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Species difference in the fibrinogenolytic effects of α- and β-fibrinogenases from Trimeresurus mucrosquamatus snake venom

Species difference in the fibrinogenolytic effects of α- and β-fibrinogenases from Trimeresurus mucrosquamatus snake venom

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ra~. va. z3. No . s. ~. ~n-~ai, i9as. rrmc°a m oreu a~mo.

ao4i-otovas s3.ao+ .oo ® tses t~,mon vr<.. ua.

SPECIES DIFFERENCE IN THE FIBRINOGENOLYTIC EFFECTS OF a- AND ß-FIBRINOGENASES FROM TRIMERESURUS MUCROSQUAMATUS SNAKE VENOM CHE-MIND TEND, CHAOHO OUYANO and SONG-CHOW LIN Pharmacological Institute, College of Medicine, National Taiwan University, Taipei, Taiwan, R.O .C . (Acceptedfor publirntion 29 January 1983) C. M. Ttovo, C. OuYwwa and S. C. LAN. Species difference in the fibrinogenolytic effects of sand ß-fibrinogenases from 7ümeresurus mucrasquamatus snake venom. Toxicon 23, 777-782, 1983 . - Alpha- and beta-fibrinogenases prepared from 7Yimeresurus mucrosquamatus venom digested specifically the a(A) and ß(B) chains of the fibrinogen moleaile, respectivdy. a-Fibrinogenase digested bovine fibrinogen more markedly than human fibrinogen, while ß-fibrinogenase digested human fibrinogen more markedly than bovine fibrinogen. Human fibrin was also digested by both enzymes. Plasma fibrinogens of 4 animal species were digested by a-fibrinogenese to the same degree, while those by ß-fibrinogenaae in the following order: human > dog > guinea-pig > rabbit . The fibrinogenolydc effects of a-fibrinogenaee on human fibrinogen were strongly inhibited by sera of the 4 animal species, while those of ß-fibrinogenase were inhibited in the following order: rabbit > guinea-pig > dog > human . It was concluded that the different activities of the protease inhibitors in the plasma of animal species are mainly responsible for the sensitivity differences.

INTRODUCTION

$NAIL v>3NOMS contain many biologically active proteins which affect different steps in blood coagulation. In our laboratory we have purified some coagulants and anticoagulants from Crotalidae and Viperidae venoms and have studied their modes of action . These are thrombin-like enzymes, fibrinogenases, prothrombin activation inhibitors and platelet aggregation inducers and inhibitors (for review, see OUYANO et al., 1982). From Trimer+esurus mucrosquamatus venom we have purified 2 fibrinogenolytic enzymes with different modes of action . The a-fibrinogenase digested specifically the a(A) chain of fibrinogen, while ß-fibrlnogena.4e digested preferentially the ß(B) chain (OUYANO and TEND, 1976; OUYANG et al., 1977). a-Fibrinogenase inhibited platelet aggregation, while ßfibrinogenase did not (OUYANG et al., 1979). In this paper we report the fibrinogenolytic activities of both enzymes in several animal species. MATERIALS AND METHODS The venom of 7Yimerrsurus mucrn~squamatus wss oollxted at our laboratory, centrifuged, lyophilized and stored in a deaiccator below -20°C. 1?Ybrlnogenases (a and ~ of the venom were purified by the procedure of OUYANCi and Tt~ta (197 . T)u 2 eazymea were homogeneous as judged by disc dectrop~horais on polyacrylamide gels. Bovine fibrinogen was purchased from Peatex Co . (Kankakee, IL, U.S .A .) and human fibrinogen from Cutter Laboratories Inc. (Berkeley, CA, U.S .A .) . Bovine thrombin purchased from Parko-Davis Co . (Detroit, MI, U.S .A .) was dissolved in SOSi glycerol-saline to give a stock solution containing 1000 NIH units/ml . Plasma was prepared by mis ng blood with 3.8sFs sodium citrate (9 :1 v/v) and centtifugiug at 1300 f and 3°C for 20 min. Serum was prepared es above without amicoagulant during blood withdrawal . 777

77s

CHE-MING TENG, CHAOHO OUYANG and SONG-CHOW LIN

Fibrinogenolytic activity was measured as described previously (OUYANG and TENC, 1976). For the effect of fibrinogenases on bovine or human fibrinogen, an equal volume of fibrinogen solution (2%) and fibrinogenases of various concentrations (in imidazole/saline buffer, pH 7.4) were mined and incubated at 37°C for 30 min. Aliquots (0.2 ml) of the above incubation mixture were withdrawn for the assay of clottable fibrinogen (WARE et al., 1947) . For the study of the inhibitory effect of serum on fibrinogenolytic activity, fibrinogenase was preincubated with serum for S min and then an equal volume of human fibrinogen (2%) was added and the mixture incubated at 37°C for another 30 min before the assay of dottable fibrinogen . For the study of the fibrinogenolytic activity of fibrinogenase in plasma, 0.5 ml of plasma was incubated with 0.05 ml of fibrinogenase at 37°C for 30 min and then the residual clottable fibrinogen was determined.

RESULTS

Effects offibrinogenases on human and bovine fibrinogen and fibrin It was reported previously (OUYANG and TEND, 1976) that the specific activities of sand ß-fibrinogenases were 100 and 17 mg fibrinogen/min/mg of enzyme, respectively, using bovine fibrinogen as substrate. We compared the %brinogenolytic activities of both enzymes on human and bovine fibrinogen. a-Fibrinogenase degraded bovine fibrinogen more markedly than human fibrinogen, while ß-fibrinogenase degraded human fibrinogen more markedly than bovine fibrinogen in 30 min (Fig . 1). The SDS-polyacrylamide gel electrophoretic patterns of human fibrinogen after incubation with a- and ß-fibrinogenases were similar to those of bovine fibrinogen (OUYANG and TEND, 197, i.e. a-fibrinogenase completely degraded the a(A) chain and concommitantly two bands of split products appeared, while ß-fibrinogenase digested the ß(B) chain first and the a(A) chain only if the incubation time was prolonged. Human cross-linked fibrin (polymerization of a-chains to a-polymer and dimerization of 2 r-chains to y-duner), formed by thrombin and Cat* ion, were digested completely within 6 hr by a- and ß-fibrinogenases (100 ~tg/ml) . The SDSpolyacrylamide gel electrophoresis showed that a-polymer of human fibrin was degraded by a-fibrinogenase, while ß~hain and a-polymer were degraded by ß-fibrinogenase. y-Dimer was not affected by both enzymes.

loo~ Enzyme Concer~tration(Nglml) FIü. 1 . COMPARI90N OF TFm FIBRIN008NOLYTIC AGTIViTISS OF a- AND ß-FIBaINOC1ENASH.4 ON HUMAN AND BOVINE FIHRINODEN .

Equal volumes of fibrinogen (2%) and various concentrations of fibrinogenases were incubated at 37°C for 30 min and then residual dottable fibrinogen was assayed. Means t S.E . of 3 experiments are presented.

Effect of a- and ß-Fibrinogenases

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TABLE 1 . COMPARISON OF THE EFFECTS OF a- AND ß-FIHRQdOOENASES ON PLASMA FIBRINOGENS OF RABBIT, GUINEA-PIG, DOG AND HUMAN

Enzyme concentration (kg/ml) a-F7biinogenase 0 3.2 6.3 12 .3 25 SO 100 ~~}brinogenase 0 3.2 6.3 12 .3 25 SO 100 200

Rabbit

Residual plasma fibrinogen (R of control) Guinea-pig Dog Human

100 91t 6 87t 7 73t 6 70t10 63t S 41t14

100 100t 7 93t 6 89t10 74t14 31t13 0

100 97f 6 99t 8 97t 9 86t10 27t18 0

100 96f 8 96t S 95t 7 88t S 49t11 0

100 103~ S 99t 6 96t 3 94~ 8 90t 6 70t14 64t13

100 96t 89f 89f 91t 88t 43t 0

100 92t4 89t 8 33t14 36f15 19t 9 0

100 94t 8 71t 6 33t11 22t17 0

6 4 8 6 S 8

Means t S.E . of 3-4 experiments are presented.

Fffect offibrinogenases on plasma fibrinogen of various animal species The fibrinogenolytic activities of a- and ß-fibrinogenases were tested on the plasma of rabbit, guinea-pig, dog and human. It is shown in Table 1 that the activity of a-fibrinogenase was much decreased in plasma compared to fibrinogen solution . Only when the concentration of a-fibrinogenase was higher than 100 pg/ml did the plasma fibrinogen of guinea-pig, dog and human become unclottable. Forty one per cent of fibrinogen in rabbit plasma remained clottable after its incubation with a-fibrinogenase (100 ~g/ml) for 30 min. Table 1 also compares the activity of ß-fibrinogenase on plasma fibrinogen of these four species . Sixty four per cent of fibrinogen in rabbit plasma remained clottable after its incubation with ß-fibrinogenase at a concentration of 200 !~g/ml for 30 min. Under the same conditions, plasma fibrinogen of guinea-pig, dog and human became unclottable after incubation with ß-fibrinogenase at concentrations of 200, 100 and SO ~g/ml, respectively . Inhibitory effect of serum on the fibrinogenolytic activity of fibrinogenases In order to see if the susceptibility differences of plasma of various animal species to fibrinogenases were due to the effect of protease inhibitors present in sera, the inhibitory effects of the sera of these four animals on the fôrinogenolytic action of both fibrinogenases on human fibrinogen were compared . As shown in Fig. 2, the fibrinogenolytic activity of a-fibrinogenase (6.25 pg/ml) on human fibrinogen was almost completely inhibited by sera of dog and human, and 80% inhibited by sera of rabbit and guinea-pig. The higher the dilution of the sera, the lower were the inhibitory effects. At 64 times dilution of the original sera, their inhibitory effects were lost. Figure 3 shows that the sera of the four animal species had different potencies of inhibition on the fibrinogenolytic activity of ßfibrinogenase (12.5 Ng/ml) . At the original concentration of sera (without dilution with imidawle/saline buffer), the potencies of the inhibitory effects. of sera were in the following order : rabbit > guinea-pig > dog > human.

CHE-MING TENG, CHAOHO OUYANG and SONG-CHOW LIN 100

Seru m

FIG .

2.

THE INHIHTI'ORY EFFECTS OF RAHBIT, GUINEA-P10, DOO AND HUMAN SERA ON THE FIBRINOGENOLYTIC ACTIVITY OF a-FIHRINOGENASE . a-Fibrinogenase (12.5 hg/ml) was incubated with sera at different dilutions fot 5 min and then an equal volume of human fibrinogen (2qo) was added and incubated for another 30 min before clottable fibrinog~ was assayed. Means t S .E . of 3 -4 experiments are presented. ~-p Rabbit, " - " guinea-pig, x-x dog, p-O human .

Serum

FIG .

3.

THE INHIHiTORY EFFECTS OF RABHIT, GUINEA-P1G, DO(i AND HUMAN SERA ON THE FIHRIIi00ENOLYTIC ACT'IVIT'Y OF ~-FIHRINOOENASE . ß-Fibrinogenase (25 Ng/ml) was incubated with sera at different dilutions for 5 min and then an oqual vohTme of human fibrinogel (24ti) was added and incubated for another 30 min before clottable fibrinogen was assayed . Means t S .E. of 3-4 experiments are presented . Rabbit, " - " guinea-pig, x-x dog. C--p human .

lrp

Effoct of a- and ß-Fibrinogenases

78 1

DISCUSSION

Snake venons cause hypofibrinogenemia by directly destroying fibrinogen (OUYANG, 1957; PHILLIPS et al., 1973) or indirectly through the activation of plasminogen (FORBES et al., 1%S) or through defibrination by procoagulants, e.g. the thrombin-like enzymes (RBID et al., 1%3; ASHFORD et al., 1968). Hypofibrinogenesis might be useful in the treatment of thrombosis . For example, the thrombin-like enzymes of snake venons, especially Arvin from Agkistrodon rhodostoma, have been extensively studied and appear to be valuable as antithrombotic agents for human use (for review see SEEGERS and OUYANG, 1979). Snake venom contains two kinds of fibrinogenolytic enzymes: (1) a-fibrinogenase which possesses caseinolytic activity but is devoid of Tos-Arg-0Me esterase activity, and its activity is inhibited by EDTA ; (2) ß-fibrinogenase possesses not only caseinolytic activity but also Tos-Arg-0Me esterase activity, and its activity is not inhibited by EDTA (OUYANG et al., 1977). Proteolytic enzymes, such as porcine plasmin (STORM et al., 1974) and brinase (from Aspergillus oryzae) (FITZGBRAI,D, 1972) have been used in some thromboembolic disorders. However, their clinical applications are limited because their fibrinogenolytic effects in vivo are inhibited by an excess of protease inhibitors, either antiplasmin (GAUROT, 1%7) or a3-macroglobulin (NxNIAN and DUCIG?RT, 1975). In the preliminary in vivo experiments for evaluating the fibrinogenolytic action of ß-fibrinogenase we found that the fibrinogen level of the rabbit was not affected significantly, while that of the dog decreased to SO-70% of the control after a single dose of 1 mg/kg. In this study we obtained evidence that this difference was mainly due to the amount or potency of the protease inhibitors present in the sera of different species. a-Fibrinogenase was inhibited to the same degree by the sera of the four species tested . However, ß-fibrinogenase was inhibited by the sera in the following order: rabbit > guinea-pig > dog > human. The different inhibitory potencies of the sera explain the differences of the fibrinogenolytic activities of both enzymes on plasma fibrinogen of various species. However, substrate (fibrinogen) specificity of the enzymes in different animal species could not be ruled out. For example, ß-fibrinogenase digested human fibrinogen more markedly than bovine fibrinogen (Fig . 1). a-Fibrinogenase was not expected to be an antithrombotic enzyme because it caused hemorrhagic effects (OUYANG and HUANG, 1977) and was inhibited markedly by protease inhibitors in plasma . ß-Fibrinogenase did not possess a hemorrhagic effect and had a stronger hydrolytic effect toward human fibrinogen and was less suppressed by the protease inhibitors of human plasma . It is worthwhile evaluating the possible application of ßfibrinogenase as an antithrombotic agent. Ackrtowkdgemertt-This work was supported by a research grant of the National Science Council of the Republic of China (NSC71-0412-H002-13) .

REFERENCES ASt~oxn, A ., Ross, J . W . and SovrHCnrE, P . (1968) Pharmacology and toxicology of a defibrinadng substance from Malayan pit viper venom . Lancet 1, 486 . F~1zaeAwt.n, D . E ., Fxiscrt, E . P . and THORNES, R . D . (1972) Thrombolytic therapy with brinase of chronic peripheral arterial disease . Preliminary report . J. Ir. Coil. Phys. Surg. 1, 123 . FORGES, C . D ., TuRE~e, A . G . G . and Douons, A . S . E . (1965) S. .~fr. mad. J. 42, 565 . GAUROT, P . O . (1967) Inhibition of plasmin activity by a,-macroglobulin . Clinics chin . Acts 16, 328 . Nvnt~w, D . and DUCKERT, F . (1975) Assay of brinase inhibitors with soluble and insoluble substrates . Thromb . Diath. haemorrh . 33, 221 .

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Effect of a- and ß-Fibrinogeaases

C. (1957) The effects of formosan snake venons on blood coagulation in vitro. J. Formosan med. Ass. 56, 435. OuYwrre, C. and HUANG, T. F. (1977) The properties of the purified fibrinolytic principle from Agkistrodon status snake venom. Toxirnn 15, 161. OIJYANO, C. and Tertc, C. M. (1976) Fibrinogenolytic enzymes of Trimeresurus mucrosquamatus venom. Biochim . biophys. Acts 42(1, 298. Otl3rntvc, C., TErtG, C. M. and CHErI, Y. C. (1977) Physicochemical properties of a- and ß-fibrinogenases of Trimeresurus mucrasquamatus venom. Biochim . biophys. Acts 481, 622. OUYANO, C., Tnva, C. M. and Curt, Y. C. (1979) Properties of fibrinogen degradation products produced by a- and ß-fibrinogenases of Trlmeresurus mucrasquamatus snake venom. Toxicon 17, 121. Olnwrte, C ., Ttave, C. M. and HUwnte, T. F. (1982) Characterization of the purified principles of formosan snake venons which affect blood coagulation and platelet aggregation . J. Formosan med. Ass. 81, 781. PHILLIPS, L. L., WEtss, H. J. and CHRI3TY, N. P. (1973) Effects of puff adder venom on the coagulation mechanism. II . in vitro 77u+omb. Dlath . haemorrh . 30, 499. ltatn, H. A. (I%3) Treatment of snake-bite poisoning. Br. mcd. J. 1, 1675 . SEeoexs, W. H. and OuYnxc, C. (1979) Snake venons and blood coagulation . In : Snake venons, Handbook of Experimental Pharmacology, Vol . 52, p. 684 (l.ee, C. Y., Ed .) . Berlin : Springer-Verlag . S~rotent, O., Ou.r~tnoeFP, P., DxevvsEta, E. and T~wc, P. (1974) Acute deep vein thrombosis treated with porcine plasmin. A double blind trial. T7tromb. Diath. haemorrh. 32, 486. W~, A. G., GUPSr, M. M. aad Se>?cPats, W. H. (1947) Fibrinogen : with special reference to its preparation and certain properties of the product. Archs Biochem . Biophys . 13, 231 . OuYwrtc,