Inhibition by ticlopidine of Paf-acether-induced in vitro aggregation of rabbit and human platelets

THROMBOSIS RESEARCH 34; 463-471, 1984 0049-3848184 $3.00 + .OO Printed in the USA. Copyright (c) 1984 Pergamon Press Ltd. All rights reserved.

INHIBITION BY TICLOPIDINE OF PAF-ACETHER-INDUCED IN VITRO AGGREGATION OF RABBIT AND HUMAN PLATELETS C. LALAU KERALY *, D. DELAUTIER *, D. DELABASSEE + , M. CHIGNARD" and J. BENVENISTE" * INSERM U 200, Universitr?Paris-Sud 32 rue des Carnets, 92140 Clamart, France t Ligne H&nobiologie, Sanofi Recherche, 31036 Toulouse Cedex, France (Received 2.5.1983; Accepted in revised form 12.3.1984 by Editor D. Meyer) ABSTRACT Ticlopidine was incubated in vitro with rabbit or human washed were submaximal and triggered by platelets aggregations concentrations of adenosine-5'-diphosphate (ADP), arachidonic acid (AA) and Paf-acether (platelet-activating factor), the mediators of the three known pathways of platelet activation. Inhibition of Paf-acether-induced rabbit platelet aggregation was proportionnal to the concentrations of Ticlopidine used. The same range of inhibition by Ticlopidine was observed when aggregations were triggered by the two other agonists. Human platelet aggregation induced by Paf-acether was also inhibited by Ticlopidine. Inhibition was increased when platelets were rendered insensitive to ADP and AA. Our results show that Ticlopidine inhibits human and rabbit platelet aggregation triggered by Paf-acether through a mechanism not related to the inhibition of the ADP and prostaglandin pathways.

INTRODUCTION Ticlopidine, a thienopyridine derivative (1,2) is a potent and long acting inhibitor of platelet aggregation. Yet the mechanism of action of this drug is not fully understood. It has been proposed that Ticlopidine could act through an increase of the platelet cyclic-AMP content (3,4). Other evidences were given for a direct interaction between the drug and the platelets, leading to a reduction of their responsiveness to adenosine-5'-diphosphate (ADP) (5) and moreover it has also been shown that Ticlopidine-treatment decreases the binding of ADP to platelets (6). An inhibitory effect of Ticlopidine on the fibrinogen binding has also been reported (7). Whatever the mechanism(s) involved, the inhibitory effect of this drug has been demonstrated using

KEY WORDS : Paf-acether, Ticlopidine, Platelet aggregation. 463

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platelets aggregated with ADP, collagen, thrombin, arachidonic acid (AA) and adrenaline in rat and healthy man (5,6,8-13). Thus far, only one study has been published (14) on the inhibitory effect of Ticlopidine on Paf-acether (Platelet-activating factor)-induced human platelet aggregation in plasma. This mediator, identified as a I-O-alkyl-Z-O-acetyl-sn_glyceryl-3is the most potent known platelet agonist. It was phosphorylcholine (15,16), first described in rabbit circulating basophils, and causes the aggregation of rabbit platelets and the release of their vasoactive amines (17). Aggregation observed on rabbit platelets stimulated by Paf-acether is independent of the Paf-acether is also known to trigger ADP and atachidonic acid pathways (18). of human platelets in plasma (19-24) and in aggregation and secretion on Paf-acether-activated human artificial medium (21,25-28). Many studies platelets have shown that the irreversibility of the aggregation was dependent on the cyclooxygenase and/or secretion of ADP. It is noteworthy that, for these studies, titrated plasma and/or calcium-free buffer were used as extracellular medium (14,19-24,27). In these conditions, ADP itself induced a release reaction which can be inhibited by aspirin (29). By contrast, aspirin failed to reduce Paf-acether-induced aggregation of platelets in heparinized plasma (26) as well as in buffer containing calcium (25,26,28), In addition, it has been described that human plasma-free platelets can also respond to Paf-acether through a mechanism independent from ADP secretion (26,28). Keeping in mind these conflicting results we used washed platelets rendered refractory to ADP and AA, in order to examine a pure effect of Ticlopidine on Paf-acether-induced This activity was studied on rabbit and human platelets, and aggregation. compared to the activity of Ticlopidine on aggregation induced by the two other agonists, ADP and AA.

MATERIALS AND METHODS The following buffers and reagents were used : Buffers and reagents. Tyrode’s buffer devoid of CaCl containing (mM concentrations) NaCl 1.37 ; KC1 2.68 ; NaHC03 11.90 j MgC12 1.&I ; dextrose 0.55. Tyrode’s devoid of CaC12 with GFR) f?d 0.25 % gelatrn (Merck, Darmstadt, 0.2 mM ethyleneglycol-tetraacetic acid (EGTA) (Tyrode’s gelatin without Ca ). Tyrode ‘s devoid of CaCl with 5 mM i%S without HE$$S (Sigma, St-Louis, MO, USA) and 0.41 mM NaH PO (Tyrode’s-HE citrate Ca ). Acid-citrate dextrose (ACD) (citric acid.2 8.8 X, trisodium Ethylenediamine-tetraacetic acid (EDTA) dextrose 2.45 X, pH 4.8). 2.2 x, (Merck). Bovine serum albumin (BSA) (Armour Co., Phoenix, AZ, USA). Human Sweden) treated with diisopropylfluorophosphate fibrinogen (Kabi, Stockholm, (DFP) to remove coagulant contaminants, was a gift from B.B.BVargaftig was from (Institut Pasteur, Paris, France). Aspirin as a lysin salt (Aspegic phosphate (CP), creatine Egic, Laboratoire France ). Creatine Amilly, phosphokinase (CPK), and ADP were purchased from Sigma Chemicals and dissolved Arachidonic acid (AA) (Sigma) was dissolved in ethanol 60 %. l-Oin saline. (Paf-acether), provided octadecyl-2-0-acetyl-sn-glyceryl-3-phosphorylcholine by Prof. J.J. Godfrzd (Universite Paris VII, France), was obtained by acetylation of the 1-O-octadecyl-sn-glyceryl-3-phosphorylcholine synthesized by Rhane-Poulenc (Vitry, France) according to the method described by Heymans et al. (30). This batch of Paf-acether was found to be 100 % pure as liquid chromatography (HPLC), mass established performance high jy H-NMR spectra and optical rotation. Paf-acether was used after spectrometry, in saline containing 0.25 4; fatty acid-free bovine serum its dissolution Sanofi Recherche (Toulouse, (BSA) (Sigma). Ticlopidine was from albumin France) and dissolved in saline.

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Briefly, platelets were prepared Preparation of washed rabbit platelets. centrifugations and dilutions from blood collected in EDTA (52$4) by successive to the method of Ardlie -et in Tyrode’s-gelatin without Ca , pH 6.5, according al. (31) modified by Benveniste et al. (17). The suspension (2/3, v/v) of Meshed platelets was preincubated for 30 min at room temperature with 0.1 mM The fifzl platelet suspensions (1.8 x aspirin; before the last centrifugation. 10 platelets/ml Tyrode’s gelatin without Ca , pH 6.5) were kept at room temperature before use.

Ninety ml of human blood were taken Preparation of washed human platelets. from a forearm vein into 10 ml of ACD. Human platelets were prepared according as described by Lalau Keraly et to the method of Lagarde et al. (32) modified al. (28). Blood was centrifuged at 100 x g during 15 min. Platelet-rich plasma ERP) was removed, acidified at pH 6.4 with citric acid 0.15 M, separated into 4 fractions, and then centrifuged at 900 x g during 10 min. The supernatant from each tube was rem ved and the pellet was resuspended in 6 ml of S+ containing 1 volume of ACD for 9 volumes of buffer Tyrode ‘s-HEPES without Ca (pH 6.4). Platelet suspensions were centrifuged at 900 x g for 10 min, in 6 ml of the same supernatants were removed and pellets were resuspended buffer. The first aliquot of the platelet suspension was treated with 100 pM aspirin, ; the second one with 0.1 mM ADP ; the third a cyclooxygenase blocker one with both aspirin and ADP, and the fourth one was kept untreated. After 1 hr of incubation, the four fractions were centrifuged (900 g ; 10 min). The supernatants were completely removed a the pellets were resuspended in the “8 same buffer in order to obtain 1.8 x 10 platelets per ml. The final platelet suspensions were kept at room temperature before use.

Aggregation studies. Rabbit platelets we v diluted with Tyrode’s gelatin without Ca L , pH 7.4, in order to obtain 3 x 10 platelets per ml, and incubated at room temperature for 20 min with Ticlopidine (1 ; 10 ; 50 and 100 pMM)or the solvent . Human platelets were diluted and insybated with Ticlopidine in the same way except that Tyrode’s-HEPES without Ca , pH 7.4 was used as suspending medium. These incubations were performed in the aggregometer tubes. Aggregations were then performed after addition of CaCl (1.3 mM) to the rabbit and human preparations, and DFP-treated fibrinogen ( 02.16 mg/ml) to the human one. The concentration of each agonist was chosen in order to obtain a submaximal aggregation of the platelets in each particular experiment, i.e. an extent of aggregation which became maximal by a slight increase of the agonist concentration (see the concentrations in caption of figures). ADP-induced aggregation was tested on platelets which were preincubated with aspirin. AAinduced aggregation was performed in rabbit platelets in the presence of CP/CPK (1 mM ; 10 U/ml) complex, and in ADP-treated human platelets. It was verified that, under these conditions, either rabbit or human platelets did not aggregate with ADP stimulation (60 PM). Rabbit aspirin-treated platelets in the presence of CP/CPK and human aspirinand ADP-treated platelets were used for the Paf-acether stimulation. In some experiments, the effect of Ticlopidine on ADP, AA and Paf-acether-induced aggregation was studied in untreated human platelets.

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RESULTS Effects of Ticlopidine on rabbit platelet aggregation. A 20 minincubation of aspirin-treated platelets with Ticlopidine (100 $I) strongly inhibited the submaximal aggregations induced by ADP as well as those induced, in the presence of CP/CPK, by Paf-acether. The submaximal aggregations induced by AA in the presence of CP/CPK were also strongly inhibited after the 100 ,JJM Ticlopidine treatment (fig. 1). The anti-aggregatory effect of Ticlopidine decreased when 50 J.IMof this drug were used. The variations of the responses between platelet preparations were too large for the construction of mean concentration-effect curves. However concentration-related activities were easily observed in most of the experiments (Fig. 1) demonstrating that Ticlopidine exhibited an equipotent inhibition of platelet aggregation induced by the three agonists.

100

r

ADP

f

0 Ticlopidine concentrations (pM) FIG.

1

Each point represents the percentage Effect of Ticlopidine on rabbit platelets. of inhibition of aggregation from one experiment after 20 min of incubation with Ticlopidine. Aggregations were triggered by ADP (5.5 + 4.3 @4) on aspirin+ 0.55 pMM) in the presence of CP/CPK, and treated platelets, AA (0.49 Paf-acether (0.22 + 0.10 nM) on-aspirin-treated platelets in the presence of CP/CPK (n=7).

Incubation with Effects of Ticlopidine on human platelet aggregation. inhibited aggregation of ADP-induced the complete ly 50 PM Ticlopidine untreated and aspirin-treated human platelets. (Fig. 2A and B). Similarly Ticlopidine inhibitory effects were obtained with untreated platelets or ADP2A and B). Submaximal aggregations as treated platelets stimulated by AA (Fig.

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a result of the stimulation of aspirin and ADP-treated platelets (Fig. 2A) and untreated platelets (Fig. 2B) by Paf-acether, were also strongly inhibited after 20 min incubations of these platelets with 50 pM Ticlopidine. Inhibition for Paf-acether than for the two other agonists was less pronounced particularly when untreated platelets were used.

-

ADP

-AA

o-

-PALACETHER

O1

I

J

I

I

10

50

100

10

50

J 100

Ticlopidine concentrations (pM)

FIG. 2 Effect of Ticlopidine on human platelets. Each point represents the mean + 1 SD (n=3-4) of the percentage of inhibition of human platelets after 20 mk of incubation with Ticlopidine. (A) M ADP(13.1 + 9.0 uM) induced aggregations on aspirin-treated platelets, +-+ AA- (0.9 + 0.6 @tM) induced aggregations on ADP-treated platelets, H Paf-acether- (0.56 2 0.24 nM) induced aggregations on ADP and aspirin-treated platelets. (B) e-~ ADP- (5.8 + 1.5 uM), % AA- (0.9 + 0.1 @lM) and A-A Paf-acether- (0.39 2 0.01 nM) Induced aggregations were carried out on untreated platelets.

DISCUSSION Ticlopidine was found to be a potent inhibitor of washed rabbit and human platelet aggregation. The inhibitory effect of this compound covered platelet aggregation induced by various stimulants including ADP, AA and Paf-acether. Previous studies, performed with platelet-rich plasma, have shown inhibition of aggregation (6,10,11,13) and of the release reaction and promotion of disaggregation (11-13) when Ticlopidine was orally administered to healthy humans. Our results show that Ticlopidine is active in vitro, indicating that its biotransformation in the body is not necessary for its anti-aggregatory effect. However, for the in vitro effect, higher doses of Ticlopidine are required than the recommended therapeutic doses. This, therefore, warrants

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studies using metabolites of this drug which may be more effective at lower concentrations. Results contradictory to ours were obtained by Ashida et al. who failed to find an in vitro effect possibly because they used rat platelets instead of human or rabbit platelets (8). Thus, Lee et al. (7) obtained an in vitro inhibitory effect of Ticlopidine on washed human platelets when studying fibrinogen binding. Furthermore, under our experimental conditions, human platelets seemed to be more sensitive than rabbit platelets to the antiaggregatory effect of Ticlopidine. Although the action of Ticlopidine on ADP, collagen and thrombin-induced conflicting aggregations is 1 imi ted yielding we 1 l-documented, studies, results, were published on the effects of Ticlopidine on AA-induced platelet aggregation (8,33,34). But, when an inhibitory effect was observed, the selective alteration of prostaglandin synthesis by Ticlopidine seemed not to be the only direct cause of this inhibition (8,33,35). Our present results show that Ticlopidine could inhibit AA-induced aggregation of rabbit and human platelets in vitro. Furthermore, we demonstrate that this inhibitory effect is Thus human independent from the one observed on ADP-induced aggregation. platelets made refractory to ADP by preincubation with this nucleotide, exhibited the same sensitivity to the Ticlopidine anti-aggregatory effect than ADP-untreated platelets, when stimulated by AA. Other work has shown the inhibitory potency of Ticlopidine on Paf-acetherhuman titrated platelet-rich plasma (14). Our induced aggregation using present study demonstrates the non-involvement of the ADP and prostaglandin pathways in the inhibitory effect on Paf-acether aggregation. Aggregations induced by Paf-acether or by the two other agonists could be inhibited by agents which cause or mimic elevation of the intracellular concentrations of cyclic-AMP (36). Our results suggest that if Ticlopidine could act by increasing the platelet cyclic-AMP content of platelets, it is probably more by a direct effect on adenyl cyclase, as it was suggested by Bonne et al. (41, than by the activation of the prostaglandin El -stimulated activity of the cyclase On the other hand, an overall effect of Ticlopidine on platelet (3). the inhibition of fibrinogen binding (71, cannot be dismissed aggregation, i.e. on human of Ticlopidine at least to explain the antiaggregatory effect platelets. Thus, Ticlopidine is a strong antagonist of the platelet activation since it is able to inhibit aggregations triggered by the mediators of the different three known pathways of platelet aggregation, namely ADP, AA and Paf-acether. respect, Ticlopidine is more effective than the non-steroidal In this antiinflaursatory drugs which are ineffective on ADP and Paf-acether-induced aggregation (18).

Aknowledgements This

work was supported

by a generous

grant

from

Sanofi

(France).

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