Toxleon, 1977, Vol. 15, pp. 107-114. PerQamon Pray . Printed in Great ltritaid.
FACTOR X CONVERTING AND THROMBIN-LIKE ACTIVITIES OF BOTHROPS JARA RAGA SNAKE VENOM YOSHIKO FURUKAWA ând KYOZO HAYASHI Department of Biological Chemistry, Faculty of Pharmaceutical Sciences, Kyoto University, Kyoto, Japan (Acceptedjor pub/lcatlon 25 Artgast 197 Y. Ftrttuluwn and K. HwYes~n. Factor X converting and thrombin-like activities of Botkrops Jararaca venom . Toxicos 1S, 107-114, 1977 .-Bothrops Jararaca venom is able to activate factor X and to convert fibrinogen to fibrin by a thrombin-like activity . The two activitks behaved an if they wero dueto distinct enzyme proteins as determined by chromatography on Sephadez G-100, iscelectric focusing, disc gel electrophoresis and heat-treatment.
TrI>: sLOOn clotting ability 1904 ; EAGLE and HARRIS,
of Crotalidae venoms has been known for many years (Noc, 1937 ; KELLAWAY, 1937 ; CESARt and BoQu>;r, 1937; VELLARD, 1938 ; PoRaES,1953). LuaK (1935) demonstrated that Bothrops venoms (Bothrops alternatus, B. neuwtedt and B. jararaca) have a proteolytic and thrombin-like action (conversion of fibrinogen to fibrin). EAGLE (1937) reported that Bothrops atrox, B. jararaca and B. nummifera clot plasma by a thromboplastin-like action (conversion of prothrombin to thrombin) in addition to the thrombin-like action, and this was confirmed by many investigators (JANZKY, 1950; BREDA et al., 1951 ; MICxL, 1954). Later, as the study of the mechanism of blood coagulation developed, NAHA3 et al. (1964) showed that B. jararaca and B. atrox venom do not have the ability to activate prothrombin but activate factor ~, thus .resembling Russell's viper venom (MACFARLANE, 1961). However, the previous preparation of prothrombin was contaminated by small amounts of factor X. B. jararaca venom contains two clotting activities, factor X converting activity and thrombin-like activity ; NAxAS et al. (1964) suggested that only one enzyme was responsible for both activities, while DErrsox and ROUSSEAU (1970) described two coagulant enzymes present in B. jararaca venom. In the present paper we compare the molecular weight, isoelectric point, electrophoretic behavior and héat inactivation of the two clotting activities of B. jararaca venom, and show that they are catalyzed by two different enzyme proteins . MATERIALS AND METHODS Matertats Bothrops Jararaca venom was kindly provided by Dr . S. Schenberg, Institute Batsman, Säo Paulo, Brazil . Bovine factor X, was a generous gift of Dr. K. ~,jikawa, University of Washington, Seattle, U.S.A . The following proteins were purchased from the indicated sources : plasminogea free bovine ßbrinogen (Mike Laboratorien, Inc., K~n_kakrr, Illinois, U.S .A .) ; thrombin (Mochida Pharmaceutical Co ., Ltd., Tokyo, Japan) ; rß,-Cephalin (Sigma Chemical Co ., St . Louis, Missouri, U.S.A .) ; bovino serum albumin, ovalbumin, chymotrypsinogen and eytochrome c (Boehringer Mannheim Gmbh, Mannheim, Germany) . The factor X converting enzyme of Russell's viper venom wan purified as described previously (FURURAwA 107
YOSHIKO FURUKAWA and KYOZO HAYASHI
et ai., 1976). Sephadox G-100 was obtained from Pharmacia Fine Chemicals, Uppsala, Sweden . All other reagents were tho best available. Assays for clotting activities The factor X converting activity was assayed by the method of Williams and Esnouf, with thefollowing modifications (FuRUiuw~ et al ., 1976). Citrated plasma (0 "1 ml) was incubated with 0'1 ml of sample and 0"1 ml of 001 M Tris-HCI buffer, pH 7"3, containing 0" 15 M NaCI (iris-saline) at 37°C for 1 min. To the preincubation mixture was added 0"1 ml of 0"OS M calcium chloride, and the clotting time of the plasma recorded . A linear relationships between logclotting time and logconcentration of venom proteinwas obtained over the range 10-0 "1 kg per ml . Thrombin-like activity was determined as follows: 0"2 ml of 0"S~ fibrinogen solution and 0"1 ml of 0"015 M CaCI, were incubated at 37°C for 1 min, then 0"1 ml of sample (200-20 Vg per mI) was added and the clotting time recorded . One unit of clotting activity was defined as the activity possessed by 1 pg of the crude venom. Factor X activity was assayed by the method of Fu~ncwwa et al. (1972). Colu»va chromatography Crudo venom (21~7 mg) was dissohred in 0"OS M Tris-HCI buffer, pH 7~3, and applied to a column (1 "5 x 132 cm) of Sephadex G-100 previously equilibrated with the same buffer. A calibration curve to estimate the molecular weight was prepared using the standard proteins ; bovine serum albumin (67,000), ovalbumin (45,000), chymotrypsinogen (25,000) and cytochrome c (12,500) . Isoelectric focusi~tg The isoelectric pointwas determined by themethod of VESrensexc and SvsrrsoN (19C~. Carrier ampholytes in the pH range 3-10 were used at 0"5 ~ concentration. The pH gradient was stabilized by a linear sucrose gradient . Fraction G-I (6 "02 mg) was subjected to electrofocusing at 800 V for 20 hr . Gel electrophoresis Disc gel electrophoresis in T5 ~ gels, 10 cm in length, was performed at pH 9"4 according to the method of Dwts (1964). Samples (50 Rg) were applied to the gel and stained with coomassie brilliant blue. Densitometric tracing of the destained gel was made with a Joyce-Loebl microdensitotneter MK III. A second set of gels with 200 Fig of sample were cut into sections 2 mm thick after electrophoresis. Gel slices were immersed overnight at 0°C in 1 ml of 0"01 M Tris-HCl buffer, pH 7"3, containing 0"15 M NaCI and then assayed. Sodium dodecyl sulfate disc gel electrophoresis was carried out by the method of Wit and OsnoRN (1969). Activation ojfactor X Activation of factor X with the venom proteins was performed by the method of Fu~txewa et al. (1974) . RESULTS
Ge! filtration of crude venom A typical elution pattern is shown in Fig . 1 . The venom was separated into five peaks. The factor X converting activity and the thrombin-like activity appeared in the elution volume which corresponded to a molecular weight of 83,000 and 71,000, respectively. The tubes No. 36-54 (fraction G-I) were pooled and desalted by dialysis, recovering 6'8 mg of protein . The specific factor X converting activity and the specific thrombin-like activity of this fraction were 1~6 and 2"5 times higher than that of the crude venom, respectively. Compared to the standard thrombin, this fraction possessed 25 NIH thrombin unit per mg. Isoelectric focusing of the fraction G-I Figure 2 shows the distribution of the two clotting activities in the pH range 3-10. The maximum thrombin-like activity was found at pH 4" 0, while the factor X converting activity was found at pH 4"9. Electrophoresis of the fraction G-I Disc gel electrophoresis showed a major fast-moving band and a number of distinct bands (Fig. 3). The thrombin-like activity migrates slightly ahead of the factor X converting activity, but neither activity corresponded to a major protein band.
Coagulant Activities in B. Jararaca Venom
0o m o .a 1
3 R s'. c
F1a. 1. GEL FILTRATION OP CRUDE Bothropa Jararaca vErroea orl3erHnnEx G-100. The crude venom (21"7 mg) was applied to a column of Sephadex G-100 (1 "S x 132 cm). Flution was carried out with 0"OS M Tris-HCI buffer, pH 7"S, and 2"1 ml fractions were collected at a flow rate of 14 ml per hr . -, absorbante at 280 nm ; x"" ~"""" ~x, factor X converting activity ; O---O, thrombin-like activity .
O .D .zao
,10 PH 5
. -~~' S~"tow
Tube No .
Fia. 2. I90ELECIRIC FocusIrAa of TFIB FRACnox G-I (Toes No. 3o--54 Ix Fio. 1) . Sample (6 "02 mg) was subjaxed to elearofocusing (4°C) for 20 hr at 800 V. Two-ml fractions were collected and assayed. -, absorbance at 280 nm ; x. .".. .. .x, factor X converting activity ; C----O, thrombin-like activity ; ---, pH .
YOSHIKO FURUKAWA and KYOZO HAYASHI
FIO. 3. DISC GEL ELECTROPHORESIS OF FRACTION G-I.
The patterns show the distribution of protein stain absorbance at 620 nm (-), factor X converting activity (x" """"" ""x) and t~rombin-like activity (O- ---0). Pleat-treatment of crude rerlom
The venom solution (2 mg per ml) was subjected to heat-treatment at 60°C for 10-60 min at various pH values . The factor X converting activity was heat labile over a pH range from 3 to 9 (Fig. 4A). The thrombin-like activity was stable to heat-treatment (60°C) at pH 7"0 for 60 min (Fig. 4H).
FIO. 4. HEAT INACITVATION OF CRUDE Bothrops jararaca vENO~.
A 2 mg per ml solution was heated at 60°C for 10, 30 and 60 min. Immediately cooled in as ice bath, the factor X converting activity (A). and thrombin-like activity (B) was assayed. O-O, în0"1 M sodium acetate, pH 3 ; x-x, in 0" 1 M sodium acetate, pH 5 ; p-p, in 0" 1 M Tris-HCl buffer, pH 7; "-", in 0"1 M Tris-HCI buffer, pH 9.
Coagulant Activities in B. Jararaca Venom
Activation offactor X Time curves of factor X activation by the venom proteins are shown in Fig . 5. The weight ratio of substrate to the venom protein was 100 : 1 . The activation reaction by the too
Qula et al., 1960; BAxeitt~e, 1960; FicxIKAx and Ht:NRIQuss, 1962) and some properties in common with thrombin were noted (BLOMHÀCK and VFSTERMARK, 1958 ; CorLEY et al., 1973). However, the coagulant factors) have not yet been purified to homogeneity. The present work was undertaken in order to investigate a molecular relationship between the factor X converting activity and thrombin-like activity. In 1970, DFNSON and ROUSSEAU reported on the partial separation of the coagulant components of B. jararaca venom using a column of DEAF-cellulose or paper elet~ro-
YOSHIKO FURUKAWA and KYOZO HAYASHI
phoresis. However, on the DEAE-cellulose, factor X converting and thrombin-like activities were not separated completely, each was contaminated with the other to some extent . Also, electrophoresis of the venom showed no clear-cut separation of the two coagulant components . From these results, they suggested that the venom of B. jararaca contained factor X converting and thrombin-like activities . Our results agree with a suggestion by DENSON and ROUSSEAU (1970). In the present investigation, factor X converting activity and thrombin-like activity behaved differently on chromatography on Sephadex G-100, isoelectric focusing, disc gel electrophoresis and heat-treatment, suggesting each activity is exhibited by a distinct protein . However, the activities were not completely separated from each other by chromatography on Sephadex G-100, iscelectric focusing or disc gel electrophoresis. Also, we attempted to separate the activities by means of chromatography on DEAF-cellulose and DEAE-Sephadex, but could not separate them completely. The difference between the two proteins appears to be small. Russell's viper venom is a potent factor X activator, and converts factor X (mol. wt ; 55,000) to factor Xaa (mol. wt : 44,000) by the cleavage ofa NHQ-terminal fragment (mol. wt ; 11,000) from the heavy chain (mol. wt ; 37,000) . Factor Xaa undergoes autocatalytic release of a COOH-terminal fragment, giving rise to a lower molecular weight form of equal clotting activity, factor Xaß (mol wt ; 40,000) . The rate of this autocatalytic reaction is very slow (FUJIKAWA et al., 1972, 1974; JES~rx et al., 1974, 1975 ; FURIE et al., 1974) . In contrast, B.jararaca venom initially forms inactive intermediate (factor Xß) by the cleavage of a COOH-terminal region, then slowly converts it to activated form (Fig. 6). It is not known whether the releasing of a COOH-terminal fragment is displayed by the factor X converting enzyme itself or by another protease present in the fraction G-I. The factor X converting enzyme of Russell's viper venom has a molecular weight ofabove 100,000 and an 1SOeleCtC1C pOlIIt of pH 6~3 (WILLIAMS and EsNOUF, 1962 ; $CHiFFMAN et al., 1969 ; HANAHAN et al ., 1972 ; FURUKAWA et al., 1976) . The factor X converting activity of B. jararaca venom was concentrated in fractions with a molecular weight 83,000 (Fig . 1) and an iscelectric point pH 4~9 (Fig. 2). Thus, the enzyme protein of B. jararaca venom is more acidic and smaller . The thrombin-like enzymes were purified from Bothrops atrox venom (HOLLEMAN and WEISS, 1974) and the Agkistrodon venoms, A. halys blornho~i (SATO et al., 1965) ; A. rhodostoma (EsrroIlF and TUNNAH, 1967) ; A. status (OUYANG et al., 1971) . These enzymes have a molecular weight of about 30,000. The thrombin-like activity of B. jararaca venom was found to have a molecular weight of 71,000 (Fig. 1). The enzyme of B. jararaca venom may be a dimer, though we have not examined the subunit structure because of nonhomogeneous preparation of this enzyme . Acknowledgements-The authors are indebted to Professor Ixvo YAINASFiINA of our laboratory for his interest and valuable advice in this work . We are also grateful to Dr . S. Sc~rn;exa, Institute Batsman, Sâo Paulo, Brazil, for the sample of the venom of Bothrops Jararaca, and to Dr. K. Fvnxwww, University of Washington, Seattle, U.S .A., for the gift of bovine factor X, . REFERENCES A. and Swxxwx, N. (1960) Isolation and purification of a coagulant from snake venom of the species Bothrops jararaca and the study of its properties . Thromb . Diath . haemarrh . S, 296. BrA~wc~c, H. and VESTewKwxe, A. (1958) Studies on the action of thrombic enzyme on bovine fibrinogen as measured by N-terminal analysis . Ark. Kemie 12, 321. Bxt~w, R., BERNARDI, R. and Swi.w, F. (1951) Coagulating effect of Bothrops Jararaca venom. Giorn. C/tn. Med. 32, 882. BwrrEx~ee, R ., Devi,
FIG . 6 . SODIUM DODECYL SULFATE DISC GEL ELECTROPHORESIS OF FACTOR X AC7IVATED BY THE FACTOR X CONVERTING ENZYME FROM RUSSELL'S VIPER VENOM OR BY FRACTION G-I FROM BothrOps jaTQraCa VENOM .
Each gel contains 20111 of mixture shown in Fig. 5 (IO kg of protein) . A, activation of factor X with factor X converting enayme from Russell's vipervenom; B, activation of factor X with the fraction G-I from Bothrops jararaca venom.
Coagulant Activities in B, jararaca Venom
Ceswxi, E. and BoQua~r, P. (1937) Recherches sur les antigenes den venins et les anticorps des serums antivenimeux. Arnr%t . Inst . Pastew, Parts 58, 6. Cor~v, A. L., Bwxen~ES, S. and Dsvi, A. (1973) Studies of snake venom on blood coagulation.-I . The thromboserpentin (thrombin-lr7ce) enzyme in the venons . Tlrromb . Res. 2, 487. Dwvts, B. J. (1964) Disc electrophoresis. II . Method and application to human serum proteins . Ann. NY. Acod. Sci. 121, 404. DExsox, K. W. E. and RovsaEwU, W. E. (1970) Separation of the coagulant components of BothropsJararaca venom. Toxicon 8, 15 . EACiIB, H. (1937) The coagulation of blood by snake venons and its physiologic significance. J. exp. Med. 66, 613. Ewai e, H. and Hwiuus, T. N. (1937) Studies in blood coagulation-V. The coagulation of blood by proteolytic enzyme (trypsin, papain). l. gai. Phystol. 20, 543. Esxotrn, M. P. and Turrxwa, ~. W. (1967) The isolation and properties of the thrombin-like activity from Agkistrodon rhodostoma venom. Br, J. Haemat . 13, 581 . ~c~wx, M. and Hrxs~Quss, O. B. (1962) Further studies on the purification of the blood clotting enzyme from the venom of Bothrops Jararaca. Archs Btochan. Biophys. 98, 95 . Fu~ncwww, K., LEowz, M. E. and Dwvta, E. W. (1972) Bovine factor X, (Stuart factor); mechanism of activation by a protein from Russell's viper venom. Biochemistry 11, 4892 . Fvnxwww, K., Coax, M. H., D$awz, M. E, and Dwvm, E. W. (1974) The *.+~~:an;a+ of activation of bovine factor X (Stuart factor) by intrinsic and extrinsic pathways. Biochemistry 13, 5290 . Ftrx~, B. C., Fuam, B., CioTrtgs, A. J. and Wu.uwr.~s, W. J. (1974) Activation of bovine factor X by the venom coagulant protein of Vipera rrrssellit; complex formation of the activation fragment. Btochim. btophys. Acte 366, 121. FURUKAWA, Y., MAT9UNAQA, Y. and Hwvwsm, K. (1976) Purification and characterization of a coagulant protein from the venom of Russell's viper. Btochim. btophys. Acte 453, 48 . HwHetuHwxx, E. (1958) Thrombin-like active principle of Jararaca poison. Arch . exp. Path . Pharmak. 234, 291. Hwxwxwx, D. J., Ror xs, M. R. and DAY, W. C. (1972) Observation on the factor V activator present in Russell's viper venom and its reaction on factor V, Biochim. btophys. Acte 286, 205. H~Ques, O. B., F~cf~wx, M. and HsxtesQUes, S. B. (1960) Partial purification and some properties of the blood lotting factor from the venom of Bothrops Jararaca . Btochem. J. 75, 551 . Hot*Rw "N, W. H. and Wstss, L. J. (1974) Properties of the thrombin-like enzyme from Bothrops atrox venom purified by affpity chromatography on p-aminobenzamidine-Agaroso. Fedn . Proc . Am. Socs . exp. Btol . 33, 1474 (abstr .) . HoL~rz, P. and RALJDONAT, H. W. (1956) Relation between proteolytic activity and blood coagulating action and bradykinin liberating action of snake venom. Nawryn-Schmiedebags Arch, exp. Path . Pharnrak . Z29, 113. JANZKY, B. (1950) The relation between the proteolytic and blood clotting activity of snake venons. Archs Biochem. 28, 139. Jester, J., Srsxcex, A. K. and N~xsox, Y. (1974) The mechanism of activation of factor X; kineticcontrol of alternative pathways leading to the formation of activated factor X. J. biol. Chem . 249, 5614. JF.st~, J., SPEN(,ER, A. K., NwxwsmMw, Y., N~eKSOx, Y. and Koxtasesxa, W. (1975) . The activation of coagulant factor X; identity of cleavage sites in the alternative activation pathways and characterisation of the COOH-terminal peptide . J. biol. Chem. 250, 4497 . KflLLAWAY, C. H. (1937) Snake venons-I . Their constitution and therapeutic applications . II. Their peripheral action . Bull. John Hopktns Hosp . 60, 1 . Ltxx, T. (1935) Der Einguss der Schlangengifte auf die Blutgerinnung. Z. Immwi%rsch . 85, 504, MwcFwtet .wxe, R. (3 . (1961) The coagulant action of Russell's viper venom; the use of antivenom in defining its reaction with serum factor . Br. J. Haemat. 7, 496. MtCm., H. (1954) The venom of Bothrops jararaca. Morratsh. Chem. 85, 1240. Nwtiws, L., Dtit~sox, K. W. E. and MwcF.~.wxe, R. D. (1964) A study of thecoagulant action of eight snake venons . Thromb. Dioth. haemonh. II2, 355. Noc, F. (1904) Des différents venins der serpents. Anrds, Inst, Pastew, Parts 18, 387. Ottvwxa, C., Hoxa, J.~ . and Terra, C:M. (1971) Purification and properties of the thrombin-lie principle of Agkistrodon acutus venom and its comparison with bovine thrombin. T7rromb. Dtath. haemorrh . 26, 224. POR(iE$, N. (1953) Snake venons, their biochemistry and mode of action. Sctarce 117, 47. Sw~ro, T., Iwwrtwaw, S., Mr7ne.**u ", Y. and Strzvl