Influence of various doses of aspirin (in vivo) on platelet arachidonic acid metabolism (ex vivo) and function

PROSTAGLANDINSLEUKOTRIENES ANDESSENTIALFATTYACIDS

Influence of Various Doses of Aspirin (In Vivo) on Platelet Arachidonic Acid Metabolism (Ex Vivo) and Function G. H. R. Rao and J. G. White Depurtment of Lahomtor-y Medicine and Pathology. (Reprint requests to GHRR)

University of Minnesota,

Minneapolis,

Mimewttr

55455, USA

ABSTRACT. The influence of various doses of enteric-coated aspirin was evaluated for its in vivo effect on ex vivo platelet arachidonic acid metabolism and function. 24 h after ingestion of the drugs compromised platelet response to the action of agonists such as epinephrine and arachidonate could be demonstrated with as low a dose as 50 mgs aspirin. However, platelets with compromised function were still capable of producing significant quantities of thromboxane. On the other hand, platelets with almost total inhibition of cyclooxygenase activity were capable of aggregating irreversibly when challenged with epinephrine and arachidonate.

INTRODUCTION

low dose aspirin (15. 16). In a recent study we demonstrated that 50 mg of enteric-coated aspirin taken daily over a period of 2 weeks significantly compromised the ability of circulating platelets to convert arachidonic acid to eicosanoids ( 17). In this paper we report our findings on the effect of various doses of enteric-coated aspirin on ex vivo platelet function.

Aspirin (acetyl salicylic acid [ASA]) is one of the oldest and relatively most cost effective drugs for the prophylaxis and therapy of clinical cardiovascular complications associated with platelet hyperfunction (I-3). It is an irreversible inhibitor of cyclooxygenase, an enzyme that converts arachidonic acid to prostaglandin (PG) endoperoxides PGG? and PGH2 (4, 5). Formation of these novel transient metabolites is essential for further transformation to thromboxane, a potent vasoconstrictor, by thromboxane synthetase in platelets and vasodilatory prostacyclin by prostacyclin synthetase in vascular tissues (6). Endothelial cells possess cyclooxygenase and can synthesize prostaglandin endoperoxides (7). Therefore. aspirin can inhibit arachidonic acid conversion to vasoactive metabolites in both these cell types. However, endothelial cells can regenerate this enzyme, whereas platelets cannot (7). In view of this difference in vascular and platelet biology, there is considerable interest in the use of low dose aspirin to achieve a ‘favorable disposition’ for the production of vasodilatory prostacyclin (Y-13). Studies from our laboratory demonstrated that as low a dose as X0 mgs of aspirin can significantly lower platelet thromboxanc production (14). Since then two major clinical trials have demonstrated the beneficial effects of

MATERIALS Arachidonic acid as sodium salt was obtained from NuChek Prep, Elysian, MN and made up in 0.1 M Tris buffer, pH 7.4. Radio-labelled ( ‘-14C) arachidonic acid was from New England Nuclear. Boston. MA. Entericcoated aspirin (50 mg) was a gift from German Remedies, Ltd, Bombay, India. 8 1 mg HalfprinTM. a low strength enteric-coated aspirin, was donated for these studies by Kramer Laboratories, Inc, Miami. FL. Injectable adrenalin (epinephrine) (Parke Davis) was obtained from the pharmacy of the University of Minnesota Hospitals. Unless otherwise mentioned, all other chemicals were from Sigma Chemical Company. St Louis MO.

METHODS Blood for these studies was obtained from normal adult donors, after informed consent. Blood was drawn into 35 ml capacity sterile plastic syringes and mixed immediately with trisodium citrate-citric acid dextrose (CCD) anticoagulant (0.1 M citrate, 7 mM citric acid. 0.14 M

I)ats received

I? November IYY? Date accrptcd 70 December lYY3 63

64

Prostaglandins

Leukotrienes

and Essential Fatty Acids

dextrose, pH 6.5) in a ratio of 9 parts blood to 1 part CCD. Platelet-rich plasma (PRP) was separated by centrifugation of anticoagulated blood at 100 x g for 20 min at room temperature (18). The response of platelets to agonists was evaluated on a Chronolog Lumiaggregometer (Chronolog Corp, Havertown, PA). Maximum amplitude of the recording pen was preset with platelet-poor plasma (PPP) and PRP from the same source of blood. Luciferase (Chronolume, Chronolog Corp) was used for monitoring the release of adenosine triphosphate (ATP) from activated platelets (19). Human subjects who agreed to participate in this study were divided into five groups, Since the specific focus of this study was to determine the effective starting daily dose of aspirin, all donors took different doses of aspirin on day 1 and later took one 50 mg aspirin daily. The first group was given a daily dose of 50 mg EC-ASA. The second group received EC-ASA twice a day (2 x 50 mg). whereas the third group received the drug three times a day (3 x 50 mg). The fourth group was advised to take two Halfprin (2 x 81 mg) per day. The fifth group received one full strength regular aspirin (325 mg). After the ingestion of the various doses of aspirin on day 1, all participants received one 50 mg EC-ASA per day on subsequent days. Blood was drawn before aspirin ingestion, 24 h past ingestion and then 72 h following drug administration. Platelet function studies and radiolabelled arachidonic acid conversion was followed using platelets from each donor. Each study was performed at least three times from blood obtained from different donors. Tracings presented are typical findings of an individualized study. Oxidation of arachidonic acid by platelet cyclooxygenase was followed by a modification of the method of Hamberg et al (1975). Five ml volumes of platelets (3 x lo8 cells/ml) in plasma were mixed with 0.1 M EDTA (1: 10, EDTA:PRP) and centrifuged at 600 x g for 1.5min to obtain platelet pellets. Supematant was discarded and the platelet pellet was resuspended in 1 ml CCD containing adenosine (5 mM) and theophylline (3 mM). Suspensions of platelets were centrifuged again and the pellet obtained was resuspended in 1 ml of Hank’s balanced salt solution without calcium and magnesium. Each sample containing approximately 1.5 x 10” cells was stirred on the platelet aggregometer 37” C for 5 min with 1 pg of “C-arachidonic acid and 10 pl of 100 mM CaCl*. The reaction was stopped by the addition of 2 ml of ethyl acetate. After acidification with citric acid (0.5 M) to a pH of 3.5, each sample was extracted twice with ethyl acetate. Fractions of ethyl acetate from each sample were pooled, concentrated by bubbling nitrogen and placed on a silica gel G plate. The solvent system used for the separation of thromboxane was ether:methanol:acetic acid (135:2:2 v/v). Radiolabelled metabolites separated on thin layer plates were scanned for radioactive spots with a Berthold radiolabel scanner (18). Areas with radioactivity were scraped from the plates, placed in scintillation vials, and counted in a

Beckman Scintillation Counter. Standard statistical procedures were used to calculate mean, standard deviation and student’s t-test of significance.

RESULTS

Influence of various doses of aspirin on ex vivo platelet function Platelets obtained from all donors before ingestion of aspirin aggregated irreversibly when stirred with epinephrine (5 pM) and arachidonate (0.45 mM). They secreted significant quantities of ATP in response to these agonists (Fig. 1). Platelets obtained from different donors 24 h after ingestion of aspirin, irrespective of the dose used, showed a compromised response to the action of epinephrine and arachidonate (Fig. 2). Aspirin-treated platelets, when challenged with epinephrine, failed to produce a second wave of aggregation. They did not secrete ATP. Similarly, platelets exposed to aspirin did not aggregate when stirred with threshold concentration of arachidonate. Earlier studies from our laboratory demonstrated that drug-exposed platelets, although showing a compromised response when tested with single agonists, respond in a normal fashion and aggregate irreversibly when challenged with a combination of epinephrine and arachidonate (20). In this study, exposure of aspirintreated platelets to epinephrine first, and then challenged with arachidonate, restored aggregation and a partial secretory response (Fig. 3).

Influence of various doses of aspirin on ex vivo platelet arachidonic acid metabolism Platelets obtained from donors prior to aspirin ingestion, upon exposure to arachidonic acid converted approximately 25.6% of the radiolabelled substrate to thromboxane B,, the stable metabolite of thromboxane A, (Table). Platelets obtained from donors 24 h after ingestion of aspirin converted from a low of 2.6% to a high of 21.6% of the substrate to thromboxane, depending upon the dose of aspirin. Each dose used exerted significantly different inhibitory effects on the ability of platelets to metabolize arachidonic acid. Platelets obtained from donors 72 h after aspirin ingestion showed significant compromise in their ability to convert arachidonic acid. A dose of aspirin above 100 mg per day was very effective in inhibiting platelet cyclooxygenase activity.

DISCUSSION The rationale for the present study evolved from comments and relevant criticisms made in an editorial in the lrzdiarz Heart Jozu-rzal regarding the efficacy or nonefficacy of the low dose aspirin prophylaxis (21). The

Influence of Various Doses of Aspirin (In Vivo) on Platelet Arachidonic Acid Metabolism (Ex Viva) and Function

65

AA

/--

AT* change

ATP released

in light transmission

Fig. 1 Platelets obtained from normal donors aggregated irreversibly when stirred with agonists such as epinephrine (Epi), arachidonate acid (AA), and secreted significant quantities of ATP

authors of this editorial conclude that ‘there is a definite need for well designed, controlled studies of low dose regimens.’ This study is part of an ongoing study on the pharmacology of aspirin and its effects on platelet physiology and function, aimed at clarifying how low a dose is low for a start-up aspirin prophylaxis protocol. In an earlier study we demonstrated that low dose

Influence Platelet

regular aspirin (40-80 mg) significantly reduced platelet cyclooxygenase activity and effectively restrained the aggregatory response of platelet to arachidonate stimulation (14). Since then many studies have reported beneficial effects of low dose aspirin (21-25). Recently several enteric-coated low dose aspirin have been made available for commercial use. Using one such commercial preparation (EC-ASA, German Remedies, Bombay, India), studies from our laboratory demonstrated that even

of Low Dose Aspirin (in vivo) on Response (ex vivo) to Agonists Epinephrine Potentiation of Arachidonate Action on Aspirin Exposed (in vivo) Platelets t-

-~

3

1 minute

/

/

1 mrnute

A-’

Control

EY’ G4

__L,-YY1 Eprnephrrne

Platelets

AT’

HolllGPGst Aspirin

(ELI; 5flM)

-"-I~ 1

Arachldonate

(AA; 0.45mM)

.y-/Epr”ephrlne I-

(Epl. 5/(M)

Arachldonate (AA. 0.45mM)

t AT* Change

in light transmission

Fig. 2 Platelets obtained from normal donors aggregated irreversibly and hecreted ATP when stirred with epinephrine or arachidonate. However. platelets obtained from donors 24 h after ingestion of aspirin farled to respond with aggregation when tested with these agonists.

AT* change rn Irght transmrssron

Fig. 3 Platelets obtained from donors 24 h after ingestion of 325 mg aspirin failed to aggregate in response to epinephrine or arachidonate. However, when challenged with epinephrine and arachidonate, these drug-exposed. refractory platelets aggregated irreversibly with a slightly compromised secretion of ATP.

66

Prostaglandins

Leukotrienes

and Essential Fatty Acids

Table Influence of various doses of EC-ASA ingestion on ex vivo platelet arachidonic acid conversion fo thromboxane (Bz)

Aspirin dose (mg)

0 Day

1 Day

Post-Aspirin* 3 Day

50 2 x 50 3 x 50 1 x81 1 x325

25.7 23.4 23.9 21.6 27.2

21.6? 16.7 ? 15.5 + 8.2 f 2.6 *

2.6 1.4 0.32 1.7 0.3

f 3.3 i 3.0 f 3.4 I? 2.8 f 2.6

11.2* 8.9 f 6.2 + 4.8 k 3.0 f

P values:

1day

3 day

50 mgs compared to control 2 x 50 mgs compared to 50 mgs 3 x 50 mgs compared to 50 mgs 2 x 80 mgs compared to 50 mgs 1 x 325 mgs compared to 50 mgs 3 x 50 mgs compared to I x 80 mgs

< < < < < <

< < < < < <

0.09 0.07 0.06 0.003 0.001 0.015

1.9 1.7 3.0 0.36 0.52

0.001 0.02 0.04 0.003 0.001 0.039

*Percent conversion: mean and the standard deviation (n = 3). Platelets from normal donors converted significant amounts of radiolabelled arachidonic acid to thromboxane (B,). Aspirin exerted a dose-dependent inhibition of arachidonic acid.

a low dose aspirin (50 mg) taken daily over a period of time significantly lowered the ability of circulating blood platelets to convert arachidonic acid to thromboxane (17). However, studies in normal volunteers and patients with unstable angina have shown that a dose of 75 mg may not be sufficient to completely inhibit thromboxane production and platelet function (14, 17, 26). In this study, irrespective of the type or dose of aspirin administered, the drug effectively prevented secondary aggregation and secretion of ATP in response to threshold concentrations of epinephrine. Platelets exposed to various concentrations of aspirin failed to respond to arachidonate and undergo irreversible aggregation. However, as we have observed in previous studies, drugexposed platelets, when challenged with epinephrine and arachidonate, aggregated irreversibly and secreted limited quantities of ATP. Studies with radiolabelled arachidonic acid showed that each dose of aspirin tested had a significant inhibitory effect on platelet cyclooxygenase activity. As expected, a 50 mg dose had the least inhibitory effect and the full strength aspirin (325 mg) the most inhibitory effect on platelet enzymes. Results of this study suggest that greater than 100 mg of aspirin are required to rapidly reduce significant thromboxane synthesis by activated platelets, Halfprin (2 x 81 mg) seems to be more effective than (3 x 50) EC-ASA in inhibiting platelet cyclooxygenase activity when compared with results from platelets obtained 24 h post-drug ingestion. This relationship holds good even for the data after a 72 h period. Further studies are needed to clarify such subtle differences between low doses taken several times a day versus higher doses taken less frequently over a long period of time. A surprising finding of this study is that even a reduction in as low as 15% synthesis of thromboxane can induce apparently compromised platelet function as shown by the response of platelets to epinephrine and

arachidonate. However, if one looks at the ability of platelets to make thromboxane 24 h post-ingestion of 50 mg EC-ASA they are only minimally compromised. They still can make as much as 80% of their capacity (2 1.6%) compared to the activity of normal control platelets (25.7%). Therefore. just platelet function testing may not reveal the relative degree of compromise in cyclooxygenase activity. Similar findings were reported from our laboratory in an earlier study evaluating the recovery of cyclooxygenase activity and platelet function following in vivo aspirin exposure (IX). In addition to this difference, the fact that platelets exposed to even the highest concentration of aspirin used in this study (325 mg) responded to the combined action of epinephrine and arachidonate has to be taken into consideration. Further well-controlled studies are essential for designing therapeutic protocol for the treatment of platelet-related clinical complications (25-30). In conclusion the present study demonstrated that a single dose of aspirin ranging from 50 mg to 325 mg effectively lowers platelet cyclooxygenase activity and their response to the action of single weak agonists such as epinephrine and arachidonate. Although a significant compromise in platelet function can be demonstrated by platelet function studies, cells exposed to lower doses of aspirin retain their ability to synthesize significant quantities of thromboxane. Repeated doses of aspirin seem to significantly lower the ability of platelets to convert arachidonic acid to thromboxane. Finally. even with the highest dose of aspirin (325 mg) tested with very little thromboxane production capability drug exposed platelets aggregated irreversible to the action of combined epinephrine and arachidonate. Results of these studies, taken together with our earlier observations, suggest the need for further studies to find appropriate protocols for aspirin therapy in preventing or reducing platelet-related cardiovascular complications.

Acknowledgments This work was supported by grants from the National lnstitutes of Health HLI 1880 and HL49.556 (USA). The authors wish to thank Mrs Julie Lee and Mr Naveen Murthy for their technical assistance and MS Susan Schwarze for her help in the preparation of the manuscript.

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lntluence of Various Doses of Aspirin (In Viva) on Platelet Arachidonic Acid Metabolism IEX Vicol and Funclmn

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Proc Nat Acad Sci USA

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