Proaggregatory effect of epinephrine on rabbit platelets inhibited by ticlopidine

THROMBOSIS RESEARCH 57; 393-403, 1990 0049-3848/90 $3.00 t .OO Printed in the USA. Copyright (c) 1990 Pergamon Press plc. All rights reserved.

PROAGGREGATORY EFFECT OF EPINEPHRINE ON RABBIT PLATELETS INHIBITED BY TICLOPIDINE

Claudia Department

K. Derianapb, Robert J. Gould and Paul A. Friedman of Pharmacology, Merck Sharp and Dohme Research Labs, West Point, Pennsylvania 19486

USA (Received 21.8.1989; accepted in revised form 20.11.1989 by Editor John Weisel) ABSTRACT inhibitor of ADP-induced aggregation of Ticlopidine is a potent In vivo, however, multiple agonists play a rabbit platelets ex vivo. role in platelet activation. In this study, we examined the effect of epinephrine on the antiplatelet action of ticlopidine in rabbit Epinephrine reversed the inhibitory effect of drug on platelets. ADP-induced platelet aggregation. The potentiating effect of mediated a2-adrenergic receptors, epinephrine was through was Nat/H+ reversed by pretreatment with the exchange inhibitor dimethylamiloride, and was mimicked by agents that increased Ticlopidine had no effect on resting intracellular sodium or pH. that the effect of epinephrine was intracellular pH, an indication compensating for a drug-induced intracellular acidification. not While this potentiation was also found to be inhibited by aspirin, it did not involve enhanced release of thromboxane A,. Our results the inhibitory effect of demonstrate that epinephrine can overcome ticlopidine on ADP-induced aggregation through a mechanism involving activation of Nat/H+ exchange and through an as yet unidentified mechanism sensitive to aspirin.

INTRODUCTION inhibitor of platelet aggregation induced a Ticlopidine is a potent The against ADP (1). m by a number of agonists but is most effective mechanism(s) by which ticlopidine exerts this antiplatelet response is not The effectiveness of the drug is less well characterized fully understood. Rorer Central Research, 620 a Current address: Prussia, PA 19406 b Author to whom correspondence should be addressed Key words:

ticlopidine;

blood

platelets;

epinephrine

393

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Road,

King

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Epinephrine is known to agonists. combinations of synergistic with potentiate platelet responses to a number of agonists including ADP, Epinephrine factor (2,3). activating and platelet collagen thrombin, reverses the inhibitory effects of aspirin on platelet aggregation induced by It also reverses aspirin-induced acid, and thrombin (4). ADP, arachidonic inhibition of platelet thrombus formation in vivo (5,6). Recently, Lecrubier et al. (7) reported that epinephrine can potentiate the aggregatory response to platelet activating factor in human platelets following oral ticlopidine less effective when that ticlopidine may be This suggests treatment. In this report we multiple agonists, particularly epinephrine, are present. the inhibitory effect of oral provide evidence that epinephrine overcomes ticlopidine treatment in rabbits and postulate possible mechanism(s). METHODS Materials Ticlopidine hydrochloride was kindly provided by Dr. John Bruno (Syntex Human fibrinogen and monensin were obtained from Research, Palo Alto, CA). 2’,7’-bis-(2-carboxyethyl)-5(and-6)carbowCalbiochem (La Jolla, CA). acetoxymethyl ester (BCECF-AM) was purchased from Molecular fluorescein, Adenosine 5’-diphosphate (ADP), apyrase, aspirin, Probes (Eugene, OR). (PGE,), were obtained hydrochloride, and prostaglandin E, methylamine from Sigma (St. Louis, MO). Epinephrine was obtained from Parke-Davis (Morris Plains, NJ). A radioimmunoassay kit for the determination of was purchased from Advanced Magnetics (Cambridge, MA). thromboxane B, Dimethylamiloride (DMA) was kindly provided by Dr. Edward Cragoe. Drug Treatment Ticlopidine (100 rabbits (1.5-2.5 kg) collected into 0.1 vol

mg/kg, p.o.) was administered to male for 4 days. Blood obtained by cardiac 3.8% sodium citrate.

New Zealand puncture was

Preoaration of Washed Platelets and Aggregation Studies Platelets were prepared as described previously (8). Aggregation of washed platelets (5~10~ cells/ml) was assessed using a Sienco Dual Sample Aggregometer. Aliquots (0.2 ml) of cells were prewarmed to 37°C in the presence of 1 mM CaCl, and 0.1 mg/ml human fibrinogen prior to the addition of agonists. Aggregation curves were recorded for 3 minutes. The extent of aggregation was determined by calibrating 0% light transmittance to the platelet suspension and 100% to buffer. Measurement of Intracellular PH with BCECF Platelets were resuspended in 5 ml of incubation buffer (composition, mM: NaCl, 145; KCl, 5; MgCl,, 1; glucose, 5; HEPES, 10) adjusted to pH 7.4 and incubated with BCECF-AM (5 pM) for 30 min, 37OC. Cells were washed two times in buffer adjusted to pH 6.5 and then resuspended in the incubation buffer to a final concentration of 5xlOa cells/ml. Fluorescent measurements were recorded on a Perkin-Elmer LS-5 spectrofluorometer using settings of 500 nm (excitation) and 530 nm (emission). Calibration curves were generated as described by Rink et al. (9) by lysing the cells in 0.1% Triton X-100 followed by step wise additions of HCl or NaOH between pH 6 and 8. Radioimmunoassav for Thromboxane A, Platelet aggregation was recorded for 2 min at was added to the cuvette (final concentration, 50 pelleted. Supernatant fractions were frozen until generation was measured by a radioimmunoassay for thromboxane B,.

which time indomethacin uM) and the platelets assay. Thromboxane A, its stable metabolite

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RESULTS Effect

of Ticlopidine on Platelet Aggregation Ticlopidine (p.0.) significantly inhibited platelet aggregation induced by ADP ex vivo as previously reported (Figs. 1 and 2). Figure 1 illustrates typical aggregation tracings obtained for normal platelets compared to

(-E)

f

(+E)

Control

(+E)

Ticlopidine

(-E)

Control

Ticlopidine

1 Min.

ADP

FIG.

1

Representative aggregation tracings for control and ticlopidineinhibited platelets. Platelets (5 x lOs/ml) were prewarmed to 37°C for 3 minutes in the presence of 1 mM CaCl, and 0.1 mg/ml human fibrinogen. Aggregation was recorded for 3 minutes. Epinephrine (E) (0.1 uM> was added immediately prior to stimulation with 2.5 @+I ADP.

platelets from ticlopidine-treated rabbits. Ticlopidine pretreatment inhibited the maximum extent of aggregation and induced rapid disaggregation of platelets. The aggregatory response was inhibited (p < 0.001) over a wide range of ADP concentrations (0.1 - 20 @) and extents of aggregation (Fig. Aggregation responses were reduced in 80% of the animals treated and 2A). Platelets which showed the extent of inhibition ranged between 30 - 80%. greater than 40% inhibition as compared to platelets from control animals Since variation was seen from week-to-week in were used in further studies.

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untreated control animals were included in all the extent of aggregation, Results from drug-treated animals were compared only to results from groups. control assays run in parallel.

80

6ol

A

B

70 50 4

0

5

10 ADP (JLM)

FIG L

Effect

2

15

20

J-l+0

-9

-8

Log [Epinephrine]

-7

-6

(M)

The effect of epinephrine on ADP-induced platelet aggregation. Epinephrine was Platelets were prepared as described in Figure 1. added just prior to ADP stimulation. A) Dose response curve for ADP in the presence of 0.1 +@I epinephrine. Control platelets without epinephrine ( o ); ticlopidine-inhibited platelets without epinephrine (0); ticlopidine-inhibited platelets plus epinephrine (4 ). *p < 0.01 compared to control platelets at the same ADP concentration. B) Dose response curve for epinephrine in the presence of 2.5 uI4 ADP. ADP response in platelets ( 0 ); ticlopidine-inhibited control platelets (0). **p < 0.005 compared to ticlopidine-inhibited cells in the absence of epinephrine and p > 0.1 compared to control platelets. Data represent mean + S.E. for three experiments.

of Epinephrine on ADP-Induced Aggregation aggregation when administered Epinephrine (0.1 uM> did not stimulate alone to rabbit platelets but potentiated the response to ADP with both control and drug-inhibited platelets. Representative tracings demonstrating the potentiating effect of epinephrine are shown in Figure 1. Addition of

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epinephrine to platelets from drug-treated animals overcame the inhibitory effect of ticlopidine on ADP-induced aggregation. As seen in Figure 2A, addition of 0.1 pM epinephrine to platelets from treated animals resulted in a dose response curve for ADP-induced aggregation essentially indistinguishable from that of control platelets receiving only ADP. The proaggregatory effect of epinephrine was dose-dependent and at concentrations of 0.1 pM and 1.0 pM irreversible aggregation was restored to the same extent as untreated control platelets (Fig. 2B). The maximal achievable response was still slightly less than for normal platelets incubated in the presence of epinephrine plus ADP (Fig. 1). The potentiating effect of epinephrine could be inhibited by pretreatment with the ccz-adrenergic receptor antagonist yohimbine (10 uM) which had no effect on ADP-induced aggregation in the absence of epinephrine (data not shown). Bole

of Na+/H+ Exchange epinephrine has previously been shown to Since stimulate Na+/H+ exchange in human platelets, resulting in elevations in both intracellular Nat and cytoplasmic pH (10-12)) we next sought to determine the contribution of these two factors to the proaggregatory effect of epinephrine on platelets from drug-treated animals. We examined the effects of 1) the Nat/H+ exchange inhibitor dimethylamiloride influx (DMA); 2) Nat alkalinization induced induced by the ionophore monensin; and 3) cytoplasmic by methylamine. DMA (50 @I) inhibited ADP-induced aggregation of platelets from both control and ticlopidine-treated animals by 20% and 45%, respectively (data not shown). More important, DMA abolished the potentiating effect of 0.1 pM epinephrine on drug-inhibited platelets (Table 1); increasing concentrations of epinephrine could surmount this inhibitory action. Monensin, at 0.1 pM and 1 pM, potentiated the response to ADP in platelets from ticlopidine-treated animals by approximately TABLE 1. Dimethylamiloride Antagonizes the Proaggregatory Effect of Epinephrine Platelets were pretreated with 50 pM dimethylamiloride (DMA) for 10 minutes prior to the addition of either 2.5 pM ADP or ADP plus epinephrine (EPI). Data represent mean + S.E. for two experiments. a p < 0.01 compared to ADP Stimulus

ADP t 0.1 pM Epi t 1.0 pM Epi t 10 @I Epi

Control 57 64 68 74

+ + * +

5 2 4 4

% Agnrepation Ticlopidine 19 21 38 45

+ + + +

4 5 2a 2a

At these concentrations, monensin respectively (Table 2). 2- and 3-fold, cytoplasmic alkalinization Similarly, alone had no effect (data not shown). induced by pretreatment of platelets with methylamine, did not alone, but enhanced ADP-induced aggregation of stimulate platelets to aggregate drug-inhibited platelets by approximately 2-fold (Table 2).

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These results with DMA, monensin, and methylamine are consistent with the hypothesis that epinephrine activates the Na+/H+ exchanger leading to cytoplasmic alkalinization and thus sensitizing the cells to stimulation by ADP . The resting pH of platelets from control and ticlopidine treated animals TABLE 2 Monensin and Methylamine Mimic the Proaggregatory Effect of Epinephrine Platelets were prepared as described in Figure 1. Monensin or methylamine was added immediate1 prior to ADP stimulation. ap < 0.001 compared to %p < 0.05 compared to respective control platelets. cells in the absence cp < 0.05 compared to control platelets in the absence of of monensin. Data represent mean + S.E. for two experiments. monens in. Stimulus 5 pM ADP + Monensin Monens in Monens in Methylamine

Control 0 0.1 uM 1.0 pM 20 lJM

47 53 56 49

* + + *

5 4 gb 5

% Aggregation Ticlopidine 12 + 3a 22 + lb

was therefore measured to determine if treatment with ticlopidine leads to acidification of cytoplasm. There was, however, no significant difference in resting pH between control platelets and platelets from ticlopidine-treated rabbits, 7.06 i 0.11 (mean + S.E.M., n=32) and 7.06 + 0.08 (mean + S.E.M., n=17), respectively. Effect

of Aspirin on the Proagaresatorv Response of Epineuhrine Epinephrine-induced activation of the Na+/H+ exchanger has been postulated to lead to the release of membrane bound arachidonic acid and its subsequent metabolism to thromboxane A, in human platelets (11,12). Since agonist-induced thromboxane A, formation provides an important amplification signal for platelet aggregation, we next examined the effect of the cyclooxygenase inhibitor aspirin on aggregation. Pretreatment with aspirin did not affect ADP-induced aggregation of control platelets when examined in the absence or presence of 0.1 uM epinephrine (Fig. 3). While aspirin had no direct effect on ADP-induced aggregation of ticlopidineinhibited platelets, it significantly inhibited (p < 0.01) the proaggregatory effect of 0.1 pM epinephrine (Fig. 3). The inhibitory action of aspirin could be overcome by increasing the epinephrine concentration lo-fold (Fig. This data suggested that the proaggregatory effect of epinephrine might 3). be mediated through enhanced thromboxane A, formation. Thromboxane A, formation was therefore measured directly after ADP and epinephrine stimulation in the absence or presence of aspirin pretreatment. Contrary to results obtained with human platelets, ADP and epinephrine did not stimulate thromboxane A, formation in platelets from either control or drug-treated rabbits. Aspirin did however significantly inhibit (>90%) the low basal thromboxane A, release (data not shown).

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100 90 1

0 m

Untreated Aspirin Treated

80

(1l)(9) T

j

60-

;

50-

2

40-

$?

30-

(1l)(7)

(3) (3)

20 lo0 A

C

Control FIG. 3

II

Ticlopidine

Effect of aspirin on the proaggregatory action of epinephrine. Platelets were preincubated in the absence or presence of 100 pM aspirin for 15 minutes prior to stimulation with either 5 pM ADP ADP plus 0.1 pM epinephrine ADP plus 1.0 pM (A), (D), or epinephrine (C). Data represent mean * S.E. The number of replications per data point is indicated in parentheses. *p < 0.001 compared to control platelets. ** p < 0.01 compared to respective untreated platelets. DISCUSSION

Our results confirm previous observations that orally administered ticlopidine is a potent inhibitor of ADP-induced platelet aggregation when examined (1). While most studies have reported significant ex viva inhibition to single agonist challenges, the effectiveness of ticlopidine against multiple stimuli, a more probable situation during a thrombotic episode, has not been well characterized. We demonstrate in this study that epinephrine exerts a proaggregatory effect on ADP-induced aggregation of platelets isolated from drug-treated rabbits. This potentiating effect of epinephrine was mediated through ar-adrenergic receptors, was reversed and was mimicked by agents that increase by dimethylamiloride pretreatment, intracellular sodium or pH. Epinephrine therefore exert its may proaggregatory effect on drug-inhibited platelets via a mechanism involving Aspirin was also an effective inhibitor activation of Na+/H+ exchange.

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of the proaggregatory effect of epinephrine on ADP-induced aggregation of However, it is unlikely that drug-treated animals. from platelets thromboxane A, production plays a significant role in this proaggregatory effect because we were unable to detect significant increases in thromboxane stimulation. This is ADP and/or epinephrine levels following Bz agonists do not stimulate reports that these consistent with previous in rabbit platelets (13,14). Bomalaski et al. thromboxane A, formation (15) recently reported that aspirin inhibits phospholipase C activity in human monocytes and U937 cells through induction of an inhibitory protein. Thus, while the exact mechanism by which aspirin inhibits the proaggregatory effect we cannot rule out the of epinephrine remains to be determined, possibility that aspirin has multiple effects in rabbit platelets. Since epinephrine can increase fibrinogen binding to both human (16) and interaction with ADP, it is rabbit (17) platelets through a synergistic epinephrine effect of on drug-inhibited probable that the potentiating enhanced exposure. ADP-mediated involves fibrinogen receptor platelets induces the exposure of fibrinogen receptors activation of platelets a prerequisite for platelet resulting in increased fibrinogen binding; its structural analogue PCR 4099 Ticlopidine and aggregation (18,19). inhibit fibrinogen receptor exposure induced by a number of different reduced including ADP as demonstrated by [1251]fibrinogen agonists binding to activated cells (20,21). The mechanism(s) by which agonists induce fibrinogen receptor exposure are not fully understood. However, it has recently been shown that one mechanism of receptor exposure involves activation of G-protein dependent phosphoinositide hydrolysis The products of phosphoinositide (22) metabolism, inositol trisphosphate and diacylglycerol, stimulate an increase in cytosolic free Ca2+ and protein kinase C activity, respectively (23). Activation of kinase C leads to an enhancement of protein calcium mobilization stimulation of Nat/H+ exchange via (24,25). In permeabilized human platelets, increasing the intracellular pH results in enhanced calcium mobilization in response to inositol trisphosphate stimulation Ticlopidine and PCR 4099 inhibit ADP-mediated (26). intracellular calcium mobilization in rabbit (8) and rat (27) platelets, respectively, inhibition of phospholipase possibly through C activation Potentiation of both calcium influx and mobilization by epinephrine (27). has recently been shown to occur via a cyclooxygenase-independent mechanism in human platelets (28). Lalau-Keraly et al. (29) have demonstrated in rabbit platelets that epinephrine potentiates the effect of ADP on inositol monophosphate and inositol bisphosphate formation without affecting formation of inositol trisphosphate. It is conceivable, therefore, that subsequent to activation of Nat/H+ exchange, alterations in phosphoinositide metabolism may play a role in the potentiating effect of epinephrine (8). Ticlopidine thus blocks aggregation by a mechanism sensitive to platelet Nat/H+ exchange. Ticlopidine may also affect the induction of fibrinogen receptor exposure by altering signal transduction mediated by the ADP receptor. Platelets from rats that have been pre-treated with ticlopidine showed a similar profile of aggregation as platelets, from control rats, that were treated with the ADP metabolizing enzyme creatine phosphokinase (30). The conclusion drawn from these was studies that ticlopidine specifically inhibits the effects of released ADP. Since epinephrine increases the affinity of the ADP receptor for ADP ten-fold (31), it is possible that the proaggregatory effects of epinephrine are due to an increase in affinity of

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the ADP receptor on ticlopidine-pretreated platelets. This may be secondary to the putative alkalinization induced by epinephrine. Regardless of the exact mechanism by which epinephrine exerts its proaggregatory response, these studies emphasize the importance of examining aggregation induced by multiple agonists when evaluating the overall efficiency of antiplatelet agents. REFERENCES 1.

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