A heparin-like compound isolated from a marine crab rich in glucuronic acid 2-O-sulfate presents low anticoagulant activity

ContentslistsavailableatSciVerseScienceDirect

Carbohydrate

Polymers

j our na l h o me p ag e : w w w . e l s e v i e r . c o m / l o c a t e / c a r b p o l

A

heparin-like

compound

isolated

from

a

marine

crab

rich

in

glucuronic

acid

2-

O

-sulfate

presents

low

anticoagulant

activity

Giulianna

P.V.

Andrade

_,

_Marcelo

_A.

_Lima

_,

_Airton

_A.

_de

_Souza

_Junior

_,

_Jawed

_Fareed

_,

Debra

A.

Hoppensteadt

_,

_Elizeu

_A.

_Santos

_,

_Suely

_F.

_Chavante

_,

_Fernanda

_W.

_Oliveira

_,

Hugo

A.O.

Rocha

_,

_Helena

_B.

_Nader

a_{DepartamentodeBioquímica,UniversidadeFederaldoRioGrandedoNorte,Natal,RN,Brazil} b_{DepartamentodeBioquímica,UniversidadeFederaldeSãoPaulo,SãoPaulo,SP,Brazil}

c_{DepartmentofStructuralandChemicalBiology,UniversityofLiverpool,CrownStreet,LiverpoolL697ZB,UK} d_{DepartamentodeBiologia,InstitutoFederaldoRioGrandedoNorte,Natal,RN,Brazil}

e_{DepartmentofPathology,LoyolaMedicalSchool,Maywood,IL,USA}

a

r

t

i

c

l

e

i

n

f

o

Articlehistory:

Received27November2012

Receivedinrevisedform21January2013 Accepted22January2013

Available online 29 January 2013

InhonorofProf.CarlP.Dietrich.

Keywords: Heparin Heparansulfate

Nuclearmagneticresonance Heparinases

Anticoagulantactivity Hemorrhagiceffect

a

b

s

t

r

a

c

t

Anaturalheparin-likecompoundisolatedfromthecrabGoniopsiscruentatawasstructurally character-izedanditsanticoagulantandhemorrhagicactivitiesweredetermined.Enzymaticandnuclearmagnetic resonanceanalysisrevealedthatitsstructureisrichindisulfateddisaccharides,possessingsignificant amountsof2-O-sulfated-␤-d_{-glucuronicacidunits.Furthermore,lowamountsoftrisulfateddisaccharide}

unitscontaining2-O-sulfated-␣-l-iduronicacidweredetected,whencomparedtomammalianheparin.

Inaddition,thisheparin-likestructureshowednegligibleinvitroanticoagulantactivityandlowbleeding potency,factsthatmakeitasuitablecandidateforthedevelopmentofstructure-driven,heparinbased therapeuticagentswithfewerundesirableeffects.

© 2013 Elsevier Ltd.

1. Introduction

Heparinandlowmolecularweightheparinsarethemain anti-coagulantandantithromboticdrugscurrentlyusedinmedicine. Besidesits well-describedanticoagulant/antithrombotic actions, heparinand heparin-like moleculesare knowntointeractwith multipleproteinsmodulating severalbiologicalprocesses (Brito etal.,2008;Dreyfussetal.,2010;Paredes-Gameroetal.,2010), however,itsfurtherclinicaluseisimpairedbyitsstrong anticoag-ulantactivityandhemorrhagiccomplications.

Heparinandheparansulfatesharestructuralfeatures,yet,they canbedifferentiatedbythelevelsofglucosamineN-acetylation, totalsulfateandglucuronic/iduronicacidratio(Casu,Naggi,&Torri, 2010).Furthermore,heparansulfatesareubiquitouscomponents ofalltissue-organizedanimallifeforms(Cassaro&Dietrich,1977; Medeirosetal.,2000;Sampaioetal.,2006;Toledo&Dietrich,1977),

∗_{Correspondingauthorat:MolecularBiology,DepartamentodeBioquímica,} Uni-versidadeFederaldeSãoPaulo,RuaTrêsdeMaio,100,CEP04044-020,SãoPaulo, SP,Brazil.Tel.:+551155703175;fax:+551155736407.

E-mailaddress:hbnader.bioq@epm.br(H.B.Nader).

whereasheparinshowsapeculiardistributioninmammalianand othervertebrates,aswellasininvertebrates(Nader,Lopes,Rocha, Santos,&Dietrich,2004).

Ininvertebrates,heparinisfoundinsomespeciesofmollusk, crustacean,annelid,echinodermate,tunicateandurochordatelife (Cassaro&Dietrich,1977;Cavalcanteetal.,2000;Dietrichetal., 1985;Luppi,Cesaretti,&Volpi,2005;Medeirosetal.,2000;Pejler etal.,1987;Sampaioetal.,2006).Insomeinvertebrates,the pres-enceofheparin-like structureswithsimilarities toheparinbut, withsomestructuralpeculiarities,havebeendescribed(Britoetal., 2008;Chavante etal.,2000;Dietrichet al.,1999a;Naderetal., 2004).Thesepreviousstudieshave shown thattheirstructures vary according tothe species and that such differences reside, mainly,intherelativeabundanceofthedifferentdisaccharideunits (Naderetal.,2004).Additionally,theseheparin-likecompounds showvariablebiologicalactivities(Boucasetal.,2006;Britoetal., 2008; Cassaro&Dietrich, 1977;Chavante et al., 2000;Dietrich etal.,1999a;Dreyfussetal.,2010;Medeirosetal.,2000;Santos et al., 2007). Thus, each heparin/heparin-like compound from invertebratetissuestendstobeahithertounknowncompound withuniquestructuralfeaturesandapotentialnoveltherapeutic agent.

0144-8617© 2013 Elsevier Ltd.

http://dx.doi.org/10.1016/j.carbpol.2013.01.069

Open access under the Elsevier OA license.

2. Materialsandmethods

2.1. Materials

Heparansulfatefrombovinepancreasandheparinfrombovine intestinalmucosaweregiftsfromthelateDr.P.Bianchini(Opocrin ResearchLaboratories,Corlo,Modena,Italy).Heparinfromporcine intestinalmucosa wasobtainedfromKin Master (PassoFundo, RS,Brazil)andenoxaparin(lowmolecularweightheparin)from Sanofi-Aventis (Maison-Alford, France). Chondroitin 4- and 6-sulfate and dermatan sulfate were purchased from Seikagaku Kogyo(Tokyo,Japan).Heparinase(HeparinaseI,EC4.2.2.7), hep-aritinasesIandIIwerepreparedfromFlavobacteriumheparinum as previously described (Nader et al., 1990). Ethylenediamine (1,2-diaminoethane)andpropylenediamine(1,3-diaminopropane) werepurchased fromSigma–AldrichCo. (Milwaukee, WI,USA). Low-Mr agarose was purchased from Bio-Rad (Richmond, CA, USA). Maxatase, a protease from Sporobacillus, was purchased from Biocon do Brasil Industrial Ltd. (Rio de Janeiro, RJ, Brazil).

2.2. Extractionandpurificationofcrabheparin-like

TheAnimal Ethics AdvisoryCommitteeapproved all experi-mentsinvolvinganimals inaccordance to theBrazilian Federal Law(11,794/2008)fortheuseand careof animalsfor scientific purposes.Adult specimens ofG. cruentata (Latreill,1803) were collectedatPotengiriverestuary(Macaíba,RN,Brazil), immedi-atelykilledandstoredat−20◦_{C.Sulfatedglycosaminoglycanswere} extractedafterproteolysisandionexchangefractionation.Ten kilo-gramsofcrabweregroundwith2volumesofcold0.5MNaClin ablender.ThepHofthemixturewasadjustedto8.0withNaOH andMaxatasewasadded(3.5mg/kgwetweight).After incubat-ingfor24hat60◦_{C,withagitationandperiodicpHadjustments,} themixturewasfilteredandLewatitionexchangeresin(Bayer, SP,Brazil) wasadded (60mg/kgwetweight), and theresulting mixturewasagitatedfor24hat60◦_{Cundera layerof toluene.} Thesuspensionwasagainfilteredandtheresinretained,washed with10 volumes of water at 60◦_{C and, subsequently, washed} with10volumesof0.5MNaClatroomtemperature.Thewashed resinwasthensuspendedin2volumesof1MNaCl,agitatedfor 3hand filteredagain. Thisprocedurewasrepeated usingwith 2 and 3M NaCl.The filtrates were maintainedfor 24hat 4◦_C after the addition of 2 volumes of methanol and the precipi-tateformedwascollectedbycentrifugation(10,000×g,20min),

driedundervacuum,suspendedindistilledwater,andanalyzed byagarosegelelectrophoresis.Fractions elutedwith2 and3M NaCl,whichshowedthepresenceofcompoundsmigratingas hep-arinandheparansulfatewerepooled,dialyzedanddried.These compoundswere furtherdissolvedin 0.15MNaCl and 0.5 vol-umesofice-coldacetonewasaddedtothesolutionundergentle agitation and maintained at 4◦_{C for 24h. The precipitate was} collectedbycentrifugationat4◦_{C. Thisprocedurewasrepeated} successivelybyadditiontothesupernatantof0.6,0.7,0.8,0.9,1.0 and2.0volumesofacetoneaccordingtothevolumeofthe ini-tialsolution.Theresultingprecipitatesweredialyzed,driedand analyzed. Fractions precipitatedwith 0.6 volumes and 0.7 vol-umesof acetone correspondedto90% of thetotal heparin-like compound.

ent buffers: 0.05M1,3-diaminopropane–acetate buffer, pH 9.0, discontinuous buffer 0.04M barium acetate, pH 4.0/0.05M diaminopropane–acetate,pH9.0or0.06MTris–acetatebuffer,pH 8.0,aspreviouslydescribed(Bianchinietal.,1980).Aliquotsofthe fractions(about10␮g)wereappliedtothegelandsubjectedto electrophoresis.Thegelswerefixedwith0.1% cetyltrimethylam-moniumbromide(CETAVLON)solutionfor4h,driedandstainedfor 15minwith0.1%toluidinebluein1%aceticacidin50%ethanoland furtherdestainedwiththesamesolutionwithoutthedye. Quantifi-cationwascarriedoutbydensitometryat530nmofthetoluidine blue-stainedelectrophoreticslide.Theextinctioncoefficientsofthe GAGswerecalculatedusingstandardsofchondroitinsulfate(CS), dermatansulfate(DS)andheparansulfate(HS).Theerrorofthe methodwasintheorderof5%.IdentificationofthesulfatedGAGs wasinitiallybasedonthemigrationofthecompoundscompared withthoseofstandards.

2.4. Enzymaticdegradation

Sampleswereincubatedwithdifferentheparinlyases (hepari-naseI,heparitinaseIandII,2.5mIUeach)aswellaswithamixture ofallenzymes.Thedisaccharidesproducedbytheenzymaticaction wereresolvedona150×4.6mmPhenosphereSAXcolumn

(Phen-omenex,Torrance,CA,USA)usingaNaClgradientof0–1Mduring 30minwitha1mL/minfluxandUVdetectionat232nm.

2.5. NMRspectroscopy

NMRspectrawererecordedeitherusingaBrukerDRX600witha tripleresonance5-mmprobeorinanAgilent600MHzSystemwith 5-mmColdProbe.Thespectrawererecordedateither60or25◦_C withHODsuppressionbypre-saturation.COSY,TOCSYand1_H/13_C

heteronuclear correlation (HSQC) spectra were recorded using states-timeproportionphaseincrementforquadraturedetection intheindirectdimension.Allchemicalshiftswererelativeto exter-naltrimethylsilyl-propionicacidand[13_C]-methanol.

2.6. Invitroanticoagulantactivity

Allcoagulationassays(aPTT,PT,TTandHEPTEST®_)were per-formedusingacoagulometerasdescribedearlier(Dietrichetal., 1999a)usingcitratednormalhumanplasma.Allassayswere per-formedinduplicateandrepeatedatleastthreetimesondifferent days(n=6).GenerationofthrombinandfactorXawasmeasured by amidolyticassays using thespecific chromogenic substrates (SpectrozymeTHandSpectrozymeFXa,AmericanDiagnosticaInc., Stamford,CT)accordingtoamethodpreviouslydescribed(Kaiser etal.,1992).Allassayswereperformedonafastkineticcentrifugal analyzer(ACL-300,Lexington,MA,USA).

2.7. Hemorrhagiceffect

94 (2013) 647–654 649

Fig.1.Electrophoreticbehaviorofthecrabsulfatedglycosaminoglycansinagarosegels.About5–20␮goftheglycosaminoglycanspurifiedfromcrabtissuesweresubjected toelectrophoresisindifferentbuffers.Aftertherun,thecompoundswereprecipitatedandthegelsdriedandstainedwithtoluidineblue.(a)Fractionselutedfromtheion exchangechromatographywereevaluatedusing1,3-diaminopropane-acetate(PDA).1M,2Mand3Mfractionswereelutedfromionexchangechromatographywith1.0, 2.0and3.0MofNaCl,respectively.(b)Fractions2Mand3Mwerepooledandfractionatedwithacetone,andtheelectrophoreticmobilityofthecompoundsevaluatedusing thediscontinuousbufferbariumacetate/PDA.Electrophoreticmobilityoftheheparin-likecompoundsprecipitatedwith0.6and0.7volumesofacetoneindifferentbuffers: (c)PDAbuffer,(d)tris-acetateand(e)discontinuousbufferbariumacetate/PDA.M,mixtureofstandardsulfatedglycosaminoglycanscontaining5␮gofchondroitinsulfate (CS),dermatansulfate(DS)andheparansulfate(HS);Hep,porcineintestinalmucosaheparinandS,I,F,slow,intermediateandfastmovingcomponents,respectively;Or, origin.

2minandwashedextensivelywithsaline.Thetreatedtailswere then immersed in isotonic saline solution, and the amount of bloodoozedwasmeasuredbyproteincontent.Thebleedingwas observedwiththeuseofastereoscope.Allexperimentswere per-formedat37◦_{C. Thebleeding wascalculated asthesumofthe} proteinvaluesofeachtubeminustheamountofproteinpresent beforetheexposuretothetestsubstances.Bleedingpotencywas expressedasthecumulativeamountofproteinreleasedfromthe woundsafterexposuretothecompoundsrelativetothecontrol (absenceofdrug).

2.8. Othermethods

Hexosamine wasdetermined afteracid hydrolysis (4M HCl, 100◦_{C,6h)bytheRondle–Morganprocedure(Rondle&Morgan,}

1955)and uronicacidbythecarbazolereaction(Dische,1947). Total sulfate was measuredby a method previously described (Dodgson &Price,1962).For molecularweightanalysis,300␮g ofeachsamplewasanalyzedbyGPC-HPLCona300mm×7.8mm

BioSepSECTM_{S-2000LCColumn.Thesamplesweresubmittedto}

anisocraticelution(0.3MNa2SO4 mobilephase)ata flowrate

of1mL/minandUVdetectionat205nm.Thecolumnwas previ-ouslycalibratedwithpolysaccharidesofknownmolecularweights (1.7kDa,4kDa,10kDa,16kDaand20kDa).

2.9. Statisticalanalysis

TheSPSSsoftware package (release16.0; SPSSInc.,Chicago, IL,USA)wasusedforstatisticalanalysis.Thedifferencebetween thegroups wasevaluated using thenon-parametric two-tailed

Mann–WhitneyU-test.P<0.05wasconsideredstatistically signif-icant.

3. Resultsanddiscussion

3.1. Purificationofthecrabheparin-likecompound

UA-NAc UA-NS UA-NAc(6S) UA(2S)-NAc UA-NS(6S) UA(2S)-NS UA(2S)-NAc(6S) UA(2S)-NS(6S)

≤1 45.9 8.1 7.5 5.1 18.0 3.0 12.0

Fig.2.1_{Hspectraat600MHzofthecrabheparin-like,heparansulfateandheparinrecordedat60}◦_Cand25◦_{Crespectively.(a)Crabheparin-likecompound(Hepn)}1_H

spectrum;(b)1_{HspectraanomericregionofcrabHepn,heparansulfatefrombovinepancreasandheparinfromporcineintestinalmucosa.A}

NS,N-sulfatedglucosamine;

ANAc,N-acetylatedglucosamine;G,glucuronicacid;G2S,2-O-sulfatedglucuronicacid;I2S,2-Osulfatediduronicacid;I,iduronicacid;G,glucuronicacid.

crustaceanArtemiafranciscana,anunusualheparansulfatewith similarelectrophoreticbehaviortothecrabcompoundthat pre-sentedahighdegreeofN-sulfation.Ontheotherhand,acompound fromtheshrimpPenaeusbrasiliensiswithsimilarelectrophoretic behaviorwasisolatedandcharacterizedasanaturallowmolecular weightheparin(Dietrichetal.,1999a).

3.2. Chemicalanalysisandmolecularmass

Themolecularmassandthemolarratiosofhexosamine,uronic acidand sulfate present in the heparin-like compounds, mam-malianheparinandheparansulfatewerealsoanalyzed.Thecrab compoundsshowedanaveragemolecularmass(∼19kDa)similar

toporcineheparin(∼16kDa)andlowerwhencomparedtobovine

heparansulfate(∼25kDa).Furthermore,bothcompoundsshowed

similarmolarratioofsulfate/hexosamineof2.1:1.0,respectively. Theseratiosarebetweenthevaluesdescribedforheparansulfate (sulfate/hexosamine,1.6:1.0)(Dietrich&Nader,1974)andheparin (sulfate/hexosamine,2.8:1.0)(Dietrichetal.,1985).Neutralsugars werenotdetected.Overall,thedatasuggestedthatthetwo frac-tionscorrespondedtothesamecompound.Thetwofractionswere pooledandhereafterisreferredtoasHepn.

3.3. Enzymaticanalysis

Some structural characteristics of the Hepn were exam-ined with enzymes purified from F. heparinum. Heparitinase I, which acts upon N-acetyl or N-sulfate glucosamine-glucuronic acid linkages (Desai, Wang, & Linhardt, 1993; Dietrich et al., 1999a; Silva & Dietrich, 1974), degraded less than 5% of the

Fig.3. 1_H/13_CHSQC(b)and1_H/1_{HCOSY(b)spectraofthecrabheparin-likecompoundrecordedat60}◦_C.A

94 (2013) 647–654 651

Table2

1_{Hchemicalshiftsfortheheparin-likecompoundofthecrabG.cruentata.}

Unit 1_{Hchemicalshifts}a

Uronicacid Glucosamine

H1 H2 H3 H4 H5 H1 H2 H3 H4 H5 H6

a_{(␤-GlcA)→(␣-d-GlcNS-6S) (I)→(A) 4.60 3.31 3.71 3.88 3.79 5.57 3.18 3.65 3.85 4.04 4.14} b_{␤-GlcA→␣-d-GlcNS-6S 4.58 3.36 3.82 3.84 3.78 5.56 3.27 3.67 3.70 3.96 4.37/4.17} a_{(␤-GlcA)→(␣-}d-GlcNS) (I′_)→(A′_{) 4.58 3.28 3.70 3.88 3.79 5.57 3.18 3.65 3.65 3.75 3.80}

b_{␤-GlcA→␣-d-GlcNS 4.55 3.41 3.83 3.90 3.83 5.58 3.27 3.68 3.68 3.79 3.81/3.83} a_{(␤-GlcA-2S)→(␣-}d-GlcNS) (H)→(B) 4.67 4.12 ∼4.03 ∼3.97 – 5.55 3.24 3.62 3.86 3.72 3.92

c_{␤-GlcA-2S→␣-}d-GlcNS 4.69 4.12 3.99 3.83 – 5.45 3.23 3.75 3.64 3.93 3.88

a_{(␤-GlcA-2S)→(␣-}d-GlcNS-6S)(H′_)→(B′_{) 4.70 4.12 ∼4.03 ∼3.97 – 5.55 3.24 3.62 3.86 4.04 4.14}

b_{␤-GlcA-2S→␣-}d-GlcNS-6S 4.74 4.14 3.98 3.85 3.88 5.44 – – – – –

a_{(␣-IdoA-2S)→(␣-d-GlcNS-6S)(F)→(C) 5.20 4.28 4.22 4.08 5.01 5.48 3.19 – – – ∼4.40} d_{␣-IdoA-2S→␣-}d-GlcNS-6S 5.23 4.37 4.22 4.14 4.82 5.42 3.31 3.69 3.79 4.05 4.42/4.30

a_{(␤-GlcA-2S)→(␣-d-GlcNAc-6S)(G)→(D) 4.73 4.08 – – – 5.32 3.83 3.92 3.77 4.15 4.25} e_{␤-GlcA-2S→␣-d-GlcNAc-6S 4.76 4.14 3.92 – – 5.42/5.32 4.03/3.93 3.85 3.57 4.04 4.48/4.22} e_{(␤-GlcA)→(␣-}d-GlcNAc-6S) 4.52 3.34 3.65 3.78 3.82 5.32 3.80 – – 4.15 4.47 (G′_)→(D′₎ f_{␤-GlcA→␣-d-GlcNAc 4.64 3.41 3.66 3.75 – 5.35 3.91 3.75 3.70 3.99 –} a_{(␤-GlcA)→(␣-}d-GlcNAc) (J)→(E) 4.49 3.34 3.65 3.78 3.82 5.28 3.85 3.98 3.48 3.90 3.89

b_{␤-GlcA→␣-}d-GlcNAc 4.48 3.37 3.69 3.78 3.78 5.36 3.89 3.86 3.64 3.82 3.84/3.84 Valuesinitalictypeindicatepositionsbearingasulfateester.AtoF,spinsystems.

a_{Datapresentedinthispaper.} b_{Casuetal.(1994).}

c _{Yamadaetal.(1995).} d _{Yatesetal.(1996).} e_{Guerrinietal.(2005).} f _{Yamadaetal.(1999).}

crab heparin-like product, producing few N-acetylated and N -sulfated disaccharides linked to non-sulfated glucuronic acid (UGlcA-GlcNSand UGlcA-GlcNAc).On the otherhand, hep-arinasedegradedthecrabcompoundproducing thesametypes of products obtained from the mammalian heparin, although in different proportions. This enzyme acts upon glycosidic linkages containing ␣-d-glucosamine-N-sulfate linked to ␣-l -iduronicacid-2-sulfateanddoesnotactwhentheuronicacidis

glucuronicacid2-Oor 3-Osulfate,or whenthe glucosamineis N-acetylated(Naderetal.,1999).Thus,an importantdifference betweenmammalianheparinandthecrabpolymerisitslowerlevel oftrisulfateddisaccharides.In addition,thehighlevelsof hexa-andtetra-saccharidespresentindicatedoligosaccharideblocksthat areresistanttotheactionofheparinase.Furthermore, hepariti-naseIIwhich actsupon glucosaminido-glucuronicacidlinkages where the N-acetyl or N-sulfate glucosamine is preferentially

Table3

13_{Cchemicalshiftsfortheheparin-likecompoundofthecrabG.cruentata.}

Unit 13_{Cchemicalshifts}a

Uronicacid Glucosamine

C1 C2 C3 C4 C5 C1 C2 C3 C4 C5 C6

a_{(␤-GlcA)→␣-}d-GlcNS-6S (I)→(A) 105.0 76.2 80.1 79.8 80.1 102.0 61.2 73.1 81.2 70.0 69.0

b_{␤-GlcA→␣-}d-GlcNS-6S 105.5 76.4 79.5 79.8 80.3 100.8 61.1 73.1 80.6 72.3 69.5

a_{(␤-GlcA)→(␣-}d-GlcNS) (I)→(A′_{) 105.0 76.2 80.1 79.8 80.1 101.5 61.2 73.1 82.1 74.5 63.2}

b_{␤-GlcA→␣-}d-GlcNS 105.1 75.6 78.7 78.8 79.3 99.9 60.5 72.4 80.7 73.3 62.4

a_{(␤-GlcA-2S)→(␣-}d-GlcNS) (H)→(B/B′_{) 103.8 83.0 77.8 77.8 – 101.8 61.0 73.0 72.0 70.0 62.0}

a_{(␤-GlcA-2S)→(␣-d-GlcNS-6S)(H)→(B/B}′_{) 103.8 83.0 – – – 101.8 61.0 73.0 72.0 74.2 69.0}

b_{␤-GlcA-2S→␣-d-GlcNS-6S 102.8 82.3 77.3 79.1 79.5 100.9 – – – – –} a_{(␣-IdoA-2S)→(␣-}d-GlcNS-6S) (F)→(C) 101.8 78.8 72.0 79.5 71.8 100.5 ∼61 – – – 70.1

d_{␣-IdoA-2S→␣-d-GlcNS-6S 102.1 78.9 72.1 79.0 72.3 99.5 60.7 72.5 78.8 72.0 69.2} a_{(␤-GlcA-2S)→(␣-}d-GlcNAc-6S)(G)→(D) 103.8 82.8 – – – 100.8 56.4 – – – 71.7

e_{␤-GlcA-2S→␣-}d-GlcNAc-6S 103.0 82.4 77.4 78.8/79.5 79.2/79.5 99.8 56.0 72.1 80.5 72.0 68.8

a_{(␤-GlcA)→(␣-}d-GlcNAc-6S) (G′_)→(D′_{) 105.5 76.2 79.0 78.9 79.7 100.8 56.8 – – 69.8 70.0}

a_{(␤-GlcA)→(␣-d-GlcNAc) (J)→(E) 105.5 76.2 79.0 78.9 79.7 101.7 56.7 77.9 73.2 72.1 63.0} b_{␤-GlcA→␣-}d-GlcNAc 105.2 76.3 78.9 79.1 79.1 99.6 56.1 73.5 81.1 72.0 62.2 Valuesinitalictypeindicatepositionsbearingasulfateester.AtoF,Spinsystems.

a_{Datapresentedinthispaper.} b_{Casuetal.(1994).}

Fig.4.Effectofthecrabheparin-likecompoundoncoagulationusingdifferentinvitroassays.(),crabHepn;(),UFH;(),enoxaparin(lowmolecularweightheparin). ResultsinaPPTandanti-IIaarestatisticallydifferentforthe3compounds(P<0.5).Significantdifferences(P<0.5)inPT,TT,HEPTESTandanti-XabetweenHepnandheparin, aswellasHepnandenoxaparin.

sulfatedattheC-6position(Dietrichetal.,1999a;Naderetal., 1990),producedmainlydisulfateddisaccharides(UA-GlcNS,6S/

UA,2S-GlcNAc,6S/ UA,2S-GlcNS). Since the useof individual enzymesledtotheformation of oligosaccharides,a mixture of allthreelyaseswasthenusedtoascertainthetotaldisaccharide compositionoftheHepn(Table1).Thisresultcontrastswithdata obtainedforheparin,whereabout80%allofdisaccharidesarethe

UA,2S-GlcNS,6S,aswellasfor heparansulfateswherearound 50–60%ofalldisaccharidesareUA-GlcNAc/UA-GlcNS(Dietrich etal.,1998;Zhang,Xie,Liu,Liu,&Linhardt,2009).

3.4. NMRspectroscopy

The1_{HNMRspectrumforHepnisshowninFig.2a.Thesignal}

at5.22ppmfromH-1of2-O-sulfatediduronicacidwasfoundin thecrabheparin-likeproduct.Theotheranomericprotonsshowed twomainregionsfrom5.28to5.57ppmand4.5to5.2ppmthat correspondtoanomericprotonsofthehexosamineanduronicacid residues,respectively(Yatesetal.,1996).Thesignalat2.04ppmdue

totheacetylgroupswasprominentintheHepn.Fig.2bshowsthe anomericregionforporcineheparin,bovineheparansulfateand thecrabHepn.Itisclearthatthesameanomericsignalspresentin heparinandheparansulfatearepresentinHepn.Nevertheless,as intheenzymaticdegradationresults,theyarepresentindifferent relativeproportions.

94 (2013) 647–654 653

respectively,andthesignalat5.2/101.8ppmindicates2-O-sulfated IdoAlinkedtoN-sulfatedglucosamine(Guerrini,Naggi,Guglieri, Santarsiero,&Torri,2005;Yatesetal.,1996).Traceablespin sys-tems arealso shown onthe homonuclearproton–proton COSY spectrumexhibitingthespin–spincoupledprotons(Fig.3b).

Thecrabheparin-likecompoundexhibitedNMRspectra con-taining similar characteristics to mammalian heparan sulfate, includingahighintensitysignalattributedtotheacetylgroups andhighintensitysignals ofH-1fromglucuronicacidresidues. Additionally, the signals attributed to the anomeric proton of 2-O-sulfatediduronicacidresidues(5.2ppm),commonto mam-malianheparin(Casuetal.,1994;Mulloyetal.,1994;Yatesetal., 1996),werealsodetected,yet,these2-O-sulfatediduronic acid residuesarelinkedeithertoN-sulfated,6-hydroxylorN-acetylated, 6-O-sulfatedglucosamine(Fig.2b).Furthermore,chemicalshifts at 4.7–4.73ppm attributed to the 2-O-sulfated glucuronic acid werealso present. Accordingto theliterature, low amounts of 2-O-sulfatedglucuronic acidresidues are foundin natural gly-cosaminoglycans, but this residue is usually not detectable in unfractionatedheparins(Guerrinietal.,2005;Yamada,Murakami, Tsuda,Yoshida,&Sugahara,1995).Tothebestofourknowledge, thisisthefirstreportofa2-O-sulfatedglucuronicacid-rich heparin-likecompound.

The NMR signals attributed toN,3,6-trisulfated glucosamine residue,atypicalmarkerofthepentasaccharidesequenceofthe activesiteofheparinandheparansulfatesforantithrombin bind-ing(Casuetal.,1996;Kusche,Torri,Casu,&Lindahl,1990;Lindahl etal.,2005;Sieetal.,1988),werenotdetectedinthespectraof thecrabheparin-likecompound,suggestingthatsuchcompound islikelytoexhibitreducedanticoagulantactivitywhencompared toheparin.

3.5. Invitroanticoagulantassays

Invitroanticoagulantactivityofthecrabheparin-likecompound isshowninFig.4.TheHepnexhibitedananticoagulantactivity around33IU/mgusingtheactivatedpartialthromboplastintime (aPTT)assay.Thismethodshowsthatthecompound,when com-paredtoheparin (193IU/mg), is atleast 5times less potentin preventinginvitroclotformationbytheintrinsicpathway.Thecrab compoundhadnoeffectintheextrinsicpathway,confirmedbythe prothrombintime(PT),dramaticallycontrastingwithmammalian heparin.Hepnhasnoeffectontheabilityofthrombintodegrade fibrinogenmeasuredbythethrombintime(TT),contrastingonce moretheeffectobservedwithheparin.TheHEPTESTtestmeasures theactionofthecompoundsuponheparincofactorII.Theresults indicatedthatHepnisatleast12timeslesspotentthanheparinin thisassay.Usingbiochemicallydefinedconditionsonafastkinetic centrifugalanalyzer,theeffectsoftheheparin-like,heparinand enoxaparinonthrombinandfactorXagenerationwerealso inves-tigated.Itisevidentthattheheparin-likecompoundismuchless potentinthedirectgenerationofthrombinandfactorXawhen comparedtoheparinandenoxaparin.Consequently,theseresults indicatedthatHepnfromG.cruentataisaless potent anticoag-ulantagentthanmammalianheparinandLMW-heparin(Fig.4). Thisloweranticoagulantactivitycanberelatedtoitslowerdegree ofsulfation,inparticular,thelackofN,3,6-trisulfatedglucosamine residues,atypicalmarkerofthepentasaccharidesequenceactive forantithrombin(Lindahletal.,2005).

3.6. Hemorrhagiceffect

CrabHepnwasalsotestedasapossibleinhibitorof hemosta-sis.Thecrabcompound,likeheparin,alsodisruptedthenormal controlofbleeding.Nevertheless,theextentofbleedinginthe ani-malsexposedtothecrabcompoundwaslesspronouncedthanthe

Fig.5.Bleedingactivityofthecrabheparin-likecompoundandporcine intesti-nalmucosaheparin.(),Hepn;(),mammalianheparin.Theresultsforthetwo compoundsarestatisticallydifferent(P<0.5).

heparintreatment(Fig.5).Thiscouldberelatedtothelower con-tent ofcriticalsulfation attheC-6positionoftheglucosamine. Studiesconductedwithdisaccharidesderivedfromheparin, hep-aransulfateandchondroitinsulfateshowedthatasulfateatthe C-6positionoftheglucosamineiscrucialfortheantihemostatic activity(Dietrich,Tersariol, Da Silva,Bianchini,&Nader,1991). Thefindingsthatothersulfateddisaccharides,withthesame sul-fate/hexosamine/uronicacidratiosbearingasulfateatadifferent position(C-2)orwithadifferentglycosidiclinkage(1–3),were inactiveasinhibitorsofhemostasisindicatingthataspecific struc-tureisneededforsuchaneffect(Dietrichetal.,1991).Ontheother hand,thisinhibitoryactivitydoesnotseemtoberelatedtothe anticoagulantactivityofthecompounds(Boucasetal.,2012)since structureswithnoanticoagulantactivityarepotentantihemostatic agents(Nader,Tersariol,&Dietrich,1989).

4. Conclusion

In summary, ourresultsindicate that theheparin-like com-poundisolated fromthecrabG.cruentatapresent intermediate structure betweenheparin andheparansulfate.In addition,the crabheparin-likeisrichin2-O-sulfatedglucuronicacidresidues, possesses low levels of trisulfated disaccharides and lacks the defined pentasaccharide structure related to the antithrombin binding site. To the best of our knowledge, this is the first studycharacterizinga natural 2-O-sulfatedglucuronicacid-rich heparin-like glycosaminoglycan using chemical, enzymatic and spectroscopicanalyses.Furthermore,concerningsome pharmaco-logicalactivities,ithasbeendemonstratedthat,thecrabcompound showednegligibleinvitroanticoagulantactivityandlowerbleeding effectcomparedtomammalianheparin.Consequently,itsunusual structuralfeatures,insignificantinvitroanticoagulantactivityand lower bleeding risk, make this compound a suitable candidate forthedevelopmentstructure-drivenheparinbasedtherapeutic agentswithlessundesirableeffects.

Acknowledgments

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