Biochimita el Biop~ysica Actg. 1074 (1991) 223-229 © 1991 Elsevier Science F'ubfsh~s BN. 0304-4165/91/$03,~0 ADONIS 03044-t6591DOIgtA

223

BBAGEN 23533

Purification and characterization of lebetase, a fibrinolytic enzyme from Vipera lebetina (snake) venom E n e Siigur a n d Jiiri Siigur Laboratory of Bioorsaaie Chemistry, Institute of Chemical Physics a~d B~ophFs~L~of tlte E~tonian Academy of Sciences, Tallitl# ~~ S. S, R.)

(Received6 Februar'y199t)

Key words: Proteinas~: Fibrinolytic e~yme: Snake venom; (V, lebefina)

An anticoagelaat proteiawe, lebetase, was isolated from the venom of gipera lebaina by gel filtration and anion-exchange chromatography. The p¢oteinase ¢mlsists of a single polypeptide chain with a melecular weight n | 23700. Amino acid analysis indicates that lebetase contains 214 residues. It consists of several isofonns ia the pH range 4.6--5.4, all having fibrinoiyfie ac6vily. Lebetase hydmlyzes casein, fibcinogen and f i ~ a , and oxidized insulin B-chain in the pns|dons AlaU-Leu Is and TyrtS-Leu It. Its fibdaolytic activity is inhibited by EDTA a,_~llost upon heating at 70°C for 10 rain. The enzyme readily byd~olyzes the Act-chain and more .,,lowly the B , ~ h a i n of fibrinog~n, in fibrin, the same chains are att~ked. Tiros, the ent~yme is m Aa, Bfl-lilwinogenase. The fibriaolyli¢ activi~j of lebetase is direct, without any plasminol~ activatinn. E ~ is 13.0 for lebetase, The enzyme show~ low ~ activity. Introduction

Snake venoms, in particular from the Crotalidae and Viperidae families, are characterized by strong effects on blood coagulation and iysis. Throughout the last | 0 - 1 5 years, the fibrino(gcuo)lytic activity of snake venoms has been intensively studied. Anticoagulant proteinascs with fibrino(gcno)lytic activity have b e ~ isolated and purified from the venoms of Agkistrodon acutus [1,21, A, eontortrix comortrix (fibrolase) [3-6], A piscivorus piscisorus [3], A. rhodos:oma [7], Crotalus atrox (atroxase) [8], C adamnnteus and C. ~iridis [91, Bothrops asper [10], Cerastes cerastes (cerastase) [11-13], Vipera aspis [14-15], etc. These enzymes have direct fibrinolyfic activity and arc all Aa-fibria0genas~ with a minor effect on the B/I-chain of fibrinogen. There are some references to snake venoms which have definite ability to activate plasmiaogen and thus show an indirect fibrinolytic acdvlty [16,17], but until now no such enzyme has been isolated from snake

Abbttviations: SDg, sodium dodecyl sulfate: EACA, ¢-aminoc,aproi¢ acid: PMSF, phenylme~hyhulfonyl fluodch~; g-2251, I~-Val-Lcx1-Lysp-nitrmnilide; MHD, minimal hcanonhagic dose. CorreSl~ndcnc~: F- Siig~, Labora~,W of Bioocg~ic Chemistry, Institute of ChemicalPhysicsand Biophysicsof ~c EstonianAcademy

of Sciences.200001 Leniai pui~t¢~ !0, Tallian, U.S.S.R.

venom. According to Komalik [17], the crude Vipera lebetma venom has both direct and indirect fihrlnolytic activity. This report deals with the purification and characterization of a direct acting fibrinohttic enzyme from V. lebetina venom. Malerials and Methods

Materialx Vipera lebetina venom was obtained from Tashkent integrated Zoo Plant (Uzbek S..q.R.). Scphadex C~100, molecular weight and p l standards were from Pharmacia Fine Chemicals (Oppsala, Sweden). Ampho|ytes in the pH range of 5-7 and 4--6 were purchased from LKB Produkter AB (Bromma, Sweden). DEAE-eellulos¢ DE-52 was from Whatman Biochemicals. Bovine fibrinoge~, plasminogen and plasmin were from Sigma Chemicals (St, Louis, U.S.A.), thrombin from Kaunas Experimental Plant (Lithuania), I>-ValLeu-Lys-pNA (S-2251), oxidized insulin B-chain and D-glucose from Scrva (Heiddbmg, F.R.G.). Uitrafihration membranes PM-10 were from Amicon B.V. (Oostcrhout, Holland). All other reagents were of or.:.lytical grade. Gel/iltratio~. Crude V. lebctina venom (4.1 g) was dissolved in 25 ml of 0.2 M ammonium acetate (pH 6.7), and the insoluble material was removed by contrifugafion (5000 × g for 15 rain). The supematant was applied to a column (4.8 × 130 cm) of Sephadex G-100

224 superfine equilibrated with 0.2 M ammonium acetate, The ehtion of venom components was effeeted at the rate of 5.5 ml/h and 8.2 ml aliquots were collected. The fractions were tested for easeinolytic and fibrinolytic activities. Fibrinolytic fractions were combined and concentrated by ultrafiltration on PM-10 membranes. DEAE-cellulose chromatography. Chromatography was performed on a column of DE-52 cellulose (1.5 × 12.5 cm) equilibrated with 0.08 M ammonium acetate (pH 8.3). Aliquots from the concentrated fibrinolytie fraction from Sephadex G-100 were diluted to 0.08 M of ammonium acetate, and the pH brought to 8.3 with 25% ammonia. The column was washed with equilibration buffer to date the non-adsorbed protein, Bound proteins were dated uzmg a linear gradient from 0.08 M to 0.15 M of ammonium acetate (pH 8.3). The flow rate was 16 ml/h; fractions of 4 ml were collected. Protein assay, Protein was assayed by the methods of IBradford [181 and Lowry et at. [191, Bovine serum albumin was used as a standard, Carbohydrate analysis. Neutral sugars were de. termined by the phenol-sulfuric acid method [20] using a standard solution containing D-glUCOse, Amino acid analysis. Purified lebetase was hydrolyzed in evacuated, sealed tubes with 6 M HCI at 105°C for 24 h. Amino acid analysis was performed on the l~io. tronik analyzer, Tryptophan was determined in the intact protein by the methods of Edelhoeh [21] and Spies and Chambers [22,23].

Molecular weight and isoeleetric point determination, SDS-polyacrylamide gel electrophoresis was carried out in 10% gels at pH 8.3 by the method of Laemmli 124]. Molecular weight standards were p!.:,~phorylase b (M r 94000), bovine serum albumin (67000), ovalbumin (43000), carbonic anhydrase (30000), soybean trypsin inhibitor (20100) and a.laetalbumin (14400). Anaiyti. cat isoelectfic focusing was performed on 5% polyacryl. amide gel plates according to the method of Vesterberg [25] in Multiphor 2117 apparatus in the pH range of 4-7. The gels were stained for proteins with Coomassie brilliant blue R.250. Enzyme assays. Caseindytic activity was assayed by the method of Kanitz as modified by Mebs [26], One unit of activity is defined as the amount of protein which causes an increase in ahsorhanee of 0.001 per 20 rain at 37°C, Fibrinolytic activity was localized by the fibrin plate technique of Astmp and Mt~llertz [28], Proteinase samples (10-30/~i) were carefully placed on the fibrin surface and incubated at 37°C for 20 h. Activity was expressed as the product of the two perpendicular diameters (ram2) of the lysed zone. For localization of lebetase after isoeleetric focusing the gel was placed for 10 rain into 0.05 M phosphate buffer (pH 7.5), and then covered with a fibrinogen-thrombin mixture according to Astrup and Miillertz [28], forming

the fibrin plate on the gel. The gel was watched until developing the visible zones appeared against an opaque background, indicating lebetase activity. Specific cleavage of fibrinogen was shown on 10% polyacrylamide gels. 0.5 ml of a 2% fibrinogen solution was incubated with 0,5 ml of enzyme (10 /zg/ml) at 37~C in 0.05 M Tris-saline buffer (pH 7.4). At various time intervals, 0.1 ml of the incubation mixture was withdrawn and added to 0,1 ml of denaturing solution (10 M urea, 4% SDS, 4% 2-mercaptoethanol). The samples were reduced and denatured overnight at 37°C before being dectrophoresed [271. Fibrin hydrolysis was shown by SDS, electrophoresis using 10% polyacrylamide gels. 0.I n-d of 1% fibrinogan was clotted with 0.1 ml of thrombin (1 mg/ml) (both in 0.05 M Tris-saline buffer, pH 7.4). The fibrin dot was allowed to form for 1 h at room temperatare. After 1 h, 0.1 ml of lebetase (10 ~g/ml) was added to the dot and incubated at 37"C for various time intervals. Then 0.3 ml of denaturing solution was added and the mixture was incubated overnight before electrophoresis [8]. Plasminogen activation was assessed by measuring the generation of S-2251-hydrolyzing activity. Purified human plasminogen (60/xg) was incubated at 37°C with lebetase (6.5 /~g) in 1.0 ml of 0.05 M Tris.HCI buffer (pH 7.4) eomaining 0.15 M NaCI, and aliqnots were taken at various times to determine the plasmin formed. 100 #1 of the specific substrate for plasmin, S-2251, was thermostated at 37"C in 900 #1 of the Tris-saline buffer, and the reaction was initiated by adding 50 /~1 of the lcbetase-plasminogen incubation mixture. Hydrolysis was followed at 405 tun on a Specord spectrophotometer. For the control, plasrrlinOgen was activated by urokinase. Hydrolysis of oxidized insulin B-chain. Oxidized insulin B-chain, 0.6 mg in 0.6 ml of 0,01 M Tris-HCI (pH 8.0), was ineubatc,d with 10 lgl of lebetase (1,3 mg/ml) at 370C. After 0,5, 1, 3 and 6 h, 100 lal aliquots were withdrawn from the digestion mixture and the reaction was stopped by adding 10/~1 of glacial acetic acid. 50 ILl of the digests were analyzed on the column with reversed phase (Pep RPC TM HR 5/5, Pharmaeia), using the DuPont 8800 Liquid Chromatography System with peak detection at 220 nm. Buffer A: 50 mM CH3COONH# (pH 6.0), buffer B" 80% CH3OH, 205 H:O (v/v), 50 mM CH3COONH,t; linear gradient of 30-100% buffer B in 40 rain with a flow rate of 1 ml/min. Peptide fractions were collected and evaporated to dryness, liydrolyzed in 6 M HCI (24 h, I I 0 ° C ) and analyzed for amino acids on an LC2000 amino acid analyzer (Biotronik, F.R.G.). Inhibition studies. Inhibition of proteolytie and fibrinolytic activity was examined with 20 mM EDTA, 10 mM EACA, 2.5 mM L-eysteine - all preineubated 45 rain at room temperature in 0.05 M Tris-HCl (pH 7.6) with 250/~g of enzyme, and 20 mM PMSF (preineuba-

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Fig. 1. Gel flirtation pat*era of V. lebetina venom (4.1 g) on Sephadex G-t00 superfine ¢oluma (4.8× 130 cm) in 0.2 M ammonium acetate (pH 6.7). Flow rate 5.5 ml/h. Absorbance at 280 nm ( ): proteinase U/ml ( o - -o); fibrinolytic activity ( i - - @ ) .

cooled. Fibrinolytic activity was determined according to Astrup und MWlertz [28], Hemorrhagic activity. The hemorrhagic activity was tested by subcutaneous injection into the skin of the

tion 19 h). Residual proteolytic activity was determined with casein, and fibrinolytic activity on fibrin plates according to Astrup and Mfillertz [28]. H e a t treatment. The enzyme (0.2 m g / m l in 0.I M ammonium acetate, pH 6.7) was int.ubated for 1O rain at 25, 37, 50, 60, 70, 80 and 95 °C, and then quickly

b a c k s o f w h i t e m i c e . T h e s a m p l e s c o n t a i n e d 10 to 100 /.Lg o f l e b e t a s e in 100 /¢! of 0.9% salin©. A f t e r 6 h t h e

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Fig. 2, DEAE-¢ellulose cbromazo~aphy of the proteLo from the Sth gel filtration fraction (33 m~) on a Whatr~Ln DE.52 ~[lulosc colunm (1.5 × 12..E. ¢m) eqoilibrated with 0.08 M ammonium acetate (pH 8.3). Linear gradient of 0.Og-0.15 M ammordum acetate (pH 8.3). Flow rate 16 ml/h, fractions of 4 all ,,vere collected. Absorbance at 280 am ( ); prot©ina,~ U / m [ ( o - - ' - 0 ) ; fibrinoly~c ~O,vity 011 e). F~ctions 28-46 vzere pooled.

226 mice were killed and the skin was removed. The minimal hemorrhagic dose (MHD) was defined as the least amount of protein that caused a hemorrhagic reaction 5 mm in diameter [291.

"t'q" P

Results

Isolation The purification of lebetase was achieved by a twostep procedure. Gel filtration of crude venom on the Sephadex G-100 superfine gave seven fractions (Fig. 1). The fibdnolytic activity was localized in the 5th fraction, which was further fraet~onated by ion-exchange chromatography on a DEAE-celhlose coinnm (Fig. 2), This step resulted in three protein fractions, the second of them being lebetase. It hydrolyzed casein with about 1,7-times higher specific activity than the crude venom (1400 O / m B at pH 7.6). Fibdnolytic activity was about 20-times as high as that of the crude venom (1 ~g of lebetase or 20 I~g of venom lysed a 441 mm z zone on fibrin plate), From 4,1 g of retrain, 160 mg of lebetase was obtained. Fractions containing lebetase did not

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Fig. 4. Reduced SDS electrophoretlcpattern el" fibrinogenand fibrin deBtadation products by lebetase(lOCggel). Lanes 1-6: fibfinagen+ leharase(10 #g/ml) incubated for O, 5, 15, 30, 60 and 120 rain. Lanes 8-11: fibria+lebetas~ (10/~g/ml) incubatedfarO, l, 3 and 6 h~Lann 7: molecularweightstandards.

dissolve after lyophilization, thus all of the preparations had to be concentrated by nltrafiltration. Molecular weight and isoelectric point The enzyme was homogeneous by SDS-etectrophoresis. Analysis under reducing conditions [24] gave a calculated molecular weight value of 23 700 (Fig. 3A). The value obtained from the amino acid composition was 23871, Isoelectrofocusing studies revealed some heterogeneity of the enzyme, It gave several bands in the pH range from 4.6 to 5.4 (the most intensive band at pH 4.9) (Fig. 313). All of them had fibrinolytic activity as shown by hydrolysis of fibrin directly on the polyaerylamide gel.

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acrylarmde get dectrophoresis,t, phospttorylaseb; 2, bovine serum albumin; L ovalbumin;4, carbonic anhydrase; 5, soybean trypsin inhibitor; 6, a-heta|bamin, (B) Isaelectricfocusingof lebetase in the pH rangeof 4-7 (5~ polyaerylamidegel).

Chemical composition The amino acid composition was calculated on the basis of the molecular weight of 23700 obtained by SDS-dectrophoresis. It is shown in Table I in comparison with other fibrinolyti¢ enzymes. The enzyme contains 214 amino add residues. The values of the isoelectrie points of the lebetase isoforms are slightly acidic (pH 4.6 to 5,4). The extinction of the 1% solution of the purified enzyme at 280 nm is 13.0. The neutral sugar content is 2.4% for the purified anticoagulant. Heat treatment Lebetase retains its full activity until heating at 60 ° C for 10 rain (pH 6,7), At 70°C the activity is completely destroyed.

Effect on blood coagulation components Both crude venom and lebelase contain fibrincsenolytie and fibfinolytic activities. Cleavage of fibfinogen is shown in Fig, 4. From this figure one can conclude that

227 TABLE I

Properties of ~nake venom fibrlnoIjmc erl.~yme~ Amino acid cotnpositJoiIa

~ lcbetine

C. cert~tex

C atrox

A. rh~to$~:mu

A. a c u t ~

(lebetase)

(cer.~ta~)

(atroxa.~)

[71

[l.2i

ll2]

ISl

Asp Thr Ser

Tq~

39 1~. l0 21 6 14 18 14 8 3 11 20 8 a 3 9 l0 4

13 g 13 20 5 l0 14 9 lB 4 10 17 7 3 3 7 10 2

27 g 23 23 6 9 7 12 12 4 1! 17 6 8 8 8 It 3

32 11 13 23 7 ]4 g 17 8 12 L2 [3 7 12 23 8 5 0

25 II 23 21 8 12 12 10 8 6 I5 12 10 5 1l 7 1 5

Total

214

193

206

226

208

22500 5.2 6.14~

23500 9,6

25360 > 10 < lff~

24 tO0 3.g < l.~.

Glu Pro

Gly Ala VaI Cys/2

Met lie Leu T~r

Phe Lys

His Arg

M olecular weight (SDS) pl

Carbohydratecontent

23700 4,6-5.4 2.4%

a Mean values of two measurements,

iebetase is an Aa,B,8-fibrinogenase with the An-chain being degraded faster, BjS-chain is degraded more slowly and the y-chain is left intact even after 22 h hydrolysis. Hydrolysis of fibri~ b y lebetas¢ results in the degradation of the same a- and ~-chains, whereas the y-?-

chains appeared unaffected (Fig. 4). The fibrinolytie enzyme from Vipera lebetina venom appears to act directly on fibrin and does not activate plasminogen. Incubation of human plasminogen with the venom enzyme for up to 18 h gave no evidence of plasmin formation, as demonstrated by the lack of generation of S-2251-hydrolyz.ing activity. At the same time, activation of plasminogen by urokinase liberated plasmin. No hemorrhage was observed at low doses (10 pg), but at 50 pg per mouse hemorrhage occurred.

Inhibition sludies

The fibrinolyfic and caseinolytic activities o f lebetase are tot~Jly i n h i b i t e d b y E D T A but not b y P M S F , N o inhibition occurs w i t h E A C A a n d o n l y slight inhibition ( - 1 5 ~ ) c a n b a o b s e r v e d w i t h L-cysteine. Thus suggesting that the f i b r i n o l y t i c ~ x z y m e is a metalloproteinase.

Hydrolysis o/oxidized insulin B.chain The disappearance of the oxidized insulin B-chain peak and the generation of fragments were observed in the HPLC experiments after incubation with lebctase for periods tanging from 30 rain to 6 h. The sites of cleavage were determined from the amino acid composition of the degradation products observed after 30 rain of digestion (Fig. S). The enzyme cleaves oxidized insulin B-chain at the positions Alal4-Leu 15 and Tyr16Leu17. The other peptide bonds sccm to be rather resistant to the enzyme attack.

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Fig. 5. HPLC pzofde of oxidized insulin B-chain digestion by ]ebetase

(3o rain d i ~ t ) .

228 Discussion V. lebelma venom contains several proteolytic enzymes which affect blood coagulation. Some of them have procoagulant activity (Factor X activator) [30], the others promote anticoagulalion by hydrolysis of the fibrinogen An- and B/~-chains. In our previous report, the purification and characterization of a flfibrinogenase from the venom of E lebetma has been described [311. Here we div'uss the purification and properties of lebetase, a fibrinolytic enzyme from F. lebetina venom. Fibrinolytic activity is localized in the 5th gel filtration fraction. By the following ion-exchange chromatography, the enzyme is isolated in homogeneous form, as verified by SDS-dectrophorcsis, The heterogeneity of the protein in the isoelectric focusing is most likely due to the existence of enzyme isoforms, as evidenced by their fibrinolytic activity. The purified enzyme is about 20-times more active than the crude venom. It has a molecular weight of 23700 and consists of 214 amino acid residues. In this respect lebetase closely resembies cerastase, another fibdnolytic enzyme purified and characterized from Viperidae venoms [11,12]. The amino acid composition of these two enzymes is ve.ry similar in all amino acids e~cept cysteine (Table I). Both contain 28% Asx + Glx and 10.3% Lys + His + Arg. The pI values arc also rather close (5.2 for cerastase, 4.6-5.4. for lebetase). The proteolytie specificity of snake venom fibrinolyric enzymes is also quite similar. All of them hydrolyre casein, fibrinogen and fibrin and do not interact with low molecular substrates. In fibrinogen they prefer An-chains, followed by B,8-chains. The same specificity is also exhibited in fibrin hydrolysis, Lebetase is a rather active enzyme in hydrolyzing fibrin. By the semiquantitative results ( m m : / # g on fibrin plates) it seems to be much more active than the fibrinolytic enzymes from C cerastes, C atrox, A. rhodoswma and A. acutus venoms (441 mm2/#g for lebctase, 196 m m : / l . 2 5 #g for atroxase 18], approx. 320 mmZ/l,25 #g for eerastase (Ref. 12, and calculated from Fig, 5), 352 m m : / 3 #g for the enzyme from A. rhodostoma venom [7], and approx. 140 mm2/2.5 #g for the enzyme from A. acutus venom (Ref. 1, and Fig. 2). In oxidized insulin B-chain lebetase cleaves the sites Ala~4-Leu15 and Tyrlr-Leu ~7. Atroxase, the only fibrinolytic enzyme whose specificity is studied on insulin, exhibits broader specificity, hydrolyzing the four bonds of Leu, and, :n addition, the Ser~-His ~° bond 18]. EDTA inhibits both proteolytic and fibfinolytic activities. EACA - the inhibitor of plasminogen activation does not alter the proteolytie activity of lebetase. This indicates that the fibrinolytic activity is not conheeled with the activation of plasminogcn. The absence of plasminogen activation is also proved by direct de-

tion of lebetase on plasminogen. Leb¢tase is relatively heat labile, it endures heating only to 60 ° C. Fibrinolytic enzymes may be of considerable importance due to their possible therapeutic value [0,32]. The use of lebetase in this field may be restricted in connection with its hemorrhagic effect at higher doses. Hemorrhage is also characteristic of some other fihrinases. Previous studies suggest that cerastase, which shows hemorrhagic effects only at high doses, may be useful as a thrombolytic agent [I1], The fibrinolydc enzyme from A. aeutus venom is hemorrhagic already at low doses ( < 5 #g) [21. In fact, fibrinolytic and hemorrhagic protcinases from crotalid and viperid venoms are quite similar with regard to physicochemical and chemical properties and as well as in their speeificities toward the oxidized insulin B-chain. Interestingly, a-fibrinogenolyric activity is associated with venom hemorrhagirm [8]. It will be of interest to find out the determining factor among these proteinases which results in hemorrhage

IS]. Acknowledgements

The authors are thankful to Dr. Tilt Hailing for experiments with mice and to Dr. Tiiu-Mai Laht for her help by the amino acid analyses. References

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