Comparative Effects of Phenylbutazone, Naproxen andFlunixin Meglumine on Equine Platelet Aggregation and

Platelet Factor 3 Availability in vitro

I.B. Johnstone*

ABSTRACT

Nonsteroidal anti-inflamma-tory drugs are commonly usedin the treatment of inflamma-tory conditions, and have poten-tial value in the treatment ofthrombotic disease in the horse.This study compares the potencyof three nonsteroidal anti-inflammatory drugs phenyl-butazone, naproxen (equiprox-en) and flunixin meglumine(banamine) with respect to theireffects on equine platelets. Twofunctional responses of horseplatelets were evaluated in vitro:their ability to aggregate andtheir ability to make availableplatelet factor 3 procoagulantactivity.Flunixin at a concentration of

106 M significantly depressedthe maximum degree of adeno-sine diphosphate-induced (10-6M) aggregation while muchhigher concentrations of phenyl-butazone and naproxen (5 x 10-5M) were required to producesimilar effects. None of the non-steroidal anti-inflammatorydrugs significantly affected theduration of the lag phase or theinitial velocity of adenosinediphosphate-induced aggrega-tion within the range of drugconcentrations used (10-610-3M). The lag phase and initialvelocity of acid-soluble collagen-induced aggregation were signi-ficantly affected by 10-6 M flu-nixin and 10-4 M phenylbuta-zone or naproxen was required

to produce equivalent effects.Concentrations of 5 x 10-6M flu-nixin and 5 x 10-4 M phenylbut-azone or naproxen were re-

quired to significantly depressthe degree of collagen-inducedaggregation of horse platelets.Although the effects of the

nonsteroidal anti-inflammatorydrugs were qualitatively sim-ilar, flunixin was a much more

potent inhibitor of plateletaggregation than either of theother two drugs (which were

equipotent). At very high drugconcentrations (5 x 10-4 M andgreater), all three drugs pro-duced the same degree of inhibi-tion of equine platelet aggrega-tion.

Platelet factor 3 activity wasmade available by exposinghorse platelets to 10-5 M adeno-sine diphosphate or 1:800 acid-soluble collagen; but not byexposure to a suspension of kao-lin particles. Only a small por-tion of the total platelet factor 3activity was made available onstimulation with either adeno-sine diphosphate or collagen.Pretreatment of horse plateletswith any of the nonsteroidalanti-inflammatory drugs (10-4Mconcentration) had no signifi-cant effect on adenosine diphos-phate or collagen-induced pla-telet factor 3 availability.Key Words: Nonsteroidal anti-inflammatory drugs, equine pla-telets, platelet aggregation, pla-telet factor 3 availability.

RlSUMROn utilise frequemment des

drogues anti-inflammatoiresautres que des steroldes pour

traiter des conditions inflamma-toires; ces drogues offrent aussiune valeur potentielle pour letraitement de la maladie throm-botique du cheval. Cette experi-ence visait a comparer les possi-bilites de trois de ces drogues, asavoir: la phenylbutazone, lenaproxen, ou equiproxen, et lameglumine de flunixine, ou

banamine, relativement a leurseffets sur les plaquettes du che-val. On etudia in vitro les deuxreponses fonctionnelles sui-vantes de ces plaquettes: leurhabilite a former des agregats eta rendre disponible l'activiteprocoagulante du facteur #3plaquettaire.Une concentration de 10-6 M

de flunixine deprima de faconappreciable le degre maximumde l'agregation provoquee par10-6 M d'adenosine-diphosphate,tandis qu'il fallut des concentra-tions beaucoup plus elevees dephenylbutazone et de naproxen(5 X 10-5 M) pour produire deseffets similaires. Ni l'une ni l'au-tre des trois drogues experimen-tales n'affecta de facon appreci-able la duree de la phase de delaiou la v6locite initiale de l'a-gregation provoquee par l'ad&nosine-diphosphate, a l'interieurdes concentrations qu'on utilisa,i.e. 10-3-10-6 M. La phase de delaiet la velocite initiale de l'agrega-

Can J Comp Med 1983; 47: 172-179.

*Department of Biomedical Sciences, Ontario Veterinary College, University of Guelph, Guelph, Ontario NlG 2W1.

Submitted June 16, 1982.

172

tion provoquee par le collagEnesoluble dans l'acide s'averbrentaffectees de fagon significativepar 10-6 M de flunixine. II fallut106 M de ph6nylbutazone ou denaproxen pour produire deseffets equivalents. I1 fallut uti-liser 5 X 10-6 M de flunixine et5 x 104 M de ph6nylbutazone oude naproxen pour deprimer defacon significative le degr6d'agregation des plaquettesequines provoque par lecollagbne.Meme si les effets des trois

drogues exp6rimentales se r6v6-lIrent qualitativement simi-laires, la flunixine s'av6ra uninhibiteur de l'agr6gation pla-quettaire beaucoup plus puis-sant que l'une ou l'autre des deuxautres drogues, lesquelles affi-chbrent par ailleurs une puis-sance egale. A des concentra-tions aussi 6lev6es que 5 x 10-4 Mou plus, les trois drogues pro-duisirent le meme degr6 d'inhi-bition de l'agr6gation plaquet-taire Equine.

L'activitW du facteur #3 pla-quettaire devint disponible enexposant les plaquettes EquinesA 10-5 M d'ad6nosine-diphos-phate ou A 1:800 de collagOnesoluble dans l'acide, mais non enles exposant A une suspension departicules de kaolin. Seulementune petite partie de l'activitetotale du facteur #3 plaquettairedevint disponible, A la suited'une stimulation par l'ad6no-sine-diphosphate ou le collagEne.Un traitement pr6alable desplaquettes Equines avec l'une oul'autre des trois drogues exp6-rimentales, A la concentration de10-4 M, n'exerca aucun effetappreciable sur la disponibilit6du facteur #3 plaquettaire pro-voqu6e par l'ad6nosine-diphos-phate ou le collagMne.Mots cl6s: drogues anti-inflamma-toires non steroides, plaquettesequines, agregation plaquettaire,disponibilite du facteur #3 pla-quettaire.

INTRODUCTION

Nonsteroidal anti-inflammatory

drugs (NSAID) inhibit prosta-glandin synthesis in many biologi-cal systems, including blood pla-telets (1, 2, 3). The production ofspecific prostaglandins, particu-larly endoperoxides and throm-boxane A2 (TXA2), plays an impor-tant role in platelet aggregation (4,5). The NSAID impair the produc-tion of these prostaglandins byinterfering with the arachidonatepathway (6). Drugs which mark-edly affect platelet function pre-dispose to impairment of the hemo-static response.

Nonsteroidal anti-inflammatorydrugs are widely used in veteri-nary medicine particularly in thetreatment of inflammatory diseasein the equine species. Unlike theplatelets of some species (man anddog), horse platelets appear to havea much more poorly developedarachidonate pathway (7). Meyerset al (8) have shown that the aggre-gation of horse platelets is par-tially depressed by phenylbuta-zone although the skin bleedingtime was not significantly in-creased. Aspirin, another NSAID,has been shown to prolong the skinbleeding time in horses (9).The purpose of this study was to

compare the relative potency of themost commonly used NSAID,phenylbutazone, to that of twonewer NSAID, naproxen and flu-nixin meglumine (banamine), withrespect to their effects on horseplatelet reactivity in vitro. Al-though therapeutic concentrationsof NSAID may not significantlyimpair hemostasis in healthyanimals, they may cause a signifi-cant clinical bleeding tendency inanimals with an already compro-mised hemostatic response (due toother disease). Certain NSAID,because of their antiplatelet activ-ity, may be useful in the treatmentof thrombotic conditions which arefrequently encountered in theequine species (9). Our resultscould prove useful in selecting anNSAID with high/low platelet-inhibitory activity.Two different functional respon-

ses of platelets were studied: theability of horse platelets to aggre-gate and the ability of horse pla-telets to make available platelet

factor 3 (PF 3) procoagulant activ-ity. Platelet aggregation responsesto adenosine diphosphate (ADP)and collagen were studied sinceADP and collagen are generallyconsidered to be important stimu-lants of platelet clumping in vivo.-This ability of platelets to aggre-gate in vivo is critical in theresponse of platelets at sites of vas-cular injury and in the subsequentformation of the primary hemo-static plug (10). Platelets must alsobe capable of making available,when stimulated, PF 3 activitywhich acts as a catalyst in fibrinproduction thus contributing tothe stabilization of the primaryplatelet plug (11). Impaired PF 3availability could also contributeto defective hemostasis.

MATERIALSANDMETHODS

The animals used in this studywere clinically normal adult Stan-dardbred horses housed at theUniversity of Guelph. They hadreceived no drugs for at least twoweeks prior to use.Blood was collected by jugular

puncture using 16 gauge needlesand plastic syringes. Blood wasimmediately dispensed into sili-conized glass tubes containing 1/9volume of 3.8% trisodium citrate.Each tube was sealed with Para-film (American Can Co., Neenah,Wisconsin) and gently tilted sev-eral times to ensure complete mix-ing of the blood and anti-coagulant. All glassware used inthe handling of blood or plasmawas siliconized and all procedureswere conducted at room tempera-ture unless otherwise indicated.PREPARATION OF PLATELET-RICHPLASMA (PRP), PLATELET-POORPLASMA (PPP), AND PLATELET-FREE PLASMA (PFP)

Citrated PRP was obtained bycentrifuging the blood at 70 x g for10 min. The upper 2/3 of the PRPwas carefully removed, and theremainder ofthe blood centrifugedat 2510 x g for 15 min to obtainPPP. The PFP was obtained bycentrifuging PPP for a further 20min at 12350 x g. No platelets

173

were detected in samples of PFPexamined with phase-contrastmicroscopy.

Platelet counts of the PRP wereadjusted with PPP to 200 x 109/Lfor aggregometry and with PFP to100 x 109/L for PF 3 availabilitystudies. The PRP was stored insealed aliquots at room tempera-ture and was used within fourhours of collection.

REAGENTS AND DRUGS

Adenosine diphosphate (SigmaChemical Co., St. Louis, Missouri)was prepared as a 10-2 M stocksolution in tris-buffered saline(TBS), pH 7.4. Acid-soluble bovinecollagen (ASC) was preparedaccording to Cazenave et al (12) asa 0.25% stock solution. Kaolin(Baker Chemical Co., Phillips-burg, New Jersey) was suspendedin TBS at a concentration of 50mg/mL and calcium chloride(Fisher Scientific, Fair Lawn,New Jersey); was prepared as anM/40 stock solution. Russell's vipervenom (Wellcome Research Lab.,Beckenham, England) was used ata concentration of 10,g/mL in0.85% sodium chloride. Phenyl-butazone (Sigma Chemical Co., St.Louis, Missouri), naproxen (Syn-tex, Palo Alto, California) and flu-nixin (Schering Corp., PointeClaire, Quebec) were prepared as10-2 M stock solutions and eitherused fresh or stored up to twoweeks at -60°C. Dilutions of theNSAID stock solutions were inTBS.

PLATELET AGGREGOMETRY

Platelet aggregometry was stu-died using a dual channel aggre-gometer module model 300 BD andchart recorder model SPHSV(Payton Associates, Scarborough,Ontario). The aggregometer wascalibrated for minimal and max-imal light transmission using PRPand PPP respectively (13).The method for studying platelet

aggregation responses was as fol-lows: 0.20 mL of PRP was stirredfor 1 min at 900 RPM and 37°C inthe aggregometer cuvette to esta-blish a base line tracing; 0.025 mL

of a NSAID or its diluent was thenadded. After a 5 min incubationperiod with stirring, 0.025 mL ofthe appropriate aggregating agentwas added. The final concentra-tions of the aggregating agentswere ADP 106 M and ASC (stock)1:1000. The final concentrations ofthe NSAID ranged from 10-3 M to10-6 M.

Platelet aggregation responseswere quantitated using threeparameters: the duration of the lagphase, the initial velocity of aggre-gation and the maximum degree ofaggregation (14,15). The lag phase(LP) represented the time inseconds from the addition of theaggregating agent until the initialonset of platelet aggregation asindicated by the first evidence ofan increase in light transmissionfollowing the addition of theaggregating agent. The initialvelocity of aggregation (A,) wasdetermined by drawing a tangentthrough the steepest linear part ofthe aggregation tracing. The slopeof this tangent was expressed inchart units/min. If a biphasicaggregation response was ob-served, only the slope of the prim-ary wave was determined. Themaximum degree of aggregation(Am) was determined by measur-ing the maximum height of theaggregation wave over a 3 minperiod beginning at the onset ofplatelet aggregation (end of the lagphase). The maximum degree ofaggregation was expressed inchart units and as a percent of themaximum possible change in lighttransmission. The degree of inhibi-tion of A, and Am at each drug con-centration was expressed as a per-centage of the untreated controlvalue.

PLATELET FACTOR 3AVAILABILITY

Platelet factor 3 availability wasdetermined according to Spaetand Cintron (16). Mixtures consist-ing of 9 vol PRP and 1 vol of kaolin(5 mg/mL final concentration)suspension, ADP (10-5 M final con-centration), ASC (1:800 final con-centration), or their diluent werestirred in aggregometer cuvettes

at 300 RPM for 5 min. At the end ofthis incubation period, 0.1 mL ofthe mixture was pipetted into eachof two tubes containing 0.1 mLRussell's viper venom (RVV) and0.1 mL calcium chloride alreadywarmed to 37°C. The clottingtimes were recorded using a man-ual "tilt tube" technique. The clot-ting times for 100% PF 3 availabil-ity were determined using PRPand PFP that had been freeze-thawed five times (17, 18).

In order to determine the possi-ble effects of ADP, collagen, kao-lin, and 5x freeze-thawing on theRVV clotting time in the absenceof platelets, the procedures wererepeated using PFP (shown to beessentially free of platelets)instead of PRP in the incubationmixture.

Platelet factor 3 availability wasexpressed as a ratio of the clottingtime in the PRP to the clotting timeof the corresponding PFP treatedin the same way. The use of PF 3availability ratios for expressingPF 3 availability eliminated theeffects of variations in the clottingtimes of individual animal PFP(18). A PF 3 ratio of 1 indicates thatthere is no shortening of the RVVclotting time in PRP compared tothe PFP; in other words PF 3 activ-ity is not made available by the pla-telets. The PF 3 ratio obtainedwith freeze-thawed PRP/PFPgives an indication of the antici-pated ratio for 100% PF 3 availa-bility.The effects of the NSAID on PF

3 availability were studied usingan identical protocol except thatthe PRP and PFP were incubatedwith one of the drugs for 5 min at37°C prior to the addition of theADP or collagen. Each of theNSAID drugs was used at a finalconcentration of 104M.

STATISTICAL ANALYSIS

The results of this study wereexpressed as the mean ± standarddeviation of the mean. Statisticalcomparisons between untreatedand a drug-treated group wereperformed by Student's t test. A pvalue of< 0.05 was considered to bestatistically significant.

174

RESULTSADP AND COLLAGEN-INDUCEDAGGREGATION OF UNTREATEDHORSE PLATELETS

Typical aggregation responsesof horse platelets to 10-6M ADP areshown in Fig. 1 (upper tracing ofeach set). The aggregation respon-ses to 104M ADP were irreversibleand on occasion, distinctly bi-phasic. The lag phase was shortand there was a transient decreasein light transmission following theaddition of the ADP. This tran-sient change is generally consi-dered to reflect a change in plateletshape (13, 20).

Iz

00i02

I-1--2a-J

0

106.

lo-4

TABLE I. Quantitative Parameters forADPand Collagen-Induced Aggregation ofUntreated Horse Platelets

ADP 104 M ASC 1:1000(21)' (21)

Lag phase(s) 8 ± lb 46 ± 5Initial velocity of aggregation

(chart units/min) 21.8 ± 4.0 23.3 ± 3.7Maximum aggregation in 3 min(chart units) 23.3 ± 3.7 26.7 ± 4.4(% of maximum possible change in

optical density) 64.3 ± 10.6 70.3 ± 12.5a(n)

bMean ± 1 SD

Typical aggregation responsesto collagen (1:1000) are shown inFig. 2 (upper tracing of each set).

a

r

-r

z0

0)z

4ccc

c

-r-

Fig. 1. Typical tracings illustrating theeffect of a) phenylbutazone, b) naproxenand c) flunixin on ADP-induced aggre-gation of horse platelets. The asterixindicates the addition of the aggregatingagent (10-6M ADP) after a 5 min incuba-tion period with the appropriateNSAID. The final drug concentrationsgiven in b) also apply to a) and c).

b _

104M

-r~~ ~ ~~~~~oG

100

I,rX , -j

Fig. 2. Typical tracings illustrating theeffect of a) phenylbutazone, b) naproxenand c) flunixin on collagen-inducedaggregation of horse platelets. Theasterix indicates the addition of theaggregating agent (1:1000 ASC) after a5 min incubation period with theappropriate NSAID. The final drugconcentrations given in b) also apply toa) and c).

Collagen-induced aggregation wascharacterized by a much longerlag phase but, as with ADP, irre-versible aggregation.The calculated values for LP, A,

and Am are shown in Table I.

EFFECTS OF THE NSAID ON ADP-INDUCED PLATELETAGGREGATION

Typical effects of the NSAID onADP-induced aggregation of horseplatelets are shown in Figs. 1 and3. None of the drugs, at the concen-trations used had any significanteffect on the LP or A, of ADP-induced aggregation (i.e. the firstphase of platelet aggregation).The Am of ADP-induced aggre-

gation was significantly depressedby all three NSAID and high con-centrations of these drugs pro-duced reversible platelet aggrega-tion (Figs. 1 and 3). Flunixin wasvery potent in this respect, produc-ing a significant and nearly max-

30r

it A,m

.... ..........

."' *------ --. .;/ ~~~~~~~~~~----Fln..... n=-8

-NaPro-*n n-=7

...........n~~~~~~~~~~~~~~-S; A 3DRUG CONCENTRATION {M}

Fig. 3. Percent inhibition of ADP-induced aggregation of horse plateletsby phenylbutazone, naproxen and flu-nixin (Mean values). A, = initial velocityof aggregation; Am = maximum degreeof aggregation in 3 min. Asterix signifiesthe lowest drug concentration at whichthere is significant inhibition.

175

*0

imal depression in Am at the lowestconcentration of the drug used (10-6M). Naproxen and phenylbutazonewere equipotent producing signif-icant depression of ADP-inducedAm at concentrations of 5 x 10-5 M.At higher concentrations, thedegrees of depression of Aminduced by naproxen and phenyl-butazone were equal to that pro-duced by flunixin.

EFFECTS OF THE NSAID ONCOLLAGEN-INDUCED PLATELETAGGREGATION

Each of the NSAID studied hadsignificant effects on collagen-induced aggregation of horse pla-telets; the drugs however were notall equipotent.

Flunixin markedly prolongedthe LP of collagen-induced aggre-gation by an average of 20 s at thelowest concentration used (10-6 M).Higher concentrations of phenyl-butazone and naproxen (10-5 and5 x 10-5 M respectively) werenecessary to significantly prolongthe LP of the aggregation responseinduced by collagen. At concentra-tions of 5 x 10-4 and higher, allthree NSAID prolonged the lagphase by an average of 35 s.Flunixin significantly reduced

A, of the aggregation responseinduced by collagen at even thelowest drug concentration used(Fig. 4). There was a further dose-related reduction in A, as the con-centrations of flunixin wereincreased to 5 x 10-5 M; at this con-centration, the degree of inhibitionof A, appeared to be maximal (Fig.4). Phenylbutazone and naproxenproduced significant inhibition ofcollagen-induced A, at drug con-centrations of 10-5M and 5 x 10-5Mrespectively; however, the degreesof inhibition were significantlyless than those observed with flu-nixin. With increased concentra-tions of naproxen and phenylbuta-zone, the degree of inhibition of A,increased to a degree comparablewith flunixin.The Am of collagen-induced

aggregation was significantlyinhibited by flunixin at a 5 X 106M concentration of the drug. Max-imal inhibition of Am was attained

5.0-

4.5-

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4.0-

2.5-

2.0-

1.5-

(a)

I:'

5.5

HhI9rr1.0 -,

5.0

4.5

0

m

-

4

I?

4.0

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1.5 -

TBS ADP+

DIL

(b)

ADP ADP ADP F/T+ + +

FLUN NAP PHEN

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.4

CO,

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1.5-

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1.0 . - .1 _ . .

h h eh1.0 D - - - - -

TBS ASC ASC ASC ASC FIT+ + + +

DIL fLUN NAP PHEN

Fig. 4. Percent inhibition of collagen-induced aggregation of horse plateletsby phenylbutazone, naproxen and flu-nixin (Mean values). Av = initial velocityof aggregation; Am = maximum degreeof aggregation in 3 min. Asterix signifiesthe lowest drug concentration at whichthere is significant inhibition.

with a flunixin concentration of10-4 M. Phenylbutazone andnaproxen were equipotent withrespect to their effects on collagen-induced Am. Only at high concen-trations (5 x 10-4M and higher) didthey significantly depress Am.

In summarizing the effects ofthe NSAID drugs on ADP andcollagen-induced aggregation,flunixin is considerably morepotent than the other drugs withrespect to its inhibitory effects onequine platelet aggregation. Phe-nylbutazone and naproxen areessentially equipotent in thisrespect.

TBS KAOL ADP ASC FIT

Fig. 5. Platelet factor 3 availabilityratios for horse platelets exposed to kao-lin (Kaol), adenosine diphosphate (ADP)acid-soluble collagen (ASC) or their dil-uent (TBS). Ratio for freeze-thawed pla-telets (F/T) represents 100% PF 3 avail-ability (Mean ± 1 SD; n = 5).

PF 3 AVAILABILITY INUNTREATED HORSE PLATELETS

Platelet factor 3 availability wasdetermined by the ability of "stim-ulated" platelets to shorten theRVV time of plasma.The PF 3 availability ratios

shown in Fig. 5 indicate that somePF 3 activity (PF 3 ratio = 1.317) ismade available from control horseplatelets, suggesting that thepreparation, handling and/or thestirring of the platelets is suffi-cient to cause some PF 3 availabil-ity. Compared to this "basalrelease" of PF 3 activity however,exposure of platelets to ADP or col-lagen increased the availability ofPF 3 as indicated by the signifi-cant increase in the PF 3 availabil-ity ratio (p< 0.02 and p< 0.05respectively). Kaolin particles didnot increase PF 3 availabilitycompared to control (TBS exposed)platelets. One hundred percent PF3 availability was represented by aPF 3 availability ratio of 4.725.

It was determined that underthe conditions of this technique fordetermining PF 3 availability,neither ADP, collagen, kaolin orfreeze-thawing produced any sig-

176

I III

II

.wPo

101.

nificant changes in the clottingtimes of individual PFP.

EFFECTS OF NSAIDON PF 3 AVAILABILITY

The PF 3 availability ratios forPRP/PFP exposed to variousNSAID then stimulated witheither ADP (10-5 M) or ASC (1:800)are shown in Figure 6. The resultsindicate that none of the drugs, atconcentrations of 10-4 M, had anysignificant effect on PF 3 availa-bility from horse platelets asinduced by stimulation with ADPor collagen.

DISCUSSION

Our aggregometric studies onuntreated horse platelets are ingeneral agreement with similarstudies on normal horse plateletsconducted by Sinakos and Caen(21), Calkins et al (22), and morerecently Meyers et al (23). Equineplatelets, like those of manymammals, aggregate in responseto either ADP or collagen, produc-

z

z

DRUG CONCENTRATION IM)

Fig. 6. Platelet factor 3 availabilityratios for horse platelets stimulated witha) ADP 10-5 M, or b) acid-soluble colla-gen (ASC) 1:800, after exposure to flu-nixin, naproxen or phenylbutazone(final concentrations 10-4 M) or their dil-uent. Control platelets were stimulatedwith buffer (TBS) or were repeatedlyfreeze-thawed (F/T) (Mean ± 1 SD;n = 5).

ing typical platelet aggregationprofiles (24). Like Meyerset al (23),we observed a biphasic irreversi-ble aggregation response in PRPfrom some horses when stimulatedwith 106 M ADP. However, inmost platelet preparations, amonophasic irreversible aggrega-tion response was observed. Lowconcentrations of collagen consist-ently produced irreversible ag-gregation following a long delayperiod. This long lag phase is typi-cal of collagen-induced aggrega-tion in many species (24).

All of the NSAID studied pro-duced qualitatively similar effectson the aggregation of horse pla-telets. The drugs differed in theirrelative potency. The range ofNSAID concentrations used in thisstudy was selected in order toinclude concentrations that mightbe anticipated in plasma followingtherapy with these drugs. It hasbeen shown that following thera-peutic doses of phenylbutazone,naproxen and flunixin, peakplasma levels in the horse arelikely to reach approximately30 ,ug/mL (104 M), 25 /Ag/mL (10-4M) and 1.5 - 3.0 ,4g/mL (about 5 x10- M) respectively (8, 25).Adenosine diphosphate is

thought to initiate platelet aggre-gation through a direct interactionwith specific receptors on the pla-telet surface to induce intracellu-lar calcium flux and subsequentplatelet contraction (5, 26). Theaggregation response is thenenhanced by secretion of productsof arachidonic acid metabolismand byADP release (11, 13,20,27).Nonsteroidal anti-inflammatorydrugs such as phenylbutazone andnaproxen interfere with the ara-chidonate pathway and plateletsecretion (2, 28, 29).Our studies using horse platelets

indicated that the LP and A, ofADP-induced aggregation werenot affected by any of the NSAIDover the wide range of concentra-tions used. This suggested that theprimary response to ADP wasnormal in the treated horse pla-telets. The maximum degree ofADP-induced aggregation (Am)was significantly depressed by allthree NSAID suggesting that the

secondary response had been inhi-bited. This lack of a secondaryresponse was further substan-tiated by the reversible aggrega-tion response observed with thehigher concentrations of all thesedrugs. Our observations with phen-ylbutazone are in agreement withresults published by Meyers et al(8) in which they described theeffects of 33,g/mL phenylbuta-zone (approximately 10-4M) on theADP-induced aggregation re-sponse of horse platelets. Naproxenand phenylbutazone were equipo-tent with respect to their effects onADP-induced aggregation ofequine platelets; flunixin wasmuch more potent.

In this study all of the NSAIDmarkedly affected collagen-in-duced aggregation of horse pla-telets; their potency however var-ied. Flunixin produced significanteffects at relatively lower concen-trations than either phenylbuta-zone or naproxen. The latter drugswere nearly equipotent.The long lag phase seen with

collagen-induced aggregation isthought to reflect the timerequired for structural changes inthe collagen to occur and for pla-telets to adhere to the collagen (11,14). According to Henry (20), pla-telets will adhere to the primarystructure of collagen monomersbut polymer collagen must form ifaggregation is to occur. In humanplatelets, the generation of TXA2through the arachidonate pathwayplays an important role in col-lagen-induced aggregation andrelease, and it is likely that therelease phenomenon is necessaryfor maximal aggregation (20, 27,28). In fact, with low concentra-tions of collagen not all plateletswill react with collagen directlyand a large part of the aggregationresponse will depend on the secondorder effect of substances releasedfrom the collagen-stimulated pla-telets (27).Our results indicate that it is the

LP and A., that are first affected byNSAID and that higher concen-trations of the drugs are necessaryto reduce Am. Our results are inagreement with observations byMeyers et al (8) who studied the

177

effects of 33,g/mL phenylbuta-zone on the collagen-inducedaggregation of horse platelets, andsuggest that the three NSAID mayinterfere with the structuralchanges of the collagen and/or theadhesion of the horse platelets tocollagen. Naproxen and phenyl-butazone have both been shown toinhibit the adhesion of human pla-telets to collagen (5, 30).The progressive reduction in A,

(and to a lesser extent Am) whenhorse platelets are treated withincreasing concentrations of theseNSAID may reflect a progressivesuppression of prostaglandin syn-thesis and platelet secretion. We,like others (8) observed that, evenwith high concentrations of theNSAID, collagen-induced aggre-gation in equine PRP was nevertotally inhibited. There is now evi-dence that collagen-induced aggre-gation may be mediated, at least inpart, by nonthromboxane depen-dent mechanisms (8, 26). Suchmechanisms would not likely beinhibited by NSAID. They may beparticularly important in horseplatelets where the arachidonatepathway is not well developed (7).

Platelet factor 3 activity is aproperty of platelets which repre-sents their contribution to theinteraction of factors X, V, and IVin the activation of prothrombin.This procoagulant activity of pla-telets only becomes available whenplatelets are stimulated and thisactivation induces structuralchanges which can provide a phy-siological surface for optimalinteraction and binding of clottingfactors with enzymatic activitynecessary for coagulation (18, 31).Platelet factor 3 activity can bemeasured by its ability to shortenthe RVV (Stypven) time of plasma(13, 16,32). A small amount of PF 3activity was made available fromhorse platelets stirred with TBS.Similar observations have beenmade in studies using human pla-telets (33). We attributed the"basal release" to some activationof platelets due to the processing,handling, and possibly mechanicalstirring of platelets in the aggre-gometer cuvette.Adenosine diphosphate, kaolin

and collagen have been shown toinduce PF 3 availability in PRPfrom man and some animal species(17, 32, 33, 34, 35). Stirring the pla-telets with ADP or collagenresulted in a significant increase inthe availability of PF 3 from thehorse platelets. However, com-pared to the total PF 3 content ofthe platelets (as measured byrepeatedly freeze-thawing the pla-telets), only a relatively small pro-portion of the total PF 3 activitywas made available. This is con-sistent with studies on human pla-telets in which it was determinedthat usually less than 20% of thetotal PF 3 content of platelets wasmade available during activation(17, 36).

In this study, kaolin did not sig-nificantly increase the availabilityof PF 3 from horse platelets. Thekaolin concentration was compar-able to that used in studies in man,although a shorter incubationperiod was used (17). The shortercontact time (5 min) was not animportant factor in these observa-tions since other studies in ourlaboratory, using incubation peri-ods of up to 20 min, still failed toproduce a significant shortening ofthe RVV time of equine PRP.Physical adhesion of platelets tokaolin particles appears to beimportant in initiating plateletactivation and PF 3 availability(16). It is possible that the failure ofkaolin particles to induce PF 3activity availability from horseplatelets was somehow related to afailure of horse platelets to adhereto kaolin particles.The results of our study of the

effects of phenylbutazone, na-proxen and flunixin on PF 3 avail-ability indicate that none of theseNSAID, at a concentration of 10-4M, have any significant effect onthe ability of horse platelets to pro-vide this essential procoagulantactivity when stimulated witheither ADP or collagen. It is of par-ticular interest that flunixin at thisconcentration, produced markeddepression of collagen-inducedplatelet aggregation (compared toeither naproxen or phenylbuta-zone) yet no inhibition of PF 3availability.

The results of this in vitro studysuggest that flunixin is a muchmore potent platelet-inhibitorydrug than either naproxen or phe-nylbutazone, significantly de-pressing the ADP-and collagen-induced aggregation responses ofhorse platelets. None of theseNSAID affect the ability of horseplatelets to make available PF 3activity when stimulated in vitroby either ADP or collagen.Although the effects of drugs on

platelets in vitro may not always beindicative of their effect in vivo,often there is a good correlationbetween the therapeutic level ofthe drug and the effectiveness ininhibiting platelet function in vitro(37). There is always the possibilityof further effects from metabolitesof the drug; these effects cannot bereadily evaluated in vitro.

ACKNOWLEDGMENTS

This study was supported in partby grants from the Ontario Minis-try of Agriculture and Food andthe Ontario Racing Commission.The flunixin meglumine was a

gift from the Schering Corpora-tion, Pointe Claire, Quebec. Thenaproxen was provided as a gift bySyntex Corporation, Palo Alto,California. These contributionsare gratefully acknowledged.

I am indebted to Mr. S. Crane forhis technical assistance andexpertise.

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