Research Article Purification and Characterization of ... · characterization of a new toxin from B. moojeni venom that inhibits platelet aggregation. e fractionation of the B. moojeni

$Page 1: Research Article Purification and Characterization of ... · characterization of a new toxin from B. moojeni venom that inhibits platelet aggregation. e fractionation of the B. moojeni$
Research ArticlePurification and Characterization of BmooAiA New Toxin from Bothrops moojeni Snake Venom ThatInhibits Platelet Aggregation

Mayara Ribeiro de Queiroz12 Carla Cristine N Mamede12 Nadia Cristina G de Morais12

Kelly Cortes Fonseca12 Bruna Barbosa de Sousa12 Thaiacutes M Migliorini3

Deacuteborah Fernanda C Pereira1 Leonilda Stanziola23 Leonardo A Calderon4

Rodrigo Simotildees-Silva4 Andreimar Martins Soares4 and Faacutebio de Oliveira23

1 Instituto de Genetica e Bioquımica Universidade Federal de Uberlandia 38400-902 Uberlandia MG Brazil2 Instituto Nacional de Ciencia e Tecnologia em Nano-Biofarmaceutica (N-Biofar) 31270-901 Belo Horizonte MG Brazil3 Instituto de Ciencias Biomedicas Universidade Federal de Uberlandia 38400-902 Uberlandia MG Brazil4 Centro de Estudos de Biomoleculas Aplicadas a Saude (CEBio) Fundacao Oswaldo Cruz Rondonia(Fiocruz Rondonia) e Nucleo de Saude Universidade Federal de Rondonia (UNIR) 76812-245 Porto Velho RO Brazil

Correspondence should be addressed to Fabio de Oliveira foliveiraumuaramaufubr

Received 1 March 2014 Revised 5 May 2014 Accepted 6 May 2014 Published 29 May 2014

Academic Editor Phillip I Bird

Copyright copy 2014 Mayara Ribeiro de Queiroz et al This is an open access article distributed under the Creative CommonsAttribution License which permits unrestricted use distribution and reproduction in any medium provided the original work isproperly cited

In this paper we describe the purificationcharacterization of BmooAi a new toxin from Bothrops moojeni that inhibits plateletaggregationThe purification of BmooAi was carried out through three chromatographic steps (ion-exchange on a DEAE-Sephacelcolumn molecular exclusion on a Sephadex G-75 column and reverse-phase HPLC chromatography on a C2C18 column)BmooAi was homogeneous by SDS-PAGE and shown to be a single-chain protein of 15000 Da BmooAi was analysed byMALDI-TOF Spectrometry and revealed two major components with molecular masses 78244 and 74092 as well as a trace ofprotein with a molecular mass of 152374 Da Sequencing of BmooAi by Edman degradation showed two amino acid sequencesIRDFDPLTNAPENTA and ETEEGAEEGTQ which revealed no homology to any known toxin from snake venom BmooAishowed a rather specific inhibitory effect on platelet aggregation induced by collagen adenosine diphosphate or epinephrine inhuman platelet-rich plasma in a dose-dependent manner whereas it had little or no effect on platelet aggregation induced byristocetin The effect on platelet aggregation induced by BmooAi remained active even when heated to 100∘C BmooAi could beof medical interest as a new tool for the development of novel therapeutic agents for the prevention and treatment of thromboticdisorders

1 Introduction

Snake venoms are a complex mixture of various proteinsenzymes and other substances with toxic properties Amongthe complex pool of proteins (more than 90 of the dryweight) are included enzymes such as acetylcholinesterasesaminotransferases phosphoesterases ADPases phospholi-pases hyaluronidases L-amino acid oxidases (LAAOs) andproteases (metalloproteinases and serinoproteases) [1ndash5]

Protein C activators growth factors (NGF VEGF) lectinsprecursors of bioactive peptides vonWillebrand factor bind-ing proteins disintegrins and bradykinin potentiators aresome representatives of nonenzymatic components fromsnake venom [2 6]

Snake venoms contain a wide variety of nonenzymaticand enzymatic components that have very specific effects onplatelet aggregation [7] Some phospholipases A

2(PLA2) can

affect platelet function usually due to phospholipid hydrolysis

Hindawi Publishing CorporationBioMed Research InternationalVolume 2014 Article ID 920942 7 pageshttpdxdoiorg1011552014920942

2 BioMed Research International

and the formation of metabolites of arachidonic acid [8ndash10]Serinoproteases can activate platelet aggregation directly byproteolytic cleavage of protease-activated receptors (PARs)or by binding to the GPIb receptor [6 9 11] Several snakevenom metalloproteinases (SVMPs) have been shown tointerfere with platelet function through specific structural orenzymatic effects on platelet receptors or their ligands [6ndash8] The effects on platelet aggregation caused by LAAOs aregenerally related to platelet exposure to hydrogen peroxidewhich is generated by the enzymatic activity of the toxin[9 12 13] Disintegrins are usually nonenzymatic inhibitorsof platelet aggregation which typically inhibit 120573

1 1205733 and 120573

5

integrins A common feature of disintegrins is the presenceof the arginine-glycine-aspartate tripeptide sequence (RGD)or a homologous non-RGD sequence in their integrin-binding sites Due to their binding to the fibrinogen receptorGPIIbIIIa (120572IIb1205733) disintegrins inhibit platelet aggregationinduced by a wide range of agonists [14ndash17] Snake venom C-type lectins are also able to affect platelet function by bindingto Von Willebrand factor (vWF) or receptors such as GPIb12057221205731 and GPVI [18 19] In contrast inhibitory effects of

51015840-nucleotidases on platelet aggregation probably occur viacatalytic activity that causes the degradation of ADP a plateletaggregation agonist [6]

In this paper we describe the purification of BmooAifromBmoojeni venomand its characterization as a new toxinthat inhibits platelet aggregation

2 Materials and Methods

21 Material Desiccated B moojeni venom was purchasedfrom Bioagents Serpentarium (Batatais SP Brazil) Acetoni-trile acrylamide ammonium bicarbonate ammonium per-sulphate bromophenol blue bovine fibrinogen glycine 120573-mercaptoethanol 1198731198731015840-methylene-bis-acrylamide sodiumdodecyl sulphate (SDS) 1198731198731198731015840 1198731015840-tetramethylethylene-diamine (TEMED) trifluoroacetic acid and Tris were pur-chased from Sigma Chemical Co (St Louis MO USA)Molecular mass markers for electrophoresis and all chro-matographic media (DEAE-Sephacel Sephadex G-75 andC2C18 columns) were purchased from GE Healthcare Tech-nologies (Uppsala Sweden) All the agonists used in theplatelet aggregation assays (collagen adenosine diphosphateepinephrine and ristocetin) were purchased from HelenaLaboratories (Beaumont Texas USA) All other reagentsused were of analytical grade

22 Blood Collection Human blood was obtained fromvolunteer-donors The experiments reported here follow theguidelines established by the Human Research Ethics Com-mittees of Universidade Federal de Uberlandia (CEPUFU)Minas Gerais Brazil (Protocol number 05511)

23 Purification of BmooAi BmooAi was first purified usingthe methodology previously described [20] with modifica-tions Crude venom from the B moojeni snake (400mg)was dissolved in 50mmolL ammonium bicarbonate buffer(pH = 78) and clarified by centrifugation at 10000timesg for

10 minutes The supernatant solution was fractionated in aDEAE-Sephacel column (25 times 200 cm) previously equili-brated with 50mmolL ammonium bicarbonate (AMBIC)pH = 78 Elution was carried out at a flow rate of 20mLhwith a concentration gradient (50mmolLndash06molL) ofthe same buffer Fractions with 30mLtube were collectedand their absorbance was recorded at a wavelength of280 nm Fractions corresponding to peak DS4 were pooledlyophilized dissolved in 50mmolL AMBIC pH 78 andthen applied to a Sephadex G-75 column (10 times 1000 cm)previously equilibrated with the same buffer The flow ratewas 20mLhour fractions of 30mL were collected and theirabsorbance was recorded at a wavelength of 280 nm FractionDS4G2 showing antiplatelet activity was pooled lyophilizeddissolved in solvent A (01 trifluoroacetic acid) and thensubjected to reverse-phase chromatography in a C2C18 col-umn (46 times 100mm) using the AKTApurifier HPLC systemThe column was equilibrated with solvent A and elutedapplying a concentration gradient toward solvent B (01trifluoroacetic acid containing 80 acetonitrile) from 0 to100 for column volume at a flow rate of 05mLmin at roomtemperature Absorbance was monitored at wavelengths of214 and 280 nm and 1mL fractions were collected

24 Estimation of ProteinConcentration Theprotein concen-tration of the fractions was determined using a UV absorp-tionmethod that calculates concentration from absorbance at214 nm using a BioSpec-mini spectrophotometer (ShimadzuBiotech Japan)

25 Electrophoretic Analysis Electrophoresis using poly-acrylamide gel (SDS-PAGE) was performed as previouslydescribed [21] using 14 gels Electrophoresis was carriedout at 20mAgel in Tris-glycine buffer pH 83 containing001 SDS The molecular mass standard proteins used werephosphorylase b (97000) bovine serum albumin (66000)ovalbumin (45000) carbonic anhydrase (30000) soybeantrypsin inhibitor (20000) and 120572-lactalbumin (14000) Gelswere stained with Coomassie blue R-250 02 (wv)

26MALDI-TOFMass Spectrometry Analysis Themolecularmass of BmooAi was analyzed by MALDI-TOF mass spec-trometry using a 4800MALDI TOFTOFmass spectrometer(Applied Biosystems Foster City California USA) as previ-ously described [22] with modifications

27 N-Terminal Sequence Determination The N-terminalsequence of BmooAi was determined by Edman degrada-tion [23] performed on an automated sequencer (Procisemodel 494 Applied Biosystems) The identity of the primarysequence of BmooAi was compared with other proteins usingBLAST (Basic Local Alignment Search) (httpblastncbinlmnihgovBlastcgi)

28 Peptide Synthesis of N-Terminal Sequences Both the Ile-Arg-Asp-Phe-Asp-Pro-Leu-Thr-Asn-Ala-Pro-Glu-Asn-Thr-Ala and Glu-Thr-Glu-Glu-Gly-Ala-Glu-Glu-Gly-Thr-Gln

BioMed Research International 3

sequences were synthesized using GenScript (PiscatawayNew Jersey USA)

29 Proteolytic Activity on Fibrinogen Fibrinogenolytic ac-tivity was assayed as previously described [24] with mod-ifications Fibrinogen (15mgmL) and samples (5 120583g) weremixed 1 100 (ww) and themixture was incubated at 37∘C for120minThe reaction was stopped by the addition of an equalvolume of a denaturing buffer containing 2 sodium dodecylsulphate (SDS) and 10 120573-mercaptoethanol Reaction prod-ucts were analyzed using 14 (wv) SDS-PAGE

210 Platelet Aggregation Platelet aggregation assays wereperformed in human platelet-rich plasma (PRP) and meas-ured using an automated 4-channel Aggregometer (AggRAMversion 11 Helena Laboratories USA) Human blood col-lected in sodium citrate (32) was centrifuged at 100timesgfor 12min at room temperature to obtain PRP Platelet-poorplasma (PPP) was obtained from the residue by centrifu-gation of citrated blood at 1000timesg for 15min Assays werecarried out using 200 120583L of PRP maintained at 37∘C undercontinuous stirring in siliconized glass cuvettes Aggrega-tion was triggered with collagen (10120583gmL) ADP (20120583M)ristocetin (15mgmL) or epinephrine (300 120583M) after theincubation of platelets with different doses of BmooAi (0610 and 14 120583g) One hundred percent (100) aggregationwas expressed as the percentage absorbance relative to PPPaggregation Control experiments were performed using onlyplatelet agonists All experiments were carried out in tripli-cate

3 Results and Discussion

In this study we describe the purification and partialcharacterization of a new toxin from B moojeni venomthat inhibits platelet aggregation The fractionation of theB moojeni venom was carried out by three chromato-graphic steps involving ion-exchange chromatography on aDEAE-Sepharose column molecular exclusion chromatog-raphy on a Sephadex G-75 column and reverse-phaseHPLC chromatography on a C2C18 column The fraction-ation of B moojeni venom by ion-exchange chromatog-raphy resulted in five major protein fractions named DS1through DS5 (Figure 1(a)) Fraction DS4 was further frac-tionated by size exclusion chromatography (Sephadex G-75) and three fractions were collected (DS4G1 throughDS4G3) (Figure 1(b)) All fractions were tested on collagen- ristocetin- epinephrine- or ADP-induced platelet aggre-gation in human plasma Fraction DS4G2 (80120583g) inhibitedaround 85 of collagen-induced and around 75 of ADP-induced platelet aggregation it was also able to degradeboth the A120572 and B120573 chains of bovine fibrinogen (results notshown) Even when preincubated at 100∘C fraction DS4G2maintained its inhibitory effect on platelet aggregation butlost its fibrinogenolytic activity (results not shown) Theseresults suggest that the effect on aggregation induced byfraction DS4G2 is not dependent on enzymatic action sincethe proteins present in this fraction were denatured by a high

temperature Fraction DS4G2 was lyophilized and subjectedto reverse-phase HPLC chromatography on a C2C18 column(Figure 1(c)) This procedure resulted in four major proteinfractions at 214 nm but only two at 280 nm These resultssuggest that the first two peaks at 214 nm are composed ofproteins poor in aromatic amino acids The first peak at214 nm was also analyzed by SDS-PAGE and showed a singlepolypeptide chain around 15 kDa (Figure 1(d))This peak wasable to interfere with platelet aggregation and was namedBmooAi (Bothrops moojeni platelet aggregation inhibitor)BmooAi showed no fibrinogenolytic activity

BmooAi seems to have low expression in B moojenisnake venom since it represented sim0005 (ww) of theinitial crude venom It is not advantageous when comparedto the overall yield of other protein molecules with in-hibitory effects on platelet aggregation such as atroxlysin-I from B atrox [25] or Bl-LAAO from B leucurus [26]which represent around 51 and 37 (ww) of their crudevenoms respectively In this study we had to repeat the pu-rification steps several times in order to obtain sufficientmaterial for an initial characterization of this new toxinFor this reason BmooAi concentration determinations wereperformed by a UV absorption method thus there was nosample waste from using traditional methods to determineprotein concentration The low recovery of BmooAi likelydiscouraged other researchers from investigating this proteinmainly due to the challenges of its purification In spite of thedisadvantages BmooAi has high antiplatelet activity thatmaycontribute significantly to the overall effects of envenomationby B moojeni

Mass spectrometry analysis of BmooAi indicated twomajor components with molecular masses (M + H) 78244and 74092 (Figure 2(a)) These two compounds are alsoseen as doubly charged ions (119872119885 = 39105 and 37034resp) Figure 2(b) shows traces of a protein with a molec-ular mass (152374) similar to that found via SDS-PAGE(Figure 1(d)) Based on the analysis by SDS-PAGE (singleband) and reverse-phase chromatography (symmetric peak)we suggest that BmooAi is a unique protein that undergoesautolysisproteolysis releasing two peptides of molecularmass around 75 kDa Indeed some snake venom toxinscan undergo proteolysisautolysis under nonphysiologicalconditions in vitro such as in the presence of reducing agentsalkaline pH or low calcium concentration [27] Additionallythe presence of two peptides composed of different aminoacids corroborates the suggestion that they originate fromthe autolysishydrolysis of BmooAi since peptides that differin hydrophobicity should elute in different peaks in reverse-phase chromatography

BmooAi was subjected to N-terminal sequencing byEdman degradation and revealed two amino-acid sequencesIRDFDPLTNAPENTA and ETEEGAEEGTQ Both N-termi-nal sequences were submitted to BLAST but neither sharedhomology with other snake venom protein Interestingly theprimary sequence of BmooAi has the sequence APEN inthe same position (residues 10ndash13) occupied by the identicalsequence in Insularin a disintegrin from B insularis venomthat inhibits platelet aggregation induced by ADP [28] Thisfinding deserves attention but more studies are needed to


0

05

1

15

2

25

3

1 25 49 73 97 121 145 169 193 217 241Fraction number

DS1DS2

DS3DS4

DS5

Abso

rban

ce (2

80nm

)

(a)

0010203040506070809

1

1 8 15 22 29 36 43 50 57 64 71 78 85 92Fraction number

DS4G1

DS4G2DS4G3

Abso

rban

ce (2

80nm

)

(b)

800

600

400

200

0

(mAU

)

0 50 100 150

(min)

100

80

60

40

20

0

B(

)Reverse phase UV1 280nmReverse phase UV2 214nmReverse phase concentration

(c)

9700066000

45000

30000

20100

14400

1 2 3

(d)

Figure 1 Sequential purification steps of BmooAi from Bothrops moojeni venom (a) Ion-exchange chromatography on a DEAE-Sephacelcolumn crude venom (400mg) was applied to the column (25times20 cm) and elution was carried out at a flow rate of 20mLh with ammoniumbicarbonate gradient buffer (50mmolLndash06molL) Fractions of 30mLtube were collected and their absorbance read at 280 nm (b)Molecular exclusion on a Sephadex G-75 column the active fraction (DS4) was applied to the column and eluted with 50mmolL ammoniumbicarbonate buffer at pH 78 with a flow rate of 20mLhour (c) Reverse-phase HPLC chromatography on a 20 times 25 cm C2C18 column (GEHealth Care) equilibrated with solvent A (01 trifluoroacetic acid) and eluted with a concentration gradient of solvent B (80 acetonitrileand 01 trifluoroacetic acid) from 0 to 100 at a flow rate of 05mLmin at room temperature (d) SDS-PAGE in a 14 (wv) gel Lanes 1standard proteins 2 reduced BmooAi fraction 3 nonreduced BmooAi fraction The gel was stained with Coomassie blue R-250

elucidate the importance of this sequence for inhibition ofplatelet aggregation

Platelets play an essential role in hemostasis Alterationsin normal platelet function are involved in various throm-botic and cardiovascular disorders [29 30] Modulation ofplatelet activation and aggregation are currently applied totreat and prevent cardiovascular disorders and stroke [6 2931 32]

In this study we characterized the interference ofBmooAi with agonist-induced platelet aggregation (colla-gen ADP epinephrine and ristocetin) Our results showedthat BmooAi inhibited collagen-induced (10120583gmL) plateletaggregation in a concentration-dependent manner Com-plete inhibition of collagen-induced platelet aggregation wasobtained with only 14 120583g of BmooAi (Figure 3) Even afterheating to 100∘C BmooAi maintained its inhibitory activitysupporting the hypothesis that its inhibition of platelet aggre-gation is nonenzymatic in nature (data not shown)The assays

of inhibition of ADP- epinephrine- and ristocetin-inducedplatelet aggregation were performed using a dose of 06 120583gdue to the low amount obtained from purification BmooAiinhibited over 80 of epinephrine-induced (300120583M) plateletaggregation and around 30 of ADP-induced aggregation(20120583M) (Figure 4) Under the same conditions BmooAidid not show any inhibitory effect on ristocetin-induced(1mgmL) aggregation

Platelet aggregation is characterized by the accumulationof platelets into a hemostatic plug The GPIIbIIIa receptorplays a central role in linking activated platelets Independentof the initial stimulus blocking integrin 120572IIb1205733 preventsplatelet aggregation and subsequent thrombus formationby preventing binding to fibrinogen The participation ofintegrin 120572IIb1205733 in platelet aggregation whatever the initiatingevent or agonist justifies the interest in the therapeuticblockade of this receptor since all routes of platelet activationconverge on to this final common pathway [7 31 33ndash35]


0102030405060708090

100

Inte

nsity

()

78244

74092

39105

37034

7753552780 73389616492387737683 15237450180 10041420709

11E + 4

4700 linear spec 1 MC ge BC ge SM5[BP = 78232 10534]

200570164454128338922225610619990Mass (mz)

(a)

3241

0102030405060708090

100

Inte

nsity

()

152374156533


161690158254154818151382147946144510Mass (mz)

(b)

Figure 2 Mass determination of BmooAi by MALDI-TOF mass spectrometry (a) The fraction consisted of two major components withmolecular masses (M + H) of 78244 and 74092 These two compounds are also seen as doubly charged ions (119872119885 = 39105 and 37034resp) (b) Expansion of the area around 15 kDa to better visualize the presence of a trace of this protein

0102030405060708090

100110

0 40 80 120 160 200 240 280 320 360 400 440 480 520 560 600

Plat

elet

aggr

egat

ion

()

Time (s)

Collagen control

minus20minus10

06 120583g

10 120583g

14 120583g

Figure 3 Effect of BmooAi (06 10 and 14120583g) on collagen-induced platelet aggregation Human PRP was preincubated withthe indicated doses of BmooAi for 8min at 37∘C before addingcollagen (10120583gmL) Platelet aggregation was recorded for 10minin an AggRAM platelet aggregation system with four-channel laseroptics (Helena Laboratories EUA) Results were expressed as anincrease in light transmission where PPP represents the maximumresponse (100) Control experiments were performed in theabsence of BmooAi

Here we show that BmooAi can inhibit ADP- epinephrine-and collagen-induced platelet aggregation at low concentra-tions which suggests that this toxin may act on integrin120572IIb1205733 On the other hand the lack of an inhibitory effect onristocetin-induced platelet aggregation suggests that BmooAidoes not interfere with von Willebrand factor

Disintegrins are a family of cysteine-rich low-molecular-mass polypeptides (40ndash100 amino acids) present in viperidvenoms that are usually nonenzymatic inhibitors of plateletaggregation [6 14 15 17] They typically have an RGDsequence that binds to integrin 120572IIb1205733 and other integrinsinhibiting their functions [15 28 36] The RGD sequencepresents inhibitory activity on platelet aggregation inducedby several agonists [6] However the Gly position can beoccupied by other individual amino acid residues or even

0102030405060708090

100110

Plat

elet

aggr

egat

ion

()

ADP control

Epinephrine control

0 40 80 120 160 200 240 280 320 360 400 440 480 520 560 600Time (s)

minus20minus10

06 120583g with ADP

06 120583g with epinephrine

Figure 4 Effect of BmooAi (06 120583g) on ADP- and epinephrine-induced platelet aggregation Human PRP was preincubated withthe indicated dose of BmooAi for 8min at 37∘C before adding ADP(20 120583molL) or epinephrine (300120583molL) Platelet aggregation wasrecorded for 10min in an AggRAM platelet aggregation systemwith four-channel laser optics (Helena Laboratories EUA) Resultsare expressed as an increase in light transmission where PPPrepresents the maximum response (100) Control experimentswere performed in the absence of BmooAi

by two residues in conformationally restrained peptides andstill retain integrin-binding activity [36] In the venom ofthe same snake species there are disintegrins that exhibit aconserved RGD-motif and disintegrins with variable non-RGD sequences such MLD MGD VGD KGD WGD orRTSKTS [16 17] However the short N-terminal sequenceof the BmooAi obtained in this work was not sufficient toshow the presence or absence of RGD or variable non-RGDsequences

In order to determine the influence of the N-terminalregion of the peptides found we synthesized the two peptidesand evaluated their effect on aggregationNeither synthesizedpeptides (500 120583g) showed any inhibitory effect on ADP-epinephrine- ristocetin- or collagen-induced platelet aggre-gation These results suggest that the antiplatelet action of


BmooAi depends not only on its N-terminal but also on otherregions formed by adjacent amino acids and the C-terminalmay be essential for its activity [16 17 37]

4 Conclusion

In conclusion we describe a new toxin from snake venomthat inhibits platelet aggregation The reported toxinBmooAi has a molecular mass around 15000Da andshowed no homology with any other snake venom toxinBmooAi has great potential for pharmacological studies dueto the low dose used to inhibit platelet aggregation and canbe of medical interest as a new tool for the development ofnovel therapeutic agents to prevent and treat patients withthrombotic disorders

Conflict of Interests

The authors declare that there is no conflict of interests

Acknowledgments

The authors gratefully acknowledge the financial supportof Fundacao de Amparo a Pesquisa do Estado de MinasGerais (FAPEMIG) Conselho Nacional de DesenvolvimentoCientıfico e Tecnologico (CNPq) Coordenacao de Aper-feicoamento de Pessoal de Nıvel Superior (CAPES) andMinisterio de Ciencias e Tecnologia (MCT) of Brazil Fi-nanciadora de Estudos e Projetos (FINEP) Fundacao deTecnologia do Acre (FUNTACFDCT) Projeto NanoBiotecRede de Biodiversidade e Biotecnologia da Amazonia Legal(BIONORTECNPqMCT) Instituto Nacional para Pesqui-sa Translacional em Saude e Ambiente na Regiao Am-azonica (INCT-INPeTAmCNPqMCT) e Instituto Nacionalpara Pesquisa em Toxinas (INCT-Tox) and Secretaria deDesenvolvimento do Estado de Rondonia (SEPLAN-ROPRONEXCNPq) AmyGrabner provided the English editingof the paper

References

[1] E M Singletary A S Rochman J C A Bodmer and C PHolstege ldquoEnvenomationsrdquo Medical Clinics of North Americavol 89 no 6 pp 1195ndash1224 2005

[2] O H P Ramos and H S Selistre-De-Araujo ldquoSnake venommetalloproteasesmdashstructure and function of catalytic and dis-integrin domainsrdquo Comparative Biochemistry and Physiology CToxicology and Pharmacology vol 142 no 3-4 pp 328ndash3462006

[3] Y Angulo and B Lomonte ldquoBiochemistry and toxicology oftoxins purified from the venom of the snake Bothrops asperrdquoToxicon vol 54 no 7 pp 949ndash957 2009

[4] M S R Gomes M M Mendes F de Oliveira et al ldquoBthMPa new weakly hemorrhagic metalloproteinase from Bothropsmoojeni snake venomrdquo Toxicon vol 53 no 1 pp 24ndash32 2009

[5] J D O Costa K C Fonseca M S Garrote-Filho et alldquoStructural and functional comparison of proteolytic enzymesfrom plant latex and snake venomsrdquo Biochimie vol 92 no 12pp 1760ndash1765 2010

[6] T Sajevic A Leonardi and I Krizaj ldquoHaemostatically activeproteins in snake venomsrdquo Toxicon vol 57 no 5 pp 627ndash6452011

[7] A S Kamiguti ldquoPlatelets as targets of snake venommetallopro-teinasesrdquo Toxicon vol 45 no 8 pp 1041ndash1049 2005

[8] F S Markland ldquoSnake venoms and the hemostatic systemrdquoToxicon vol 36 no 12 pp 1749ndash1800 1998

[9] Q Lu JM Clemetson andK J Clemetson ldquoSnake venoms andhemostasisrdquo Journal of Thrombosis and Haemostasis vol 3 no8 pp 1791ndash1799 2005

[10] N A Santos-Filho L B Silveira C Z Oliveira et al ldquoAnew acidic myotoxic anti-platelet and prostaglandin I2 induc-tor phospholipase A2 isolated from Bothrops moojeni snakevenomrdquo Toxicon vol 52 no 8 pp 908ndash917 2008

[11] B F Santos S M T Serrano A Kuliopulos and S Niewi-arowski ldquoInteraction of viper venom serine peptidases withthrombin receptors on human plateletsrdquo FEBS Letters vol 477no 3 pp 199ndash202 2000

[12] M A Belisario S Tafuri C Di Domenico et al ldquoH2

O2

activity on platelet adhesion to fibrinogen and protein tyrosinephosphorylationrdquo Biochimica et Biophysica Acta vol 1495 no2 pp 183ndash193 2000

[13] K J Clemetson Q Lu and J M Clemetson ldquoSnake venomproteins affecting platelets and their applications to anti-thrombotic researchrdquo Current Pharmaceutical Design vol 13no 28 pp 2887ndash2892 2007

[14] A S Kamiguti M Zuzel and R D G Theakston ldquoSnakevenom metalloproteinases and disintegrins interactions withcellsrdquo Brazilian Journal of Medical and Biological Research vol31 no 7 pp 853ndash862 1998

[15] S Braud C Bon and A Wisner ldquoSnake venom proteins actingon hemostasisrdquo Biochimie vol 82 no 9-10 pp 851ndash859 2000

[16] J J Calvete CMarcinkiewicz DMonleon et al ldquoSnake venomdisintegrins evolution of structure and functionrdquo Toxicon vol45 no 8 pp 1063ndash1074 2005

[17] J A Eble ldquoMatrix biology meets toxinologyrdquo Matrix Biologyvol 29 no 4 pp 239ndash247 2010

[18] K J Clemetson Q Lu and J M Clemetson ldquoSnake C-typelectin-like proteins and platelet receptorsrdquo Pathophysiology ofHaemostasis andThrombosis vol 34 no 4-5 pp 150ndash155 2006

[19] T Morita ldquoStructures and functions of snake venom CLPs (C-type lectin-like proteins) with anticoagulant- procoagulant-and platelet-modulating activitiesrdquo Toxicon vol 45 no 8 pp1099ndash1114 2005

[20] C P Bernardes N A Santos-Filho T R Costa et al ldquoIsolationand structural characterization of a new fibrin(ogen)olytic met-alloproteinase from Bothrops moojeni snake venomrdquo Toxiconvol 51 no 4 pp 574ndash584 2008

[21] U K Laemmli ldquoCleavage of structural proteins during theassembly of the head of bacteriophage T4rdquo Nature vol 227 no5259 pp 680ndash685 1970

[22] A Vilca-Quispe L A Ponce-Soto F V Winck and SMarangoni ldquoIsolation and characterization of a new serineprotease with thrombin-like activity (TLBm) from the venomof the snake Bothrops marajoensisrdquo Toxicon vol 55 no 4 pp745ndash753 2010

[23] P Edman ldquoMethod for determination of the amino acidsequence in peptidesrdquo Acta Chemica Scandinavica vol 4 pp283ndash293 1950

[24] W Edgar and C R M Prentice ldquoThe proteolytic action of an-crod on human fibrinogen and its polypeptide chainsrdquoThrom-bosis Research vol 2 no 1 pp 85ndash95 1973


[25] E F Sanchez F S Schneider A Yarleque et al ldquoThe novelmetalloproteinase atroxlysin-I from Peruvian Bothrops atrox(Jergon) snake venom acts both on blood vessel ECM andplateletsrdquo Archives of Biochemistry and Biophysics vol 496 no1 pp 9ndash20 2010

[26] G B Naumann L F Silva L Silva et al ldquoCytotoxicity and inhi-bition of platelet aggregation caused by an l-amino acid oxidasefrom Bothrops leucurus venomrdquo Biochimica et Biophysica Actavol 1810 no 7 pp 683ndash694 2011

[27] J W Fox and S M T Serrano ldquoInsights into and specula-tions about snake venom metalloproteinase (SVMP) synthesisfolding and disulfide bond formation and their contribution tovenom complexityrdquo FEBS Journal vol 275 no 12 pp 3016ndash3030 2008

[28] M S Della-Casa I Junqueira-de-Azevedo D Butera et alldquoInsularin a disintegrin from Bothrops insularis venom inhibi-tion of platelet aggregation and endothelial cell adhesion by thenative and recombinant GST-insularin proteinsrdquo Toxicon vol57 no 1 pp 125ndash133 2011

[29] L K Jennings ldquoMechanisms of platelet activation need for newstrategies to protect against platelet-mediated atherothrombo-sisrdquo Thrombosis and Haemostasis vol 102 no 2 pp 248ndash2572009

[30] C Y Koh and R M Kini ldquoFrom snake venom toxins to thera-peuticsmdashcardiovascular examplesrdquo Toxicon vol 59 no 4 pp497ndash506 2012

[31] H E Speich A D Earhart S N Hill et al ldquoVariability ofplatelet aggregate dispersal with glycoprotein IIb-IIIa antag-onists eptifibatide and abciximabrdquo Journal of Thrombosis andHaemostasis vol 7 no 6 pp 983ndash991 2009

[32] A D Michelson ldquoAntiplatelet therapies for the treatment ofcardiovascular diseaserdquo Nature Reviews Drug Discovery vol 9no 2 pp 154ndash169 2010

[33] E J Topol T V Byzova and E F Plow ldquoPlatelet GPIIb-IIIablockersrdquoThe Lancet vol 353 no 9148 pp 227ndash231 1999

[34] K Jurk and B E Kehrel ldquoPlatelets physiology and biochem-istryrdquo Seminars inThrombosis and Hemostasis vol 31 no 4 pp381ndash392 2005

[35] R Shlansky-Goldberg ldquoPlatelet aggregation inhibitors for usein peripheral vascular interventions what canwe learn from theexperience in the coronary arteriesrdquo Journal of Vascular andInterventional Radiology vol 13 no 3 pp 229ndash246 2002

[36] E Ruoslahti ldquoRGD and other recognition sequences for inte-grinsrdquo Annual Review of Cell and Developmental Biology vol12 pp 697ndash715 1996

[37] C Barja-Fidalgo A L J Coelho R Saldanha-Gama E Helal-Neto A Mariano-Oliveira and M S de Freitas ldquoDisintegrinsintegrin selective ligands which activate integrin-coupled sig-naling and modulate leukocyte functionsrdquo Brazilian Journal ofMedical and Biological Research vol 38 no 10 pp 1513ndash15202005

Submit your manuscripts athttpwwwhindawicom

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Volume 2014

ToxinsJournal of

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Autoimmune Diseases


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Pharmaceutics


MEDIATORSINFLAMMATION

of


and the formation of metabolites of arachidonic acid [8ndash10]Serinoproteases can activate platelet aggregation directly byproteolytic cleavage of protease-activated receptors (PARs)or by binding to the GPIb receptor [6 9 11] Several snakevenom metalloproteinases (SVMPs) have been shown tointerfere with platelet function through specific structural orenzymatic effects on platelet receptors or their ligands [6ndash8] The effects on platelet aggregation caused by LAAOs aregenerally related to platelet exposure to hydrogen peroxidewhich is generated by the enzymatic activity of the toxin[9 12 13] Disintegrins are usually nonenzymatic inhibitorsof platelet aggregation which typically inhibit 120573

1 1205733 and 120573

5

integrins A common feature of disintegrins is the presenceof the arginine-glycine-aspartate tripeptide sequence (RGD)or a homologous non-RGD sequence in their integrin-binding sites Due to their binding to the fibrinogen receptorGPIIbIIIa (120572IIb1205733) disintegrins inhibit platelet aggregationinduced by a wide range of agonists [14ndash17] Snake venom C-type lectins are also able to affect platelet function by bindingto Von Willebrand factor (vWF) or receptors such as GPIb12057221205731 and GPVI [18 19] In contrast inhibitory effects of

51015840-nucleotidases on platelet aggregation probably occur viacatalytic activity that causes the degradation of ADP a plateletaggregation agonist [6]

In this paper we describe the purification of BmooAifromBmoojeni venomand its characterization as a new toxinthat inhibits platelet aggregation

2 Materials and Methods

21 Material Desiccated B moojeni venom was purchasedfrom Bioagents Serpentarium (Batatais SP Brazil) Acetoni-trile acrylamide ammonium bicarbonate ammonium per-sulphate bromophenol blue bovine fibrinogen glycine 120573-mercaptoethanol 1198731198731015840-methylene-bis-acrylamide sodiumdodecyl sulphate (SDS) 1198731198731198731015840 1198731015840-tetramethylethylene-diamine (TEMED) trifluoroacetic acid and Tris were pur-chased from Sigma Chemical Co (St Louis MO USA)Molecular mass markers for electrophoresis and all chro-matographic media (DEAE-Sephacel Sephadex G-75 andC2C18 columns) were purchased from GE Healthcare Tech-nologies (Uppsala Sweden) All the agonists used in theplatelet aggregation assays (collagen adenosine diphosphateepinephrine and ristocetin) were purchased from HelenaLaboratories (Beaumont Texas USA) All other reagentsused were of analytical grade

22 Blood Collection Human blood was obtained fromvolunteer-donors The experiments reported here follow theguidelines established by the Human Research Ethics Com-mittees of Universidade Federal de Uberlandia (CEPUFU)Minas Gerais Brazil (Protocol number 05511)

23 Purification of BmooAi BmooAi was first purified usingthe methodology previously described [20] with modifica-tions Crude venom from the B moojeni snake (400mg)was dissolved in 50mmolL ammonium bicarbonate buffer(pH = 78) and clarified by centrifugation at 10000timesg for

10 minutes The supernatant solution was fractionated in aDEAE-Sephacel column (25 times 200 cm) previously equili-brated with 50mmolL ammonium bicarbonate (AMBIC)pH = 78 Elution was carried out at a flow rate of 20mLhwith a concentration gradient (50mmolLndash06molL) ofthe same buffer Fractions with 30mLtube were collectedand their absorbance was recorded at a wavelength of280 nm Fractions corresponding to peak DS4 were pooledlyophilized dissolved in 50mmolL AMBIC pH 78 andthen applied to a Sephadex G-75 column (10 times 1000 cm)previously equilibrated with the same buffer The flow ratewas 20mLhour fractions of 30mL were collected and theirabsorbance was recorded at a wavelength of 280 nm FractionDS4G2 showing antiplatelet activity was pooled lyophilizeddissolved in solvent A (01 trifluoroacetic acid) and thensubjected to reverse-phase chromatography in a C2C18 col-umn (46 times 100mm) using the AKTApurifier HPLC systemThe column was equilibrated with solvent A and elutedapplying a concentration gradient toward solvent B (01trifluoroacetic acid containing 80 acetonitrile) from 0 to100 for column volume at a flow rate of 05mLmin at roomtemperature Absorbance was monitored at wavelengths of214 and 280 nm and 1mL fractions were collected

24 Estimation of ProteinConcentration Theprotein concen-tration of the fractions was determined using a UV absorp-tionmethod that calculates concentration from absorbance at214 nm using a BioSpec-mini spectrophotometer (ShimadzuBiotech Japan)

25 Electrophoretic Analysis Electrophoresis using poly-acrylamide gel (SDS-PAGE) was performed as previouslydescribed [21] using 14 gels Electrophoresis was carriedout at 20mAgel in Tris-glycine buffer pH 83 containing001 SDS The molecular mass standard proteins used werephosphorylase b (97000) bovine serum albumin (66000)ovalbumin (45000) carbonic anhydrase (30000) soybeantrypsin inhibitor (20000) and 120572-lactalbumin (14000) Gelswere stained with Coomassie blue R-250 02 (wv)

26MALDI-TOFMass Spectrometry Analysis Themolecularmass of BmooAi was analyzed by MALDI-TOF mass spec-trometry using a 4800MALDI TOFTOFmass spectrometer(Applied Biosystems Foster City California USA) as previ-ously described [22] with modifications

27 N-Terminal Sequence Determination The N-terminalsequence of BmooAi was determined by Edman degrada-tion [23] performed on an automated sequencer (Procisemodel 494 Applied Biosystems) The identity of the primarysequence of BmooAi was compared with other proteins usingBLAST (Basic Local Alignment Search) (httpblastncbinlmnihgovBlastcgi)

28 Peptide Synthesis of N-Terminal Sequences Both the Ile-Arg-Asp-Phe-Asp-Pro-Leu-Thr-Asn-Ala-Pro-Glu-Asn-Thr-Ala and Glu-Thr-Glu-Glu-Gly-Ala-Glu-Glu-Gly-Thr-Gln












0

05

1

15

2

25

3

1 25 49 73 97 121 145 169 193 217 241Fraction number

DS1DS2

DS3DS4

DS5

Abso

rban

ce (2

80nm

)

(a)

0010203040506070809

1

1 8 15 22 29 36 43 50 57 64 71 78 85 92Fraction number

DS4G1

DS4G2DS4G3

Abso

rban

ce (2

80nm

)

(b)

800

600

400

200

0

(mAU

)

0 50 100 150

(min)

100

80

60

40

20

0

B(


(c)

9700066000

45000

30000

20100

14400

1 2 3

(d)








0102030405060708090

100

Inte

nsity

()

78244

74092

39105

37034

7753552780 73389616492387737683 15237450180 10041420709

11E + 4


200570164454128338922225610619990Mass (mz)

(a)

3241

0102030405060708090

100

Inte

nsity

()

152374156533


161690158254154818151382147946144510Mass (mz)

(b)


0102030405060708090

100110

0 40 80 120 160 200 240 280 320 360 400 440 480 520 560 600

Plat

elet

aggr

egat

ion

()

Time (s)

Collagen control

minus20minus10

06 120583g

10 120583g

14 120583g




0102030405060708090

100110

Plat

elet

aggr

egat

ion

()

ADP control

Epinephrine control

0 40 80 120 160 200 240 280 320 360 400 440 480 520 560 600Time (s)

minus20minus10

06 120583g with ADP







4 Conclusion




Acknowledgments


References













O2
































Volume 2014

ToxinsJournal of

VaccinesJournal of















AddictionJournal of






Autoimmune Diseases




Journal of


Pharmaceutics



of












0

05

1

15

2

25

3

1 25 49 73 97 121 145 169 193 217 241Fraction number

DS1DS2

DS3DS4

DS5

Abso

rban

ce (2

80nm

)

(a)

0010203040506070809

1

1 8 15 22 29 36 43 50 57 64 71 78 85 92Fraction number

DS4G1

DS4G2DS4G3

Abso

rban

ce (2

80nm

)

(b)

800

600

400

200

0

(mAU

)

0 50 100 150

(min)

100

80

60

40

20

0

B(


(c)

9700066000

45000

30000

20100

14400

1 2 3

(d)








0102030405060708090

100

Inte

nsity

()

78244

74092

39105

37034

7753552780 73389616492387737683 15237450180 10041420709

11E + 4


200570164454128338922225610619990Mass (mz)

(a)

3241

0102030405060708090

100

Inte

nsity

()

152374156533


161690158254154818151382147946144510Mass (mz)

(b)


0102030405060708090

100110

0 40 80 120 160 200 240 280 320 360 400 440 480 520 560 600

Plat

elet

aggr

egat

ion

()

Time (s)

Collagen control

minus20minus10

06 120583g

10 120583g

14 120583g




0102030405060708090

100110

Plat

elet

aggr

egat

ion

()

ADP control

Epinephrine control

0 40 80 120 160 200 240 280 320 360 400 440 480 520 560 600Time (s)

minus20minus10

06 120583g with ADP







4 Conclusion




Acknowledgments


References













O2
































Volume 2014

ToxinsJournal of

VaccinesJournal of















AddictionJournal of






Autoimmune Diseases




Journal of


Pharmaceutics



of


0

05

1

15

2

25

3

1 25 49 73 97 121 145 169 193 217 241Fraction number

DS1DS2

DS3DS4

DS5

Abso

rban

ce (2

80nm

)

(a)

0010203040506070809

1

1 8 15 22 29 36 43 50 57 64 71 78 85 92Fraction number

DS4G1

DS4G2DS4G3

Abso

rban

ce (2

80nm

)

(b)

800

600

400

200

0

(mAU

)

0 50 100 150

(min)

100

80

60

40

20

0

B(


(c)

9700066000

45000

30000

20100

14400

1 2 3

(d)








0102030405060708090

100

Inte

nsity

()

78244

74092

39105

37034

7753552780 73389616492387737683 15237450180 10041420709

11E + 4


200570164454128338922225610619990Mass (mz)

(a)

3241

0102030405060708090

100

Inte

nsity

()

152374156533


161690158254154818151382147946144510Mass (mz)

(b)


0102030405060708090

100110

0 40 80 120 160 200 240 280 320 360 400 440 480 520 560 600

Plat

elet

aggr

egat

ion

()

Time (s)

Collagen control

minus20minus10

06 120583g

10 120583g

14 120583g




0102030405060708090

100110

Plat

elet

aggr

egat

ion

()

ADP control

Epinephrine control

0 40 80 120 160 200 240 280 320 360 400 440 480 520 560 600Time (s)

minus20minus10

06 120583g with ADP







4 Conclusion




Acknowledgments


References













O2
































Volume 2014

ToxinsJournal of

VaccinesJournal of















AddictionJournal of






Autoimmune Diseases




Journal of


Pharmaceutics



of


0102030405060708090

100

Inte

nsity

()

78244

74092

39105

37034

7753552780 73389616492387737683 15237450180 10041420709

11E + 4


200570164454128338922225610619990Mass (mz)

(a)

3241

0102030405060708090

100

Inte

nsity

()

152374156533


161690158254154818151382147946144510Mass (mz)

(b)


0102030405060708090

100110

0 40 80 120 160 200 240 280 320 360 400 440 480 520 560 600

Plat

elet

aggr

egat

ion

()

Time (s)

Collagen control

minus20minus10

06 120583g

10 120583g

14 120583g




0102030405060708090

100110

Plat

elet

aggr

egat

ion

()

ADP control

Epinephrine control

0 40 80 120 160 200 240 280 320 360 400 440 480 520 560 600Time (s)

minus20minus10

06 120583g with ADP







4 Conclusion




Acknowledgments


References













O2
































Volume 2014

ToxinsJournal of

VaccinesJournal of















AddictionJournal of






Autoimmune Diseases




Journal of


Pharmaceutics



of



4 Conclusion




Acknowledgments


References













O2
































Volume 2014

ToxinsJournal of

VaccinesJournal of















AddictionJournal of






Autoimmune Diseases




Journal of


Pharmaceutics



of



















Volume 2014

ToxinsJournal of

VaccinesJournal of















AddictionJournal of






Autoimmune Diseases




Journal of


Pharmaceutics



of





Volume 2014

ToxinsJournal of

VaccinesJournal of















AddictionJournal of






Autoimmune Diseases




Journal of


Pharmaceutics



of

Documents

Research Article Purification and Characterization of ... · characterization of a new toxin from B. moojeni venom that inhibits platelet aggregation. e fractionation of the B. moojeni