Journal of Biochemistry and Molecular Biology, Vol. 35, No. 2, March 2002, pp. 199-205 © BSRK & Springer-Verlag 2002 A Novel Anticoagulant Protein from Scapharca broughtonii Won-Kyo Jung, Jae-Young Je, Hee-Ju Kim and Se-Kwon Kim* Department of Chemistry, Pukyong National University, Busan 608-737, Korea Received 24 September 2001, Accepted 26 November 2001 An anticoagulant protein was purified from the edible marine organisms have rarely been isolated, except for several portion of a blood ark shell, Scapharca broughtonii, by anticoagulant proteoglycans and polysaccharides from marine ammonium sulfate precipitation and column algae (Chargaff et al., 1936 Kindness et al., 1980; Maimone chromatography on DEAE-Sephadex A-50, Sephadex G- and Tollefsen, 1990; McLellan & Jurd, 1991; Jurd et al., 75, DEAE-Sephacel, and Biogel P-100. In vitro assays with 1995) and ascidian tunic (Lee et al., 1998). human plasma, the anticoagulant from S. broughtonii, During screening of the anticoagulant activity in marine prolonged the activated partial thromboplastin time animals, we recently detected anticoagulant activity from (APTT) and inhibited the factor IX in the intrinsic soluble extracts of the blood ark shell, Scapharca broughtonii. pathway of the blood coagulation cascade. But, the fibrin In the present paper, we report the purification and properties plate assay did not show that the anticoagulant is a of the first anticoagulant protein from marine bivalves. fibrinolytic protease. The molecular mass of the purified S. broughtonii anticoagulant was measured to be about 26.0 Materials and Methods kDa by gel filtration on a Sephadex G-75 column and SDS- PAGE under denaturing conditions. The optimum activity Materials Fresh blood ark shells, Scapharca broughtonii, were o in the APTT assay was exhibited at pH 7.0-7.5 and 40-45 C obtained from a blood ark shell aquafarm (Tongyoung, Korea), and 2+ in the presence of Ca . kept under −20oC until use. HEMOLAB Thrombomatt, HEMOLAB Silimat, and HEMOLAB Cofac reagent for inhibitory Keywords: Purification, Anticoagulant protein, Scapharca assay were obtained from the BioMerieux Co. (Marcy-I’Etoile, broughtonii, Factor IX France). BioMerieux Coagulometer Option 8 was a product of Behnk Electronic Co. (Norderstedt, Germany). Fibrinogen (from bovine) and urokinase (from human urine) were purchased from the Introduction Sigma Chemical Co. (St. Louis, USA). Molecular weight markers Blood coagulation is processed by coagulation factors in order for gel filtration and SDS-polyacryl amide gel electrophoresis were products of the Sigma Chemical Co. (Hercules, USA). DEAE- to stop the flow of blood through the injured vessel wall Sephadex A-50, Sephadex G-75, DEAE-Sephacel, and Biogel P- whenever an abnormal vascular condition and exposure to 100 were purchased from the Sigma Chemical Co. (St. Louis, non-endothelial surfaces at sites of vascular injury occur USA). ACE homogenizer AM-6 was a product of Nihonseiki Kaisa (Guyton, 1982; Grey and Meyer, 1988). As endogenous or Ltd. (Tokyo, Japan). The ultramembrane filter, SM165 (Molecular exogenous anticoagulants interfered with the coagulation weight, 10 kDa cut-off), for the concentration of protein solutions factors, the blood coagulation can be prolonged or stopped was obtained from the Sartorious Co. (Göttingen, Germany). All of (MacFarlane, 1964; Davie and Ratnoff, 1964; Esmon, 2000). the other chemicals that were used in this study were of analytical These anticoagulants have been used as convenient tools for grade and the highest purity. the exploration of the complex mechanisms of coagulation cascade. Coincidentally, the importance of research for Purification of the anticoagulant from S. broughtonii $OORIWKH anticoagulants also arose with therapeutic purposes, for RSHUDWLRQV ZHUH FDUULHG RXW DW R&XQOHVV VWDWHGRWKHUZLVH $OO RI example, a cure for hemophilia. While it was studied for WKH HGLEOH SDUWV J RI 6 EURXJKWRQLL H[FHSW WKH VKHOO ZHUH various exogenous anticoagulants (Woo et al., 1996; Waidhet- PLQFHGDQGKRPRJHQL]HGDW USPIRU PLQLQ PORI Kauadio et al., 1998; Kim et al., 1999), anticoagulants from P07ULV+&OWKDWFRQWDLQHGP0('7$S+ 7ULV+&O ('7$ EXIIHU DIWHU ZDVKLQJ LQ WKH VDPH EXIIHU WR UHPRYH WKH *To whom correspondence should be addressed. EORRG7KHLQVROXEOHPDWHULDOVZHUHUHPRYHGE\FHQWULIXJDWLRQDW Tel: 82-51-620-6375; Fax: 82-51-628-8147 × JIRU PLQDQGWKHVXSHUQDWDQWZDVWUHDWHGE\ PORI R E-mail address: [email protected] &&O DIWHUFRROLQJWR− & WRUHPRYHOLSLG&UXGHH[WUDFWLQ 200 Won-Kyo Jung et al. WKH ZDWHU OD\HU ZDV GLDO\]HG DJDLQVW P0 7ULV+&O EXIIHU S+ Fibrinolytic activity )LEULQRO\WLFDFWLYLW\ZDVDVVD\HGXVLQJWKH IRU K DQG WHVWHG IRU WKHLU DQWLFRDJXODQW DFWLYLW\ XVLQJ D ILEULQSODWHPHWKRG -HVSHUVHQDQG$VWUXS 6L[PLOOLOLWHUVRI SURWKURPELQ WLPH 37 DQG DFWLYDWHG SDUWLDO WKURPERSODVWLQ WLPH ERYLQHILEULQRJHQ PJPOLQ P0EDUELWDOEXIIHUS+ $377 DVVD\ VHH EHORZ 7KH H[WUDFW ZDV GHVDOWHG RXW LQ WKH FRQWDLQLQJ P0 &D&O DQG JHODWLQ ZHUH SODFHG LQ D SHWUL UDQJH RI ZLWK DPPRQLXP VXOIDWH $IWHU FHQWULIXJDWLRQ GLVKDQGFORWWHGE\DGGLQJ1,+8RIERYLQHWKURPELQ$IWHU6 × J PLQ WKH SUHFLSLWDWH ZDV FROOHFWHG 7KH SUHFLSLWDWH EURXJKWRQLL VDPSOHV µO UHSOLFDWHV SODWH ZHUH DSSOLHG LW ZDV GLVVROYHG LQ D 7ULV+&O('7$ EXIIHU WKHQ WKH GLVVROYHG ZDVLQFXEDWHGIRU KDWR&7KHQWKHO\VHGDUHDV PP ZHUH VROXWLRQZDVGLDO\]HGDJDLQVWDP07ULV+&OEXIIHU S+ IRU PHDVXUHGDVWKHSURGXFWRIWZRSHUSHQGLFXODUGLDPHWHUV8URNLQDVH K7KHGLDO\]HGVROXWLRQZDVORDGHGRQWRD'($(6HSKDGH[$ µOXQLWVPO ZDVXVHGDVDSRVLWLYHFRQWURO FROXPQ × FP WKDWZDVSUHYLRXVO\HTXLOLEUDWHGZLWKD7ULV +&O('7$ EXIIHU ,W ZDV WKHQ HOXWHG POPLQ ZLWK D OLQHDU Measurement of molecular weight 7KHPROHFXODUZHLJKWRI6 JUDGLHQWIURPWR01D&OLQWKHVDPHEXIIHU7KHHOXWHG EURXJKWRQLL DQWLFRDJXODQW ZDV PHDVXUHG E\ JHO ILOWUDWLRQ RQ D IUDFWLRQV DW HDFK SXULILFDWLRQ VWHS ZHUH WHVWHG DV WR WKHLU 6HSKDGH[ * FROXPQ × FP WKDW ZDV SUHYLRXVO\ DQWLFRDJXODQWDFWLYLW\XVLQJDQ$377DVVD\7KHDFWLYHIUDFWLRQZDV HTXLOLEUDWHGZLWKD7ULV+&O('7$EXIIHU7KHSURWHLQZDVHOXWHG SRROHGGLDO\]HGDJDLQVWDP07ULV+&OEXIIHUDQGVXEVHTXHQWO\ POPLQ ZLWKWKHVDPHEXIIHU7KHPROHFXODUZHLJKWVWDQGDUGV ORDGHGRQWRD6HSKDGH[*FROXPQ × FP POPLQ WKDW ZHUH ERYLQH VHUXP DOEXPLQ N'D FDUERQLF DQK\GUDVH ZDV SUHYLRXVO\ HTXLOLEUDWHG ZLWK D 7ULV+&O('7$ EXIIHU 7KH N'D F\WRFKURPH F N'D DQG DSURWLQLQ N'D $ HOXWHGIUDFWLRQZDVSRROHGGLDO\]HGDQGUHFKURPDWRJUDSKHGRQD 6'6SRO\DFU\ODPLGHJHOHOHFWURSKRUHVLVZDVSHUIRUPHGRQ '($(6HSKDFHO FROXPQ × FP WKDW ZDV SUHYLRXVO\ VODEJHOE\WKHPHWKRGRI/DHPPOL 7KHSURWHLQEDQGVZHUH HTXLOLEUDWHGZLWKD7ULV+&O('7$EXIIHU,WZDVWKHQHOXWHG VWDLQHGZLWK&RRPDVVLH%ULOOLDQW%OXH57KHPROHFXODUPDVV POPLQ ZLWKDOLQHDUJUDGLHQWIURPWR01D&OLQWKHVDPH VWDQGDUGV ZHUH IXPDUDVH N'D WULRVHSKRVSKDWH LVRPHUDVH EXIIHU 7KH IUDFWLRQ VKRZLQJ WKH KLJKHVW DFWLYLW\ ZDV SRROHG N'D F\WRFKURPHF N'D DQGDSURWLQLQ N'D GLDO\]HG DQG UHFKURPDWRJUDSKHG RQ D %LRJHO 3 FROXPQ × FP WKDW ZDV SUHYLRXVO\ HTXLOLEUDWHG ZLWK D 7ULV+&O('7$ Amino acid composition The anticoagulant was hydrolyzed with EXIIHU ,W ZDV DQG WKHQ HOXWHG POPLQ ZLWK WKH VDPH EXIIHU 6 N HCl at 110R& for 24 h in vacuum-sealed ampoules. The amino 7KH PDMRU SHDNV ZLWK DQWLFRDJXODQW DFWLYLW\ ZHUH FROOHFWHG acids were analyzed with an amino acid analyzer (Biochrom 20, GLDO\]HG DJDLQVW GLVWLOOHG ZDWHU FRQFHQWUDWHG XVLQJ WKH Biochrom Ltd., Cambridge, UK). Cysteine residues were estimated XOWUDPHPEUDQH ILOWHU PROHFXODU ZHLJKW N'D FXWRII by the method of Spencer and Wold (1969). PHPEUDQH DQGWKHQO\RSKLOL]HG Effects of pH, temperature, and cations on anticoagulant Measurement of APTT and PT $QLQYLWURFRDJXODWLRQDVVD\RI activity 7KH HIIHFWV RI S+ WHPSHUDWXUH DQG FDWLRQV RI WKH 6 $377 DQG 37 ZDV SHUIRUPHG DFFRUGLQJ WR WKH PDQXIDFWXUHUV EURXJKWRQLL H[WUDFW µO µJPO ZHUH PHDVXUHG RQ WKH LQVWUXFWLRQV 7KH FLWUDWHG QRUPDO KXPDQ SODVPD µO DQG 6 DQWLFRDJXODQWDFWLYLW\ EURXJKWRQLLVDPSOHV µO ZHUHLQFXEDWHGIRUPLQDWR&7KH The pH effect of the anticoagulant protein was measured after the PL[WXUH ZDV DFWLYDWHG ZLWK µORI+(02/$%6LOLPDWWKDW protein was incubated in various buffers for 48 h at 4R& and FRQWDLQHG UDEELW EUDLQ FHSKDOLQH RU µO RI NDROLQVDOLQH PJ neutralized. The following buffers were used: 10 mM glycine-HCl, PO IRU PLQ DW R&,QWKH37DVVD\µO RI +(02/$% pH 2.5-3.5; 10 mM acetate, pH 4.0-5.5; 10 mM phosphate, pH 6.0- 7KURPERPDWW FRQWDLQHG UDEELW EUDLQ WKURPERSODVWLQ 7.5; 10 mM Tris-HCl, pH 8.0-9.0; 10 mM glycine-NaOH, pH 9.5- WKURPERSODVWLQF $IWHU µORI P0&D&OZDVDGGHG7KH 11.0. The residual activity was measured by an APTT assay. SURORQJHGWLPH VHF ZDVPHDVXUHGDVDQLQKLELWRU\DFWLYLW\XVLQJD The effect on temperature of the anticoagulant was examined &RDJXORPHWHU2SWLRQ after the protein was incubated in a Tris-HCl/EDTA buffer for 15 min at various temperatures. After cooling to room temperature, the Inhibitory activity for the activated coagulation factors ,QWKH residual anticoagulant activity was examined by an APTT assay. LQKLELWLRQDVVD\IRUDFWLYDWHGIDFWRU,; µORIWKH+(02/$% To test the dependency of the anticoagulant activity on cations, &RIDF,; FRQWDLQLQJIDFWRU,;GHILFLHQWKXPDQSODVPDDQG µO the sample solution was dialyzed against 25 mM EDTA in a Tris- RI +(02/$% 6LOLPDW FRQWDLQLQJ UDEELW EUDLQ FHSKDOLQH ZHUH HCl/EDTA buffer at 4R& overnight. Then the solution was dialyzed LQFXEDWHG IRU PLQ DW R& 7KHQ WKH PL[WXUH RI WKH FLWUDWHG against deionized water for 24 h in order to remove EDTA. The QRUPDO KXPDQ SODVPD µO DQG 6EURXJKWRQLL VDPSOHV µO effect of the cations on APTT was assayed in the presence of SUHYLRXVO\LQFXEDWHGIRU PLQDWR& ZDVDGGHG6XEVHTXHQWO\ various metal cations. µO RI P0 &D&OZDVDGGHGDQGWKHFORWWLQJWLPHZDV