Biochimica et Biophysica Acta, 1074(1991)136-143 © 1991ElsevierSciencePublishersB.V.0304-4165/91/$03.50 ADONIS 030441659100166N

136

Purification and characterization of an antiplatelet peptide, arietin, from Bitis arietans venom T u r - F u H u a n g 1, W e n - J e n g W a n g , C h e - M i n g Teng, C h e n - S h e n g Liu 2 a n d C h a o h o Ouyang t Pharmacological Institute, college of Medicine, National Taiwan University, Taipei (Taiwan) and 2 Institute of Biological Chemistry, Academia Sinica. Taipei (Taiwan)

(Received12 December1990) Key words: Snakevenompeptide,Arg-Gly-Asp-containing;Antiplateletaggregation;Fibrinogenbinding By means of Fractogel TSK-50, CM-Sephadex C-50 column chromatography, gel filtrations on Sephadex G-75 and Sephacryl S-200 columns and reverse.phase HPLC, an antiplatelet peptide, arietin, was purified from venom of Bitis arietans. Arietin was shown to be an Arg-Gly-Asp-containing peptide with a NH2-terminus, Ser-Pro-Pro-Val-Cys-GlyAsn-Lys- ( M r 8500). Arietin dose-dependently inhibited aggregation of human platelet suspension stimulated by ADP, thrombin, collagen and U46619 with ICso, 1.3-2.7 • 10 -7 M, while it had no effect on the initial shape changes and only slightly affected ATP release of platelets caused by thrombin and collagen. Arietin also Mocked platelet aggregation in platelet-rich plasma and whole blood, and inhibited thrombin-induced clot retraction of platelet-rich plasma. Furthermore, arietin (6.5-10 -s M) completely blocked the fibrinogen-lnduced aggregation of elastase-treated platelets, indicating that arietin interferes with the fibrinogen binding to fibrinogen receptors on platelet membranes, in conclusion, arietin, an Arg.Gly-Asp-eontaining peptide, inhibits platelet aggregation Wohably through the blockade of fibrinogen binding to the activated platelets. Introduction Snake venoms not only affect blood coagulation in a complicated manner but also profoundly affect platelet function [1-3]. Some components induce aggregation and release reaction [4-10], however, some inhibit these reactions [11-13]. We have previously reported that there are basically three kinds of antiplatelet components existing in hemorrhagic snake venoms [14-22], namely, ADPase or 5'-nucleotidases [12,16,24], afibrinogenases [25] and trigramin-like peptides, the specific fibrinogen receptor antagonist [22,23]. It is well established that the binding of fibrinogen with its receptors associated with glycoprotein Ilb-llIa complex results in platelet aggregation [26]. In normal circulation, fibrinogen molecules do not bind to the platelets unless platelets are. activated by some physicochemical factors, such as ADP, thrombin, epinephrine, prostagiandin endoperoxides, exposed collagen or Abbreviation:RGD, Arg-Gly-Asp. Correspondence:T.-F. Huang, PharmacologicalInstitute,Collegeof Medicine,NationalTaiwan University,Taipei,Taiwan.

endothelial injury [27-34]. The exposure of fibrinogen receptor is the common pathway of platelet aggregation caused by these factors. Therefore, a new strategy against arterial thromboembolism is being proposed, i.e., the development of drugs which are fibrinogen receptor antagonists, including the synthetic Arg-GlyAsp (RGD)-containing peptides [35-39], monocional antibodies against glycoprotein llb-llla complex [40-48] and Arg-Gly-Asp-containing peptides from natural sources [22,23,49,61]. In this paper, we purified an antiplatelet peptide, arietin, from B i a s arietans snake venom and characterized this peptide, and found that it was an RGD-containing trigramin-like peptide, acting as fibrinogen receptor antagonist. Materials and Methods Materials Bitis arietans

venom was purchased from Sigma Chemicals, U.S.A. and stored at - 2 0 ° C. CM-Sephadex C-50, Sephadex G-25, -75 and Sephacryl S-200, were obtained from Pharmacia, Sweden. Fractogei TSK HW50 and trifluoroacetic acid (TFA) were purchased from Merck, F.R.G. Human thrombin, adenosine monophos-

137 phate (AMP), adenosin diphosphate (ADP), acid molybdate, Fiske and Subbarrow reducing agent, collagen (bovine tendon, type l), prostaglandin E~ (PGE~), apyrase, bovine serum albumin, elastase (Type VI), luciferase-luciferin, ovalbumin, /3-1actoglobumin, lysozyme, acrylamide, heparin, tris(hydroxymethyl) aminoethane HCI (Tris-HCl), tosyl-L-arginine methyl ester, EDTA, ethylene glycol-bis-(aminoethyl ester) N, N'-tetraacetic acid (EGTA), sucrose, deoxycholate and L-aphosphatidylcholine (dipalmitoyl) were purchased from Sigma Chemicals, U.S.A. H u m a n fibrinogen (Kabi. Sweden), acetonitrile (L.C. grade, Alps Chemicals), and U46619 (Biomol Research Laboratories) were used.

Methods Fractionation of crude venom of Bitis arietans. 1.0 g of the crude venom dissolved in 0.1 M NaCI was applied to the column (3.6 × 66 cm) packed with Fractogel TSK HW-50, pre-equilibrated in 0.1 M NaCI. The eluates were monitored continuously at 278 nm at 4 ° C by LKB Uvicord. The flow rate was adjusted to 30 m l / h and the eluates collected by the Fraction collector, 4 ml per tube. The antiplatelet fraction was desalted by Sephadex G-25 column, lyophilized and further fractionated by CM-Sephadex C-50 column chromatography. The antiplatelet fraction was dissolved in small volume of 0.05 M a m m o n i u m acetate (pH 5.0) and applied to a column (1.2 × 70 cm) packed with CM-Sephadex C-50, preequilibrated with 0.05 M a m m o n i u m acetote. Gradient elution was carried out in three stages; (i) 0.05 M ammonium acetate (pH 5.0), 250 ml; (ii) 0.05 M ammonium acetate (pH 5.0), 150 ml vs. 0.6 M a m m o n i u m acetate (pH 8.5), 150 ml; and (iii) 0.6 M a m m o n i u m acetate (pH 8.5), 100 mi vs. 1.0 M a m m o n i u m acetate (pH 9.0), 100 mi. The flow rate was adjusted to 20 m l / h and ehiate collected every 3 ml per tube at 4 ° C , Gel filtration of crude arietin. The main antiplatelet fraction obtained was applied to a column (1.8 × 100 cm) packed with Sephacryl S-200 pre-equilibrated in 0.01 M a m m o n i u m bicarbonate and eluted with the same buffer at 4 " C . The flow rate was adjusted to 20 m l / h and eluate collected every 3 ml per tube. Finally, the collected fraction with antiplatelet activity was refractionated twice by Sephadex G-75 columns (2.8 × 100 era; 1.2 x 76 cm). The last step of the purification was conducted by t, sing reverse-phase HPLC (Cts column, 4.0 x 300 ram, Waters laBondapak). The HPLC system consists of a two pumps system (Waters, 501), Injector (U6K), detector (Waters, 490E) and monitored by a computer software (NEC, Baseline 810). The column was pre-equilibrated with 0.1% T F A and flow rate adjusted to 1 mi per rain. Crude arietin in 200 #! buffer A (400 #g) was injected into the column and eluted by a two-pump system during a 50 min period, consisting of buffer A and B. Buffer A, 100% distilled water with 0.1% T F A and buffer B, 80% acetonitrile with 0.1%

TFA. The elution gradient was performed according to the preset scheme shown in Fig. 3. The elution was monitored at 208 nm, and at room temperature and eluates were collected manually. The eluates were further dried with a Savant Speed Vac Centrifuge, then redissolved in distilled water and lyophilized. This lyophilized powder with antiplatelet activity was named as arietin. Protein assay. This was performed according to the method of Lowry et al. [50], using bovine serum albumin as standard. Electrophoresis. SDS-polyacrylamide electrophoresis in a system of Laemmli [51] was used to estimate the purity and molecular weight of the purified arietin. An 18% gel was used. After electrophoresis, the gel was prefixed by 10% glutaraldehyde for 30 min and stained with Coomassie brilliant blue for 1 h. then destained. The standard proteins used were lysozyme, fl-lactoglobulin, a-chymotrysinogen, ovalbumin, serum albumin, phosphorylase B and myosin (H-chain). Amino acid anal.vsis. This was performed by a Beckman Model 121 analyzer using a two column system. The hydrolysis was carried out in a l l 0 ° C oven under a nitrogen vaccum atmosphere for 22 h. Preparation of human platelets. Blood was obtained from healthy individuals who did not take drugs within 2 weeks. Blood collected in acid citrate dextrose (9 : 1, v / v ) or in sodium citrate (3.8%, 9 : 1, v / v ) was centrifuged at 100 × g at room temperature for 10 rain to obtain platelet rich plasma (PRP). H u m a n washed platelet suspension was prepared according to the methods of Mustard et al. [52] and Komecki et al. [29], and suspended in Tyrode's solution (pH 7.35) containing 3.5 m g / m l bovine serum albumin (Sigma, Fraction V). Elastase-treated platelets were prepared according to the method of Kornecki et al. [53,54]. The concentration of elastase in the incubation mixture was 1.25 U per l0 s platelets and the incubation was conducted in the presence of apyrase (0.5 U / m i ) at 3 7 ° C for 50 rain. After incubation, the platelet suspension was centrifuged at 170 × g for 10 rain and the pellets obtained were washed once, recentrifuged and resuspended. Platelet aggregation and A TP release reaction. Platelet aggregation and release reaction was performed at 37 o C by the turbidimetric method [55] using a Lumi-Aggregometer (Chrono-Log) in the case of platelet suspension and platelet-rich plasma. The platelet count was adjusted to 3 . 0 - 6 . 0 - l 0 g platelets per ml by a Coulter counter (Model ZM). Usually, platelet suspension or platelet-rich plasma in 0.4 ml was added to siliconcoated cuvette and incubated at 3 7 ° C with an appropriate amount of Tyrode's solution, arietin or other inhibitory reagents were then added 1 min prior to the addition of aggregation agonist. The extent of aggregation was expressed in light transmission unit, When measuring the ATP release reaction, 20 ILl of luciferase-

138 luciferin mixture was added 1 rain prior to the addition of agonist and the amount of ATP released was quantitatively measured by the peak of the fluorescence change compared to control. Whole blood aggregation was measured by the impedance method using whole blood aggregometer (Chrono-Log). Clot retraction. Patelet-rich plasmas (0.6 ml) were added to clean, uncoated glass tubes in the absence or presence of arietin, incubated at 37 ° C for 3 rain, before adding 0.2 ml human thrombin (final concentration, 4 U / m l ) , mixing well and incubating at 3 7 ° C for 2 h. Finally, the residual volume of serum was measured as an index of clot retraction. % retraction = serum volume (test)

~~

0

6 Forcotgel CTSKHW-50

040

020

OC0

50

100 150 Tubenumber

200

250

Fig. I. Fractogel TSK HW-50 column chromatographof B. arietans venom. The venom (1 g) was applied to a column (3.4 era×66 cm) with bed volumeof 400 ml. 0.l M NaCI was used as eluent. The flow rate was adjusted to 30 ml/h and 4 ml per tube was collected.

/ s e r u m volume (control) x 100.

Esterase activity. The method of Habermann I56] was followed. Phospholipase A activity. This was estimated by indirect hemolysis method following the method of Brown and Bowles [57] using phosphatidyl choline as the substrafe. Fibriogenolytic activities. The fibrinogenolytic activity was determined according to the method described by Ouyang and Huang [581. Fibrinolytic activity. This was determined using the method of Astrup and Mullertz [59]. Nucleotidase and ADPase activity. This was assayed according to the method of Dixon and Purdon [60]. 0.25 mM AMP or A D P was used as substrate, respectively. Automated NI1,-terminal sequencing. This was performed on a gas-phase sequencer (Apllied Biosystems, model 477A). The instruments are operated routinely Mr. Chen, S.W. at the Institute of Biological Chemistry, Academia Siniea, Taipei. Standard protocols of the manufacturer were followed with regard to both Edman degradation and separation of PTH-amino acids by HPLC. Cysteine was detected as S-(pyridylethyl) cysteine. Pyridylethylation of arietin. This was carried out by adding 1 p.l of vinylpyridine to the reduced protein (50 /tg in 99 /tl of 6 M guanidine hydrochloride, 4 m M EDTA, 0.1 M Tris-HCl (pH 8.5) and 4.5 m M dithiothreitol). The reaction mixture was incubated for 2 h at 22°C in the dark, under argon first for reduction, and then a further 2 h for pyridylethylation. Mofified protein was isolated free of reagents by reverse-phase HPLC in 0.1% trifluoroacetic acid with acetonitrile as organic modifier. Partial sequencing of a, ietin. S-Pyridylethyl arietin was incubated at 37°C in the presence of lysyl-endopeptidase (Wako, Japan) (final concentrations was given): Endoproteinase LYS-C, 3% (w/w) in 0.05 M Tris-HCl (pH 9.0) for 20 h. The digests were fractionated

by a nucleosil C~g column. The elution was carried out by a two buffer system, consisting of buffer A and B with the gradient, 0-60% acetonitrile in 60 rain.

Results Fractionation of crude venom of Bit& arietans By means of Fractogel TSK HW-50 column chromatography, the crude venom was separated mainly into six fractions (Fig. 1). A m o n g them, fraction I (tube number, 38-80) exhibited a potent inhibitory activity on collagen (10/~g/ml)-induced platelet aggregation of h u m a n washed platelet suspensions, while other fractions showed little effect. Therefore, this fraction was further fractionated on CM-Scphadex C-50 column and eight subfractions were obtained (Fig. 2). The antiplatelet activity distributed mainly at fraction 1-4 (tube number, 93-111). The desalted, lyophilized fraction I-4 was then further refractionated on Sephacryl S-200 col-

Q05N' DHSO

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Fig. 2. Chromatograph of fraction I (obtained from Fractogel TSK HW-50 column chro,, ,ograph) on CM-SephadexC-50 column (1.2 cmx70 era, bed volume 120 ml). Ammonium acetate was used as eluent in gradient as indicated.The flow rate was adjusted to 20 ml/h and 3 ml per tube was collected.

139 umn, we found that the subfraction I-4-2 (tube number, 26-38) was active in inhibiting platelet aggregation. Finally, this fraction was purified by Sephadex G-75 columns twice successively and a symmetric peak was obtained (not shown). In order to get a highly purified component, we purified crude arietin by the reversephase HPLC (Cts column). Essentially six subfractions were obtained, eluated at different time periods (Fig. 3). Among them, two fractions with ehition times of 27 and 29 min, were the active components with about equal potency in inhibiting platelet aggregation, whereas other fractions, eluted at 9, 36 and 38 rain. were inactive. Therefore, we named the fraction eluted at 27 rain as arietin as its yield was higher than the fraction eluted at 29 min. The yield of this purified arietin was about 1% (w/w).

Control

Arletln (~g/rnl)

Aop B

Physicochemical properties of arietin SDS-polyacrylamide gel electrophoresis showed that the /3-mercaptoethanol-treated arietin migrated as a single component and its molecular weight was estimated to be around 8500. However, the non-reduced arietin was not stained by Coomassie brilliant blue. The purified arietin was demonstrated to have a single NH2-terminus of Ser-Pro-Pro-Val-Cys-Gly-Asn-Lys-lleLeu-Glu-Gln-Gly-Glu-Asp. We also sequenced one of the eleven fragments obtained from the digests of the reduced S-pyridyethyl arietin with lysyl-endopeptidase, and found that this fragment had the following sequence, Ala-Leu-Thr-VaI-Cys-Arg-lle-AlaArg-Gly-Asp-Ser-Asn-Asp-Asp-. Although its complete sequence has not been established, arietin is surely an Arg.Gly-Asp (RGD)-containing peptide. From the amino acid analysis, arietin was shown to be a peptide, rich in aspartic acid, glutamic acid, glycine and halfcystine (data not shown). At 10 /tg/ml, arietin was devoid of the following enzymatic activities, including fibrino(geno)iytic, phospholipase A, AMPase, ADPase and esterase activities. 2 5O

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Fig. 3. Purificationof arietin usingreversephase HPLC. An aliquotof 200-400 .al of crude arietin (200-400/tg) was injected into a column (4 ram×300 ram) of .aBondapak Cts equlibrated in 0.1% trifluoroacetic acid at a flow rate of 1.0 ml/min. Fractionswere eluted during a 50 rain period with a gradient of 0-80% acetonitrile (-- -- --). The elution time of arietin was around 27 rain ( * ).

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Fig. 4. Effect of arietin on ADP (20 .aM, A), U46619 (S .aM, B), collagen (10 .ag/ml, C~indueed agglegation of human platelet-rich plasmas. Platdets (3.10S/ml) were preincubated with arietin (l-t0 .ag/ml), respectively, and stirred for l rain, then ADP, U46619, or collagen was added to trigger the aggregation.The aggregation was measured by turbidimetfic method(changesin transmission,AT).

Effect on platelet aggregation In human platelet-rich plasma, arietin dose-dependently inhibited platelet aggregation caused by ADP. At the concentration of 4 u g / m l , arietin completely abolished aggregation (Fig. 4A). On the other hand, arietin also dose-dependently inhibited platelet aggregation induced by prostaglandin endoperoxide analogue, U46619 (5/tM) (Fig. 4B) and collagen (10 p.g/ml) (Fig. 4C). However, arietin even at 8-10 lag/ml did not completely block aggregation induced by U46619 or collagen, The residual aggregation was around 10-20%. The initial shape change of platelets caused by all these agonists were not affected in the presence of arietin, comparing the amplitude of initial decrease of transmittance upon the addition of agonist (Fig. 4). The IC50 of arietin on platelet aggregation caused by collagen, U46619 and ADP was estimated to be 6.1 t~g/ml (0.8 /.tM), 4.1 p,g/mi (0.5 p,M) and 2.2 p,g/ml (0.3 p,M) (n = 4), respectively.

140

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(.ug/ml)

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0.125 O25 05 Ar~et~n (,uglml)

Fig. 5. Dose-response curve of arietin on the aggregation of human plalelet suspension induced by thrombin (0.1 U/ml; B), collagen (10 yg/ml; o). U46619 (l #M: A) and A D P (20 #M; v). Values are presented as means _+S.E. (n = 3-6).

In washed platelet suspension, arietin dose-dependently inhibited fibrinogen-induced aggregation of ADP-stimulated platelets (data not shown). Similarly, arietin also dose-dependently inhibited aggregation of platelets stimulated with thrombin (0.1 U/ml), collagen (10 /~g/ml) and U46619 (1 /tM) (Fig. 5). The ICs0 of arietin on platelet aggregation caused by thrombin, collagen, U46619 and ADP was estimated to be 1.2 /tg/ml (0.16 p,M), 2.2 /tg/ml (0.30 #M), 1.1 # g / m l (0.15 #M) and 1.4/zg/ml (0.19 #M), respectively (n = 4-6) (Fig. 5). However, arietin only showed a slight inhibition on ATP release reaction of platelets stimulated by thrombin (0.1 U / m l ) (30 4-127o inhibition, n = 4) and this effect was not dose-related for arietin (0.5-2 /tg/ml) exhibited similar inhibitory effect on release reaction (Fig. 6). Similar results were obtained in case of collagen- and U46619-induced release reaction

iI / Control

J ~ Arletln ADP Fig. 7. Effect of arietin on the aggregationof wholeblood induced by ADP. Human whole blood was incubated with saline (control) or arietin (0.125-0.5/zg/ml) and stirred for 1 min, then ADP (50 /zM) was added to trigger the aggregation.Typical tracingsof three similar experiments were shown. The aggregation was monitored by the impedance method(AI2). (data not shown). In the meantime, arietin also did not affect the initial platelet shape change caused by thrombin as reflected by the observation that the initial decrease of transmittance upon the addition of thrombin was not inhibited in the presence of arietin (Fig. 6). The platelet aggregation of whole blood is more physiological an in vitro model, therefore, we tested if arietin also exhibited antiplatelet activity in this preparation. As measured by impedance method, arietin (0.5 # g / m l ) completely blocked ADP (50/tM)-induced platelet aggregation of whole blood (Fig. 7). The ICs0 was around 0.2 /Lg/ml (0.026 #M), indicating that arietin apparently was not inactivated or highly bound to other blood cells or plasma proteins.

Ar~et~n

(~Jg/rnl)

T~rornb,n

Fig. 6. Typical patterns of antiplatelet effects of arietin on thrombin (0A U/ml)-inducedaggregation and ATP release of washed human platelets. Platelets (3.10S/ml) were pre-ineubated with arietin (final concentrations indicated) at 37°C for 1 rain, then thrombin (0.1 U/ml) was added to trigger the aggregation (/tT, upward trancings) and ATP release (downwardtrancings). 20 pd of luciferase-lueiferin mixture was added I rain prior to the addition of agonist, in order to monitorATP releasereaction.

7~

a

b

c

~

~

Arlentin ( J~l/ml) Fig. 8. Effect of arietin on thromhin-inducedclot retraction of human platelet-rich plasma. Plasma was mixed with 0.2 ml of arietin (l, 2. 4. 8 and 16 .ag/ml) and saline in a non-coated glass tube. 0.2 ml of thrombin(20 U/ml) was added to each tube, mixedgently and left to stand at 37°C for 2 h. The serumvolumeof each tube was measured and compared to that of a control. Pictures were taken 2 h after the addition of thrombin (a, control: b. in the presence of arietin l #g/mR e, 2#g/ml: d, 4/~g/ml; e. 8/~g/ml: and f, 16/~g/ml).

141

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"~r'ge/IIron. . . . . . 26 rain

025

F*brinogen Fig. 9. Effect of arietin on fibrinogen-induced

aggregation of

elastase-treated platelets. Supensionof elastase-treatedplatelets (1.25 U elastase/lOa platelets) were preincubatedwith arietin (0A25, 0.25, 0.5 ttg/ml; O), respectively, and stirred for 1 min. then fibrinogen (200 ttg/ml) was added to trigger the aggregation.

Effect on clot retraction Arietin dose-dependently inhibited clot retraction of platelet-rich plasma caused by thrombin (5 U / m l ) . The % inhibition of arietin at 2, 4, 8 and 16 / t g / m l was 10.5 4- 3.4, 12.6 4-1.9, 38.4 4- 8.2 and 67.0 _ 2.7% (n = 3), respectively (Fig. 8).

Effect on fibrinogen-induced aggregation of elasfasetreated platelets Proteinases such as pronase, a-chymotrypsin and elastase cause the exposure of fibrinogen receptors which are inaccessible to fibrinogen when platelets are intact [29,53,54]. Thus, the addition of fibrinogen caused aggregation of elastase-treated platelets (Fig. 9). This aggregation was not affected by PGE t (5 p.M), indicating that aggregation results from the direct interaction of fibrinogen with its receptors on platelet surface. Owing to the similar ICs0 of arietin on platelet aggregation induced by different agonists, a common site of action may be involved in this aggregation process. Arietin dose-dependently inhibited fibrinogen (200 p.g/ml)-induced aggregation of elastase-treated platelets (Fig. 9). At 0 . 5 / t g / m l , arietin completely inhibited aggregation in this preparation, indicating that arietin directly interferes with the interaction of fibrinogen with its receptors. Discussion

By means of gel filtration, CM-Sephadex C-50 colu m n chromatography and reverse-phase HPLC, a potent antiplatelet peptide, areitin, was purified from venom of Bitis arietans. It is an Arg-Giy-Asp-containing peptide, rich in glutamic acid, aspartic acid, glycine and haifcystine and its molecular weight was estimated to be 8500. The N H 2 terminal sequence of arietin was shown to be Ser-Pro-Pro-Val-Cys . . . . . . Currently, Shebuski et al. reported an RGD-containing antithrombotic peptide, bitistatin, purified from the same snake venom [61]. When comparing the partial sequence of arietin with the sequence of bitistatin, ariefin seems to be very similar to bitistatin as they share almost the same

sequence'except for a few residues. Therefore, arietin may be the isoform of Bitistatin reported by Shebuski et al. [61]. The purified arietin was devoid of phospholipase A, fibrino(geno)lytic, esterase or ADPase activities, Arietin was shown to be very active in inhibiting aggregation not only in washed platelet suspension but also in platelet-fich plasma and whole blood. This fact indicates that afietin would not be easily inactivated or highly bound to blood cells and plasma proteins other than platelets. Ari,etin inhibited thrombin-induced clot retraction of platelet rich plasma, indicating that the interaction of fibrinogen and its receptors associated with glycoprotein l l b / l l l a complex is essential for clot retraction [62.631. The IC5o of arietin on platelet aggregation induced by agonists tested in the preparations of platelet-rich plasma, washed platelet suspension and whole blood was 2 - 6 txg/mi (2.7-8.0-10 -7 M), 1 - 2 / t g / m l (1.3-2.7 • 10 _7 M) and 0.2 / t g / m l (2.7-10 -~ M), respectively. The ICso of arietin (300 nM) in inhibiting ADP-induced aggregation of h u m a n platelet-rich plasma is about the same as compared to 237 nM reported by Shebuski et at. [61]. That arietin exhibited a weaker activity in platelet-rich plasma may be chiefly due to the presence of a large amount of fibrinogen in plasma. Arietin completely blocked aggregation induced by ADP, whereas a residual aggregation (around 10-15%) was resistant to arietin in case of collagen-, thrombin-, and U46619-induced aggregation. We think that collagen, thrombin and U46619 are more potent inducers causing platelet release reaction including some adhesive proteins (such as fibronectin and thrombospondin) on which arietin has little effect• Therefore, arietin as well as Arg-Gly-Asp-Ser is a partial antagonist in the case of platelet aggregation induced by those agonists other than A D P [64]. Arietin apparently had no effect on the initial shape change of platelet caused by collagen, thrombin and U46619. Meanwhile, arietin only slightly decreased ATP release reaction (less than 30%) caused by these agonists and this action was not dose-dependent (Fig. 6). This result may be explained by the fact that A D P release reaction may reinforce the aggregation through a positive feedback mechanism especially when platelets are stimulated by low concentration of agonists [65]. Arietin directly inhibits aggregation, resulting in 'indirect" inhibition on the release reaction. The exposure of fibrinogen receptor is believed to be the common step of platelet aggregation induced by several agonists [27-34]. In view of the results showing that arietin inhibits platelet aggregation with a similar IC5o (especially in platelet suspension) regardless of agonists used and it does not affect the shape change and has only slight effect on release reaction, more direct evidence shows that arietin completely inhibited

142 f i b r i n o g e n - i n d u c e d a g g r e g a t i o n o f elast-ase-treated platelets (Fig. 9). Therefore, we c o n c l u d e t h a t arietin, a n A r g - G l y - A s p - c o n t a i n i n g peptide, a c t i n g as t r i g r a m i n like peptide [22.23,611, inhibits platelet a g g r e g a t i o n b y interferrino with the interaction between f i b r i n o g e n a n d its receptors on platelet surface. Its detailed m e c h a n i s m and binding properties toward fibrinogen receptors w o u l d be elucidated in the following p a p e r . Acknowledgements This p r o g r a m w a s financially s u p p o r t e d b y N a t i o n a l Science council o f the R e p u b u l i c of C h i n a ( N S C 7 7 0412-B002-129). W e also express o u r sincere t h a n k s to Mr. I.S. Peng for his excellent t y p i n g o f this m a n u s c r i p t . References 1 Seegers. W.H. and Ouyang, C. (1979) in Snake Venoms (Lee, C.Y., ed.), Handbook of Experimental Pharmacology, Vol. 52, pp. 684750. Springer-Verlag, Berlin. 20uyang, C., Teng, C.M. and Huang, T.F. (1982) J. Formosan Med. Assoc. 81,781-790. 30uyang, C.. Teng, C.M. and Huang. T.F. (1987) Asia Pac. J. Pharmacol. 2, 169-179. 4 Davey, M.C. and Luseher, E.R. (1967) Nature 216, 857-858. 5 Kirby. E.P.. Niewiarowski. S. Stocker, K., Kettner. C.. Shaw, E. and Brudzynski. T.M. (1979) Biochemistry 18, 3564-3570. 6 Schmaier, A H., Claypool, W. and Colman, R.W. (1980) Blood 56, 1013-1019. 7 Ouyang, C., Wang. J.P, and Teng, C.M. (1980) Biochim. Biophys. Acta 630, 246-253. 8 Vargaftig, B.B.. Prado-Francesehi, J.. Chignard. M., Lefort. J. and Marlas, G. (1980) Eur. J. Pharmacol. 68, 451-464. 9 Ouyang, C. and Huan8, T.F. (1983) Biochim. Biophys. Acta 761, 126-134. l0 Teng, C.M,. Hung, M.L., Huang, T.F. and Ouyang. C. (1989) Biochim. Biophys. Aeta 992, 258-264. II Marney, S.R. (1977) J. Immunol. 106, 82-90. 12 Boffa, M.C. and Boffa, G.A. (1974) Biochim. Biophys. Acta 354, 275-290. 13 Biran, H.. Dvilansky, A., Nathan, I. and Live, A. (1974) Thromb. Diathes. Haemorrh. 30,191-198. 14 Ouyang, C. and Huang, T.F, (1983) Biochim. Biophys. Aeta 757. 332-341. 15 Huang, T.F. and Ouyang. C. (1984) Biochim. Biophys. Acta 33, 125-138. 16 Ouyang, C. and Huang, T.F. (1983) Toxicon 21,491-501. 17 Ouyang, C., Yeh, H.I. and Huang, T.F. (1983) Toxieon 21,797804. 18 Huang. T.F., Yeh. HT and Ouyang, C. (1984) Toxicon 22, 243251. 19 Ouyang, C., Ma, Y.H., Jih, H.C. and Teng, C.M. (1985). Biochim. Biophys. Acta 841, 1-7. 20 Teng, C.M., Ma. Y.H. and Ouyang, C. (1985) Biochim. Biophys. Acta 841, 8-14. 21 Huang, T.F., Wu, Y.J. and Ouyang, C. (1987) Biochim. Biophys. Acta 925, 248-259. 22 Huang, T.F., Holt. J,C., Lukasiewicz. H. and Niewiarowski, S. (1987) J. Biol. Chem. 262, 16157-16163. 23 Gan. Z.R.. Gould, R,J., Jaeobs, J.W.. Friedman, P.A. and Polokoff, M.A. (1988) J. Biol. Chem. 263, 19827-19832.

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Purification and characterization of an antiplatelet peptide, arietin, from Bitis arietans venom.

By means of Fractogel TSK-50, CM-Sephadex C-50 column chromatography, gel filtrations on Sephadex G-75 and Sephacryl S-200 columns and reverse-phase H...
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