A potent platelet aggregation inducer from Trimeresurus gramineus snake venom

126

Biochimica etBiophysicaActa. 761 (1983) 126-134

Elsevier BBA21612

A P O T E N T PLA T E L E T A G G R E G A T I O N I N D U C E R F R O M T R I M E R E S U R U S G R A M I N E U S SNAKE VENOM CHAOHO OUYANG and TUR-FU HUANG Pharmacological Institute, College of Medicine, National Taiwan University, TaipeL Taiwan (China)

(Received May 10th, 1983) (Revised manuscript received August 29th, 1983)

Key words: Platelet aggregation inducer," Snake venom," Serotonin release,"(Trimeresurus gamineus)

A potent platelet aggregation inducer (platelet aggregoserpentin) was purified from Trimeresurus gramineus snake venom by DEAE-Sephadex A-50 and Seplmcryl S-300 column chromatography. It was homogeneous as judged by sodium dodecyi sulfate-polyacrylamide gel electrophoresis. It elicited dose-dependently platelet aggregation and serotonin release reaction in rabbit platelet-rich plasma and platelet suspension. Exogenous calcium was required for its activity. Creatine phosphate/creatine phosphokinase and apyrase showed no significant inhibitory effect on aggregoserpentin-induced platelet aggregation in platelet suspension. Aggregoserpentin induced aggregation in ADP-refractory platelet-rich plasma. It caused no detectable malonic dialdehyde formation in the process of platelet aggregation. Indometlmcin did not inhibit aggregoserpentin-induced platelet aggregation. Mepacrine abolished preferentially its aggregating activity, while prostoglandin E 1 completely blocked both aggregoserpentin-induced aggregation and release reaction. Furthermore, platelet aggregoserpentin lowered basal and prostnglandin El-stimulated cAMP levels in platelet suspension. Nitroprusside inhibited both its aggregating and releasing activity, while verapamil preferentially blocked its aggregating activity. It is concluded that aggregoserpentin activated platelets through lowering cAMP levels or the activation of endogenous phospholipase A 2, resulting in the formation of platelet activating factor, but not of prostnglandins. Introduction Snake venoms affect platelet aggregation in various aspects. Some induce platelet aggregation and release reaction [1,2], while some inhibit these reactions [3-5]. Recently, several groups have purified and characterized the platelet aggregating principles from different snake venoms [2,6-11]. These platelet activating priciples can be classified into two groups: Firstly, serine proteases with amidolytic activity such as thrombocytin [6,7],

crotalocytin [10,11], secondarily, non-enzymatic ones such as the platelet aggregoserpentin from Trimeresurus mucrosquamatus [9] and a proteinpolysaccharide complex from Trimeresurus okinavensis [2]. However, the action mechanisms of these platelet activating principles are still obscure. Recently, we purified a potent platelet aggregation inducer from Trirneresurus gramineus, characterized its properties and investigated its action mechanism.

Materials and Methods Abbreviations: EGTA, ethyleneglycol bis(fl-aminoethylether)N,N'-tetraacetic acid; PMSF, phenylmethylsulfonylfluoride. 0304-4165/83/$03.00 © 1983 Elsevier Science Publishers B.V.

Materials. T. gramineus venom was collected at our laboratory, centrifuged, lyophilized and stored in a desiccator below - 2 0 ° C . Bovine thrombin

127 was purchased from Parke, Davis and Co., U.S.A. Adenine nucleotides (AMP, ADP, ATP), prostaglandin El, adenosine, indomethacin, mepacrine, theophylline, sodium arachidonate, apyrase, bovine serum albumin, ovalbumin, pepsin, trypsinogen, fl-lactoglobulin, lysozyme (egg-white), creatine phosphate (sodium salt), creatine phosphokinase (Type I), EDTA (disodium salt), EGTA, pbromophenacyl bromide and 2-thiobarbituric acid were purchased from Sigma, U.S.A. Sephadex G25, Sephacryl S-300 and DEAE-Sephadex were purchased from Pharmacia, Sweden. Other reagents: S-2238 (H-o-Phe-Pip-Arg-p-nitroaniline. 2HC1, from Ortho-Diagnostics, U.S.A.

DEAE-Sephadex A-50 column chromatography. This procedure was carried out by the method of Ouyang and Huang, [12]. Gel filtration. Sephacryl S-300 was prepared in 0.01 N ammonium bicarbonate (pH 7.8) and packed in columns of various sizes. The flow rate was adjusted to 18 m l / h and eluates of 3 ml per tube were collected. Desalting was performed with Sephadex G-25, eluted with distilled water. SDS-polyacrylamide gel electrophoresis. The method of Weber and Osborn [13] was followed. A 7.5% polyacrylamide gel was used. The freshly prepared aqueous sample solution (1 mg/ml) was mixed with equal volume of 0.02 M phosphate buffer (pH 7.4) containing 10 M urea, 4% SDS in the presence or absence of 4% fl-mercaptoethanol and incubated at 37°C for 1 h. Then the treated samples (50/~g) were applied to each gel column. The electrophoresis was performed just the same as the method of Weber and Osborn [13] except bovine serum albumin, ovalbumin, pepsin, trypsinogen, fl-lactoglobulin and lysozyme were used as standard proteins. Amino acid analysis. This was done on a Beckman 121 M Analyzer using a two column system. The hydrolysis was carried out in a 110°C oven under a nitrogen vaccum atmosphere for 22 h. Estimation of carbohydrate. This was determined by the method of Dubois et al. [14] using dextrose as standard. Amidolytic activity. The reaction mixture consisted of appropriate amount of Tris-saline buffer (0.05 mM Tris/0.10 M NaC1, pH 7.4), test solutions (25 /~l at various concentrations) and the substrate S-2238 (1 mM) in a total volume 1.0 ml.

The mixture was incubated at 25°C and the liberated p-nitroaniline was read at different time intervals over a 10 min period. Test solutions included platelet aggregoserpentin or phenylmethylsulfonyl fluoride (PMSF)-treated aggregoserpentin which was obtained by incubating with PMSF (0.1 mM) at 37°C for 6 h, dialysed against Tris-saline, pH 7.4 for 20 h. Preparation of platelet-rich plasma. Blood was collected from rabbit marginal ear vein through a 19-gauge needle and was mixed with 3.8% sodium citrate (14: 1, v/v). Citrated blood was immediately centrifuged for 10 min at 150 × g and room temperature. After removal of the plateletrich plasma, the remaining blood was then recentrifuged at 1500 × g for additional 20 min, and the platelet-poor plasma was obtained and mixed with platelet-rich plasma to give a platelet count about 300000/mm 3. Preparation of platelet suspension. A modified method of Mustard et al. [15,16] was followed. The composition of Tyrode's solution was prepared in the following (mM): 136.8 NaC1, 2.8 KCL, 11.9 NaHCO 3, 1.1 MgC12, 0.33 NaH2PO4, 11.2 glucose. The final concentration of Ca 2+ in the Tyrode's solution was 1.0 mM. The washed platelet suspension was finally suspended in Tyrode's solution containing bovine serum albumin (3.5 mg/ml) and apyrase (0.1 mg/ml). The platelet count was adjusted to about 1 • 108 platelets/ml.

Preparation of aggregoserpentin-treated platelet suspension. Prostaglandin E 1 (10 -5 M)was added to the platelet aggregates 1 min after the addition of aggregoserpentin (0.5/~g/ml), and the reaction mixture was incubated at 37°C for 30 min. After the platelet aggregation disaggregated, it was centrifuged at 500 x g for 15 rain. The platelet pellets were washed and resuspended in a Ca 2+-free Tyrode's solution containing apyrase (0.1 mg/ml) and bovine serum albumin (3.5 mg/ml). This washing procedure was repeated once and finally the pellets were suspended in the same Tyrode's solution, containing 1 mM of Ca 2+.

Preparation of formalin-fixed p latelet suspension. It was prepared according to the method described by Grant et al. [17]. Platelet aggregation. This was measured by the turbidimetric method [18,19], using a dual channel-Payton aggregometer (Ontario, Canada). Gen-

128

erally, 0.40 ml of platelet-rich plasma or platelet suspension was pre-incubated with aggregation inhibitor or Tyrode's solution (0.05 ml) at 37°C for 2 min before the addition of platelet aggregoserpentin (0.05 ml) or aggregation inducer. Platelet release reaction. EDTA-platelet-rich plasma was labeled with 5-hydroxy[side-chain 2~4C]tryptamine creatinine sulfate (0.6 /~Ci/108 platelets), following the method described by Ardlie et al. [20]. After incubation at room temperature for 30 min, the platelet-rich plasma was washed twice with Tyrode's solution containing albumin (3.5 m g / m l ) and apyrase (0.1 mg/ml). The release reaction was terminated by adding 0.5% glutaraldehyde at 5 min after the addition of the inducer. Then the suspensions were centrifuged immediately at 5000 x g for 5 rain, An aliquot of the supernatant (100/~1) was added to counting vial containing 10 ml of Aquasol-2 solution, then the radioactivity was counted by a Beckman scintillation counter. % release was expressed as (A - B ) / C - B) x 100 where A = radioactivity of a test sample. B = radioactivity of a control sample. C = total radioactivity of a control sample (i.e. 0.5% Triton X-100-treated suspension).

Cyclic adenosine monophosphate determination. The cAMP levels were determined using a radioimmunoassay kit (Amersham). Determinations of basal and prostaglandin El-stimulated cAMP were conducted at the same time. 0.8 ml of platelet suspension was incubated with platelet aggregation inducer at 37°C for 1.5 min in the presence or absence of the prostaglandin E l (6/~M). 0.2 ml of ice-cold 30% trichloroacetic acid was added to stop the reaction. After centrifugation (3000 x g, 10 min), the supernatant was extracted four times successively with ether to remove trichloroacetic acid, and the residual ether was evaporated off in water bath at 37°C. The samples were frozen and lyophilized overnight. A certain amount of Tris buffer (pH 7.4) containing EDTA (0.2 mM) was added to dissolve the sample, and the levels of cAMP were assayed according to the procedure recommended by the manufacturer. Malonic dialdehyde assay. This was measured according to the method of Smith et al. [21]. Lactate dehydrogenase assay. This was carried out according to the method of Wroblewski and LaDue [22].

Results

Purification of platelet aggregation inducer from Trimeresurus gramineus oenom. By means of DEAE-Sephadex A-50 column chromatography, T. gramineus venom was separated into twelve fractions. The platelet aggregating activity was detected chiefly in Fraction XI (Fig. 1). Upon refractionation of Fraction XI on Sephacryl S-300 column, the platelet aggregating fraction was eluted before the thrmobin-like enzyme which coagulated fibrinogen solution (0.1%). The refractionation was repeated twice on a Sephacryl S-300 column (50 ml) until a single symmetric peak was obtained. The yield of this platelet aggregating component was about 0.5% of the crude venom on weight basis. Since the potency ratio between platelet aggregoserpentin and crude venom was approx. 10-15 (Fig. 2), the platelet aggregoserpentin yield on an activity basis would be 5-7.5%.

Homogeneity and physicochemical properties of the purified platelet aggregoserpentin. The purified platelet aggregoserpentin showed one band with a molecular weight of about 43 300 in SDS-polyacrylamide gel electrophoresis. It showed the same mobility in the presence or absence of 2% fl-

Ok41

- o.m

X

Y 330

350 Tu~e ~umber

4~

370

390

Fig. 1. Fractionation of T. gramineus. This crude venom was fractionated into twelve fractions. Fractions I - I X showed no aggregating activity. Platelet aggregating component was mainly distributed in Fraction XI. Platelet aggregating activity (O O) was expressed as relative % of maximum aggregation (absolute value: 70% aggregation) induced by 10 ~tl of the eluates.

129 &

IX

--

80

TABLE I

80

0.1

1

10

i

I

100

1000

Concentretion ( / ~ g / m l / Fig. 2. Dose-response curves of aggregoserpentin and crude venom of T. gramineus on platelet aggregation and platelet []4C]serotonin release reactions in platelet suspension. % Aggregation was expressed as % of m a x i m u m aggregation (absolute value: 70% aggregation) induced by optimal concentration. % [14C]serotonin release was expressed as % of m a x i m u m release in the presence of Triton X-100 (0.5%). © ©, • • represent the % aggregation and % release of crude venom, while zx zx, • • represent % aggregation and % release of the aggregoserpentin, respectively.

mercaptoethanol, indicating that it was a single chain polypeptide. It was estimated to contain 22% of carbohydrate. Amino acid analysis showed that aggregoserpentin contained 307 amino acid residues per molecule (Table I). It contained high amount of aspartic acid, leucine, serine, glycine and glutamic acid. It did not possess 5'-nucleotidase, fibrinogenolytic or phospholipase A 2 activities even at a concention of 50/~g/ml. However, aggregoserpentin showed a weak amidolytic activity upon the substrate S-2238 with a K m of 2.2. 10 -4 M and Vma~ of 4.5 g m o l / m i n per mg. Phenylmethylsulfonyl fluoride-modified aggregoserpentin had a reduced Vm,x (1.0/tmol/min per mg) but still kept its full aggregating activity (0.5 /tg/ml). Dose-response curves of aggregoserpentin and crude oenom on platelet aggregation and 5-[ ~4C]hydroxytryptamine release reaction. Platelet aggregoserpentin showed a greater potency in platelet suspension than in platelet-rich plasma. Aggregoserpentin elicited irreversible aggregation at a con-

AMINO ACID COMPOSITION OF THE PURIFIED PLATELET AGGREGOSERPENTIN FROM T. GRAMINEUS V E N O M A m i n o acid

Residues/molecule

Lys His Arg Asp Thr Set Glu Pro Gly Ala 1 / 2 Cys Val Met Ile Leu Tyr Phe Total

19 10 14 37 17 26 23 11 25 14 13 19 11 19 27 11 11 307

centration of 0.5 /~g/ml (0.01 #M). It also dosedependently induced []4C]serotonin release (Fig. 2). At concentration of 1/~g/ml, it induced maximum aggregation (70% aggregation) and release reaction (% release: 80-85%). The crude venom showed a biphasic effect on platelet aggregation. It showed a peak activity at 10-100 # g / m l , while higher concentrations of the crude venom ( > 100 /~g/ml) showed a decreasing activity. During this 'decreasing response', no platelet lysis occurred, because no lactic dehydrogenase was found in the supernatant of platelet suspension. However, the crude venom-induced release reaction was increased gradually when the concentration was increased. No aggregation response was observed with crude venom or aggregoserpentin in the nonstirred platelet suspension, but the release reaction still proceeded just about the same as in a stirred condition. Effect of adenine nucleotides and adenosine on aggregoserpentin-induced responses. In platelet-rich plasma, adenosine showed a more potent effect on inhibiting aggregoserpentin (1 /~g/ml)-induced platelet aggregation (ICso 4/~M) than AMP (ICs0 0.5 mM) and ATP (IC_50 1 mM) did. However, in

130 platelet suspension, these adenine nucleotides did not inhibit aggregoserpentin-induced aggregation even at a high concentration of 2 mM. The ADP-removing system, creatine phosphate (5 mM)/creatine phosphokinase (8 U / m l ) slightly inhibited the platelet aggregation induced by a lower concentration of aggregoserpentin (0.2 ftg/ml) (22 _+ 5% inhibition, n = 4). However, creatine phosphate/creatine phosphokinase had no significant effect on platelet aggregation when induced by a higher concentration of aggregoserpentin (0.5 # g / m l ) ( < 10% inhibition). Apyrase (100 # g / m l ) also showed no significant effect on aggregoserpentin-induced aggregation.

Effect of aggregoserpentin in ADP-refractory platelet-rich plasma. The platelet-rich plasma became refractory to a second challenge of ADP after preincubating platelet-rich plasma with high concentration of ADP (4 mM) at 37°C for 30 min. However, aggregoserpentin (1 # g / m l ) still elicited platelet aggregation in this preparation, although a slight reduction was observed (20 + 7%). Calcium dependency of aggregoserpentin. At a low concentration (0.2 /~g/ml), aggregoserpentin did not elicit platelet aggregation under a Ca 2+-free condition (in the presence of 1 mM of EGTA or EDTA) except exogenous Ca 2+ (2 mM) was added. EDTA (2 mM) did not inhibit aggregoserpentin (> 0.5/~g/ml)-induced platelet aggregation and release reactions. However, at a higher concentration (5 mM), EDTA completely inhibited these reactions. On the other hand, 2 mM EDTA completely blocked the thrombin (0.1 U/ml)-induced aggregation. The thrombin-induced release reaction was rather resistant to EDTA (30% of control). Similarly, EDTA (5 mM) completely blocked both reactions.

Effect of cyclic AMP elevating agents on aggregoserpentin-induced platelet aggregation. At a concentration of 0.5 #M, prostaglandin E~ completely inhibited the aggregoserpentin-induced aggregation and release reaction in both platelet-rich plasma and platelet suspension. When prostaglandin E 1 (10 /.tM) was added after aggregoserpentin, it also disaggregated the platelet aggregates. Papaverine (0.5 mM) and theophylline (2.5 mM) also showed the same effect.

Effect of aggregoserpentin in formalin-fixed platelet suspension. At a concentration of 5/xg/ml,

aggregoserpentin still had no agglutinating activity in formalin-fixed platelet suspension, while polylysine (10 -4 g/ml) did.

Effects of other inducers on aggregoserpentin-treatedplatelet suspension. A second challenge of aggregoserpentin (0.5 /~g/ml) showed no response in aggregoserpentin-treated platelet supension unless a higher concentration ( > 5 /~g/ml) was added. The aggregation activity of aggregoserpentin ( 5 / t g / m l ) in the aggregoserpentin-treated platelet suspension was only 32% of that of aggregoserpentin (1 /~g/ml) in the platelet suspension not treated with aggregoserpentin. Other inducers, ADP (10 -5 M), thrombin (0.1 U / m l ) and collagen (50 ftg/ml) still exhibited their aggregating activities (Fig. 3) in this preparation. Sodium arrachidonate showed no aggregating activity unless fibrinogen (0.125%) was present in the medium. Effect of prostaglandin synthesis inhibitor. Indomethacin (50 #M) showed no significant effect on aggregoserpentin-induced platelet aggregation ( < 10%, n = 5), but a partial blockade of aggregoserpentin-induced release reaction (34 _ 8%, n = 6). Indomethacin (50/tM) also exerted no significant effect on thrombin-induced aggregation, and it slightly inhibited thrombin-induced release reaction (39 + 6%, n = 4).

c. -

•o

-

"C¢

~g¢

Thr,

d. $ ~ , , ~ , , ~ ¢

•

. b. ADP

],dT,10% I

rain

~• ,,,,,,4%__........... g ¢ e, °

C O I . ~

Fig. 3. The aggregatingactivitiesof aggregoserpentin(PAS) (0.5 #g/ml, lower tracing and 5 #g/ml, upper tracing in (a), ADP (10-5 M, b), thrombin(Thr. 0.1 U/ml, c), sodiumarachidonate (SA, 100 pM, d) and collagen (Col, 50 pg/ml, e) in aggregoserpentin-treated platelet suspensions were tested in the presence (~) or absence (g) of fibrinogen(~, 0.125%).

131 TABLE II EFFECT OF MEPACRINE ON AGGREGOSERPENTININDUCED PLATELET AGGREGATION AND RELEASE REACTION Mepacrine (# M)

% Aggregation

% Release

10 25 50 100 50 a

69_+6 (3) 57_+9 (3) 2 _+1 (8) 0 83 -+4 (3) a

72+7 (3) 55_+4 (3) 42 + 6 (5) 48 -+5 (3) 82 _+7 (3) a

a The % aggregation and release reaction represent the thrombin (0.1 U/ml)-induced reactions in the presence of mepacrine (50/~M). The values represent means + S.E. (n).

Independency of malonic dialdehyde formation. Platelet aggregoserpentin (5 #g/ml) still produced no detectable amount of malonic dialdehyde even when it induced a maximum aggregation. On the other hand, sodium arachidonate (125 #M) and thrombin (0.1 U/ml) produced an indomethacinsensitive malonic dialdehyde formation ( > 90% inhibition). Effect of phospholipase A inhibitors. Mepacrine dose-dependently (10-100 #M) inhibited aggregoserpentin (1 /~g/ml)-induced platelet aggregation and release reaction (Table II). Mepacrine (50/~M).completely blocked the aggregoserpentininduced aggregation, but only partially inhibited aggregoserpentin-induced release reaction (58 _+ 6%). Similar results with p-bromophenacyl bromide (0.1 mM) were obtained. Mepacrine (50/zM)

only slightly inhibited thrombin (0.1 U/ml)-induced aggregation and release reactions (17 + 7% and 18 + 7%, respectively). Effects of verapamil and nitroprusside. Veraparail ( _ 110 #M) completely inhibited the aggregoserpentin (0.5 #g/ml)-induced platelet aggregation, but only partially blocked aggregoserpentininduced release reaction (Table III). On the contrary, verapamil (275 #M) only showed a slight inhibition on the thrombin-induced aggregation. Nitroprusside (0.1 mM) completely blocked aggregoserpentin-induced platelet aggregating and releasing reactions, while a higher concentration (0.5 raM) was required to inhibit the thrombin-induced ones (Table III).

Effect of aggregoserpentin on basal and prostaglandin Et-stimulated adenylate cyclase activity. Platelet aggregoserpentin (1 /~g/ml) lowered the cAMP levels in both basal and prostaglantlin E 1 (6 /xM)-stimulated platelet suspension. The cAMP levels in basal and prostaglandin E 1 stimulated platelet suspension were determined to be 13.3 + 2.5 and 59.5 + 3.3 pmol/5 • 107 platelets (% inhibition, 49 + 6 and 38 + 6, n = 6) respectively. Discussion

A potent platelet, aggregating component (platelet aggregoserpentin) has been purified from Trimeresurus gramineus snake venom. It was a single chain peptide complexed with polysaccharide with a molecular weight of 43 400. It has 307 amino acid residues per molecule which was

T A B L E III

EFFECTS OF VERAPAMIL AND NITROPRUSSIDE ON AGGREGOSERPENTIN-INDUCED PLATELET AGGREGATION AND [14C]SEROTONIN RELEASE REACTION % Aggregation and % release reactions represent % of control. These values represent means 5: S.E. (n)

Verapamil (# M) 110 275

Aggregoserpentin (0.5 #g/ml)

Thrombin (0.1 U/ml)

% Aggregation

% Release

% Aggregation

% Release

0(3) 5 + 3 (5)

62+8 (3) 55 + 8 (4)

104+ 4(3) 63 + 24 (4)

109 _+21 (4)

3+2 (3) 0 (3)

92+ 8(3) 0 (3)

98_+ 2(3) 0 (3)

Nitroprusside (~ M) 100 500

0(3) 0 (3)

132

rich in aspartic acid, leucine, serine, glycine and glutamic acid (Table I). It possessed no phospholipase A 2 and 5'-nucleotidase or fibrinogenolytic activities. Platelet aggregoserpentin possessed a weak amidolytic activity. However, PMSF-modified aggregoserpentin lost amidolytic activity (residual activity: one fifth of the control), but still showed full aggregating activity. It indicates the amidolytic activity may be dissociated from its platelet-activating property. Platelet aggregoserpentin showed no procoagulant activity toward fibrinogen and could be separated from the thrombin-like component. The potency ratio between crude venom and the purified aggregoserpentin on platelet aggregation was about 1:10-15. Platelet aggregoserpentin (0.1-1 /~g/ml) dose-dependently elicited platelet aggregation and [14C]serotonin release. At concentrations of less than 100/~g/ml, the crude venom elicited a dose-dependent aggregation. But, when its concentration was higher than 100 /~g/ml, its aggregating activity was progressively reduced. However, the crude venom-induced release reaction was dose-dependently increased in spite of its reduced aggregating activity at higher concentrations. This may result from the effect of an acidic phospholipase A in the crude venom which was shown to inhibit the aggregoserpentin-induced platelet aggregation without affecting the aggregoserpentin-induced release reaction (unpublished data). Without stirring, aggregoserpentin did not elicit platelet aggregation, but it still induced [laC]serotonin release (80% of control). Therefore, the aggregoserpentin-induced platelet aggregation could be dissociated with its [14C]serotonin releasing activity. Platelet aggregoserpentin (5/xg/ml) showed no agglutinating activity in formalin-fixed platelet suspension. Therefore, its action mechanism was not due to a lectin-like or polylysine-like effect, ~ but due to the transmembrane induction leading to platelet aggregation. Thrombin exhibited the aggregating activity in aggregoserpentin-treated platelet suspension. Therefore, the site of action of aggregoserpentin was different from that of thrombin. Similarly, collagen also showed aggregating activity in aggregoserpentin-treated suspension in which the second challenge of aggregoserpentin (0.5/~g/ml) showed a 'desensitizing' effect.

Therefore, the site of action of aggregoserpentin was also different from that of collagen. Whether a specific receptor exists on platelet membrane remained to be clarified. The ADP removing system, creatine phosphate (5 mM)/creatine phosphokinase (8 U/ml) slightly inhibited aggregoserpentin-induced platelet aggregation only when low concentration of aggregoserpentin was used (22% inhibition). Its inhibitory effect was not significant when a high concentration of aggregoserpentin was used (< 10% inhibition). In addition, aggregoserpentin (0.5 #g/ml) also showed aggregating activity in ADP-refractory platelet-rich plasma. Therefore, it is infer'red that the ADP-releasing effect of aggregoserpentin play only a minor role in its platelet aggregating activity and aggregoserpentin receptor is also different from that of ADP. Platelet aggregoserpentin-induced platelet aggregation showed a lesser dependency on exogenous Ca 2+ than thrombin did. The thrombin-induced platelet aggregation and release reaction were more susceptible to EDTA. However, EDTA (5 mM) completely inhibited both aggregoserpentin- and thrombin-induced platelet aggregation and release reactions. It is accepted that agents raising the cAMP levels would inhibit the platelet activity. Prostaglandin E 1 (5- 10 -7 M) completely blocked aggregoserpentin-induced platelet aggregation and release reactions. Theophylline (2.5 mM) and papaverine (0.5 mM) had the same effects. Furthermore, aggregoserpentin was shown to lower the cAMP levels in both resting and prostaglandin El-stimulated platelet suspensions (49 + 6 and 38 + 6, respectively). The thromboxane A 2 formation was one of the main pathway to platelet activation [23]. However, indomethacin (50 /~M) didn't affect aggregoserpentin (1 #g/ml)-induced platelet aggregation, but inhibited partially the release reaction. In.case of thrombin-induced aggregation and release reaction, indomethacin (50 #M) had a similar effect on them. In addition, aggregoserpentin itself produced no detectable malonic dialdehyde, the end metabolic product of thromboxane A 2 and prostaglandin endoperoxides. Therefore, it is inferred that aggregoserpentin-induced platelet aggregation is independent of prostaglandin synthesis. This

133

was consistent with thrombocytin, a purified platelet activating component from Bothrops atrox [6,7], but was different from the aspirin-sensitive crotalocytin from Crotalus horridus horridus [10,11]. Mepacrine and p-bromophenacyl bromide blocked aggregoserpentin activity on platelet aggregation. The platelet aggregating activity of aggregoserpentin was more susceptible to these phospholipase A 2 inhibitors than thrombin. However, aggregoserpentin-induced release reaction was only partially inhibited (50%). Therefore, the release reaction of aggregoserpentin was partially elicited through a pathway other than the activation of phospholipase A 2. Recently, a third pathway leading to platelet aggregation was proposed that the platelet activating factor may activate platelets via an indomethacin-insensitive but mepacrine-sensitive pathway [24,25]. In addition, pathways involving the formation of phosphatidic acid and lysophosphatidic acid via activation of phospholipase C may also operate [26-28]. Verapamil (100 #M) completely blocked the aggregoserpentin-induced platelet aggregation, but only partially blocked the release reaction (45%). Thrombin (0.1 U/ml) was less susceptible to the inhibitory effect of verapamil (275 /~M, 37% inhibition). Verapamil has been reported to inhibit Ca 2+ influx [29] in several smooth muscle preparations. Nitroprusside completely inhibited aggregoserpentin-induced release reaction in contrast to the partial blockade of verapamil and mepacrine. Nitroprusside has been proven to prevent Ca 2÷ mobilization from intracellular stores [30]. Therefore, internal calcium mobilization may be in part responsible for the aggregoserpentin-induced release reaction. It is consistent with the idea that mobilization of Ca 2÷ from intracellular stores was involved in the platelet release reaction [31]. In summary, aggregoserpentin may activate platelets through the following sequence, (1) binding on platelet membrane ~ increasing the influx of calcium (verapamil-sensitive) or lowering the cAMP levels (prostaglandin El-sensitive ) activation of endogenous phospholipase A 2 (mepacrine-sensitive) ---, formation of platelet activating factor or phosphatidic acid and lysophosphatidic acid or other unknown mediators ---, mobilization of calcium from intracellular stores (nitroprusside-sensitive) ~ release reaction and platelet aggregation.

Acknowledgements This work was supported by the National Science Council Research Grant of China (NSC-700412-B002-10). The authors wish to express their heartfelt thanks to Drs. Walter H. Seegers and Lowell E. McCoy, Department of Physiology, Wayne State University, Detroit, for their cooperation in amino acid analysis on the protein.

References 1 Davey, M.G. and Liischer, E.F. (1967) Nature 216, 857-858 2 Davey, M.G. and Esnouf, M.P. (1969) Biochem. J. 111, 733-743 3 Marney, S.R. (1971) J. Immunol. 106, 82-90 4 Boffa, M.C. and Boffa, G.A. (1974) Biochim. Biophys. Acta 354, 275-290 5 Biran, H., Dvilansky, A., Nathan, I. and Live, A. (1974) Thromb. Diathes. Haemorrh. 30, 191-198 6 Kirby, E.P., Niewiarowski, S., Stocker, K., Kettner, C., Shaw, E. and Brudzynski, T.M. (1979) Biochemistry 18, 3564-3570 7 Niewiarowski, S., Kirby, E.P., Brudzynski, T.M. and Stocker, K. (1979) Biochemistry 18, 3570-3577 8 0 u y a n g , C. and Teng, C.M. (1979) Thromb. Haemostas. 41, 475-490 9 0 u y a n g , C., Wang, J.P. and Teng, C.M. (1980) Biochim. Biophys. Acta 630, 246-253 10 Schmaier, A.H. and Colman, R.W. (1980) Blood 56, 1020-1028 11 Schmaier, A.H., Claypool,'W. and Colman, R.W. (1980) Blood 56, 1013-1019 12 Ouyang, C. and Huang, T.F. (1979) Biochim. Biophys. Acta 571,270-283 13 Weber, K. and Osborn, M. (1969) J. Biol. Chem. 244, 4406-4412 14 Dubois, M., Gilles, K.A., Hamilton, J.K., Roberts, P.A. and Smith, F. (1956) Anal. Chem. 28, 350-356 15 Mustard, J.F., Perry, D.W., Ardlie, N.G. and Packham, M.A. (1972) Brit. J. Hematol. 22, 194-204 16 Ardlie, N.G., Perry, D.W., Packham, M.A. and Mustard, J.F. (1971) Proc. Soc. Exp. Biol. Med. 136, 1021-1023 17 Grant, R.A., Zucker, M.B. and Mcpherson, J. (1976) Am. J. Physiol. 230, 1406-1410 18 O'Brien, J.R. (1962) J. Clin. Pathol. 15, 452-455 19 Born, G.V.R. and Cross, M.J. (1963) J. Physiol. 168, 178-195 20 Ardlie, N.G. and Han, P. (1974) Brit. J. Haematol. 26, 331-356 21 Smith, J.B., Ingerman, C.M. and Silver, M.J. (1976) J. Lab. Clin. Med. 88, 167-172 22 Wroblewski, F. and LaDue, J.S. (1955) Proc. Soc. Exp. Biol. Med. 90, 210-213 23 Kinlough-Rathbone, M.A., Packham, M.A., Reimers, H.J., Cazenave, J.,P. and Mustard, J.F. (1977) J. Lab. Clin. Med. 90, 707-718

134 24 Chignard, M., Le Couedic, J.P., Tence, M., Vargaftig, B.B. and Benveniste, J. (1979) Nature 279, 799-800 25 Vargaftig, B.B., Chignard, M. and Benvensite, J. (1981) Biochem. Pharmacol. 30, 263-272 26 Billah, M.M., Lapentina, E.G. and Cuatrecasas, P. (1979) Biochem. Biophys. Res. Commun. 70, 92-98 27 Otnaess, A-B. and Holm, T. (1976) J. Clin. Invest. 57, 1419-1425

28 Schick, P.K. and Yu, B.P. (1974) J. Clin. Invest. 54, 1032-1039 29 Meyer, C.J., Van Breemen, C. and Casteels, R. (1972) Pfluegers Arch. 337, 333-350 30 Verhaeghe, R.H. and Shepherd, J.T. (1976) J. Pharmacol. Exp. Ther. 199, 269-277 31 Detwiler, T.C., Charo, I.F. and Feinman, R.D. (1978) Thromb. Haemostas. 40, 207-211