I

Asiatic Herpetological Research

June 1995

Fibrinogenase from theJIAN-PING MAO

Venom of Trimeresurus mucrosquamatus

WAN-YU WANG YU-LlANG XlONG AND LIANG LU 211,Vol. 6, pp. 78-84

1,'Kunming Institute of Zoology, Academia Sinica, Kunming, 650223 China
2Yunnan Provincial Hospital, Kunming, 650021, China
Abstract. -A new fibrin(ogen)olytic protease (FP) was purified from the venom of Trimeresurus
mucrosquamatus by DEAE-Sephadex A50, Sephadex-G75, CM-Sepharose CL-6B and mono-s (FPLC)
column chromatography. The molecular weight was 22,000 Da and the isoelectric point was 9.2. It was aglycoprotein composed of 194 amino acid residues. FP could hydrolyze casine, finbrin, fibrinogen and also
showed hemorrhagic subcutaneously, no phospholipase A activity, arginine esterase activity which existed inthe crude venom. The enzyme could be inhibited by ethylenediamine tetra-acetate (EDTA) and cysteine, but
not by phenylmethyl sulfonyl fluoride (PMSF). FP cleaved the BB-chain of fibrinogen first following theAa-chain. In vivo, thrombolytic activity was tested on artificial thrombus placed in the cerebral artery ofrabbits. Thrombolysis was then characterized by angiographic techniques over several intervals. The
fibrinolytic activity resulted in thrombolytic recanalization of two dosage groups. Of four rabbits of 0.2
mg/kg, one achieved recanalization in 12 hrs. and three in 24 hrs. Of another four under the dosage of 0.4

mg/kg, three recanalized successfully in 5 hrs. and one in 9 hrs.

Keywords: Venom, Fibrin(ogen)olytic Protease, Thrombolysis.

Introduction

Materials

Fibrinolytic and fibrinogenolytic activity
had been described in the venoms of anumber of snake species, including
members of the Crotalinae, Viperinae, andElapidae families (Ouyang and Teng, 1976;

LyophilysedAnd Methods
Trimeresurus

mucro-squamatus venom was obtained fromYuanlin Farm (Hunan, China) and stored at-20°C. Human thrombin was purchasedfrom Shanghai Hospital.

Fibrinogen,1988; Daoud et al., 1987;
TheBarrett,1988).

BAEE (N- benzoyl- L- arginine ethyl ester),PMSF were from the Shanghai Institute offuibrin(ogen)olytic enzymes in snakevenoms had also been reviewed previouslyBiochemistry, Academia Sinica. Urokinase(UK) came from Nanjing University.

(Seegers and Ouyang, 1979; Hellmann,1968; Markland, 1988; Markland, 1991).
Several fibrin(ogen)olytic enzymes wereisolated from the venom of Trimeresurus

DEAE-Sephadex A50, Sephadex-G75, CMSepharose CL-6B and mono-s HR5/5

Willis etEvansal.,andtwo fibrinogenases(Ouyang and Teng, 1976), two hemorrhagic
principals HT-a and HT-b (Nikai et al.,1985) and three proteinases (Sugihara andmucrosquamatus:Mori, 1985). Willis, et al. (1989) evaluatedthe thrombolytic potential of anticoagulant
proteases in Crotalus atrox venom usingrats.

In the present study,

new fibrinogenase fromChinese

habusnakewe purified avenom oftheandstudied

characteristics.

its(FPLC) were purchased from Phrmacia Fine
Chemicals (made in Uppsala, Sweden).
The other chemicals used were analyticalgrade from commercial sources.
Isolation Procedure: Isolation of FP
was achieved by a combination of gel
filtration and ion-exchange chromatography
at 4°C (Fig. 1). One gram of crude venom
was dissolved in 5 ml of 50Tris, pHmM

The insoluble material was removed bycentrifugation (2000 g) for 10 min. The
supernatant was fractionated thus: first, on
DEAE-A50 (3 X 100 cm), 50 mM Tris-HCl
pH 8.5; second, Sephadex -G75 (2 X 1008.8.

mM Tris-HCl pH 7.5; third, CMCL-6B (2 X 30 cm), 10 mMcm), 20Sepharose© 1995 by Asiatic Herpetological ResearchVol. 6, p. 79

Asiatic Herpetological Research

June 1995

CVscIII*M

10!030

40

iJO!040

FPLC Mono-S

50 l a k, Nui

40

10Tubr Nuaber

FIG.

1.

Fractionation of Trimeresurus

mucrosquamatus venom.

1st,DEAE-A50 (3X100 cm) anion

exchanging, 50mM Tris-HCL pH 8.5; 2nd, Sephadex G-75 gel filtration (2X100 cm), lOmM ammonium
acetate pH 7.0; 4th, mono-s cation exchanging, lOmM sodium acetate pH 5.8.

ammonium acetate pH 7.0; and fourth, on
mono-s HR 5/5, 10 mM sodium acetate pH

qualitatively assayed with the substrate of5.8.

yolk.

Characterization of FP:

Assay forfor

gross
hemorrhagic activity assay
observation was performed as reported
previously (Bjarnason and Fox, 1983).
Proteolytic activity was assayed by a methodTheusing casine of Kunitz (1947).
inhibition of EDTA, Cysteine, and PMSF
was also tested with this method..
Fibrinogenase activity was measured by themethod of Ouyang and Huang (1979).
Fibrinolytic activity was tested with thefibrin plate method of Astrup and Mullertz(1952), and also with fibrin clot fromfibrinogen with thrombin. Arginine esterhydrolytic activity was assayed using BAEE
as a substrate. BAEE 50(containing 1with50
Triswas
CaC12)
preparedHC1 (pH 8.0) buffer. Trypsin was taken asmMmMmM

the control.

The phospholipase activity wasThe pH ofmodulated to 8.0;
added, the pH

thesubstrate

after the fraction

waswasdecreased by thephospholipase activity, and was adjusted tothat of the original by 10NaOH. The
values of sodium hydroxide was taken tomM

Amino acidpresent the activity.
werecarriedon
a Model 835compositions
50 Hitachi high speed automatic analyzer by
the method of Simpson et al. (1976).
Twenty-four h, 48 h hydrolysates wereused.
Phanolanaline was used as theminimum residue to calculate the number ofamino acid residues.

Thrombosis

assay by angiography: For FP dosage determination, 0.2
ml rabbit plasma made clot with 3U
thrombin, 50(lg FP was used to test in vitro
activity.

In 4.5-5.5 hours, the milky white

Vol. 6, p. 80

Asiatic Herpetological Research

TABLE 1 Summary of purified FP from T. mucrosquamatus venom (n=6).
.

Recovered protein (mg)Hemorrhage (mm X mm)(25±3 gm mice 100 ug sample)
Caseinolytic activity

Units/mg.min
Fibrinolytic activity

mm 2(Fibrin heated plate
Arginie esterase activity

Phospholipase A activity)

June 1995

June 1995

Asiatic Herpetological ResearchfVol.6, p. 81iu-nai

FIG. 3. Aniographs of in vivo thrombolysis (from left). Control group! A, Normal graph. B, Thrombosis
performed (X). C, Treated with saline, 24 hours. FP group: D and G, Normal graphs. E and H, Thrombosis
performed (X). F, Treated with FP at 0.2 mg/kg dosage, recanalization occured in 24 hrs. I, Treated with FP
at 0.4 mg/kg dosage, recanalization occured in 5 hrs. U. K group (positive control): J, Normal graph. K,Thrombosis performed (X). L, Recanalized within 9 hours of U. K administration (1 ,000 IU/kg).

obtained in this step yielded fibrinolyticactivity. This fraction was dialyzed against
10
aminium acetate (pH 7.0) and loadedon the CM-Sepharose CL-6B column andmM

was separated

into three fractions, thefibrinolytic activity was located in the 2nd,was rechromatographied on
FPLC mono-s column and eluted with threephases of gradient: 0-30 ml (0 M NaCl);
30-80 ml (0-0.1 M NaCl) and 80-100 ml
this fraction

Vol. 6, p. 82

Asiatic Herpetological Research

TABLE 3. Proteases from the venom of T. mucrosquamatus.
AminoJune 1995

June 1995

Asiatic Herpetological Research

phospholipase A activity. The enzyme
also had hemorrhagic activity (Table 1).

HeatandpH

stability:

The

proteinase, at a concentration of 40 |ig/ml
in 10
acetate buffer (pH 5.8)

mM

mM

Vol. 6, p. 83

Discussion

The venom of the Crotalinae species
contained much proteinases, which hadproteolytic and esterase activities. Several
enzymes were isolated from Trimeresurus

50°C, and almost lost complete activity at55°C. Incubation of FP at pH values

mucrosquamatus venom (Table 3 and 4).
Our results showed that FP is a new
fibrinogenase existing in Trimeresurus
mucrosquamatus venom. Compared withthe others, this enzyme, is a metalloproteinase which attacks the Bb-chain offibrinogen preferentially. As we know,
the enzymes which had fibrinogenolytic

below 5.0 and above 10.0 for 30 min leadto a sudden decrease in proteolytic

activity were classified as a-fibrinogenases
and b-fibrinogenases; most of the a-NaCl, were incubatedcontaining 10for 30 minutes at various temperatures andthen quickly cooled down to room
temperature. The caseinolytic activity wasthen determined. The enzyme was fully
active at 37°C,

showed little activity at

Biological activity: The effects ofsome reagents on the proteolytic activity of
FP were examined. This activity wasfibrinogenases can degrade Aa-chain offibrinogen and usually are metalloOf most of the bproteinases.
fibrinogenases degrading the Bb-chain offibrinogen, little of them could also

inhibited by ethylenediamine tetraacetic

degrade the Aa-chain inhibited by DFP oracid (EDTA), cysteine but not by PMSF.
The effects of some divalent and trivalentions on the proteolytic activity of FP werealso assayed. The enzyme (40 |ig/ml) andin 10
ions at a concentration of 10
acetate buffer were first incubated atPMSF. They are serine proteinases. HT-activity.

mM

mM

37°C for 30 min. before the proteolytic
was assayed. The proteolytic

activity

activity increased in the presence ofbivalent ions in the following order: Ca++of which hydrolyzed
Bb-chain of fibrinogen and was inhibitedby EDTA. The followed FP was thesecond report of these enzymes. Three
proteinases from the Agkistrodon halys
blomhoffii were activated by Ca++ anda, the first example

Co++ (Satake et al., 1963). Ca++ andZn++ were also needed for the proteolytic
activity of protein

G from Bothrops asper< Cu++ < Zn++ < Co++. The increase
by Fe+++ and A1+++ were lower than thatof Zn++ but higher than that of Cu++,
Ca++ (Table 2). When fibrinogen wasincubated with FP, this enzyme cleaved

FP was(Ortiz and Gubensak, 1987).
activated by Co++ and Zn++. For these
metallo-proteinases, these bivalent cations

the Bb-chain of fibrinogen and followed

plasma clot in vitro, also 0.4 mg FP/kg
dosage occurred thrombolysis in 3/4 in

the Aa-chain as shown in Figure 2.

Thedegrading of fibrinogen by FP wasmeasured as 36.5 mg per mg enzyme inone minute.

Thrombolysis:

None of the four

rabbits of saline administration reachedrecanalization.

Of the FP 0.2 u.g/kg

group, one achieved recanalization in 12hrs. and three in 24 hrs.; of the other fourof 0.4 mg/kg, three recanalized
successfully in 5 hrs. and one in 9 hrs. Of
the four rabbits treated with 1,000 IU/kgof U.K., thrombolytic recanalization
occurred in two in 6 hrs. and two in 9 hrs.
(Fig. 3).

were important for their stability and theiractivities. FP had marked activity on thevivo.

ItwasagoodtrialforFP

thrombolytic potential. Hemorrhage wascaused when injected subcutaneously, but
did not occur within the heart, liver,kidney and lung after FP injection inrabbits at the dosage of 1 mg/kg in mice.

Vol. 6, p. 84

Asiatic Herpetological Research

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June 1995

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