Tozkon. 1977, Vol. 13, pp. 161-167. Paryamon Prep . Printed 1n Great Britain.
THE PROPERTIES OF THE PURIFIED FIBRINOLYTIC PRINCIPLE FROM AGKISTRODON ACUTUS SNAKE VENOM CHAOHO OUYANG arid TUR-FU HUANG Pharmacological Institute, College of Medicine, National Taiwan University, Taipei, Taiwan, Republic of China (Accepted jor publication S October 1976)
C. OaYwxa and T:F. HUANG. The properties of the purified fibrinolytic principle from Agklstrodon status snake venom. Toxlcon IS, 161-167, 1977 .-In addition to fibrinolytic, fibrinogenolytic and caseinolytic activities, the purified fibrinolytic principle of Agkistrodon status venom possessed hemorrhagic activity. Trasylol had a much higher inhibitory action on the fibrinolytic activity of the fibrinolytic principle of the venom than did s-aminocaproic acid . Thus, the fibrinolytic action of the fibrinolytic principle was chiefly due to a direct action on fibrin . Both EDTA (S x 10 - ' M) and cysteine (S x 10- ' M) completely inhibited the fibrinolytic, fibrinogonolytic, hemorrhagic and caseinolytic activities of the fibrinolytic principle of the venom. Disulfide bonds might be essential for the biological activities of the ßbrinolytic principle. INTRODUCTION
venoms are known to contain proteolytic enzymes, while elapid venoms are practically devoid of such professes with viperid venoms being in an intermediate position (Kocxol.ATY, 1971). A large number of studies were devoted to these proteolytic activities, including fibrinolytic, fibrinogenolytic, hemorrhagic, esterolytic, caseinolytic, bradykininreleasing and thrombin-like activities (DIMITxoz, 1971 ; Tv et al., 1969 ; DEhPn~, 1968 ; OHSAI{A et al., 1973 ; ROCHA and SILVA, 1949 ; COHEN et al., 1970; OUYANG et al., 1971 ; OUYANG arid YANG, 1974; DBUTI3CH and DINIZ, 19$5 ; OÜYANG, 1957 ; OÜYÀNG arid SHIAiJ, 1970). The authors (OUYANG and HUANG, 197 have purified the fibrinolytic principle of Agkistrodon status venom, studying its physicochemical properties . This paper reports on the biological activities of the purified fibrinolytic principle and also the effects of various inhibitors on these activities . CROTALID
MATERIAL-S AND METHODS Tho venom of Agkistrodon status was collected, centrifuged, lyophilized and stored in a dessiwtor containing anhydrous CaCI,. Its fibrinolytic principle was purified by the procedure of OUYANG and Hu~NG (1976) . This principlewas homogeneous as judged by electrophoresis on cellulose acetatomembrane, disc electrophoresis on polyacrylamide gel and ultracentrifugation . Bovine fibrinogen (90~ clottable) was purchased from Pentea Co., U.S .A . Bovine thrombin topical was purchased from Park-Devil Co ., U.S .A. DEAF-Sephadex A-SO and Sophadex G-7S were purchased from Phan~nacia, Sweden . Human urokinase (1000 Ploug units per vial) was generously supplied by Green-Cross Co ., Japan. Trasylol (100,000 k.i .u . per ampoule) was purchased from FBA Pharmac. Inc. Plasmin, t:-aminocaproic acid (EACA), trypsin, soybean trypsin inhibitor (SBTn collagen, cysteine and EDTA 2 NA were purchased from Sigma Chemical Co ., U.S.A . FYbrinolytic activity
Fibrinolytic activity was determined by the method of ASIRUP and MOLtBßrz (1952) . Ten ml of the fibrinogen solution (0"2 %~ in imidazole-salino buffer (pH 7"4, O~OS M imidazole bu$'er mixed with 0"8S saline in 1 :9 v/v) was placed in Petri dishes . Tile dishes were placed on a horizontal surface and the fibrinogen
162
CHAOHO OUYANG and TUR-FU HUANG
solution was clotted by the addition of 0"2 ml of thrombin (100 units per ml). After shaking the mixture for S sec, a clot was allowed to form . To this clotted surface was then added, with a micropipette, 0023 ml of the test solution . Usually, triple points were applied on each plate. After the plates were incubated for 20 hr at 37°C, the lysed zones were measured . The activity was expressed as the product of the two perpendicular diameters in square millimetres. For each experiment, each test solution was run in triplicate . EACAcontaining plates were made by mixing S ml of the fibrinogen solution (0 "4;~) with S ml of EACA of various concentrations before the addition of thrombin . The inhibitory effect of EACA was more marked in the EACA-incorporated plates than that of EACA preincubated with urokinase or venoms and applied on to the plates . The other inhibitors such as EDTA, cysteine, Trasylol and SBTI were preincubated with the fibrinolytic agents at 37°C for 15 min. Then the preincubated mixtures were applied on to the fibrin plates . A-B Inhibition = ;c 100. A is the lysed zone of test solution in the absence of inhibitor while B A is that of test solution in the presence of inhibitor. Fibrlnogenolytic activity Fibrinogenolytic activity was measured by the modified meWod of Wnee et al . (1947) . The fibrinogen solution (20 mg per ml) and test solution (selected concentrations in imidazole-saline buffer, pH 7"4) were preincubated separately at 37°C for 3 min . Then various volumes of the test solution was pipetted into an equal volume of fibrinogen solution and onset of incubation time recorded . At various time intervals (5, l0, 15, 30 and 60 min) samples (0 " 2 or 0" 5 ml) were withdrawn into a 50 ml-tube containing 30 ml of imidazolesaline buffer and mixed. Then, the clottable fibrinogen was detettttined as follows : to the mixture in the 50 ml tube containing the incubated solution, 0"l ml of thrombin (1000 units per ml) was added, mixed, and the mixture left to stand at room temperature. After the formation of the fibrin net was completed, the tubes were centrifuged at 1500 rev/min for 20 min. The fibrin clot was removed carefully with a rod, blotted on filter paper and washed with saline . The fibrin was put in a tube containing 1 ml of 10% NaOH and boiled for 30 min. To this were added S ml of water, 3 ml of 20~ NaCO, and 1 ml of phenol reagent . After mixing and standing for 20 min, the absorbance was measured at 540 nm (Bausch & Lomb colorimeter) . According to a tyrosine titration curve, the quantity of the clottable fibrinogen was determined . Hemorrhagic activity T'he method of Koxno et al . (1960) was used . All tests for determination of the hemorrhagic activity were made on albino rabbits of either sex weighing about 2 kg . The day before testing, the fur over the dorsal area was gently clipped and then depilated with barium sulfide. The depilated skin was rubbed with silicone oil. Test solutions, in a volume of 0" 1 ml, were injected intradermally in the depilated area . The injectionsites were spaced at distance of about 2~5 cm in a checker-board arrangement . Jn order to minimize the possible vâriations in response depending upon the site of injection or on individual rabbit, each test solution was injected into two rabbits at twodifferent sites. After 24 hr, the skin wasremoved, spread and fixed to maintain its original size on a transparent glass plate-covered box, containing a lamp . The cross diameters of hemorrhagic spots were measured from the inside of the skin and the mean of the diameters mere taken as the intensity. Minimum hemorrhagic dose was defined as the amount causing a 10 mm cross-diameter lesion according to the definition of Korrno et al . (1960) . Caseinolytic activity The modified method of MAENO and Mrrsutt.~sEn (1939) was used . One-tenth milliliter of test solution dissolved in 0~1 M sodium phosphate buffer (pH 7"4) was added to a tube containing 0~4 ml of sodium phosphate buffer (ph 7"4). To the mixture, warmed at 37°C for 3 min, was added 1 ml of Gaseine (1 ~) and this was incubated at 37°C for exactly 13 min. Immediately after the incubation, 2 ml of 0" 4 M trichloroacetic acid was added to stop the reaction . After centrifugation at 2000 rev/min for 20 min, the reaction mixture was filtered through a Whattnan No. 42 filter paper. To thefiltrate were added with mixing 2 ml of 20 ~ Na,CO, and 0"5 ml of phenol reagent, and the mixture was left to stand for 20 mia at room temperature . The absorbance was then measured at 540 nm with a colorimeter. According to a tyrosine titration curve, we can calculate the specific activity of the test material . The specific activity of caseinolysis (units per mg) wasdefined as the number of itmoles tyrosine released permin at 37°C by 1 mg of proteolytic enzyme . The inhibitory e,~ects ojEDTA, cystelne, EACA, Trasylol and SBTI on biological activities These experiments were performed as follows. The test materials were preincubated with inhibitors at 37°C for 15 min and percentage of inhibition of the fibrinolytic, fibrinogenolytic, hemorrhagic and caseinolytic activities was measured . Collgqenase activity This was measured by the method Of NEUMAN and TYTELL (1950) .
Fibrinolytic Principle from A. acutus Venom
163
TABLE I . ~ INHIBCriON HY EACA, TRASYLOL, AND SBTI OF FIBRINOLYSI3 LNDUCED BY CRUDE VENOM, THE PURIFIED FIHRINOLYTIC PRINCIPLE, PLASMIN, UROKINASE AND TRYPSIN . THE VALUES ARE EXPRESSED AS MEANS f S .E . (n = 5)
Inhibitor (M) : Test solution Crude venom Fibrinolytic principle Plasmin Urokinase Trypsin
EACA
Trasylol
10'' 41 24 4l 80 56
f ~f f f
7 2 ]0 13 6
10 - ' 18 23 42 20 40
f4 ~4 f 12 ~8 f8
10 - ' 67 f 6 63 s 13 82 f 4 100 100
SBTI 10-'
63 f 7 65 f S 79 f 3 100 100
S x 10 -° 64 ~ 5 61 i 16 57 :t 12 100 100
The concentrations of test solutions used were as follows : crude venom, 400 Ng per ml ; fibrinolytic principle, 100 hg per ml ; plasmin, 100 pg per ml ; urokinase, 1 U per ml ; trypsin, 5 ug per ml . The lysed zones caused by these test solutions were about 200-300 mm'. RESULTS The effects of the fibrinolytic blhibitors on fibrinolysis induced by the purified fibrinolytic principle, crude venom, plasmin, urokinase and trypsin
As shown in Table 1, the fibrinolytic activities of crude venom, the fibrinolytic principle, plasmin, urokinase and trypsin were partially inhibited by EACA. BothTrasylol (10 -6 M) and SBTI (5 x 10 -6 M) completely inhibited urokinase and trypsin activity, but did not completely inhibit the activity of crude venom, the fibrinolytic principle or plasmin . The effects of cysteine and EDTA or:fibrinolysis induced by crude venom, the purifiedfibrinolytic principle, plasmin, trypsin and urokinase Cysteine (5 x 10'' M) and EDTA (5 x 10 - ' M) completely inhibited the fibrinolytic
activities induced by crude venom, the fibrinolytic principle and plasmin, but they did not affect significantly the trypsin and urokinase activities except that cyteine showed only a weak (25 ~) inhibitory action on urokinase activity. The concentrations of test solutions were the same as in Table l . . ; The effects of cysteûle, EDTA, EACA, Trasylol and SBTI on the fibrinogenolytir activities inducedby the purifiedfibrinolytic principle, plasmin and trypsin As shown in Fig . 1, both cysteine (5 x 10 - a M) and EDTA (5 x 10 - ° M) inhibited the
fibrinogenolytic activities induced by the purified fibrinolytic principle and plasmin, but trypsin was not inhibited . EACA (2 x 10 - ! M) and Trasylol (2 x 10 -' M) had no sig nificant effects on the fibrinogenolysis induced by thè fibrinolytic principle and plasmin ; however, Trasylol and SBTI (5 x 10 -6 M) blocked the action oftrypsin . The hemorrhagic activities of cnrde venom and the purified fibrinolytic principle
The hemorrhagic activity of the. purified fibrinolytic principle was more potent than that of the crude venom at lower doses (< 5 lrg), while they were equipotent at higher doses (> 10 ltg) (Fig. 2). The minimum hemorrhagic dose of the purified fibrinolytic principle was 2~6 ~ 1~6 pg, while that ofthe crude venom was 9~4 ~ 0~5 ltg . Trypsin (1001tg), plasmin (1001tg) and urokinase (1 unit) had no apparent hemorrhagic effect . The effects of cysteine, EDTA, EACA, Trasylol and SBTI on the hemorrhage induced by the purifiedfibrinolytic principle As shown in Fig . 3 cysteine (5 x 10 - ' M) and EDTA (5 x 10 - ' M) completely inhibited
164
CHAOHO OUYANG
and
TUR-FU HUANG
the hemorrhagic activity of the purified fibrinolytic principle, while EACA, Trasylol and 5BTI had no effect . The e,(ject ofcations (Mge ~ and CaY+) on the EDTA-induced inhibitory action on lremorrlrage andfibrinolysis induced by tire purifiedfibrinolytic principle Neither Ca'+ nor Mgs+ (2 x 10 -8 M) had any inhibitory effect on hemorrhage or fibrinolysis. The optimal pH for the hemorrhagc activity of the purified fibrinolytic principle was quite broad (5~0-9~0). The effects of cysteine and EDTA on casei~rolytic activities induced by the purified,fibrinolytic principle, plasmin and trypsin Both EDTA (5 x 10 - a M) and cysteine (5 x 10-8 M) completely inhibited the caseinolysis induced by the purified fibrinolytic princple (1001Ig per ml), and induced, respectively, 60 ~ 20 and 87 ~ 13 ~ inhibition of plasmin (301tg per ml), while the activity of trypsin (51Lg per ml) was not affected. Results were each based on four experiments .
Trypsin
FIG. 1 . THE EFFECTS OF EDTA, CYSTEINE, EACA, TRASYLOL AND SBTI ON FIBRINOGBNOLYSIS INDUCED BY THE PIIRIFlI~ FLßRINOLYTIC PRINCIPLE OF TAB VENOM (FP), PLA9MIN AND TRYPSIN.
The abscissa represents the incubation time of the fibrinogenolytic agents with fibrinogen. The ordinaterepresents the percentage of the clottable fibrinogen remainingafter the incubation. D), cysteine; (" "), EACA; (O O). central ; ("-~- "), EDTA ; (p ([]- - - - ~, Trasylol ; (" /), SBTI . The concentrations of the fibrinogenolytic agents were the same as in Table 1, and those of the inhibitors were the same as in the fibrinolysis experiment.
Filxinolytic Principle from A . acutus Venom
165
20
E E
vd
IS
w 4
E0
v
c 0
~n 4
10
S 0.6
I. S
I 2 .5
I 5
I 10
Dose, N.q
I 20
I 40
FIG . Z . THE LOG DOSE-RESPONSE CURVE FOR HEMORRHAGE INDUCED BY PRINCD'LE (FP) AND CRUDE VENOM.
Tf~
PURIFIED FIBRINOLYIIC
The effects offibrinolytic inhibitors on caseinolysis induced by thepurifiedfibrinolytic principle, plasmin and trypsin The caseinolytic activity of the purified fibrinolytic principle (100 ~Ig per tnl) was not inhibited by EACA (2 x 10-' M), Trasylol (2 x 10 -' M) or SBTI (5 x 10 -6 M). The activity of plasmin could be inhibited to 17 ~ 7 and 38 ~ 12 ~ of the original values by Trasylol and SBTi, respectively, but not by EACA. The activity of trypsin could be inhibited completely by Trasylol (2 x 10 -' M) and SBTT (5 x 10 -6 M), but not by EACA (2 x 10 - s M). DISCUSSION
It is known that the antifibrinolytic activity of EACA is chiefly due to the inhibitory action on plasminogen activation and that of Trasylol is chiefly due to the direct inhibitory action on plasmin (ALKJAERSIG et al., 1959 ; Txnursxotn et al., 1967; AMBRUS et al., 1968) . Since the inhibitory action of Trasylol on the fibrinolysis induced by the purified fibrinolytic principle was much higher than that of EACA (Table 1), the fibrinolytic activity of the fibrinolytic principle might be due to a direct action on fibrin . Trasylol (10 -~ M) having no inhibitory effect on urokinase still had about 50~ inhibitory action on the fibrinolytic principle. SBTI (5 x 10-6 M) could completely inhibit the fibrinolysis induced by trypsin and urokinase, but only had about 60 ~ inhibitory activity on the fibrinolytic principle and plasmin . From all these findings we may draw a conclusion that the fibrinolytic principle is a plasmin-like enzyme rather than an indirectly-acting activator (plasminogen activator) like urokinase . Concerning the hemorrhagic action of the fibrinolytic principle, it was similar to HRI of T. ,~iavoviridis purified by OMORI-$ATUIi and OHSAKA (1970). Both of them were acidic proteins . At lower doses ( < S lIg) the hemorrhagic activity of the fibrinolytic principle was about three times higher than that of crude venom, but they were equivalent at higher doses, because the effect of anticoagulant principle would be magnified at higher doses of crude venom which produced a more profound hemorrhagic effect . No collagenase activity
166
CHAOHO OUYANG and TUR-FU HUANG
was detected in the fibrinolysic principle. That trypsin (100 Itg), plasmin (100 itg) and urokinase (1 unit) caused no apparent hemorrhage indicated that the causes of local hemorrhage may be a phenomenon which cannot be induced solely by the caseinolytic, fibrinolysic or fibrinogenolysic action . In many organ systems, normally occurring plasminogen activators appear to sustain local hemorrhage but do not induce hemorhage (AxnEltsox et al., 1968). Tryasylol and EACH were effective in several fibrinolysic bleedings (AxDHRSON et al., 1968), while they could not inhibit the hemorrhage induced by the fibrinolysic principle of the venom given intradermally (Fig. 2). The causes of hemorrhage may be the damage of basement membrane and surrounding fibrils as suggested by OHSAKA et al. (1973) . EDTA and cysteine completely inhibited the activities tested, namely fibrinolysis, fibrinogenolysis, caseinolysis and hemorrhage . This implies that cysteine and EDTA induce a chemical modification in the structure of the proteolytic enzymes (the fibrinolysic principle and plasmin) . Disulfide bond might be indispenable for the structure of the proteases. That EACA was an effective inhibitor for fibrinolysis but not for fibrinogenolysis, caseinolysis and hemorrhage induced by plasmin, trypsin and the fibrinolysic principle of the venom (Figs. 1 and 3, Table 1) agreed with those previous reports on plasmin and trypsin (AMHRUS et al., 1968 ; OKAMOTO, 1962). Trasylol is a competitive inhibitor to plasmin (IVIAKI and BE1.1.ER, 1966) and also a nonspecific inhibitor to trypsin, chymotrypsin and kallikrein (TRAUTSHOLD et al., 1967), It inactivated fibrinolysis, fibrinogenolysis and caseinolysis by trypsin and it also inactivated fibrinolysis by urokinase (Table 1). SBTI inhibited all actions of trypsin. This may have resulted from the formation of an inactive complex of SBTI with trypsin (SIEDL and LINER, 1972). SBTI was as nonspecific as Trasylol ; it also inactivated plasmin and the fibrinolysic principle of the venom.
Acknowledgements-This work was supported in part by the National Science Council Research Grant of the Republic of China. REFERENCES At-tupt:xstG, N., Ftrct~tt, A. P. and SHERRY, S. (1959) E-ACA : An inhibitor of plasminogen activation . J. Biol. Chem. 234, 832. Anteßus, C. M., Aatexus, J. L., LASSMAN, H. B. and MtNx, L. B. (1968) Studies on the mechanism of action of inhibitors of the fibrinolysin system . Arm. N.Y. Acad. Sci. 146, 430. ANDER40N, L., Nttsoox, 1. M ., COLLEEN, S., Gnnnsrturm, B. and Miz.nxn$n, B. (1968) Role of urokinase and tissue activator in sustaining bleeding and the management thereof with E-ACA and AMCHA. Arm. N.Y . Acad. Sci. 146, 642 . AsTaur, T. and Mtita,exTZ, S. (1952) The fibrin plate method for the estimation of fibrinolytic activity . Archs Biochem. Blophys. 40, 346. Cot~N, L, Zux, M., ICnMtrrsxv, E. and Dt: VItIE3, A. (1970) Isolation and characterization of kinin-releasing enzyme of F .clris coloratus venom. Biochem. Pharnrac . 19, 785. DELPIERRI:, G. R. (1968) The proteolytic activities of some Africa Viperidae venoms . Toxicwt 5, 233. DEUTSCH, H . F. and Dtxtz, C. R. (1955) Some proteolytic activities of snake venoms. J. blol . Chem. 216, 17 . DtMtTxoz, G. D. (1971) Purification and partially characterization of two proteolytic enzymes from the venom of Vipera russellü . Toxicon 9, 33 . KocrtoLaTV, W. F. (1971) Toxicity and some enzymatic properties and activities in the venom of Crotalidae, Elapidae and Viperidae. Toxicon 9, 131. Koxno, H., Koxno, S., IKEZAWA, H., MutmTa, R. and Ot~tu, A. (1960) Studies on the quantitative method for determination of hemorrhagic activity of Habu snake venom. Jap. J. med. Sci . Biol .13, 43 . MAENO, H . and MtTSUFIA3HI, S . (1959) Procedure for proteolytic activity determination (2). Studies on Habu snake venom. Jap. J. Microbiol. 3, 131 . Mntct, M . and BF.LLER, F. K. (1966) Comparative studies of fibrinolytie inhibitors in vitro. Thronrb. Diatlr . haemorrh . 16, 668. NEUMAN, R. E. and TrTELL, A. A . (1950) Action of proteolytic enzyme on collagen . Proc . Soc. exp. Biol . Med. 73, 409. OHSAKA, A., JvsT, M. and HasExhtArrrr, E. (1973) Action of snake venom hemmorhagic principles on isolated glomerular basement membrane . Blochim. blophys. Acta 323, 415. ORAMOTO, S. (1962) Influence of AMCHA on the activity of fibrinolysin . Keio J. Med. 11, 117. OMORI-SATOH, T. and OIjSAKA, A. (1970) Purification and some properties of hemorrhagic principle I in the venom of 7ilmeresurus,llavovlrldls . Blochim. blophys. Acta 207, 432.
FIG . 3 . THE EFFECTS OF CYSTEINE, EDTA, EACA, TRASYLAL, ANTIVENIN AND SBTI HAGE INDUCED BY THE PURIFIED FiHRiNOLYTIC PRINCIPLE .
On HEMORR-
Patterns of hemorrhage observed from inside of the removed skin . (1) Control, (2) cysteine, (3) EACA, (4) EDTA, (5) Trasylol, (~ anitvenin and (7) SBTI .
Fibrinolytic Principle from A. acutus Venom
167
Ovverra, C. (1957) The effects of Formosan snake venoms on blood coagulation In vitro. J. Fornwsan nred. Assoc. 56, 435. Ouv~xa, C., Hoxc, J. S. and Terta, C. M. (1971) Purification and properties of thrombin-h7ce principle of Agklstrodon acutus venom and its comparison with bovine thrombin. Thromb . Dlath.hacmorrh . 26, 224. Ovv,uva, C. and Huerrc, T. F. (1976) Purification and characterization of the fibrinolytic principle of Agkistrodon acutus venom. Blochim. blophys. Acts 439, 146. Ousr~ro, C. and Sauu, S. Y. (1970) Relationship between pharmacological actions and enzymaticactivities of the venom of 7}imeresurus gra»tlneus. Toxlcon 8, 183. OUYLNCi, C. and TExa, C. M.(1972) Purification and properties of the anticoagulant principle of Agkistrodon aeutus venom. Bioehlm. btophys. Acts 278, 155. Ousr~xc, C. and YANC3, F. Y. (1974) Purification and properties of the thrombin-like enzyme from T. gramlneus venom. Biochim. biophys. Acts 331, 354. Rocx~, E. and Su.vn, M. (1949) Bradykinin, a hypotensive and smooth muscle stimulating factor released from plasma by snake venom and trypsin. Am. J. Physiol.156, 261. SrEnt D. S. and Lt1VER, I. E. (1972) Isolation and properties of complexes of the Bowman-Birk Soybean inhibitor with Trypsin and chymotrypsin . J, biol. Chem . 247, 3533. TRAITISHOLD, L, Wenr~, E. and GErnun, Z. R. (1967) Trasylol . Biochem. Pharmac.l6, 59. Tu, A. T., How+.+, M. and HONG, B. S. (1969) Hemorrhagic, myoneurotic, thrombotic and proteolytic activities of viper venoms . Toxocon 6, 175. W~, A. J., GuFSr, M. M. and SescEns, W. H. (1947) Fibrinogen : with special reference to its preparation and certain properties of the product . Arch Biochem. 13, 231 .
THE PROPERTIES OF THE PURIFIED FIBRINOLYTIC PRINCIPLE FROM AGKISTRODON ACUTUS SNAKE VENOM CHAOHO OUYANG arid TUR-FU HUANG Pharmacological Institute, College of Medicine, National Taiwan University, Taipei, Taiwan, Republic of China (Accepted jor publication S October 1976)
C. OaYwxa and T:F. HUANG. The properties of the purified fibrinolytic principle from Agklstrodon status snake venom. Toxlcon IS, 161-167, 1977 .-In addition to fibrinolytic, fibrinogenolytic and caseinolytic activities, the purified fibrinolytic principle of Agkistrodon status venom possessed hemorrhagic activity. Trasylol had a much higher inhibitory action on the fibrinolytic activity of the fibrinolytic principle of the venom than did s-aminocaproic acid . Thus, the fibrinolytic action of the fibrinolytic principle was chiefly due to a direct action on fibrin . Both EDTA (S x 10 - ' M) and cysteine (S x 10- ' M) completely inhibited the fibrinolytic, fibrinogonolytic, hemorrhagic and caseinolytic activities of the fibrinolytic principle of the venom. Disulfide bonds might be essential for the biological activities of the ßbrinolytic principle. INTRODUCTION
venoms are known to contain proteolytic enzymes, while elapid venoms are practically devoid of such professes with viperid venoms being in an intermediate position (Kocxol.ATY, 1971). A large number of studies were devoted to these proteolytic activities, including fibrinolytic, fibrinogenolytic, hemorrhagic, esterolytic, caseinolytic, bradykininreleasing and thrombin-like activities (DIMITxoz, 1971 ; Tv et al., 1969 ; DEhPn~, 1968 ; OHSAI{A et al., 1973 ; ROCHA and SILVA, 1949 ; COHEN et al., 1970; OUYANG et al., 1971 ; OUYANG arid YANG, 1974; DBUTI3CH and DINIZ, 19$5 ; OÜYANG, 1957 ; OÜYÀNG arid SHIAiJ, 1970). The authors (OUYANG and HUANG, 197 have purified the fibrinolytic principle of Agkistrodon status venom, studying its physicochemical properties . This paper reports on the biological activities of the purified fibrinolytic principle and also the effects of various inhibitors on these activities . CROTALID
MATERIAL-S AND METHODS Tho venom of Agkistrodon status was collected, centrifuged, lyophilized and stored in a dessiwtor containing anhydrous CaCI,. Its fibrinolytic principle was purified by the procedure of OUYANG and Hu~NG (1976) . This principlewas homogeneous as judged by electrophoresis on cellulose acetatomembrane, disc electrophoresis on polyacrylamide gel and ultracentrifugation . Bovine fibrinogen (90~ clottable) was purchased from Pentea Co., U.S .A . Bovine thrombin topical was purchased from Park-Devil Co ., U.S .A. DEAF-Sephadex A-SO and Sophadex G-7S were purchased from Phan~nacia, Sweden . Human urokinase (1000 Ploug units per vial) was generously supplied by Green-Cross Co ., Japan. Trasylol (100,000 k.i .u . per ampoule) was purchased from FBA Pharmac. Inc. Plasmin, t:-aminocaproic acid (EACA), trypsin, soybean trypsin inhibitor (SBTn collagen, cysteine and EDTA 2 NA were purchased from Sigma Chemical Co ., U.S.A . FYbrinolytic activity
Fibrinolytic activity was determined by the method of ASIRUP and MOLtBßrz (1952) . Ten ml of the fibrinogen solution (0"2 %~ in imidazole-salino buffer (pH 7"4, O~OS M imidazole bu$'er mixed with 0"8S saline in 1 :9 v/v) was placed in Petri dishes . Tile dishes were placed on a horizontal surface and the fibrinogen
162
CHAOHO OUYANG and TUR-FU HUANG
solution was clotted by the addition of 0"2 ml of thrombin (100 units per ml). After shaking the mixture for S sec, a clot was allowed to form . To this clotted surface was then added, with a micropipette, 0023 ml of the test solution . Usually, triple points were applied on each plate. After the plates were incubated for 20 hr at 37°C, the lysed zones were measured . The activity was expressed as the product of the two perpendicular diameters in square millimetres. For each experiment, each test solution was run in triplicate . EACAcontaining plates were made by mixing S ml of the fibrinogen solution (0 "4;~) with S ml of EACA of various concentrations before the addition of thrombin . The inhibitory effect of EACA was more marked in the EACA-incorporated plates than that of EACA preincubated with urokinase or venoms and applied on to the plates . The other inhibitors such as EDTA, cysteine, Trasylol and SBTI were preincubated with the fibrinolytic agents at 37°C for 15 min. Then the preincubated mixtures were applied on to the fibrin plates . A-B Inhibition = ;c 100. A is the lysed zone of test solution in the absence of inhibitor while B A is that of test solution in the presence of inhibitor. Fibrlnogenolytic activity Fibrinogenolytic activity was measured by the modified meWod of Wnee et al . (1947) . The fibrinogen solution (20 mg per ml) and test solution (selected concentrations in imidazole-saline buffer, pH 7"4) were preincubated separately at 37°C for 3 min . Then various volumes of the test solution was pipetted into an equal volume of fibrinogen solution and onset of incubation time recorded . At various time intervals (5, l0, 15, 30 and 60 min) samples (0 " 2 or 0" 5 ml) were withdrawn into a 50 ml-tube containing 30 ml of imidazolesaline buffer and mixed. Then, the clottable fibrinogen was detettttined as follows : to the mixture in the 50 ml tube containing the incubated solution, 0"l ml of thrombin (1000 units per ml) was added, mixed, and the mixture left to stand at room temperature. After the formation of the fibrin net was completed, the tubes were centrifuged at 1500 rev/min for 20 min. The fibrin clot was removed carefully with a rod, blotted on filter paper and washed with saline . The fibrin was put in a tube containing 1 ml of 10% NaOH and boiled for 30 min. To this were added S ml of water, 3 ml of 20~ NaCO, and 1 ml of phenol reagent . After mixing and standing for 20 min, the absorbance was measured at 540 nm (Bausch & Lomb colorimeter) . According to a tyrosine titration curve, the quantity of the clottable fibrinogen was determined . Hemorrhagic activity T'he method of Koxno et al . (1960) was used . All tests for determination of the hemorrhagic activity were made on albino rabbits of either sex weighing about 2 kg . The day before testing, the fur over the dorsal area was gently clipped and then depilated with barium sulfide. The depilated skin was rubbed with silicone oil. Test solutions, in a volume of 0" 1 ml, were injected intradermally in the depilated area . The injectionsites were spaced at distance of about 2~5 cm in a checker-board arrangement . Jn order to minimize the possible vâriations in response depending upon the site of injection or on individual rabbit, each test solution was injected into two rabbits at twodifferent sites. After 24 hr, the skin wasremoved, spread and fixed to maintain its original size on a transparent glass plate-covered box, containing a lamp . The cross diameters of hemorrhagic spots were measured from the inside of the skin and the mean of the diameters mere taken as the intensity. Minimum hemorrhagic dose was defined as the amount causing a 10 mm cross-diameter lesion according to the definition of Korrno et al . (1960) . Caseinolytic activity The modified method of MAENO and Mrrsutt.~sEn (1939) was used . One-tenth milliliter of test solution dissolved in 0~1 M sodium phosphate buffer (pH 7"4) was added to a tube containing 0~4 ml of sodium phosphate buffer (ph 7"4). To the mixture, warmed at 37°C for 3 min, was added 1 ml of Gaseine (1 ~) and this was incubated at 37°C for exactly 13 min. Immediately after the incubation, 2 ml of 0" 4 M trichloroacetic acid was added to stop the reaction . After centrifugation at 2000 rev/min for 20 min, the reaction mixture was filtered through a Whattnan No. 42 filter paper. To thefiltrate were added with mixing 2 ml of 20 ~ Na,CO, and 0"5 ml of phenol reagent, and the mixture was left to stand for 20 mia at room temperature . The absorbance was then measured at 540 nm with a colorimeter. According to a tyrosine titration curve, we can calculate the specific activity of the test material . The specific activity of caseinolysis (units per mg) wasdefined as the number of itmoles tyrosine released permin at 37°C by 1 mg of proteolytic enzyme . The inhibitory e,~ects ojEDTA, cystelne, EACA, Trasylol and SBTI on biological activities These experiments were performed as follows. The test materials were preincubated with inhibitors at 37°C for 15 min and percentage of inhibition of the fibrinolytic, fibrinogenolytic, hemorrhagic and caseinolytic activities was measured . Collgqenase activity This was measured by the method Of NEUMAN and TYTELL (1950) .
Fibrinolytic Principle from A. acutus Venom
163
TABLE I . ~ INHIBCriON HY EACA, TRASYLOL, AND SBTI OF FIBRINOLYSI3 LNDUCED BY CRUDE VENOM, THE PURIFIED FIHRINOLYTIC PRINCIPLE, PLASMIN, UROKINASE AND TRYPSIN . THE VALUES ARE EXPRESSED AS MEANS f S .E . (n = 5)
Inhibitor (M) : Test solution Crude venom Fibrinolytic principle Plasmin Urokinase Trypsin
EACA
Trasylol
10'' 41 24 4l 80 56
f ~f f f
7 2 ]0 13 6
10 - ' 18 23 42 20 40
f4 ~4 f 12 ~8 f8
10 - ' 67 f 6 63 s 13 82 f 4 100 100
SBTI 10-'
63 f 7 65 f S 79 f 3 100 100
S x 10 -° 64 ~ 5 61 i 16 57 :t 12 100 100
The concentrations of test solutions used were as follows : crude venom, 400 Ng per ml ; fibrinolytic principle, 100 hg per ml ; plasmin, 100 pg per ml ; urokinase, 1 U per ml ; trypsin, 5 ug per ml . The lysed zones caused by these test solutions were about 200-300 mm'. RESULTS The effects of the fibrinolytic blhibitors on fibrinolysis induced by the purified fibrinolytic principle, crude venom, plasmin, urokinase and trypsin
As shown in Table 1, the fibrinolytic activities of crude venom, the fibrinolytic principle, plasmin, urokinase and trypsin were partially inhibited by EACA. BothTrasylol (10 -6 M) and SBTI (5 x 10 -6 M) completely inhibited urokinase and trypsin activity, but did not completely inhibit the activity of crude venom, the fibrinolytic principle or plasmin . The effects of cysteine and EDTA or:fibrinolysis induced by crude venom, the purifiedfibrinolytic principle, plasmin, trypsin and urokinase Cysteine (5 x 10'' M) and EDTA (5 x 10 - ' M) completely inhibited the fibrinolytic
activities induced by crude venom, the fibrinolytic principle and plasmin, but they did not affect significantly the trypsin and urokinase activities except that cyteine showed only a weak (25 ~) inhibitory action on urokinase activity. The concentrations of test solutions were the same as in Table l . . ; The effects of cysteûle, EDTA, EACA, Trasylol and SBTI on the fibrinogenolytir activities inducedby the purifiedfibrinolytic principle, plasmin and trypsin As shown in Fig . 1, both cysteine (5 x 10 - a M) and EDTA (5 x 10 - ° M) inhibited the
fibrinogenolytic activities induced by the purified fibrinolytic principle and plasmin, but trypsin was not inhibited . EACA (2 x 10 - ! M) and Trasylol (2 x 10 -' M) had no sig nificant effects on the fibrinogenolysis induced by thè fibrinolytic principle and plasmin ; however, Trasylol and SBTI (5 x 10 -6 M) blocked the action oftrypsin . The hemorrhagic activities of cnrde venom and the purified fibrinolytic principle
The hemorrhagic activity of the. purified fibrinolytic principle was more potent than that of the crude venom at lower doses (< 5 lrg), while they were equipotent at higher doses (> 10 ltg) (Fig. 2). The minimum hemorrhagic dose of the purified fibrinolytic principle was 2~6 ~ 1~6 pg, while that ofthe crude venom was 9~4 ~ 0~5 ltg . Trypsin (1001tg), plasmin (1001tg) and urokinase (1 unit) had no apparent hemorrhagic effect . The effects of cysteine, EDTA, EACA, Trasylol and SBTI on the hemorrhage induced by the purifiedfibrinolytic principle As shown in Fig . 3 cysteine (5 x 10 - ' M) and EDTA (5 x 10 - ' M) completely inhibited
164
CHAOHO OUYANG
and
TUR-FU HUANG
the hemorrhagic activity of the purified fibrinolytic principle, while EACA, Trasylol and 5BTI had no effect . The e,(ject ofcations (Mge ~ and CaY+) on the EDTA-induced inhibitory action on lremorrlrage andfibrinolysis induced by tire purifiedfibrinolytic principle Neither Ca'+ nor Mgs+ (2 x 10 -8 M) had any inhibitory effect on hemorrhage or fibrinolysis. The optimal pH for the hemorrhagc activity of the purified fibrinolytic principle was quite broad (5~0-9~0). The effects of cysteine and EDTA on casei~rolytic activities induced by the purified,fibrinolytic principle, plasmin and trypsin Both EDTA (5 x 10 - a M) and cysteine (5 x 10-8 M) completely inhibited the caseinolysis induced by the purified fibrinolytic princple (1001Ig per ml), and induced, respectively, 60 ~ 20 and 87 ~ 13 ~ inhibition of plasmin (301tg per ml), while the activity of trypsin (51Lg per ml) was not affected. Results were each based on four experiments .
Trypsin
FIG. 1 . THE EFFECTS OF EDTA, CYSTEINE, EACA, TRASYLOL AND SBTI ON FIBRINOGBNOLYSIS INDUCED BY THE PIIRIFlI~ FLßRINOLYTIC PRINCIPLE OF TAB VENOM (FP), PLA9MIN AND TRYPSIN.
The abscissa represents the incubation time of the fibrinogenolytic agents with fibrinogen. The ordinaterepresents the percentage of the clottable fibrinogen remainingafter the incubation. D), cysteine; (" "), EACA; (O O). central ; ("-~- "), EDTA ; (p ([]- - - - ~, Trasylol ; (" /), SBTI . The concentrations of the fibrinogenolytic agents were the same as in Table 1, and those of the inhibitors were the same as in the fibrinolysis experiment.
Filxinolytic Principle from A . acutus Venom
165
20
E E
vd
IS
w 4
E0
v
c 0
~n 4
10
S 0.6
I. S
I 2 .5
I 5
I 10
Dose, N.q
I 20
I 40
FIG . Z . THE LOG DOSE-RESPONSE CURVE FOR HEMORRHAGE INDUCED BY PRINCD'LE (FP) AND CRUDE VENOM.
Tf~
PURIFIED FIBRINOLYIIC
The effects offibrinolytic inhibitors on caseinolysis induced by thepurifiedfibrinolytic principle, plasmin and trypsin The caseinolytic activity of the purified fibrinolytic principle (100 ~Ig per tnl) was not inhibited by EACA (2 x 10-' M), Trasylol (2 x 10 -' M) or SBTI (5 x 10 -6 M). The activity of plasmin could be inhibited to 17 ~ 7 and 38 ~ 12 ~ of the original values by Trasylol and SBTi, respectively, but not by EACA. The activity of trypsin could be inhibited completely by Trasylol (2 x 10 -' M) and SBTT (5 x 10 -6 M), but not by EACA (2 x 10 - s M). DISCUSSION
It is known that the antifibrinolytic activity of EACA is chiefly due to the inhibitory action on plasminogen activation and that of Trasylol is chiefly due to the direct inhibitory action on plasmin (ALKJAERSIG et al., 1959 ; Txnursxotn et al., 1967; AMBRUS et al., 1968) . Since the inhibitory action of Trasylol on the fibrinolysis induced by the purified fibrinolytic principle was much higher than that of EACA (Table 1), the fibrinolytic activity of the fibrinolytic principle might be due to a direct action on fibrin . Trasylol (10 -~ M) having no inhibitory effect on urokinase still had about 50~ inhibitory action on the fibrinolytic principle. SBTI (5 x 10-6 M) could completely inhibit the fibrinolysis induced by trypsin and urokinase, but only had about 60 ~ inhibitory activity on the fibrinolytic principle and plasmin . From all these findings we may draw a conclusion that the fibrinolytic principle is a plasmin-like enzyme rather than an indirectly-acting activator (plasminogen activator) like urokinase . Concerning the hemorrhagic action of the fibrinolytic principle, it was similar to HRI of T. ,~iavoviridis purified by OMORI-$ATUIi and OHSAKA (1970). Both of them were acidic proteins . At lower doses ( < S lIg) the hemorrhagic activity of the fibrinolytic principle was about three times higher than that of crude venom, but they were equivalent at higher doses, because the effect of anticoagulant principle would be magnified at higher doses of crude venom which produced a more profound hemorrhagic effect . No collagenase activity
166
CHAOHO OUYANG and TUR-FU HUANG
was detected in the fibrinolysic principle. That trypsin (100 Itg), plasmin (100 itg) and urokinase (1 unit) caused no apparent hemorrhage indicated that the causes of local hemorrhage may be a phenomenon which cannot be induced solely by the caseinolytic, fibrinolysic or fibrinogenolysic action . In many organ systems, normally occurring plasminogen activators appear to sustain local hemorrhage but do not induce hemorhage (AxnEltsox et al., 1968). Tryasylol and EACH were effective in several fibrinolysic bleedings (AxDHRSON et al., 1968), while they could not inhibit the hemorrhage induced by the fibrinolysic principle of the venom given intradermally (Fig. 2). The causes of hemorrhage may be the damage of basement membrane and surrounding fibrils as suggested by OHSAKA et al. (1973) . EDTA and cysteine completely inhibited the activities tested, namely fibrinolysis, fibrinogenolysis, caseinolysis and hemorrhage . This implies that cysteine and EDTA induce a chemical modification in the structure of the proteolytic enzymes (the fibrinolysic principle and plasmin) . Disulfide bond might be indispenable for the structure of the proteases. That EACA was an effective inhibitor for fibrinolysis but not for fibrinogenolysis, caseinolysis and hemorrhage induced by plasmin, trypsin and the fibrinolysic principle of the venom (Figs. 1 and 3, Table 1) agreed with those previous reports on plasmin and trypsin (AMHRUS et al., 1968 ; OKAMOTO, 1962). Trasylol is a competitive inhibitor to plasmin (IVIAKI and BE1.1.ER, 1966) and also a nonspecific inhibitor to trypsin, chymotrypsin and kallikrein (TRAUTSHOLD et al., 1967), It inactivated fibrinolysis, fibrinogenolysis and caseinolysis by trypsin and it also inactivated fibrinolysis by urokinase (Table 1). SBTI inhibited all actions of trypsin. This may have resulted from the formation of an inactive complex of SBTI with trypsin (SIEDL and LINER, 1972). SBTI was as nonspecific as Trasylol ; it also inactivated plasmin and the fibrinolysic principle of the venom.
Acknowledgements-This work was supported in part by the National Science Council Research Grant of the Republic of China. REFERENCES At-tupt:xstG, N., Ftrct~tt, A. P. and SHERRY, S. (1959) E-ACA : An inhibitor of plasminogen activation . J. Biol. Chem. 234, 832. Anteßus, C. M., Aatexus, J. L., LASSMAN, H. B. and MtNx, L. B. (1968) Studies on the mechanism of action of inhibitors of the fibrinolysin system . Arm. N.Y. Acad. Sci. 146, 430. ANDER40N, L., Nttsoox, 1. M ., COLLEEN, S., Gnnnsrturm, B. and Miz.nxn$n, B. (1968) Role of urokinase and tissue activator in sustaining bleeding and the management thereof with E-ACA and AMCHA. Arm. N.Y . Acad. Sci. 146, 642 . AsTaur, T. and Mtita,exTZ, S. (1952) The fibrin plate method for the estimation of fibrinolytic activity . Archs Biochem. Blophys. 40, 346. Cot~N, L, Zux, M., ICnMtrrsxv, E. and Dt: VItIE3, A. (1970) Isolation and characterization of kinin-releasing enzyme of F .clris coloratus venom. Biochem. Pharnrac . 19, 785. DELPIERRI:, G. R. (1968) The proteolytic activities of some Africa Viperidae venoms . Toxicwt 5, 233. DEUTSCH, H . F. and Dtxtz, C. R. (1955) Some proteolytic activities of snake venoms. J. blol . Chem. 216, 17 . DtMtTxoz, G. D. (1971) Purification and partially characterization of two proteolytic enzymes from the venom of Vipera russellü . Toxicon 9, 33 . KocrtoLaTV, W. F. (1971) Toxicity and some enzymatic properties and activities in the venom of Crotalidae, Elapidae and Viperidae. Toxicon 9, 131. Koxno, H., Koxno, S., IKEZAWA, H., MutmTa, R. and Ot~tu, A. (1960) Studies on the quantitative method for determination of hemorrhagic activity of Habu snake venom. Jap. J. med. Sci . Biol .13, 43 . MAENO, H . and MtTSUFIA3HI, S . (1959) Procedure for proteolytic activity determination (2). Studies on Habu snake venom. Jap. J. Microbiol. 3, 131 . Mntct, M . and BF.LLER, F. K. (1966) Comparative studies of fibrinolytie inhibitors in vitro. Thronrb. Diatlr . haemorrh . 16, 668. NEUMAN, R. E. and TrTELL, A. A . (1950) Action of proteolytic enzyme on collagen . Proc . Soc. exp. Biol . Med. 73, 409. OHSAKA, A., JvsT, M. and HasExhtArrrr, E. (1973) Action of snake venom hemmorhagic principles on isolated glomerular basement membrane . Blochim. blophys. Acta 323, 415. ORAMOTO, S. (1962) Influence of AMCHA on the activity of fibrinolysin . Keio J. Med. 11, 117. OMORI-SATOH, T. and OIjSAKA, A. (1970) Purification and some properties of hemorrhagic principle I in the venom of 7ilmeresurus,llavovlrldls . Blochim. blophys. Acta 207, 432.
FIG . 3 . THE EFFECTS OF CYSTEINE, EDTA, EACA, TRASYLAL, ANTIVENIN AND SBTI HAGE INDUCED BY THE PURIFIED FiHRiNOLYTIC PRINCIPLE .
On HEMORR-
Patterns of hemorrhage observed from inside of the removed skin . (1) Control, (2) cysteine, (3) EACA, (4) EDTA, (5) Trasylol, (~ anitvenin and (7) SBTI .
Fibrinolytic Principle from A. acutus Venom
167
Ovverra, C. (1957) The effects of Formosan snake venoms on blood coagulation In vitro. J. Fornwsan nred. Assoc. 56, 435. Ouv~xa, C., Hoxc, J. S. and Terta, C. M. (1971) Purification and properties of thrombin-h7ce principle of Agklstrodon acutus venom and its comparison with bovine thrombin. Thromb . Dlath.hacmorrh . 26, 224. Ovv,uva, C. and Huerrc, T. F. (1976) Purification and characterization of the fibrinolytic principle of Agkistrodon acutus venom. Blochim. blophys. Acts 439, 146. Ousr~ro, C. and Sauu, S. Y. (1970) Relationship between pharmacological actions and enzymaticactivities of the venom of 7}imeresurus gra»tlneus. Toxlcon 8, 183. OUYLNCi, C. and TExa, C. M.(1972) Purification and properties of the anticoagulant principle of Agkistrodon aeutus venom. Bioehlm. btophys. Acts 278, 155. Ousr~xc, C. and YANC3, F. Y. (1974) Purification and properties of the thrombin-like enzyme from T. gramlneus venom. Biochim. biophys. Acts 331, 354. Rocx~, E. and Su.vn, M. (1949) Bradykinin, a hypotensive and smooth muscle stimulating factor released from plasma by snake venom and trypsin. Am. J. Physiol.156, 261. SrEnt D. S. and Lt1VER, I. E. (1972) Isolation and properties of complexes of the Bowman-Birk Soybean inhibitor with Trypsin and chymotrypsin . J, biol. Chem . 247, 3533. TRAITISHOLD, L, Wenr~, E. and GErnun, Z. R. (1967) Trasylol . Biochem. Pharmac.l6, 59. Tu, A. T., How+.+, M. and HONG, B. S. (1969) Hemorrhagic, myoneurotic, thrombotic and proteolytic activities of viper venoms . Toxocon 6, 175. W~, A. J., GuFSr, M. M. and SescEns, W. H. (1947) Fibrinogen : with special reference to its preparation and certain properties of the product . Arch Biochem. 13, 231 .
