Purification and partial characterization of draculin, the anticoagulant factor present in the saliva of vampire bats (Desmodus rotundus) Citation for published version (APA): Apitz-Castro, R., Beguin, S., Tablante, A., Bartoli, F., Holt, J. C., & Hemker, H. C. (1995). Purification and partial characterization of draculin, the anticoagulant factor present in the saliva of vampire bats (Desmodus rotundus). Thrombosis and Haemostasis, 73(1), 94-100. https://doi.org/10.1055/s-0038- 1653731 Document status and date: Published: 01/01/1995 DOI: 10.1055/s-0038-1653731 Document Version: Publisher's PDF, also known as Version of record Please check the document version of this publication: • A submitted manuscript is the version of the article upon submission and before peer-review. There can be important differences between the submitted version and the official published version of record. People interested in the research are advised to contact the author for the final version of the publication, or visit the DOI to the publisher's website. • The final author version and the galley proof are versions of the publication after peer review. • The final published version features the final layout of the paper including the volume, issue and page numbers. Link to publication General rights Copyright and moral rights for the publications made accessible in the public portal are retained by the authors and/or other copyright owners and it is a condition of accessing publications that users recognise and abide by the legal requirements associated with these rights. • Users may download and print one copy of any publication from the public portal for the purpose of private study or research. • You may not further distribute the material or use it for any profit-making activity or commercial gain • You may freely distribute the URL identifying the publication in the public portal. If the publication is distributed under the terms of Article 25fa of the Dutch Copyright Act, indicated by the “Taverne” license above, please follow below link for the End User Agreement: www.umlib.nl/taverne-license Take down policy If you believe that this document breaches copyright please contact us at: [email protected] providing details and we will investigate your claim. Download date: 03 Jun. 2020 Thrombosisand Haemostasis @ F.K. SchattauerVerlagsgesellschaft mbH (Stuttgart) 73 (1) 94-100(1995) Purificotionond PortiolChorocterizolion of Dtoculin, theAnticoogulont Foctor Present in theSolivo of VompireBots (Desmodusrotundus) RofoelApitz-Costror, Suzette Beguin2, Alfonzo Toblonter, Fulvio Bortolir, John C, Holt3, H,Coenrood Hemker2 Fromthe rlnstituioVenezolono de lnvestigocionesCientificos (lVlC), Corocos, Venezuelo; 2CordiovosculorReseorch lnstutute, Moostricht, The Netherlonds; ond 3ProteinChemistry Dept, Rh6ne-PoulencRorer, Collegeville PA, USA Summory Here, we report resultson the isolation and partial characterization of the anticoagulantfactor presentin vampirebat saliva and we deter- Fromthe saliva of thevampire bar Desmodus rotundus, we isolated minedits siteof actionin the coagulationmechanism. We havepurified an unknownanticoagulant protein which we havenamed draculin. the anticoagulantfactor to apparenthomogeneity and show that it is an Its molecularmass as determined by non-reducedSDS-PAGE is about acidic(pI = 4.15) 88.5kDa glycoproteinwhich interactswith purified 83kDa Thereduced polypeptide shows a slowermigration. HPLC in factor IXa andXa and inhibits thrombin generationin humanplasma. a molecularsieve matrix yields a single,symmetrical peak comespond- ing to 88.5kDa. Isoelectricfocusing shows an acidicprotein with pl = 4.14.2. Aminoacidanalysis is compatiblewith a singiechain Moleriols ond Melhods polypeptideof about80 kDa. Cyanogenbromide cieavage yields a SephacrylS-200 (Pharmacia, Sweden), hydroxyapatite (BioGel HTP), Affi- single16-aminoacid peptide, conesponding to the amino-terminusof Gel-15,and acrylamide(BioRad Labs), Activated Thrombofax, and rabbit thenative molecule. Draculin inhibits the activated form of coagulation brainpartial Thromboplastin (Ortho Diagnostic Systems, Raritan, NJ, USA) factorsIX andX It doesnot act on thrombin, trypsin, chymotrypsin and wereobtained from commercialsources. Coagulation factors were purified doesnot express fibrinolytic activity. The inhibition is immediateand by establishedprocedures by Dr. Rob Wagenvoord(Dept. of Biochemistry, notreadily reversible, with a stoichiometryof abouttwo moleculesof Univ of Limburg,Maastricht, The Netherlands). Chromogenic substrates were ' draculinper molecule of factorIXa or Xa. Surprisingly,the inhibitory obtainedfrom Kabi (Sweden) Thiobenzyl Benzyloxicarbonyl-L-lysinate HCI . activityagainst either factor is notaffected by thepresence of theother. and p'-nitrophenyl-p-guanidobenzoateHCI were from Sigma All other reagentsused were also of thehighest quality available. The buffers used were Draculinbinds quantitatively to eitherimmobilised factor Xa or factor A: 0.05M Tris-HCl,0 I M NaClpH 7.35,with 0.5% of eggalbumin (Sigma) IXa. Our preliminaryinterpretation is that thereare two forms of andB: BufferA atpHT 9 with 20mM EDTA. draculinthat hardly differ in structure.Both bind to factorXa andto factorIXa but oneform inhibits factor Xa andthe other inhibits factor Animals IXa. Whenadded to plasma,draculin increases the lag phase as well as Vampirebats (Devnodus rotundrs,) were regularly captured from wild colo- theheight of thepeak of thrombingeneration. niesliving in a cavein thenorthwest part of Venezuela(State of Falc6n)in a regionwhere rabies is reportedlyabsent. Twenty vampires were curently kept in captivity,in individualcages of themetabolic type (Acme Metal Products, lnlroduclion Chicago,IL, USA),under controlled light and temperature (25o C). The animals Theanticoagulant propefiies of vampirebat saliva have been intui- weremaintained on bovineblood anticoagulated with 3.2%sodium citrate at a ratioof I : Foodwas given every 24 h, alwaysin thelate afternoon. Water tively knownfor manyyears as is witnessedby their name.Vampir, 9, wasgiven ad libitum (6). sincethe earlymiddle ages or evenbefore indicated blood drinking "semi-dead"in easternEuropean folk1ore. The nameevidently has beenextended to haematophagousbats after these have become known SalivaCollection to Europeans,i. e. after1492. One of thefirst documentedaccounts on For salivacollection, the vampireswere anaesthetized with a mixtureof the anticoagulanteffect of salivafrom an haematophagousbat was 2.5V02-bromo-2-chloro, 1,1,1-trifluorethane (Halothan, Hoechst), 30% nitrous donein 1932byBier, and some years later by Romana(1,2). After oxide,in oxygen(5, 6). Onceanaesthetized, 20 pl of 1%pilocarpine (Isopto these,very few papershave appeared on this subject.At present,the Carpin,Alcon Labs.,Inc, Ft Worth,TX, USA) wasplaced in the mouthin bestcharacterized factor from vampirebat saliva is a recentlyisolated orderto stimulatesalivation. It wasobserved that incidentally wounding of the plasminogenactivator from extracts of vampiresalivary glands (3). It is gingivaewith the plasticpipette tip that was usedto applythe pilocarpine notclear if thiscomesponds to the plasminogen activator from vampire causeda very minor bleedingthat stoppedalmost immediately. Saliva was plasticmicrocentrifuge tubes, placed in ice.The saliva collection batsaliva previously described by Hawkey(4) andCartwdght (5). collectedin took 30 to 40 min, with a regularyield of about0.5 ml/animal. Individual sampleswere kept at -30o C untiluse. lsolationof theAnticoagulant Factor from VampireBat Saliva Conespondenceto: Dr. H. C Hemker,Department of Biochemistry,Medi- cal Faculty,University of Maastricht,P O Box 616,N-6200MD, Maastricht, The protocolroutinely used was as follows:Saliva (=5 ml, 15mg of TheNetherlands - FAX Number:+3 1 43 670988 protein)was thawed and dialysed for six hoursagainst 10 litres of distilled 94 | .00 waterThe dialysate was centrifuged at 48,000X g during20 min andthe clear supematantwas lyophilized and redissolved in 1.5ml of distilledwater This = wasloaded on a Sephacryl5-200 column (2 5 x 30 cm) andeluted with dis- - tilled waterat a rateof 0 5 ml/min at room temperatureFractions of 2 ml werecollected and the relativeprotein concentration as well as the anti-Xa =^ >c activitywere measured every two fractionsAnti-Xa activity of the fractions =ts was assayedusing a micro-plateadaptation of the FXa assaybased on the hydrolysisof the chromogenicsubstrate 5-2222 as follows: Each microplate dE well contained6 25nM FXa, 50mM Tris pH 7.35 and an aliquotof the pl 20 pl 4 mM fractions,in a totalvolume of I 50 Reactionwas started with of E = 3-2222and after 10 min of incubationat 37" C theabsorbency at 405nm was recordedin a micro-platereader (Bio-Tek, Model EL312e). Fractions with anti-Xaactivity were pooled and lyophilizedThe lyophilizedmaterial was 25 50 75 t00 125 dissolvedin 1 mM NaCl(3-5 ml) andthe pH adjustedto 7 2 with NaOH.The FRACTIONM}I8ER samplewas loadedon a hydroxyapatitecolumn (1 X 7 5 cm, Biogel HTP) Flg.1 Proteinand anti-FXaactivity profile from fractionsobtained after equilibratedwith 1 mM NaCl,pH 7.2 Afterall of thesample was loaded, the molecularsieve of lyophilisedvampire bat saliva through sephacryl S-200 Co- columi was washedwith 6 columnvolumes of 1 mM NaCl (pH7 2) fbl- lumnsize was 2.5 X 30cm; equilibration and elution was done with delonized lowedby 2 columnvolumes of 200mM potassiumphosphate, pH 6 8 At waterand 2 ml fractionswere collected Dashed line comespondsto the inhibi-