Abstract
Biomolecules from Cerastes cerastes venom have been purified and characterized. Two phospholipases isolated from Cerastes cerastes venom share 51% of homology. CC2-PLA2 exhibits antiplatelet activity that blocks coagulation. CCSV-MPase, a non-hemorrhagic Zn2+-metalloproteinase, significantly reduced the plasmatic fibrinogen level and hydrolyzes only its Bβ chain. Serine proteinases such as RP34, afaâcytin and CC3-SPase hydrolyze the fibrinogen and are respectively α, αβ and αβ fibrinogenases. In deficient human plasma, afaâcytin replaces the missing factors VIII and IX, and activates purified human factor X into factor Xa. It releases serotonin from platelets and directly aggregates human (but not rabbit) blood platelets. RP34 proteinase also had no effect on both human and rabbit blood platelet aggregation. CC3-SPase revealed a pro-coagulant activity. However, the insolubility of the obtained clot indicates that CC3-SPase does not activate factor XIII. In addition, CC3-SPase clotting activity was carried out with human plasmas from volunteer patients deficient in clotting factors. Results showed that CC3-SPase shortens clotting time of plasma deficient in factors II and VII but with weaker clotting than normal plasma. The clotting time of plasma deficient in factor II is similar to that obtained with normal plasma; suggesting that CC3-SPase is able to replace both factors IIa and VII in the coagulation cascade and thus could be involved in the blood clotting process via an extrinsic pathway. These results imply that CC3-SPase and afaâcytin could repair hemostatic abnormalities and may replace some factors missing in pathological deficiency. Afaâcytin also exhibits α fibrinase property similar to a plasmin-like proteinase. Despite its thrombin-like characteristics, afaâcytin is not inhibited by plasmatic thrombin inhibitors. The procoagulant properties of afaâcytin might have potential clinical applications.
Cerastes cerastes venom; Proteinases; Phospholipases A2; Platelets; Blood-clotting; Hemostasis
Introduction
Serine proteases and phospholipases A2 isolated from snake venoms act on the hemostatic system as procoagulants, anticoagulants, pro- or anti-platelet aggregants. Some of these isolated molecules, mainly from Viperidae venoms, are used in diagnosis or treatment of thrombotic diseases and ischemic heart disease. Metalloproteinases can cause hemorrhage after accidental or experimental envenomation. However, some of these metalloproteinases are directly involved in the clotting of blood as they can act on fibrinogen and/or fibrin; they are called in this case fibrino(geno)lytic metalloproteinases. Their fibrinolytic activity makes them potent inhibitors of blood coagulation. Phospholipase A2 exert their anticoagulant effect by their ability to inhibit platelet aggregation due to their high affinity to bind to activated factor Stewart (FXa). All these biological effects based on their direct involvement in hemostasis, let consider these molecules as potential tools or biomarkers in blood diseases.
Review
Viperidae and Crotalidae venoms are rich sources of hydrolytic enzymes and produce a
complex pattern of clinical and toxic effects such as coagulation disorders, hemorrhage and
necrosis [ 1Rivière G, Bon C. Immunothérapie antivenimeuse des envenimations
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]. Some of venom components act at various stages of the coagulation cascade.
These components perform antagonistic functions, whilst some of them act synergistically.
Therefore, the venom toxicity cannot be attributed to only one component [ 11Laraba-Djebari F, Martin-Eauclaire MF, Mauco G, Matchot P. Afaâcytin and α,
β-fibrinogenase from Cerastes cerastes (horned viper) venom, activates purified Factor X
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]. However, most venom components produce beneficial
effects when they act alone [ 12Chérifi F, Rousselle JC, Namane A, Laraba-Djebari F. Zn2+: a required ion
for procoagulant metalloproteinase (CCSV-MPase) activities, isolated from Cerastes
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161–164. ]. Snake venom also
contains non-protein components including citrate, metal ions, carbohydrates, nucleotides as
well as low concentrations of free amino acids and lipids [ 13Freitas MA, Geno PW, Summer LW, Cooke ME, Hudenberg SA, Ownby CL. Citrate is
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Phospholipases A2 (PLA2s) represent more than 10% of the dry weight of the snake venoms
from which they are isolated. PLA2s isolated from Viperidae venoms consist of
125–130 amino acid residues cross-linked by seven disulfide bonds which confer
stability on the molecule and are calcium-dependant [ 16Kini RM. Venom phospholipase A2 enzymes: structure, function and
mechanism.England: Chichester; 1997. pp. 1–511. ]. In addition to their hydrolytic activity, PLA2s may display many other
activities, such as edematous, neurotoxic, cardiotoxic, hemolytic, convulsive, antiplatelet,
antitumoral and anticoagulant properties. Based on their anticoagulant activity, PLA2s can
be clinically useful against thrombotic diseases and for the diagnosis and treatment of
hemostatic disorders [ 17Chérifi F, Rousselle JC, Namane A, Laraba-Djebari F. CCSV-MPase, a novel
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]. The anticoagulant
activity is due to their ability to inhibit platelet aggregation through factor Xa (FXa)
blockade. Based on their direct involvement in the hemostatic cascade, PLA2 could also be
used as tools or biomarkers in blood diseases.
Metalloproteinases found in Viperidae venoms may cause local hemorrhaging following
accidental or experimental intradermal or subcutaneous injection of venom [ 18Kamiguti AS. Platelets as target of snake venom metalloproteinases.
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]. Some of them are known to display fibrino
(geno)lytic activity. Fibrino(geno)lytic metalloproteinases dissolve fibrin clots and
prevent clot formation by hydrolyzing fibrinogen, thus enhancing the toxic effect of
hemorrhagic metalloproteinases, giving rise to pathological bleeding [ 19Leonardi A, Fox JW, Trampus-Bakija A, Krizaj I. Ammodytase, a
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useful against thrombotic diseases thanks to their potential use in laboratory tests or as
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system.Blood Rev. 1993;19(3):840–855. ]. These proteinases may be useful for investigating the mechanisms of
blood coagulation and platelet aggregation [ 11Laraba-Djebari F, Martin-Eauclaire MF, Mauco G, Matchot P. Afaâcytin and α,
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,
12Chérifi F, Rousselle JC, Namane A, Laraba-Djebari F. Zn2+: a required ion
for procoagulant metalloproteinase (CCSV-MPase) activities, isolated from Cerastes
cerastes Venom. SFET Editions, Toxins and Ion transfers, E-Book RT19. 2011. pp.
161–164. ].
In addition to their beneficial effects, venom molecules are the cause of health problems
after snake envenomation. Annually, more than 100,000 deaths are recorded worldwide,
including 20,000 on African continent, while 400,000 victims retain severe and permanent
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]. Improvement of this treatment requires knowledge of
the kinetic distribution of venom in vascular and tissue compartments.
Cerastes cerastes venom is a mixture of various proteins with broad
biological and physiological activities; most of them are proteinases while some have been
well characterized. Most of these proteins act on blood coagulation, including PLA2, the
thrombin-like enzymes RP34 and afaâcytin, anticoagulant protease fraction, aggregant
serine protease, hemorrhagic metalloproteinase CcH1, and the non-hemorrhagic
metalloproteinase CCSV-MPase [ 11Laraba-Djebari F, Martin-Eauclaire MF, Mauco G, Matchot P. Afaâcytin and α,
β-fibrinogenase from Cerastes cerastes (horned viper) venom, activates purified Factor X
and induces serotonin release from human blood platelet. Eur J Biochem.1995;19(3):756–765.
doi: 10.1111/j.1432-1033.1995.756_3.x.
https://doi.org/10.1111/j.1432-1033.1995...
, 12Chérifi F, Rousselle JC, Namane A, Laraba-Djebari F. Zn2+: a required ion
for procoagulant metalloproteinase (CCSV-MPase) activities, isolated from Cerastes
cerastes Venom. SFET Editions, Toxins and Ion transfers, E-Book RT19. 2011. pp.
161–164. , 31Laraba-Djebari F, Martin-Eauclaire MF. Purification and characterization of
a phospholipase A2 from Cerastes cerastes (horn viper) snake venom.
Toxicon. 1990;19(6):637–646. doi: 10.1016/0041-0101(90)90252-3.
https://doi.org/10.1016/0041-0101(90)902...
- 35Boukhalfa-Abib H, Meksem A, Laraba-Djebari F. Purification and biochemical
characterization of a novel hemorrhagic metalloproteinase from horned viper
(Cerastes cerastes) venom. Comp Biochem Physiol C Toxicol Pharmacol.
2009;19(2):285–290. doi: 10.1016/j.cbpc.2009.05.008.
https://doi.org/10.1016/j.cbpc.2009.05.0...
]. In this paper, we report pharmacological activity
of biomolecules isolated from Cerastes cerastes on hemostasis process.
Biochemical properties of biomolecules and their proteomic identification
Several molecules from Cerastes cerastes venom act on hemostasis, such
as RP34, a serine proteinase which consists of two subunits of 48.5 kDa [ 32Laraba-Djebari F, Martin-Eauclaire MF, Marchot PA. A fibrinogen-clotting
serine proteinase from Cerastes cerastes (horned viper) with arginine -
esterase an amidase activities, purification, characterization and kinetic parameter
determination. Toxicon.1992;19(1):1399–1410. ]. Another serine proteinase, a thrombin-like
molecule denominated afaâcytin, was purified and characterized [ 11Laraba-Djebari F, Martin-Eauclaire MF, Mauco G, Matchot P. Afaâcytin and α,
β-fibrinogenase from Cerastes cerastes (horned viper) venom, activates purified Factor X
and induces serotonin release from human blood platelet. Eur J Biochem.1995;19(3):756–765.
doi: 10.1111/j.1432-1033.1995.756_3.x.
https://doi.org/10.1111/j.1432-1033.1995...
]. Afaâcytin presents caseinolytic,
arginine-esterase and amidase activities. It is a homodimeric proteinase with two
subunits, alpha and beta, which have the same apparent molecular mass (40.0 kDa for each
unit) and are indistinguishable in the absence of reduction or/and deglycosylation [ 11Laraba-Djebari F, Martin-Eauclaire MF, Mauco G, Matchot P. Afaâcytin and α,
β-fibrinogenase from Cerastes cerastes (horned viper) venom, activates purified Factor X
and induces serotonin release from human blood platelet. Eur J Biochem.1995;19(3):756–765.
doi: 10.1111/j.1432-1033.1995.756_3.x.
https://doi.org/10.1111/j.1432-1033.1995...
]. Both α and β chains are
N-glycosylated. The two chains present the same N-terminal sequence (20 residues) which is
similar to the sequence of other proteinases isolated from snake venom.
Three molecules – CC2-PLA2, CCSV-MPase and CC3-SPase – were characterized
by proteomic analysis. Results showed some sequence similarity with other homologous
enzymes isolated from several venoms [ 11Laraba-Djebari F, Martin-Eauclaire MF, Mauco G, Matchot P. Afaâcytin and α,
β-fibrinogenase from Cerastes cerastes (horned viper) venom, activates purified Factor X
and induces serotonin release from human blood platelet. Eur J Biochem.1995;19(3):756–765.
doi: 10.1111/j.1432-1033.1995.756_3.x.
https://doi.org/10.1111/j.1432-1033.1995...
, 12Chérifi F, Rousselle JC, Namane A, Laraba-Djebari F. Zn2+: a required ion
for procoagulant metalloproteinase (CCSV-MPase) activities, isolated from Cerastes
cerastes Venom. SFET Editions, Toxins and Ion transfers, E-Book RT19. 2011. pp.
161–164. , 17Chérifi F, Rousselle JC, Namane A, Laraba-Djebari F. CCSV-MPase, a novel
procoagulant metalloproteinase from Cerastes cerastes venom:
purification, biochemical characterization and protein identification. Protein J.
2010;19:466–474. doi: 10.1007/s10930-010-9273-1.
https://doi.org/10.1007/s10930-010-9273-...
, 31Laraba-Djebari F, Martin-Eauclaire MF. Purification and characterization of
a phospholipase A2 from Cerastes cerastes (horn viper) snake venom.
Toxicon. 1990;19(6):637–646. doi: 10.1016/0041-0101(90)90252-3.
https://doi.org/10.1016/0041-0101(90)902...
- 34Chérifi F, Laraba-Djebari F. Mise en évidence et Caractérisation d’une
Fraction Coagulante et Agrégante du venin de Cerastes cerastes. Toxines et fonctions
cholinergiques neuronales et non neuronales. Editions de la SFET. 2008. pp.
153–154. ].
CC2-PLA2, another PLA2 found in the same venom, presents 51% sequence homology with a
previously purified molecule from the same venom by Laraba-Djebari et al
. [ 32Laraba-Djebari F, Martin-Eauclaire MF, Marchot PA. A fibrinogen-clotting
serine proteinase from Cerastes cerastes (horned viper) with arginine -
esterase an amidase activities, purification, characterization and kinetic parameter
determination. Toxicon.1992;19(1):1399–1410. ] (accession number in NCBInr is gi
|129506|), i . e . 61 out of 120 amino acid residues are
common to the two PLA2s [ 31Laraba-Djebari F, Martin-Eauclaire MF. Purification and characterization of
a phospholipase A2 from Cerastes cerastes (horn viper) snake venom.
Toxicon. 1990;19(6):637–646. doi: 10.1016/0041-0101(90)90252-3.
https://doi.org/10.1016/0041-0101(90)902...
]. The peptide
sequence of the new PLA2 was obtained by alignment with sequences of other venom PLA2s.
Some snake venom proteinases were identified sharing sequence homology with CCSV MPase,
four of which belonged to the metalloproteinase family (Group III snake venom
metalloproteinase and Zn+2-metalloproteinase disintegrin) [ 17Chérifi F, Rousselle JC, Namane A, Laraba-Djebari F. CCSV-MPase, a novel
procoagulant metalloproteinase from Cerastes cerastes venom:
purification, biochemical characterization and protein identification. Protein J.
2010;19:466–474. doi: 10.1007/s10930-010-9273-1.
https://doi.org/10.1007/s10930-010-9273-...
]. Three of these proteins corresponded to
Cerastes vipera venom and the others to venom proteins of different
snake species [ 17Chérifi F, Rousselle JC, Namane A, Laraba-Djebari F. CCSV-MPase, a novel
procoagulant metalloproteinase from Cerastes cerastes venom:
purification, biochemical characterization and protein identification. Protein J.
2010;19:466–474. doi: 10.1007/s10930-010-9273-1.
https://doi.org/10.1007/s10930-010-9273-...
].
All of these proteins were identified with at least two unique specific peptides and presented similarities with the purified CCSV-MPase. Previous studies showed that CCSV-MPase, characterized by SDS-PAGE analysis, could be classified as a member of the high-molecular-mass metalloproteinase family, due to its molecular mass estimated at 70 kDa in both reducing and non-reducing electrophoresis conditions [ 12Chérifi F, Rousselle JC, Namane A, Laraba-Djebari F. Zn2+: a required ion for procoagulant metalloproteinase (CCSV-MPase) activities, isolated from Cerastes cerastes Venom. SFET Editions, Toxins and Ion transfers, E-Book RT19. 2011. pp. 161–164. ]. Furthermore, the partial amino acid sequence of CCSV-MPase was identified by MALDI-TOF MS/MS analysis. Based on its molecular mass and partial amino acid sequence, CCSV-MPase may be classified in the P-III class of SVMPs containing a disintegrin-like metalloproteinase, with cystein-rich domains.
It is well established that some venom components have beneficial effects when acting in
isolation. Cerastes cerastes venom is a mixture of protein components
with multiple actions including coagulation [ 11Laraba-Djebari F, Martin-Eauclaire MF, Mauco G, Matchot P. Afaâcytin and α,
β-fibrinogenase from Cerastes cerastes (horned viper) venom, activates purified Factor X
and induces serotonin release from human blood platelet. Eur J Biochem.1995;19(3):756–765.
doi: 10.1111/j.1432-1033.1995.756_3.x.
https://doi.org/10.1111/j.1432-1033.1995...
,
32Laraba-Djebari F, Martin-Eauclaire MF, Marchot PA. A fibrinogen-clotting
serine proteinase from Cerastes cerastes (horned viper) with arginine -
esterase an amidase activities, purification, characterization and kinetic parameter
determination. Toxicon.1992;19(1):1399–1410. , 36El-Asmar MF, Shaban E, Hagag M, Swelam N, Tu A. Coagulant component in
Cerastes cerastes (Egyptian sand viper) venom. Toxicon.
1986;19(11–12):1037–1044. ,
37Bazaa A, Marrakchi N, El Ayeb M, Sanz L, Calvete JJ. Snake venomics:
comparative analysis of the venom proteomes of the Tunisian snakes Cerastes
cerastes, Cerastes vipera and Macrovipera
lebetina. Proteomics. 2005;19(16):4223–4235. doi:
10.1002/pmic.200402024.
https://doi.org/10.1002/pmic.200402024...
]. These proteins may induce hemorrhage and
capillary permeability disorders, through their disintegrin domain or related proteins
that disrupt primary hemostasis by acting on platelet adhesion. Thus, a single molecule
can be endowed with several activities. The structural differences between proteins,
natural factors of hemostasis, as well as the multiplicity of target components of the
same venom, are elements that could explain the efficiency of partial immunotherapy [
15Mion G, Olive F, Hermandez E, Martin YN, Viellefosse S, Goyffon M. Action
des venins sur la coagulation sanguine: diagnostic des syndromes hémorragiques. Bull Soc
Path Exot. 2002;19(3):132–138. ]. Fibrinogenases (serine proteinases or
metalloproteinases) are widespread in Viperidae venoms. They hydrolyze fibrinogen and/or
degrade the fibrin clot, enhancing the effect of hemorrhagic metalloproteinases that give
rise to pathological bleeding.
Coagulant and fibrinogenolytic activities of isolated molecules
Fibrinogen is a glycoprotein of 340 kDa with three polypeptide chains; Aα (67 kDa), Bβ (50 kDa) and γ (43 kDa) linked by disulfide bonds. It can be hydrolyzed by thrombin, thus producing fibrin components and fibrinopeptides. Thrombin activity (control) on fibrinogen demonstrated the release of fibrinopeptide A (FpA) followed by fibrinopeptide B (FpB).
Proteolytic enzymes of Cerastes cerastes venom were identified as
α, β or γ fibrinogenases depending on their ability to hydrolyze
the fibrinogen in vitro . SDS-PAGE analysis of fibrinogen in the presence
of venom revealed two entities (55 kDa and 50 kDa) indicating activities of α- and
β-fibrinogenase. Purification and characterization of three procoagulant
proteinases (RP34, afaâcytin and CC3-SPase proteinase) showed fibrinogenolytic
activities when analyzed by SDS-PAGE, afaâcytin and RP34 displayed, respectively,
α,ß-fibrinogenase and α-fibrinogenase activity [ 11Laraba-Djebari F, Martin-Eauclaire MF, Mauco G, Matchot P. Afaâcytin and α,
β-fibrinogenase from Cerastes cerastes (horned viper) venom, activates purified Factor X
and induces serotonin release from human blood platelet. Eur J Biochem.1995;19(3):756–765.
doi: 10.1111/j.1432-1033.1995.756_3.x.
https://doi.org/10.1111/j.1432-1033.1995...
, 32Laraba-Djebari F, Martin-Eauclaire MF, Marchot PA. A fibrinogen-clotting
serine proteinase from Cerastes cerastes (horned viper) with arginine -
esterase an amidase activities, purification, characterization and kinetic parameter
determination. Toxicon.1992;19(1):1399–1410. , 34Chérifi F, Laraba-Djebari F. Mise en évidence et Caractérisation d’une
Fraction Coagulante et Agrégante du venin de Cerastes cerastes. Toxines et fonctions
cholinergiques neuronales et non neuronales. Editions de la SFET. 2008. pp.
153–154. ]. Like afaâcytin, CC3-SPase is also
characterized as an α,β-fibrinogenase due to the release of both A and B
fibrinopeptides.
Susceptibility of afaâcytin to diisopropyl fluorophosphate and benzamidine
indicates the presence of a serine and an aspartic (or glutamic) acid residues in the
catalytic site. Calcium is required for structural cohesion of the afaâcytin
molecule [ 11Laraba-Djebari F, Martin-Eauclaire MF, Mauco G, Matchot P. Afaâcytin and α,
β-fibrinogenase from Cerastes cerastes (horned viper) venom, activates purified Factor X
and induces serotonin release from human blood platelet. Eur J Biochem.1995;19(3):756–765.
doi: 10.1111/j.1432-1033.1995.756_3.x.
https://doi.org/10.1111/j.1432-1033.1995...
]. CCSV-MPase cleaves only the
Bβ chain of fibrinogen and exerted no action on Aα or γ chains.
This property contrasts with those of other SVMPs which preferentially cleave only the
Aα-fibrinogen chain. However, these metalloproteinases, belonging to the PI class
of SVMPs, present low molecular mass, with only the metalloproteinase domain, as in the
case of fibrolase purified from Akgistrodon contortrix contortrix ,
piscivorase II of Akgistrodon piscivorus piscivorus , lebetase purified
from the venom of Vipera lebetina , neuwiedase from Bothrops
neuwiedi venom, the atroxase of Crotalus atrox and
leucurolysins from venom of Bothrops leucurus [ 5Castro HC, Zingali RB, Albuquerque MG, Pujol-Luz M, Rodrigues CR. Snake
venom thrombin-like enzymes: from reptilase to now. Cell Mol Life Sci.
2004;19(7–8):843–856. , 38Retzios AD, Markland FS. Fibrinolytic enzymes from the venoms of
Agkistrodon contortrix contortrix and Crotalus basiliscus
basiliscus: cleavage site specificity towards the alpha-chain of fibrin. Thromb
Res. 1994;19(4):355–367. doi: 10.1016/0049-3848(94)90151-1.
https://doi.org/10.1016/0049-3848(94)901...
- 42Bello CA, Hermogenes AL, Magalhaes A, Veiga SS, Gremski LH, Richardson M.
Isolation and biochemical characterization of a fibrinolytic proteinase from
Bothrops leucurus(white-tailed Jararaca) snake venom. Biochimie.
2006;19(2):189–200. doi: 10.1016/j.biochi.2005.07.008.
https://doi.org/10.1016/j.biochi.2005.07...
].
Proteinases (afaâcytin, RP34, CC3-SPase and CCSV-MPase) showed caseinolytic
activity as crude venom. CC3-SPase displayed arginine ester hydrolase activity while the
CCSV-MPase does not. Both molecules presented a high amidolytic activity similar to that
of crude venom. Previous results revealed that the use of specific inhibitors for serine
proteinases and metalloproteinases showed that CC3-SPase is a thrombin-like
Ca2+-dependent serine proteinase. Afaâcytin isolated from the venom
of Cerastes cerastes showed that Ca2+ is essential for its
activity not only as a cofactor but can contribute to the stability or structural cohesion
of the enzyme [ 11Laraba-Djebari F, Martin-Eauclaire MF, Mauco G, Matchot P. Afaâcytin and α,
β-fibrinogenase from Cerastes cerastes (horned viper) venom, activates purified Factor X
and induces serotonin release from human blood platelet. Eur J Biochem.1995;19(3):756–765.
doi: 10.1111/j.1432-1033.1995.756_3.x.
https://doi.org/10.1111/j.1432-1033.1995...
]. CCSV-MPase appears to be a
zinc-dependent metalloproteinase given that metal chelators, EDTA and 1,10-phenanthroline
completely inhibited its proteolytic activity, which also suggested that unlike CC3-SPase,
Ca2+ is not required for its catalytic activity. The sensitivity of the
serine proteinase CC3-SPase, a specific inhibitor of thrombin (heparin and antithrombin
III) may indicate that the receptor of CC3-SPase is identical to that of thrombin. Given
its procoagulant properties and insensitivity to thrombin-specific plasma inhibitors,
afaâcytin might be interesting to employ as a hemostatic agent in some types of
hemorrhage, such as post-operative thrombocytopenia [ 11Laraba-Djebari F, Martin-Eauclaire MF, Mauco G, Matchot P. Afaâcytin and α,
β-fibrinogenase from Cerastes cerastes (horned viper) venom, activates purified Factor X
and induces serotonin release from human blood platelet. Eur J Biochem.1995;19(3):756–765.
doi: 10.1111/j.1432-1033.1995.756_3.x.
https://doi.org/10.1111/j.1432-1033.1995...
].
Serine-proteinases hydrolyze fibrinogen by acting on the two chains, α and
β, of this substrate thereby causing the formation of a fragile fibrin clot.
CCSV-MPase acts on the β chain of fibrinogen resulting in the release of only
fibrinopeptide B. Afaâcytin, as a component of the venom (2% w/w), hydrolyzes
fibrinogen in the same manner that CC3-SPase degrades firstly the Aα chain and
then, 24 hours later, the Bβ chain, leading to a fragile clot, which suggests that
CC3-SPase, similarly to afaâcytin, is unable to activate the factor XIII
responsible for the resistance of the fibrin clot [ 11Laraba-Djebari F, Martin-Eauclaire MF, Mauco G, Matchot P. Afaâcytin and α,
β-fibrinogenase from Cerastes cerastes (horned viper) venom, activates purified Factor X
and induces serotonin release from human blood platelet. Eur J Biochem.1995;19(3):756–765.
doi: 10.1111/j.1432-1033.1995.756_3.x.
https://doi.org/10.1111/j.1432-1033.1995...
, 34Chérifi F, Laraba-Djebari F. Mise en évidence et Caractérisation d’une
Fraction Coagulante et Agrégante du venin de Cerastes cerastes. Toxines et fonctions
cholinergiques neuronales et non neuronales. Editions de la SFET. 2008. pp.
153–154. ]. CCSV-MPase cleaves only the
Bβ chain of fibrinogen and exerts no activity on Aα and γ chains.
CCSV-MPase properties may allow its use as a therapeutic agent in some pathologies that
require anticoagulant administration.
Most thrombin-like enzymes (TLE) isolated from snake venoms act on fibrinogen by hydrolyzing one chain rather than two, although the cleavage site is the same (Arg16-Gly17) as the α chain (Arg15- Gly16) on β chain, by releasing fibrinopeptides A or B as CCSV-MPase which degrades only the β chain of fibrinogen. CC3-SPase shortened the clotting time of plasma deficient in factor VII and II with a weaker clot than that formed with normal plasma. The clotting time of plasma deficient in factor II is similar to that obtained with normal plasma after the action of serine protease CC3-SPase, which suggests that this molecule is able to replace factors IIa and VII.
Procoagulant and anticoagulant snake venom components often act at later stages of the
coagulation cascade. The main targets of these components are fibrinogen, prothrombin,
factor X and platelets [ 3Braud S, Bon C, Wisner N. Snake venom proteins acting on haemostasis.
Biochimie.2000;19(9–10):851–859. , 5Castro HC, Zingali RB, Albuquerque MG, Pujol-Luz M, Rodrigues CR. Snake
venom thrombin-like enzymes: from reptilase to now. Cell Mol Life Sci.
2004;19(7–8):843–856. , 11Laraba-Djebari F, Martin-Eauclaire MF, Mauco G, Matchot P. Afaâcytin and α,
β-fibrinogenase from Cerastes cerastes (horned viper) venom, activates purified Factor X
and induces serotonin release from human blood platelet. Eur J Biochem.1995;19(3):756–765.
doi: 10.1111/j.1432-1033.1995.756_3.x.
https://doi.org/10.1111/j.1432-1033.1995...
, 12Chérifi F, Rousselle JC, Namane A, Laraba-Djebari F. Zn2+: a required ion
for procoagulant metalloproteinase (CCSV-MPase) activities, isolated from Cerastes
cerastes Venom. SFET Editions, Toxins and Ion transfers, E-Book RT19. 2011. pp.
161–164. , 17Chérifi F, Rousselle JC, Namane A, Laraba-Djebari F. CCSV-MPase, a novel
procoagulant metalloproteinase from Cerastes cerastes venom:
purification, biochemical characterization and protein identification. Protein J.
2010;19:466–474. doi: 10.1007/s10930-010-9273-1.
https://doi.org/10.1007/s10930-010-9273-...
].
Several molecules have been purified from Viperidae and Crotalidae venoms and
characterized as FX activator factors that are used as biomarkers in many hemostatic
disorders. Indeed, thrombin-like components serve as structural models to extend our
understanding of the structure-function relationships of blood coagulation factors, some
of which have been clinically used for the treatment of thrombotic diseases, and are
employed as tools in clinical assays [ 43de Oliveira DG, Murakami MT, Cintra ACO, Franco JJ, Sampaio SV, Arni RK.
Functional and structural analysis of two fibrinogen-activating enzymes isolated from the
venoms ofCrotalus durissus terrificus and Crotalus durissus
collilineatus. Acta Biochim Biophys Sin (Shanghai) 2009;19(1):21–29. doi:
10.1093/abbs/gmn003.
https://doi.org/10.1093/abbs/gmn003...
].
Combination of gel filtration and ion-exchange chromatography proved to be successful in
obtaining milligram quantities of new pure TLEs from the venoms of Crotalus
durissus terrificus and Crotalus durissus collilineatus [
43de Oliveira DG, Murakami MT, Cintra ACO, Franco JJ, Sampaio SV, Arni RK.
Functional and structural analysis of two fibrinogen-activating enzymes isolated from the
venoms ofCrotalus durissus terrificus and Crotalus durissus
collilineatus. Acta Biochim Biophys Sin (Shanghai) 2009;19(1):21–29. doi:
10.1093/abbs/gmn003.
https://doi.org/10.1093/abbs/gmn003...
]. Functional characterization indicates that
both enzymes preferentially degrade the Bβ chain of bovine fibrinogen and present
edema-inducing and coagulant activities. However, the TLE from Crotalus durissus
collilineatus venom showed twofold higher coagulant activity with a minimum
coagulant dose (MCD) of 0.6 μg/μL, whereas the enzyme isolated from
Crotalus durissus terrificus indicated an MCD of 1.5
μg/μL [ 43de Oliveira DG, Murakami MT, Cintra ACO, Franco JJ, Sampaio SV, Arni RK.
Functional and structural analysis of two fibrinogen-activating enzymes isolated from the
venoms ofCrotalus durissus terrificus and Crotalus durissus
collilineatus. Acta Biochim Biophys Sin (Shanghai) 2009;19(1):21–29. doi:
10.1093/abbs/gmn003.
https://doi.org/10.1093/abbs/gmn003...
].
Recently, a TLE denominated BpSP-I was isolated from Bothrops pauloensis
snake venom; its biochemical, enzymatic and pharmacological characteristics were
determined. BpSP-I showed high clotting activity upon bovine and human plasma and was
inhibited by PMSF, benzamidine and leupeptin. Moreover, this enzyme showed stability when
examined at different temperatures (−70 to 37°C), pH values [ 3Braud S, Bon C, Wisner N. Snake venom proteins acting on haemostasis.
Biochimie.2000;19(9–10):851–859. - 9Hamza L, Girardi T, Castelli S, Gargioli C, Cannata S, Patamia M. Isolation
and characterization of a myotoxin from the venom of Macrovipera lebetina
transmediterranea. Toxicon. 2010;19(3):381–390. doi:
10.1016/j.toxicon.2010.04.001.
https://doi.org/10.1016/j.toxicon.2010.0...
] or in the
presence of divalent metal ions (Ca2+, Mg2+, Zn2+ and
Mn2+). BpSP-I showed high catalytic activity upon substrates, such as
fibrinogen, TAME, S-2238 and S-2288. It also showed kallikrein-like activity, but was
unable to act upon factor Xa or plasmin substrates [ 44Costa FL, Rodrigues RS, Izidoro LF, Menaldo DL, Hamaguchi A,
Homsi-Brandeburgo MI. Biochemical and functional properties of a thrombin-like enzyme
isolated from Bothrops pauloensis snake venom. Toxicon.
2009;19(6):725–735. doi: 10.1016/j.toxicon.2009.05.040.
https://doi.org/10.1016/j.toxicon.2009.0...
].
Reducing blood viscosity is often required in the treatment of thrombotic and ischemic heart diseases. Defibrinogenation of the plasma by some enzymes from snake venoms is of interest. Indeed, all of these defibrinogenating biomolecules sharing these properties could be used as tools in clinical applications and in basic research. Further studies, in pharmacology and toxicology should be undertaken to determine their mode of action in vivo .
Effect of molecules on platelet function
Biological characterization of CCSV-MPase and CC2-PLA2 has been shown to be highly
anti-aggregative in relation to human platelets. The antagonistic effect of CC3-SPase is
of interest in the context of the antiplatelet action of the hemostatic system, and may be
an effective tool for reducing blood viscosity, a property that is often necessary in the
treatment of thrombotic diseases and ischemic heart syndrome due to platelet aggregation.
Previous studies have already demonstrated that afaâcytin may replace the missing
factors VIII and IX in deficient plasmas, and activate purified human factor X into factor
Xa [ 11Laraba-Djebari F, Martin-Eauclaire MF, Mauco G, Matchot P. Afaâcytin and α,
β-fibrinogenase from Cerastes cerastes (horned viper) venom, activates purified Factor X
and induces serotonin release from human blood platelet. Eur J Biochem.1995;19(3):756–765.
doi: 10.1111/j.1432-1033.1995.756_3.x.
https://doi.org/10.1111/j.1432-1033.1995...
]. It releases serotonin from platelets and
directly aggregates human (but not rabbit) blood platelets. On the other hand, RP 34 has
no effect on platelet aggregation [ 32Laraba-Djebari F, Martin-Eauclaire MF, Marchot PA. A fibrinogen-clotting
serine proteinase from Cerastes cerastes (horned viper) with arginine -
esterase an amidase activities, purification, characterization and kinetic parameter
determination. Toxicon.1992;19(1):1399–1410. ]. Several
anticoagulant PLA2s from snake venoms have been isolated and well characterized. Recently,
two phospholipases, known as CC-PLA2-1 and CC-PLA2-2 with antiplatelet aggregation
activity, were isolated from Cerastes cerastes venom [ 45Zouari-Kessentini R, Luis-José L, Karray A, Kallech-Ziri O, Srairi-Abid N,
Bazaa A. Two purified and characterized phospholipases A2 from Cerastes
cerastes venom, that inhibits cancerous cell adhesion and migration. Toxicon.
2009;19(4):444–453. doi: 10.1016/j.toxicon.2009.01.003.
https://doi.org/10.1016/j.toxicon.2009.0...
]. An anticoagulant PLA2 was isolated and
characterized as an inhibitor of the prothrombinase complex through its specific binding
to FX [ 46Faure G, Gowda VT, Maroun RC. Characterization of a human coagulation factor
Xa-binding site on Viperidae snake venom phospholipases A2 by affinity
binding studies and molecular bioinformatics. BMC Struct Biol. 2007;19:82. doi:
10.1186/1472-6807-7-82.
https://doi.org/10.1186/1472-6807-7-82...
]. Ammodytoxin A (Atxa) and its natural
ammodytoxin isoform C were isolated from Vipera ammodytes ammodytes venom
and belong to group IIA secreted phosphoplipases. These two isoforms differ only by two
amino acid residues (Phe 124 > Ile and Lys128 > Glu), but there are
significant differences in toxicity. The mechanism by which they block coagulation has
been elucidated. Complementary experiments using surface plasmon resonance showed complete
inhibition of binding to FXa through calmodulin (CaM). The crystal structure showed that
the C-terminal region required for binding to FXa and CaM is highly exposed and accessible
for interaction with receptor proteins in the monomeric and dimeric forms of ammodytoxin [
45Zouari-Kessentini R, Luis-José L, Karray A, Kallech-Ziri O, Srairi-Abid N,
Bazaa A. Two purified and characterized phospholipases A2 from Cerastes
cerastes venom, that inhibits cancerous cell adhesion and migration. Toxicon.
2009;19(4):444–453. doi: 10.1016/j.toxicon.2009.01.003.
https://doi.org/10.1016/j.toxicon.2009.0...
].
Conclusion
Viperidae venoms, considered to be one of the most important bioresources, include
pharmacologically active molecules such as proteinases (metalloproteinases and serine
proteinases) and phospholipase A2 [ 46Faure G, Gowda VT, Maroun RC. Characterization of a human coagulation factor
Xa-binding site on Viperidae snake venom phospholipases A2 by affinity
binding studies and molecular bioinformatics. BMC Struct Biol. 2007;19:82. doi:
10.1186/1472-6807-7-82.
https://doi.org/10.1186/1472-6807-7-82...
, 47Franceschi A, Rucavado A, Mora N, Gutiérrez JM. Purification and
characterization of BaH4, a haemorrhagic metalloproteinase from the venom of snake
Bothrops asper. Toxicon. 2000;19(1):63–77. doi:
10.1016/S0041-0101(99)00127-0.
https://doi.org/10.1016/S0041-0101(99)00...
]. All of these molecules are of interest in
biotherapy as biomedicines or may be used as diagnostic tools. Proteases and PLA2 act on the
hemostatic system as procoagulants, anticoagulants, and as agents of pro- or anti-platelet
aggregation. Some of these molecules, especially those isolated from Viperidae venoms, are
used in the diagnosis and treatment of thrombotic and heart diseases. Some components act
synergistically at different stages of the coagulation cascade [ 48Marrakchi N, Barbouche R, Guermazi S, Bon C, el Ayeb M. Procoagulant and
platelet-aggregating properties of Cerastocytin from Cerastes cerastes venom.
Toxicon.1997;19(2):261–272. doi: 10.1016/S0041-0101(96)00116-X.
https://doi.org/10.1016/S0041-0101(96)00...
].
Constituents of Viperidae venoms contain two categories of components that act antagonistically through activation or inhibition of coagulation factors and platelet aggregation. These compounds, able to hydrolyze the coagulation factors with high specificity, are divided into serine proteinases and metalloproteinases. Phospholipases also display potent inhibition of platelet aggregation. Biomolecules of snake venoms are of great fundamental diagnostic and therapeutic interest. Therapeutically, proteinases from Viperidae venoms are widely used as anticoagulants. Furthermore, they are valuable tools for understanding the different mechanisms of hemostasis and are also used in the diagnosis of dysfunctions related to coagulation factors such as enzyme activity in thrombin-like venoms that are used for the fibrinogenopathy screening. Venoms are also used for diagnostic analysis of various coagulation factors (factors V, VII, X, platelet factor III, protein C and factor of Willbrand). Snake venom proteases are useful tools for studying coagulation reactions.
This work was partially supported by PNR and ANDRS (National Agency for the Development of Health Research, Oran, Algeria) projects. We are grateful to Benayad Tahar for technical assistance (Laboratory of Food Security and the Environment, Scientific Police, Algiers, Algeria), Abdelkader Namane and Jean-Claude Rousselle (Platforme of Proteomics, CNRS URA 2185, Pasteur Institute of Paris) for proteomic analyses.
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Ethics committee approval The present study was approved by the Ethics Committee.
Publication Dates
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Publication in this collection
2013
History
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Received
18 Sept 2012 -
Accepted
19 Nov 2012