Inhibition of cyclooxygenase-independent platelet aggregation by sodium salicylate

THROMBOSIS RESEARCH 54; 583-593, 1989 0049-3848/89 $3.00 t .OO Printed in the USA. Copyright

(c) 1989 Pergamon

Press plc.

All rights reserved.

INHIBITION OF CYCLOOXYGENASE-INDEPENDENT AGGREGATION

PLATELET

BY SODIUM SALICYLATE.

F. Violi, C. Alessandri, D. Pratico, A. Guzzo, A. Ghiselli and F. Balsano Institute of Clinical Medicine I, University of Rome "La Sapienza:'Policlinico Umberto I, 00161 Rome, Italy. (Received 30.10.1988; accepted in original form I.0.3.1989

by Editor P.M. Mannucci)

ABSTRACT

The effect of acetylsalicylic acid (ASA) on platelet aggregation (PA) and thromboxane A (TxA ) formation was investigated in vitro and ex vivo after 1 g or2300 rn$ASA administration to healthy subjects. 50100 uM ASA inhibited PA by single aggregating agent such as platelet aggregating factor (PAF) or epinephrine and reduced to<5% of control platelet TxB formation, but did not influence PA by epinephrine plus PAF. The lat z er was inhibited by increasing ASA concentration. In sam ples incubated with 100 uM ASA and stimulated with epinephrine plus PAF, PA could,be inhibited by the addition of 100-300 uM sodium sali cylate. After 300 mg-1 g ASA administration to healthy subjects, the inhibition of PA by epinephrine plus PAF was more marked by highest doses of ASA. This study suggests that aspirin inhibits PA with a cl clooxygenase-independentmechanism; this effect is mediated, at least

in vitro,

by salicylic

acid. INTRODUCTION

Platelet-modifying substances have been used in atherosclerotic disease with the aim of reducing thromboembolic events (1). Recent trials showed that ASA, an inhibitor of platelet cyclooxygenase enzyme which prevents the forma tion of TxA2(2, 3), a potent endogenous vasoconstrictor and aggregating agent, improves the natural course of clinical models which are complicated by throm boembolism (4-9). ASA was given

Key

in an extremely variable

words: acetylsalicylic

acid, platelet

dosage, from 300 to 1500 mgdaily,

aggregation, 583

thromboxane.

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but the choice of these pharmacological regimens was empiric. An important point not yet resolved is if the antithrombotic property of ASA depends exclu sivelyoncyclooxygenase inhibition or if ASA is capable of exerting its anti thrombotic activity with other mechanisms (1). In experimental models ASA redu ces thromboembolism with property not related to its effect on cyclooxygenasepathway (10). Furthermore benefical effects showed by clinical trials were ob tained with ASA dosage higher than that necessary to fully block cyclooxygena se pathway. In this research we investigated if ASA regimens close to those common ly used in clinical trials affect platelet function with mechanism independe? of cyclooxygenase inhibition. MATERIAL AND METHODS The study was carried out on 20 healthy volunteers (8 males, 12 females; age 19-40) who did not take any drug known to interfere with platelet function for at least 15 days. None was overweight, 10 habitually smoked 15 cigarettes/ day. All give informed consent to partecipate in this investigation. In vitro study: In vitro study was performed on all but one subject, and consisted in the evaluation of platelet sensitivity to serial concentration of ASA. Baseline PA was performed with single aggregating agent such as PAF or epinephrine and then a minimal concentration of ASA which inhibited PA and reduced to,
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oefore the addition of aggregating agents. In samples where the influence of nordihydroguairetic acid (NDGA), a lipooxygenase and cyclooxygenase inhibitor (12), on "aspirinated" platelets was investigated, PRP was incubated 10 min at 37°C with ASA plus NDGA or ASA plus ethanol as control(PRP-ethanol ratio 500 : I); this volume of ethanol did not influence PA NDGA up to 40 uM (final con centration) was not able to affect the cyclooxygenase pathway. This was checked by measuring platelet TxB2 (see below) in PRP incubated with and without NDGA and stimulated with TC of PAF. Platelet TxB2 (n=4) of control (64 t 3 ng/ml; Mean t SE) and NDGA-treated samples (58 t 4 ng/ml) were not significantly dif ferent. Thromboxane A2: TxA2 was studied on stimulated PRP as previously described (13).Briefly, PAF or epinephrine or epinephrine plus PAF were added and after 3 min the reac tion was stopped by adding absolute methanol; the mixture was centrifuged 2mc 4000 x g and the supernatant stored at -20°C. Platelet TxA2 was evaluated by measuring its metabolite TxB2 ( TxB2 1125, NEN ). Blood salicylate determination: Blood sample was taken from each subject and centrifuged IO min 2000 x g; aliquots of serum were stored at -20°C until use. The method was based on Trinder's procedure (14) which detects the purple colour produced when salicy late reacts with ferric ions in a weakly acidic medium. The reagent was prep: red by mixing 4 g HgCl in 850 ml bidistilled water with 40 g Fe2(N0 ) in3$ r and 120 ml 1 N HCL..The so1ution was raised to 1 liter with bidistilled wat stored in dark bottle at room temperature before use. Standard salicylate solu tion was prepared dissolving 200 mg salicylic acid in 100 ml bidistilled water with 5 drops of chloroform; working standard solution was 20 mg/dl. 1 ml of se rum or standard solution or bidistilled water was thoroughly mixed with 5 mlrf reagent and after 5 min centrifuged 20 min 2000 x g. The optical density of supernatant was read at 540 nm (14) Materials: PAF (Sigma, Chemical Company, USA) dissolved in chloroform-methanol (Merck)(2:1), was dried under N2 and stored at -70°C until use. PAF working so lution was prepared by diluting evapored aliquots with Hank's balanced solution Epinephrine (Stago) was dissolved in Michaelis buffer, ratio 1:lO. Aspirin as its soluble lysine salt (Flectadol R, Maggioni, Italy) and sodium salicylate (Merck) were dissolved in distilled water. NDGA (Sigma) was dissolved in absoll te ethanol (Merck). HgC12, Fe2(N03)3 (Merck). Statistical analysis: Data reported are expressed as Mean t SE, Student's "t" test was used. RESULTS In vitro sensitivity to aspirin: Before stimulating platelets with couple of aggregating agents,the mini ma1 quantity of ASA necessary to inhibit PA induced by single aggregating ager% was checked. ASA inhibited PAF or epinephrine-inducedsecondarywaveofaggrega-

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tion in a range from 50 to 100 uM (Fig.l), TxB of these samples wasG5% of con trols (Fig.1). Epinephrine plus PAF increased 6A and platelet TxB2formationanb still induced irreversible PA of aspirin-treated PRP, despite the almost fully block of TxB2 production (Fig-l). When ASA concentration was gradually increa sed PA curve returned reversible (Fig-l).

PIE P+E*A

P

P+E*3A

r-l

I1

I

FIG. 1 Representative PA tracing and platelet TxB2 formation from PRP of one healthy subject. P 4.5 nM PAF, E = 1 uM epinephrine, A = 100 uM ASA, ??

3A = 300 uM ASA.

The minimal dose of ASA necessary to prevent epinephrine plus PAF-induced PA was largely variable. Males were more sensitive than females since 75% showed PA inhibition by<300 uM ASA;), uM ASA were, on the contrary, necessary to fully inhibit PA in 75% of females. NOGA up to 40 uM did not influence PA by single or pair of aggregating agents (data not shown), but inhibited PA in a dose-dependent fashion if incuba ted with aspirin-treated platelets stimulated by epinephrine plus PAF (Fig.2).Sodium salicylate in a range of 100-300 uM did not affect either PA or TxB2 formation by single (data not shown) or couple of aggregating agents (Fig. 3). In sample incubated with 100 uM ASA and stimulated by epinephrine plus PAF,

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t

60-

CI

50LO3020lO-

FIG. 2 ?A (n=5) in aspirin (100 uM)-treated samples incubated with 0, 1, 10, 20 uM NDGA and stimulated by epinephrine (0.7-2 UM) piUS PAF (i-10 nM). * P4 O.025 ** Pd 0.01 ?A was irreversible despite an almost complete suppression of TxB2 (Fig.3). The addition of sodium salicylate (100-300 uM) to aspirin-treated sample signi ficantly reduced PA (Fig.3); this effect was mainly due to the inhibition of secondary aggregation curve. Ex vivo study I: PA by PAF or epinephrine or epinepnrine plus PAF was significantly inhi bited 2 and 24 hours after 1 g ASA intake (Fig.4). PA wave by single aggregating agent was always reversible in all subjects while PA by epinephrine plus PAFwas reversible in all subjects after 2 hours and in all but two after 24 hours. TxA2 formation by epinephrine was higher than that by PAF, but the diffe rence was not significant; TxA by pair of aggregating agents was significantlyinhibited and almost suppresse8 2 and 24 hours after ASA intake whatever stimu lus was employed (Fig.5). Ex vivo study II: PA induced by PAF or epinephrine was reversible in all subjects 2 and 24 hours after ASA ingestion. PA by epinephrine plus PAF was significantly inhi bited after 2 hours and was still reduced, but not significantly, after 24hous (Fig.6); PA was reversible in 50% (3 males and 2 females) and in only 20%

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100

90

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LT X 1

60 70

1

60

-I

50 40 30 20 10 0 PAF-I-E

90

Tx62

PAF+E+AsA

pAF+tI+ss

(ng/ml)

60 70 60 50 40 30 20 10 0

FIG. 3 PA and TXB

formation in samples (n=6) incubated with and without 106 uM ASA and/or 100-300 uM sodium salicylate (SS), stimulated with epinephrine (0.7-2 uM) plus PAF (I-10 nM). * p < 0.001

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FIG. 4 PA by 0.7-Z uM epine phrine (E), I-IO nMPAF or epinephrine plus PAF, before and after 2 and 24 hours from 1 g ASA intake. (n=ll)

LT (9.)

,oo

90 80 70

** p c 0.005 * p (0.001

60 50 40 30 20 10 0

0

2h

24h

FIG. 5 TxB2 formation by PAF (P), epinephrine (E), epinephrine plus PAF in subjects given 1 g ASA. (n=ll)

TxB2 (no/ml) 120 100

** p c 0.05 * p
0 WELINE

2h

24h

PLATELET AGGREGATION & SALICYLATE

590

LT(xl t

,oo

FIG. 6

90

PA by 0.7-I IJMepinephrine plus l-10 nM PAF beforeand after 2 and 24 hours from 300 mg ASA intake (n=lO).

80 70

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z

60 50

* p<

0.005

40 30 20 10 0 0

(1 male and 1 female) after 2 and 24 hours respectively).PlateletTxB 148 t 15 SD ng/ml, 6 t 1.2 SD ng/ml and 6 t 1.4 SD ng/ml at 0, 2and24

(n=6) was hours re

specfively. Blood salicylate: The value of blood salicylate we found are comparable with those already reported in literature (15). The blood salicylate concentration was dependent on the ASA regimen; in fact, significantly higher values were detected 2 hours after 1 g ASA than after 300 mg (Fig.7). Significant difference between males and females was not seen even if fema les tended to have higher values (2 hours after 1 g ASA: females 79 t 24 SD ug/mT males 66 t 18 SD ug/ml). SALICY

LATE 1

&g/ml)

FIG. 7 Blood salicylate

2 and 24

hours after 1 g (n=ll) and 300 mg (n=lO) ASA. * p < 0.001

2h

0

1g

l.lmnul300

ASA mg ASA

24h

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DISCUSSION Aspirin is an inhibitor of cyclooxygenase pathway and prevents the forma tion of TxA2. This should be the mechanism explaining its antithrombotic proper ty, but in experimental and clinical models the inhibition of thromboemboliccorn plications has been achieved by doses of aspirin much higher than those necessary to prevent the formation of TxA . Therefore it is not yet clear if the antithrom ootic property of aspirin is soTely related to the inhibition of cyclooxygenasepathway (4-10). Our demonstration that aspirin inhibits PA with a mechanism not relatedto the inhibition of cyclooxygenase pathway gives new insights into the antithrombo tic property of ASA. Using high concentrations of agonists or pair of aggregating agents, which stimulate PA with a cyclooxygenase-independentmechanism ((12, 13, 16), we demonstrated that ASA inhibits in vitro PA in a dose-dependent fashion. This effect should be mediated by salicylic acid; indeed in SampleswherePA by epinehrine plus PAF was irreversible despite the platelet incubation with IOOuM ASA, a significant inhibition could be achieved by the addition of 100-300 uM sodium salicylate. Our findings are apparently in contrast with previous investi gations showing that salicylate does not influence PA. However, these studiesdid not evaluate the influence of aspirin and salicylate on PA by pairs of agonists (17, 18). It is noteworthy that the concentrations of salicylate we used arecorn monly found after 300 mg - 1 g ASA intake (19). Indeed after ASA administration to healthy subjects we found salicylate blood concentrations similar to those used in vitro. The salicylate concentration was dependent on the quantity of ASA administered, reaching highest values after 1 g ASA; this could perhaps explain the more marked PA inhibition observed with this dose of ASA. Salicylate hasbeen reported to inhibit the transformation of HPETE to HETE (20), which could be in volved in the activation of platelets (12, 21). Previous studies, where a similar in vitro model was used, showed that NDGA, a lipo- and cyclooxygenase inhibitor, at concentration up to 200 uM, inhibited PA in samples pre-treated with aspirin (12, 15). Using lower NDGA concentrations which did not influence cyclooxygenase pathway, we observed similar findings and demonstrated that the inhibition of cl clooxygenase-independent PA by NDGA is dose-dependent; these data should exclude that the inhibition we observed is unspecific. Even if this finding could support the hypothesis that the inhibition of cyclooxygenase-independentPA by salicylate is due to the inhibition of lipooxygenase pathway, the direct measurement of lipo oxygenase products is necessary to support this suggestion. In conclusion this study shows that the inhibition of cyclooxygenase-inde pendent PA by aspirin is mediated by salicylate; this could contribute to further elucidate the antithrombotic property of aspirin and might have therapeutic im plications. REFERENCES I. HARKER, L.A., FUSTER, V.: Pharmacology of platelet inhibitor. J.A.C.C., 8, 21b-32b, 1986. 2. ROTH, G.L., MAJERUS, P.W.: The mechanism of the effect of aspirin on human This study is in part supported by Andrea Cesalpino Foundation, Rome, Italy.

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platelets. I Acetylation of a particulate fraction protein. J. Clin. Invest., 56, 624-632, 1975. 3. PATRIGNANI, P., FILABOZZI, P., PATRONO, C.: Selective cumulative inhibition of platelet thromboxane by low-dose aspirin in healthy subjects. J. Clin. Invest., -69, 1366-1372, 1982. 4. DALE, J., MYHRE, E., STORSTEIN, O., STORMORKEW, H., EFSKIND, L.: Prevention of arterial thromboembolism with acetylsalicylic acid. A controlled clinical study in patients with aortic ball valves. Am. Heart J., 94 > 101-111, 1977. 5. HERSHEL, R., HARTER, H.R., BURCH, J.W., MAJERUS, P.W., STANFORD, N., DELMEZ, J.A., ANDERSON, C.D., WEERTS, A.: Prevention of thrombosis in patients on hemodialysis by low-dose aspirin. N. Engl. J. Med., 301, 577-579, 1979. 6. CANADIAN COOPERATIVE STUDY GROUP: A randomized trial of aspirin and sulfin pyrazone in treatened stroke. N. Engl. J. Med., 299, 53-59, 1978. 7. LEWIS, H.D. jr., DAVIS, J.W., ARCHIBAL, D.G., STEINKE, W.E., SMITHERMAN, T. C ., DOHERTY, J.E., SCHNAPER, H.W., LEWINTER, M.M., LINARES, E., POUGET, J. M., SUBHASH, C., SABHAR, W.: Protective effects of aspirin against acute myocardial infarction and death in men with unstable angina. N. Engl. J. Med., 309, 396-403, 1983. -8. CAIRNS, J.A., GENT, M., SINGER, J., FINNIE, K.I., FROGATT, G.M., HOLDER, D. A ., JABLONSKI, G., KOSTUK, W.J., MELENDEZ, L.J., MYERS, M.G., SACKETT, W.J., SEALEY, B.J., TANSER, P.H.: Aspirin sulfinpyrazone or both in unstable angi na: results of a Canadian multicenter trial. N. Engl. J. Med., 313, 13691375, 1985. 9. LORENZ, R.L., WEBER, M., KOTZUR, J., THEISEN, K., SCHALKY, C.V., MEISTER, W., CHARDT, B.R., WEBER, P.C.: Improved aortocoronary bypass by low-dose aspirin (100 mgldaily). Effects on platelet aggregation and thromboxane for mation. -Lancet, 1, 1262-1264, 1984. lO.HANSON, S.R., HARKER, L.A., BJORNSSON, T.D.: Effect of platelet-modifying drugs on arterial thromboembolism in baboon. Aspirin potentiates the anti thrombotic actions of dypiridamole and sulfinpyrazone by mechanisms indepen dent of platelet cyclooxygenase inhibition. J. Clin. Invest., -75, 1591-1595, 1985. ll.VIOLI, F. GHISELLI, A., ALESSANDRI, C., FRATTAROLI, S., IULIANO, L., BALSA NO, F.: ctivation of platelet cyclooxygenase by red blood cells in vitro, N. Engl. J. Med., 313, 1091-1092, 1985. 12.MACCON1, D., MORGENTI, G., LIVIO, M., MORELLI, C., CASSINI, G., REMUZZI, G.:

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Acetyl glyceril phosphorylcholine aggregates human platelets through two distinct pathway, both dependent on arachidonic acid metabolism. Lab. Invest., -52, 159-168, 1985. 13.VIOL1, F., PRATICO', D., GHISELLI, A., ALESSANDRI, C., CORDOVA, C.: Human platelet aggregation by PAF and thromboxane production. Thromb. Res., _' 48 265-266, 1987. 14.TRINDER, P.: Rapid determination of salicylate in biological fluids. Biochem. J., -57, 301-303, 1954. 15.CERLETT1, C., BONATI, M., DEL MASCHIO, A., GALLETTI, F., DEJANA, E., TOGNO NI, G., DE GAETANO, G.: Plasma levels of salicylate and aspirin in healthy volunteers. Relevance to drug interaction on platelet function. J. Lab. Clin. Med., 103, 869-877, 1984. 16.CERLETl.1, C., CARRIERO, M.R., DE GAETANO, G.: Platelet aggregation response

to single or paired aggregating stimuli after low-dose aspirin. N. Engl. J. Med., 314, 316-317, 1986. 17.ROO-LEE, L.M., ELMS, M.J., CHEM, B.E., BOCHUER, F., BUNCE, I.H., EADIL, ci. J .. * Plasma levels of aspirin following effervescent and enteric-coated ta blets and their effects on platelet function. Eur. J. Clin. Pharmacol., 73 _' 545-551, 1982. 18.DE GAETANO, G., CERLETTI, C., DEJANA, E., LATINI, R.: Pharmacology of pla telet inhibition in humans: implications of the salicylate-aspirin inter%tion. Circulation, -72, 1185-1193, 1985. lg.BRANDON, R.A., EMMETT, J.A.G., EADIE, M.J.,CURRAN, A.C.W., BUNCE, 1-M.: Peripheral venous aspirin concentrations and platelet aggregation inhibition produced by enteric-coated aspirin formulations. Thromb. Haemostas., _' 55

20.SIEGEL, M.I.,MCCONNELL, R.T., CUATRECASAS, P.: Aspirin-like drugs interfe res with arachidonic metabolism by inhibition of the 12-hydroperoxy-5,8,1F 14-eicosatetraenoicacid peroxidase activity of the lipooxygenase pathway. Proc. Nat. Acad. Sci. USA, -76, 3774, 1979. 21.MEHTA, P., MEHTA, J., LAWSON, D., KROP, I., LETTS, L.G.: Leukotrienes tiate the effects of epinephrine Thromb. Res., -41, 731-738,

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poten

and thrombin on human platelet aggregation.