570164611310010003.
224. de Oliveira Junior, N.G., M.H. e Silva Cardoso, and O.L. Franco, Snake venoms:
attractive antimicrobial proteinaceous compounds for therapeutic purposes.
NU
Cell Mol Life Sci, 2013. 70(24): p. 4645-58, DOI: 10.1007/s00018-013-1345-x.
225. Costa, B.A., et al., Interaction of the rattlesnake toxin crotamine with model
membranes. J Phys Chem B, 2014. 118(20): p. 5471-9, DOI:
MA
10.1021/jp411886u.
226. San, T.M., et al., Screening Antimicrobial Activity of Venoms from Snakes
Commonly Found in Malaysia. Journal of Applied Sciences, 2010. 10: p. 2328-
2332.
227. Al-Asmari, A.K., et al., Assessment of the antimicrobial activity of few saudi
D
arabian snake venoms. The Open Microbiology Journal, 2015. 9: p. 18-25, DOI:
TE
10.2174/1874285801509010018.
228. Hakim, M. and M. Reza, In vitro Antibacterial Activity of Snake Venom, Naja
naja from Bangladesh. British Biotechnology Journal, 2015. 8(2): p. 1-5, DOI:
P
10.9734/bbj/2015/18374.
229. Ferreira, B.L., et al., Comparative analysis of viperidae venoms antibacterial
CE
profile: a short communication for proteomics. Evid Based Complement

Alternat Med, 2011. 2011: p. 960267, DOI: 10.1093/ecam/nen052.
230. Gennaro, R. and M. Zanetti, Structural features and biological activities of the
AC
cathelicidin-derived antimicrobial peptides. Biopolymers, 2000. 55(1): p. 31-49,

DOI: 10.1002/1097-0282(2000)55:1<31::AID-BIP40>3.0.CO;2-9.
231. Zanetti, M., Cathelicidins, multifunctional peptides of the innate immunity. J
Leukoc Biol, 2004. 75(1): p. 39-48, DOI: 10.1189/jlb.0403147.
232. Gomes, V.M., et al., Purification and characterization of a novel peptide with
antifungal activity from Bothrops jararaca venom. Toxicon, 2005. 45(7): p. 817-
27, DOI: 10.1016/j.toxicon.2004.12.011.
233. Sachidananda, M.K., S.K. Murari, and D. Channe Gowda, Characterization of
an antibacterial peptide from indian cobra (Naja naja) venom. Journal of
Venomous Animals and Toxins including Tropical Diseases, 2007. 13: p. 446-
461.
234. Correa, P.G. and N. Oguiura, Phylogenetic analysis of beta-defensin-like genes
of Bothrops, Crotalus and Lachesis snakes. Toxicon, 2013. 69: p. 65-74, DOI:
10.1016/j.toxicon.2013.02.013.
235. Siqueira, A.M., et al., A proposed 3D structure for crotamine based on
homology building, molecular simulations and circular dichroism. Journal of
Molecular Graphics and Modelling, 2002. 20(5): p. 389-398, DOI:
http://dx.doi.org/10.1016/S1093-3263(01)00139-5.
ACCEPTED MANUSCRIPT
44
236. Nicastro, G., et al., Solution structure of crotamine, a Na+ channel affecting
toxin from Crotalus durissus terrificus venom. European Journal of
Biochemistry, 2003. 270(9): p. 1969-1979, DOI: 10.1046/j.1432-
1033.2003.03563.x.
237. Torres, A.M. and P.W. Kuchel, The beta-defensin-fold family of polypeptides.
Toxicon, 2004. 44(6): p. 581-8, DOI: 10.1016/j.toxicon.2004.07.011.
T
238. Amer, L.S., B.M. Bishop, and M.L. van Hoek, Antimicrobial and antibiofilm
activity of cathelicidins and short, synthetic peptides against Francisella.
IP
Biochem Biophys Res Commun, 2010. 396(2): p. 246-51, DOI:
10.1016/j.bbrc.2010.04.073.
R
239. Fosgerau, K. and T. Hoffmann, Peptide therapeutics: current status and future
directions. Drug Discov Today, 2015. 20(1): p. 122-8, DOI:
SC
10.1016/j.drudis.2014.10.003.
240. Schulte, I., et al., Peptides in body fluids and tissues as markers of disease.
Expert Review of Molecular Diagnostics, 2005. 5(2): p. 145-157, DOI:
NU
10.1586/14737159.5.2.145.
241. Doery, H.M. and J.E. Pearson, Phospholipase B in snake venoms and bee
venom. Biochem J, 1964. 92(3): p. 599-602.
MA
242. Mohamed, A.H., A. Kamel, and M.H. Ayobe, Studies of phospholipase A and B
activities of Egyptian snake venoms and a scorpion toxin. Toxicon, 1969. 6(4):
p. 293-8.
243. Bernheimer, A.W., S.A. Weinstein, and R. Linder, Isoelectric analysis of some
Australian elapid snake venoms with special reference to phospholipase B and
D
hemolysis. Toxicon, 1986. 24(8): p. 841-9.

TE
244. Rey-Suarez, P., et al., Integrative characterization of the venom of the coral
snake Micrurus dumerilii (Elapidae) from Colombia: Proteome, toxicity, and
cross-neutralization by antivenom. J Proteomics, 2016. 136: p. 262-73, DOI:
P
10.1016/j.jprot.2016.02.006.
245. Chatrath, S.T., et al., Identification of novel proteins from the venom of a cryptic
CE
snake Drysdalia coronoides by a combined transcriptomics and proteomics

approach. J Proteome Res, 2011. 10(2): p. 739-50, DOI: 10.1021/pr1008916.
246. Rokyta, D.R., et al., The venom-gland transcriptome of the eastern diamondback
AC
rattlesnake (Crotalus adamanteus). BMC Genomics, 2012. 13: p. 312, DOI:

10.1186/1471-2164-13-312.
247. Aird, S.D., et al., Snake venoms are integrated systems, but abundant venom
proteins evolve more rapidly. BMC Genomics, 2015. 16: p. 647, DOI:
10.1186/s12864-015-1832-6.
248. Margres, M.J., et al., The venom-gland transcriptome of the eastern coral snake
(Micrurus fulvius) reveals high venom complexity in the intragenomic evolution
of venoms. Bmc Genomics, 2013. 14, DOI: 10.1186/1471-2164-14-531.
249. Flexner, S. and H. Noguchi, Snake Venom in Relation to Haemolysis,
Bacteriolysis, and Toxicity. J Exp Med, 1902. 6(3): p. 277-301.
250. Stephens, J.W.W. and W. Myers, The action of cobra poison on the blood: a
contribution to the study of passive immunity. J. Pathol. Bacteriol., 1898. 5: p.
279-301.
251. Birdsey, V., J. Lindorfer, and H. Gewurz, Interaction of toxic venoms with the
complement system. Immunology, 1971. 21(2): p. 299-310.
252. Smith, C.A., C.W. Vogel, and H.J. Muller-Eberhard, Ultrastructure of cobra
venom factor-dependent C3/C5 convertase and its zymogen, factor B of human
complement. J Biol Chem, 1982. 257(17): p. 9879-82.
ACCEPTED MANUSCRIPT
45
253. Vogel, C.W. and H.J. Muller-Eberhard, Cobra venom factor: improved method
for purification and biochemical characterization. J Immunol Methods, 1984.
73(1): p. 203-20.
254. Vogel, C.W. and H.J. Muller-Eberhard, The cobra venom factor-dependent C3
convertase of human complement. A kinetic and thermodynamic analysis of a
protease acting on its natural high molecular weight substrate. J Biol Chem,
T
1982. 257(14): p. 8292-9.
255. Fritzinger, D.C., et al., Primary structure of cobra complement component C3. J
IP
Immunol, 1992. 149(11): p. 3554-62.
256. Abbas, A.K., A.H. Lichtman, and S. Pillai, Cellular and molecular immunology.
R
2012, Philadelphia: Elsevier.
257. Tambourgi, D.V. and C.W. van den Berg, Animal venoms/toxins and the
SC
complement system. Mol Immunol, 2014. 61(2): p. 153-62, DOI:
10.1016/j.molimm.2014.06.020.
258. Junqueira-de-Azevedo, I.L., et al., Venom-related transcripts from Bothrops
NU
jararaca tissues provide novel molecular insights into the production and
evolution of snake venom. Mol Biol Evol, 2015. 32(3): p. 754-66, DOI:
10.1093/molbev/msu337.
MA
259. Nicolau, C.A., et al., An in-depth snake venom proteopeptidome
characterization: Benchmarking Bothrops jararaca. J Proteomics, 2016, DOI:
10.1016/j.jprot.2016.06.029.
260. Vogel, C.W., et al., Recombinant cobra venom factor. Mol Immunol, 2004.
41(2-3): p. 191-9, DOI: 10.1016/j.molimm.2004.03.011.
D
261. Vogel, C.W. and D.C. Fritzinger, Humanized cobra venom factor: experimental
TE
therapeutics for targeted complement activation and complement depletion. Curr

Pharm Des, 2007. 13(28): p. 2916-26.
262. Vejayan, J., T.L. Khoon, and H. Ibrahim, Comparative analysis of the venom
P
proteome of four important Malaysian snake species. J Venom Anim Toxins

Incl Trop Dis, 2014. 20(1): p. 6, DOI: 10.1186/1678-9199-20-6.
CE
263. Li, S., et al., Proteomic characterization of two snake venoms: Naja naja atra
and Agkistrodon halys. Biochem J, 2004. 384(Pt 1): p. 119-27, DOI:
10.1042/BJ20040354.
AC
264. Birrell, G.W., et al., The diversity of bioactive proteins in Australian snake
venoms. Mol Cell Proteomics, 2007. 6(6): p. 973-86, DOI:
10.1074/mcp.M600419-MCP200.
265. Pung, Y.F., et al., Ohanin, a novel protein from king cobra venom: its cDNA and
genomic organization. Gene, 2006. 371(2): p. 246-56, DOI:
10.1016/j.gene.2005.12.002.
266. Ogawa, Y., et al., Characterization and cDNA cloning of dipeptidyl peptidase IV
from the venom of Gloydius blomhoffi brevicaudus. Comp Biochem Physiol B
Biochem Mol Biol, 2006. 145(1): p. 35-42, DOI: 10.1016/j.cbpb.2006.05.013.
267. Aird, S.D., Snake venom dipeptidyl peptidase IV: taxonomic distribution and
quantitative variation. Comp Biochem Physiol B Biochem Mol Biol, 2008.
150(2): p. 222-8, DOI: 10.1016/j.cbpb.2008.03.005.
268. Ogawa, Y., et al., Exosome-like vesicles in Gloydius blomhoffii blomhoffii
venom. Toxicon, 2008. 51(6): p. 984-93, DOI: 10.1016/j.toxicon.2008.02.003.
269. Cardoso, K.C., et al., A transcriptomic analysis of gene expression in the venom
gland of the snake Bothrops alternatus (urutu). BMC Genomics, 2010. 11: p.
605, DOI: 10.1186/1471-2164-11-605.
ACCEPTED MANUSCRIPT
46
270. Gao, J.F., et al., Neonate-to-adult transition of snake venomics in the short-
tailed pit viper, Gloydius brevicaudus. J Proteomics, 2013. 84: p. 148-57, DOI:
10.1016/j.jprot.2013.04.003.
271. Dias, G.S., et al., Individual variability in the venom proteome of juvenile
Bothrops jararaca specimens. J Proteome Res, 2013. 12(10): p. 4585-98, DOI:
10.1021/pr4007393.
T
272. Yamasaki, S.C., et al., Aminopeptidase activities, oxidative stress and renal
function in Crotalus durissus terrificus envenomation in mice. Toxicon, 2008.
IP
52(3): p. 445-54, DOI: 10.1016/j.toxicon.2008.06.015.
273. Vaiyapuri, S., et al., Purification and functional characterisation of
R
rhiminopeptidase A, a novel aminopeptidase from the venom of Bitis gabonica
rhinoceros. PLoS Negl Trop Dis, 2010. 4(8): p. e796, DOI:
SC
10.1371/journal.pntd.0000796.
274. Zini, S., et al., Identification of metabolic pathways of brain angiotensin II and
III using specific aminopeptidase inhibitors: predominant role of angiotensin III
NU
in the control of vasopressin release. Proc Natl Acad Sci U S A, 1996. 93(21): p.
11968-73.
275. Francischetti, I.M., et al., Bitis gabonica (Gaboon viper) snake venom gland:
MA
toward a catalog for the full-length transcripts (cDNA) and proteins. Gene,
2004. 337: p. 55-69, DOI: 10.1016/j.gene.2004.03.024.
276. Qinghua, L., et al., A catalog for transcripts in the venom gland of the
Agkistrodon acutus: identification of the toxins potentially involved in
coagulopathy. Biochem Biophys Res Commun, 2006. 341(2): p. 522-31, DOI:
D
10.1016/j.bbrc.2006.01.006.
TE
277. Ichijo, H., et al., Molecular cloning and characterization of ficolin, a multimeric
protein with fibrinogen- and collagen-like domains. J Biol Chem, 1993. 268(19):
p. 14505-13.
P
278. Kakinuma, Y., et al., Molecular cloning and characterization of novel ficolins
from Xenopus laevis. Immunogenetics, 2003. 55(1): p. 29-37, DOI:
CE
10.1007/s00251-003-0552-2.
279. OmPraba, G., et al., Identification of a novel family of snake venom proteins
Veficolins from Cerberus rynchops using a venom gland transcriptomics and
AC
proteomics approach. J Proteome Res, 2010. 9(4): p. 1882-93, DOI:

10.1021/pr901044x.
280. Ching, A.T., et al., Some aspects of the venom proteome of the Colubridae snake
Philodryas olfersii revealed from a Duvernoy's (venom) gland transcriptome.
FEBS Lett, 2006. 580(18): p. 4417-22, DOI: 10.1016/j.febslet.2006.07.010.
281. St Pierre, L., et al., Identification and analysis of venom gland-specific genes
from the coastal taipan (Oxyuranus scutellatus) and related species. Cell Mol
Life Sci, 2005. 62(22): p. 2679-93, DOI: 10.1007/s00018-005-5384-9.
282. Margres, M.J., et al., Linking the transcriptome and proteome to characterize
the venom of the eastern diamondback rattlesnake (Crotalus adamanteus). J
Proteomics, 2014. 96: p. 145-58, DOI: 10.1016/j.jprot.2013.11.001.
283. Casewell, N.R., et al., Comparative venom gland transcriptome surveys of the
saw-scaled vipers (Viperidae: Echis) reveal substantial intra-family gene
diversity and novel venom transcripts. BMC Genomics, 2009. 10: p. 564, DOI:
10.1186/1471-2164-10-564.
284. Wagstaff, S.C. and R.A. Harrison, Venom gland EST analysis of the saw-scaled
viper, Echis ocellatus, reveals novel alpha9beta1 integrin-binding motifs in
venom metalloproteinases and a new group of putative toxins, renin-like
ACCEPTED MANUSCRIPT
47
aspartic proteases. Gene, 2006. 377: p. 21-32, DOI:

10.1016/j.gene.2006.03.008.
285. Chapeaurouge, A., et al., Interrogating the Venom of the Viperid Snake Sistrurus
catenatus edwardsii by a Combined Approach of Electrospray and MALDI Mass
Spectrometry. PLoS One, 2015. 10(5): p. e0092091, DOI:
10.1371/journal.pone.0092091.
T
286. Georgieva, D., et al., Pseudechis australis venomics: adaptation for a defense
against microbial pathogens and recruitment of body transferrin. J Proteome
IP
Res, 2011. 10(5): p. 2440-64, DOI: 10.1021/pr101248e.
287. Junqueira-de-Azevedo Ide, L., et al., Cloning and expression of calglandulin, a
R
new EF-hand protein from the venom glands of Bothrops insularis snake in E.
coli. Biochim Biophys Acta, 2003. 1648(1-2): p. 90-8.
SC
288. Hargreaves, A.D., et al., Restriction and recruitment-gene duplication and the
origin and evolution of snake venom toxins. Genome Biol Evol, 2014. 6(8): p.
2088-95, DOI: 10.1093/gbe/evu166.
NU
289. Calvete, J.J., P. Juarez, and L. Sanz, Snake venomics. Strategy and applications.
J Mass Spectrom, 2007. 42(11): p. 1405-14, DOI: 10.1002/jms.1242.
290. Schoni, R., The use of snake venom-derived compounds for new functional
MA
diagnostic test kits in the field of haemostasis. Pathophysiology of Haemostasis
and Thrombosis, 2005. 34(4-5): p. 234-240, DOI: 10.1159/000092430.
291. Koh, D.C.I., A. Armugam, and K. Jeyaseelan, Snake venom components and
their applications in biomedicine. Cellular and Molecular Life Sciences, 2006.
63(24): p. 3030-3041, DOI: 10.1007/s00018-006-6315-0.
D
292. Chan, Y.S., et al., Snake venom toxins: toxicity and medicinal applications. Appl
TE
Microbiol Biotechnol, 2016. 100(14): p. 6165-81, DOI: 10.1007/s00253-016-

7610-9.
293. Harvey, A.L., Toxins and drug discovery. Toxicon, 2014. 92: p. 193-200, DOI:
P
10.1016/j.toxicon.2014.10.020.
294. Reyes-Velasco, J., et al., Expression of venom gene homologs in diverse python
CE
tissues suggests a new model for the evolution of snake venom. Mol Biol Evol,
2015. 32(1): p. 173-83, DOI: 10.1093/molbev/msu294.
295. Fry, B.G., From genome to "venome": molecular origin and evolution of the
AC
snake venom proteome inferred from phylogenetic analysis of toxin sequences

and related body proteins. Genome Res, 2005. 15(3): p. 403-20, DOI:
10.1101/gr.3228405.
296. Lynch, V.J., Inventing an arsenal: adaptive evolution and neofunctionalization
of snake venom phospholipase A2 genes. BMC Evol Biol, 2007. 7: p. 2, DOI:
10.1186/1471-2148-7-2.
297. Fry, B.G., et al., Molecular evolution and phylogeny of elapid snake venom
three-finger toxins. J Mol Evol, 2003. 57(1): p. 110-29, DOI: 10.1007/s00239-
003-2461-2.
298. Casewell, N.R., et al., Complex cocktails: the evolutionary novelty of venoms.
Trends Ecol Evol, 2013. 28(4): p. 219-29, DOI: 10.1016/j.tree.2012.10.020.
299. Casewell, N.R., et al., Domain loss facilitates accelerated evolution and
neofunctionalization of duplicate snake venom metalloproteinase toxin genes.
Mol Biol Evol, 2011. 28(9): p. 2637-49, DOI: 10.1093/molbev/msr091.
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FIGURE CAPTIONS
Figure 1. Schematic representation of adenosine production/generation by snake

venom toxins. During the envenoming, toxins (in green) promote cellular lysis,
releasing nucleic acids (DNA and RNA). Nucleic acids become susceptible to the action
T
of snake venom nucleases, resulting in the production of 5’-nucleotides, which are
IP
cleaved by snake venom phosphatases, generating adenosine. Another pathway to
R
produce adenosine involves the cleavage of ATP by ATPase or phosphodiesterase,
SC
producing ADP and AMP, which are degraded by nucleotidases, yielding adenosine.
Figure 2. Minor snake venom protein classes and their main potential applications.
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The summary of the minor protein classes discussed herein are illustrated. Magenta:
antimicrobial class. Green: Proteases inhibitors class. Yellow: Growth factors class.
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Blue: other classes. The possible applications are numbered and indicated in the right
panel, accordingly.
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P TE
CE
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Figure 1
T
RIP
SC
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MA
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TE
P
CE
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Figure 2
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RIP
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MA
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TE
P
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HIGHLIGHTS
 Minor snake venom protein classes and their potential applications are still
T
poorly studied;
IP
 Improving the understanding of snake venom protein diversity is important for
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drug discovery and to better comprehend the pathophysiology of snake bites;
SC
 Minor snake venom protein classes may also provide important information
regarding the evolutionary processes that generated snake venom repertories.
NU
MA
D
P TE
CE
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Aplicacion Farmacologica Venenos

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Minor snake venom proteins: structure, function and potential applications

Johara Boldrini-França, Camila Takeno Cologna, Manuela Berto Pucca,

To appear in: BBA - General Subjects

Received date: 20 August 2016

Minor snake venom proteins: structure, function and potential applications

Johara Boldrini-Françaa; Camila Takeno Colognaa; Manuela Berto Puccab; Karla de

Phone: +55 16 33154275, Fax: +55 16 33154880, E-mail: ecabraga@fcfrp.usp.br

Camila Takeno Cologna: camilatcbio@gmail.com;

Eliane Candiani Arantes: ecabraga@fcfrp.usp.br;

Fernanda Gobbi Amorim: fernandagamorim@gmail.com;

Snake venoms present a great diversity of pharmacologically active compounds

Keywords: snake venoms; toxins; drug discovery.

injection or extraction [8-10]. However, synthesis and secretion of different protein

different stages of the venom production cycle [11].

Many regulatory mechanisms are supposed to impact protein composition in

toxin-related genes [13], post-transcriptional microRNAs regulation [14] and proteolytic

contradictory considering that the maintenance of the venom repertoire is considerably

nonvenomous tissues, the conservation degree of physiologic activities comparing with

snake venom evolution and the generation of toxins diversity.

2. Omic approaches in the identification of minor venom proteins

minor venom proteins [37-39].

Table 1 – Main minor protein classes in snake venoms.

Act in synergism with [40-42]

Cobra venom factors Mainly found in [55-59]

Kazal-type inhibitors P, T 8,3 Protease inhibition -- [71, 72]

Increases the vascular

peripheral and central envenoming. May act

Cleavage of membrane Hydrolysis of

Hypolocomotion, Ohanin presents a

T=transcriptome; P = Proteome; T.A. = traditional approach (activity-guided assays)

3. Minor venom protein classes

3.1 Growth factors

(LTBP), transforming growth factor beta-2 (TGF-β), hepatocyte growth factor-regulated

tyrosine kinase (HGF), hepatoma-derived growth factor (HDGF), delta/notch-like

binding protein 5 (IGF), epidermal growth factor (EGF-like protein), brain-derived

neurotrophic factor (BDNF), interferon (IF), platelet-derived growth factor (PDGF),

3.1.1 Nerve growth factors (NGFs)

NGFs participate in neuronal differentiation, synaptic plasticity, and

differentiation activity evidenced with sv-NGF from N. sputatrix [109] and O.

3.1.2 Vascular endothelial growth factor (VEGF)

Bothrops insularis) (19), Bothropoides pauloensis (former Bothrops pauloensis) (1),

microvascular endothelial cells. That proteoglycan may help to regulate the

D-glucuronate from hyaluronan producing tetra and hexasaccharides [69]. Therefore,

several organisms, being ubiquitous in snake venoms [137, 138].

is outstanding [136, 137, 150-156].

3.3 Cysteine-rich secretory proteins

Cysteine-rich secretory proteins (CRISPs) are non-enzymatic components

demonstrated a dose-dependent antiparasitic activity on Leishmania amazonensis and

Trypanosoma cruzi amastigote forms and Trypanosoma brucei rhodesiense metacyclic

3.4 Nucleases and nucleotidases

Nucleases and nucleotidases are hydrolytic enzymes ubiquitously distributed in

toxins (phospholipases A2, cardiotoxins, myotoxins, cytolytic peptides and

and overall pathology caused by the snake bite envenoming [37].

Nucleotidases are enzymes involved in the cleavage of nucleic acid derivatives

toxins, like phosphodiesterase, phosphomonoesterase, endonuclease, phospholipases A2,

myotoxins) is quickly hydrolyzed by ATPase, and act as a danger signal molecule

stimulating purinergic receptors, contributing to the pathophysiology of envenomation

ADPase hydrolyzes ADP, producing adenosine and orthophosphate [171, 190].

thrombin-induced aggregation in platelet poor plasma [178]. The inhibition of platelet

3.5 Protease inhibitors

Snake venoms are interesting sources of protease inhibitors, although these