Br. J. Pharmac. (1984),83,625-633

Triggering by Paf-acether and adrenaline of cyclo-oxygenase-independent platelet aggregationF. Fouque & B.B. Vargaftig

INSTITUT PASTEUR Departement de Physiopathologie Experimentale, Unit6 des Venins, 28, rue du DrRoux, 75015 Paris, France

1 Platelet-activating factor (Paf-acether, 1-alkyl-2-acetyl-sn-glycero-3-phosphorylcholine) in-duced full aggregation and a limited release reaction of human platelets in plasma or in blood.2 Cyclo-oxygenase inhibition with aspirin only reduced aggregation when induced by thresholdamounts of Paf-acether, whereas higher concentrations surmounted inhibition whether tested incitrated or in heparinized platelet-rich plasma or blood. Aspirin-induced inhibition of plateletsecretion by Paf-acether was insurmountable and independent of the anti-coagulant used.3 Paf-acether and adrenaline acted synergistically in inducing aggregation in citrate and in heparin.Aspirin in vitro or after oral ingestion at doses that suppressed aggregation induced by arachidonicacid alone, failed to reduce significantly the synergized aggregation induced by Paf-acether alone orcombined with adrenaline. Twenty-four hours after the oral ingestion of aspirin, when aggregationby arachidonic acid remained blocked, a slight inhibitory activity on the effect of Paf-acether noted4 h after aspirin, had ceased. This was probably accounted for by the synthesis of thromboxane A2 bynewly formed platelets, since the in vitro addition of aspirin, or of the thromboxane/endoperoxidereceptor inhibitor 13-azaprostanoic acid caused the 24 h platelets to behave in a manner similar toplatelets collected 4 h after aspirin.4 The c2-adrenoceptor inhibitor, yohimbine, blocked the direct effect of adrenaline as well as itssynergism with Paf-acether.5 Since the synergistic effect of Paf-acether and adrenaline was maintained when thrombin-degranulated platelets were used, and aspirin remained ineffective against it, it is clear that theaugmented platelet responsiveness is not accounted for by the platelet release reaction.6 Paf-acether and adrenaline act synergistically and stimulate platelets by cyclo-oxygenase-independent mechanisms, which may be relevant in human physiopathological conditions.

Introduction

Platelet-activating factor (Paf-acether, 1-alkyl-2-acetyl-sn-glycero-3-phosphorylcholine) is a poten-tial mediator of the activation of various cell types(Vargaftig et al., 1981; Benveniste & Vargaftig,1983) including platelets (Benveniste et al., 1975;Cazenave et al., 1979). Doubts were raised concern-ing the relevance of aggregation of human plateletsby Paf-acether, since it has been claimed that therelease of ADP (the mediator of the first pathway ofaggregation) and/or of arachidonic acid (themediator of the second pathway; Vargaftig& Zirinis,1973) fully account for aggregation (Marcus et al.,1981; McManus et al., 1981; Tsien et al., 1982;Chesney et al., 1982; Rao et al., 1982; Ostermann etal., 1983). This is an important problem, since ifindeed Paf-acether activates human plateletsthrough aspirin-inhibitable mechanisms (i.e. the for-

mation of cyclo-oxygenase arachidonate metabo-lites) and/or the release of ADP, its potential role asthe mediator of the third pathway, which was demon-strated on rabbit platelets (Chignard et al., 1979),would be strongly challenged. Most experimentsshowing that aspirin or indomethacin interfere withhuman platelet aggregation due to Paf-acether wereperformed on citrated platelet-rich plasma (citratedPRP), i.e., under conditions that favour the secon-dary secretion-dependent aggregation, which is sup-pressed by aspirin (Cazenave et al., 1981). We de-monstrated previously that platelet aggregation byPaf-acether, and particularly the synergized aggrega-tion resulting from its combination withADP or withadrenaline obtained on plasma-free platelet suspen-sions is refractory to aspirin (Vargaftig et al., 1982).We have now studied the activation by Paf-acether

© The Macmillan Press Ltd 1984

626 F. FOUQUE & B.B. VARGAFTIG

alone or with adrenaline on human platelets in plas-ma, using different anti-coagulants, and have com-pared the extent of aggregation and of secretion inthe presence and absence of aspirin. Since the ag-gregation due to the synergistic effect of Paf-acetherplus adrenaline was indeed refractory to aspirinunder conditions where aggregation by adrenalinealone was blocked, we concluded that the interactionbetween adrenaline and platelets is accounted for byat least two distinct mechanisms, one dependent andthe other independent of arachidonate cyclo-oxygenase metabolites.

Methods

Venous blood from healthy donors who had taken nomedication for at least the last ten days was obtainedfrom the Centre National de la Transfusion Sanguine(Paris). Blood (9 vol) was mixed with the differentanticoagulants (1 vol) and centrifuged (190 g; 25°Cfor 20 min) to separate the platelet-rich plasma(PRP). Platelet-poor plasma was obtained by cen-trifugation of the remaining blood for 10 min at2500 g. Platelet aggregation was monitored with awhole blood Aggregometer (Chronolog) at1000r.p.m. and at 37°C in 1 ml siliconized glasscuvettes.

Measurement ofATP

During aggregation 20 ll aliquots were collected andimmediately tested for ATP by the luciferin-luciferase technique, with an automatic device (PicoATP, Jobin Yvon, France). In a few experiments, thesimultaneous determination of aggregation and ofsecretion of ATP was performed with a Lumi-aggregometer (Chronolog).

Platelet 5-hydroxytryptamine secretion

Platelets were pre-incubated with 1 tM of [14C]-5-HT for 15 min at 37°C (50 nCi ml-1 of PRP). At least90% of the added label were taken up by the cells.Samples of prelabelled platelets were removed be-fore and 3 min after the addition of the agonists andrapidly centrifuged with a Unipan micro centrifugefor 2 min. The supernatant and the pellet were re-moved and counted in a SL 3 000 Kontron counterwith ACS II. The results are expressed as the percen-tage of the radioactivity released from the platelets.

Preparation of washed platelets

The plasma-free human platelet suspension was pre-pared according to Kinlough-Rathbone etal. (1977).Apyrase was omitted when secretion of ATP wasstudied.

Degranulation procedure

The method of Reimers et al. (1976) was used, inwhich bovine thrombin (1 u ml-') is added to thestirred platelet suspension and left for 5 min. A de-aggregating mixture of prostaglandin E1 (PGE1,10 fLM), plasmin and SBTI (0.025% of each),TAME(1 mM), heparin (10 u mlP 1) and rabbit serum (10%by vol) was then added. The platelets were washedtwice, and resuspended in autologous plasma con-taining heparin (10 u ml1). Platelet secretion ofATP, and the number of free remaining plateletswere monitored during degranulation. The final re-covery yield was of 80-90%, platelets depleted oftheir granules being unable to secrete ATP whenfurther stimulated with convulxin, as described byVargaftig etal. (1983).

Drugs and reagents

Heparin (Laboratoires Choay, Paris); bovine throm-bin (Hoffmann La Roche, Basel); ADP, ATP, ad-renaline, potato apyrase, indomethacin, arachidonicacid, p-tosyl-L-arginine methyl ester (TAME), soy-bean trypsin inhibitor (SBTI) and plasmin (Sigma);prostaglandin E1 (PGE1, Upjohn, Kalamazoo);lysine acetylsalicylate (Aspegic, Laboratoires Egic,Amilly, France); 5-hydroxytryptamine (5-HT, 5-hydroxy-[14C]-tryptamine creatinine sulphate,50 mCi mmol-1) and the scintillation counting liquidACS II (Amersham); luciferin-luciferase reagents(Lumac, Basel and Chronolume from Chronolog,according to Cargill et al., 1983). Paf-acether (theoctadecyl derivative) was a gift from Prof. J.J. God-froid (Universite de Paris VII), the low molecularweight heparins CY 216 and CY 222 (Dunn et al.,1983) were given by Dr F. Toulemonde(Laboratoires Choay, Paris), 13-azaprostanoic acid(13-APA) by Drs G. de Gaetano (Instituto MarioNegri, Milano) and Y. Remuzzi (Ospedali Riuniti,Bergamo), and convulxin, a glycoprotein extractedfrom the venom of Crotalus durissus cascavella, byDr G. Marlas (Institut Pasteur, Paris). All salts wereof analytical grade. ACD (citric acid 7 mM, sodiumcitrate 93 mm and glucose 139 mM; pH 6.5), sodiumcitrate (final concentration of 0.38% by vol), andheparin (10 u ml-) were used as anticoagulants.

Results

Comparison between the effects ofPaf-acetheroncitrated and on heparinizedplatelet-rich plasma

Paf-acether induced a dose-dependent aggregationof platelets in citrated PRP, when used at100-1000 nM, wheras 5-10 times lower concentra-tions were sufficient to aggregate the platelets col-

HUMAN PLATELET AGGREGATION BY Paf-acether 627

Citrate

ASA

ta)

a)

0

C.,

6 2 min

ta)

oo0

4--

00

Heparin

ASA

ta)-C

(L0Cv

cva-

06

ta)

a)0CvCD

oo20006

Figure 1 Interference of aspirin with platelet activation due to Paf-acether. Tracings to compare aggregation incitrated and in heparinized PRP from the same donor, in presence or absence of aspirin (ASA, 0.1 mM, 5 min,incubation). Vertical scale: % light transmission across the PRP. Horizontal scale: time (2 min).

lected from the same donor in heparinized PRP(Figure 1). When citrate was added up to 0.38% tothe heparinized PRP, more (4-8 times as much)Paf-acether was required than in its absence. Incontrast, when heparin was added up to 10 u ml-1 tocitrated PRP, the effective concentrations of Paf-acether were not modified significantly. Heparinalone, at the concentrations used to prevent clotting,can potentiate aggregation (Beck, 1977; Bygdeman& Tangen, 1977), and this might have accounted forthe lower thresholds of Paf-acether. We thus testedtwo low molecular weight heparin fragments whichprevent clotting without potentiating aggregation. Assummarized in Table 1, in the presence of heparinsCY 216 or CY 222, Paf-acether was very active ininducing aggregation, but less so than if commercialheparin were used. Figure 2 shows that aggregationby Paf-acether in citrated PRP was accompanied bythe secretion of up to 2 nmol ml- 1 of ATP,arachidonic acid being slightly more effective. Thisaccounts for 30-50% of the total releasable ATP,which amounted to 10 nmol ml- 1 as determined inseparate experiments with convulxin. Secretion was

below 0.4 nmol ml-', when heparin was used as an

anticoagulant. Similar results were obtained whensecretion was monitored on-line with the Lumi-aggregometer (Figure 3). The unexpected contrastbetween the higher efficiency of Paf-acether in in-ducing the aggregation in heparin-PRP as comparedto citrated PRP, and its lower efficiency in releasing

ATP, was confirmed for the release of [14C]-5-HT(Table 2).

Interference ofaspirin with platelet secretion andaggregation due to Paf-acether

Aggregation by sub-maximal amounts of Paf-acetherin citrated PRP was unaffected by aspirin, whereasfull aggregation induced by twice or thrice as muchPaf-acether was occasionally inhibited (Figure 1).Nevertheless, when all experiments were pooled, no

Table 1 Effective concentrations of Paf-acetherapplied to platelets in the presence of differentanti-coagulants'

Anticoagulants

Sodium citrate0.38%

HeparinlOuml-I

Heparin CY 21660igml-I

Heparin CY 22260Agml-1

ACD

Range ofconcentrations needed to aggregate

by 70%

100- 1,000 nM

20-100nM

200-800 nM

400- 1,000 nM20- 100 nM

I See Methods for details

r0

200

c0(.)

Ecn

cCD

40 k

60 F

80 L

628 F. FOUQUE & B.B. VARGAFTIG

3

-5Ea-

H4-0a)

u)a)

10 20 40

2

100 400 10 20 40 100 400

Time (min)Figure 2 Comparison between aggregation (a) and secretion ofATP (b) induced by Paf-acether applied to citrated(O), or to heparinized (O) PRP at the indicated final concentrations (10-400 nM). Closed symbols (0, *) are usedin the presence of aspirin (0.1 mM; 5 min incubation) and open symbols (0, K>) in its absence. Left hand scale (a): %aggregation of the PRP. Right hand scale (b): Release of ATP. Values are mean for n = 6; s.d. shown by vertical lines.

statistically significant inhibition was seen (Figure 2).Similar results were obtained in heparin PRP (Figure1 and 2), and here simply doubling the amounts ofPaf-acether used was enough to overcome inhibitionby aspirin. In contrast to aggregation, the secretion ofATP (Figures 2 and 3) and of [14C]-5-HT (Table 2)induced by Paf-acether were suppressed by aspirin,irrespective of the anti-coagulant used.

Comparison between the effects ofPaf-acetherassociated with adrenaline on citrated or heparinizedplatelet-richplasma: interference ofaspirin

Adrenaline and Paf-acether acted synergistically andconcentrations of each which were ineffective alone,triggered full aggregation when applied together tocitrated or to heparinized PRP (Figures 4 and 5). The

Table 2 Comparison between the release of [14C]-5-hydroxytryptamine (5-HT) or ATP by Paf-acether alone orwith adrenaline in citrated or heparinized platelet-rich plasma

Control5-HT ATP

Citrate+ ASA

5-HT ATP

HeparinControl + ASA

5-HT ATP 5-HT ATP

6% 0.6%21% 22 %

7% 0.5% 4% 2 % 6% 2 %7% 6 % 14% 9 % 14% 5 %

Adrenaline 30 nM+ Paf 20 nM

5% 0.1% 4% 0 %20% 1.3% 6% 0.5%

5% 0 % 4 % 0 %6% 0.7% 5 % 0.4%

Adrenaline 300 nM+ Paf 20 nim

11% 8 % 6% 6 % 5% 0.7% 5 % 0.2%29% 25 % 8% 15 % 9% 7.4% 9 % 3.3%

88% 100 % 73% 90 % 85% 100 % 65 % 85 %

Results are the means of 4-6 experiments.

100

c0

0)a) 50

80)

Paf 20 nM400 nM

Cx 2 nim

0

20 F

40 H

60 Fa

c80 L

2 minO r

2

b

a

c

4 -

6

Citrate Heparin

Figure 3 Tracings of platelet aggregation (upperpanel) and of the simultaneous release of ATP (lowerpanel) induced by Paf-acether applied to citrated (leftpanel) or to heparinized PRP (right panel). Additionswere as follows: (a) Paf-acether, 100 nM: (b) as (a), butin the presence of aspirin, 0.1 mm, 5 min incubation; (c)convulxin, 2 nM; (d) Paf-acether, 40 nM; (e) as (d), but inthe presence of aspirin. Scale of aggregation as in Figure1 and scale of ATP in jimol 1- 1.

JMAN PLATELET AGGREGATION BY Paf-acether 629

minimal final concentrations of Paf-acether requiredto act synergistically with adrenaline were of2-20nM in heparinized PRP, and of 4-40nM incitrated PRP. Secretion of ATP was also increasedwhen Paf-acether and adrenaline were associated,and the release was again more pronounced in citratethan in heparin (Table 2). In every instance, aspirinsuppressed secretion induced by adrenaline plus Paf-acether, whereas aggregation was only blocked iftriggered by low (0.3-3 Mm) concenitrations of ad-renaline in citrated PRP (Figure 4), higher amountsovercoming inhibition. Salicylic acid was completelyinactive against Paf-acether alone or associated withadrenaline (not shown). In the presence of lowmolecular weight heparins, adrenaline plus Paf-acether acted synergistically at concentrations inter-mediate between those needed in citrated and inheparinized PRP. Independently of the anti-coagulant used, inhibition of aggregation by aspirinwas surmounted by increasing either the amounts ofPaf-acether, or of adrenaline.When human volunteers ingested 325 mg of aspi-

rin orally, the synergism between adrenaline andPaf-acether was maintained for the 4 h and the 24 hPRP, under conditions where the effects ofarachidonic acid (0.5 mM) or of adrenaline (3MM)alone were blocked. The association of the indicatedconcentrations of arachidonic acid and adrenalinefailed to aggregate the 4 h PRP, but was effective onthe 24 h sample. In contrast, no synergism betweenarachidonic acid and Paf-acether was obtained inthe 4 and 24 h samples. The re-exposure of the 24 hPRP to aspirin (0.1 mM) or to the endoperoxide/thromboxane A2 receptor antagonist 13-APA(50 Mm) (Le Breton et al., 1979) suppressed therecovered synergism between adrenaline and

Table 3 Failure of thrombin-treatment alone or with aspirin (ASA) to interfere with the synergism betweenPaf-acether and adrenaline

Final concentrationsControl platelets

% AggregationThrombinized platelets

Paf alone'Adrenaline aloneADP aloneAdrenaline withPaf2

ADP with Paf2

(M)(10-8-10-6)

3.10-710-73.10-83.10-73.10-6

10-7

No ASA3 With ASA3 No ASA311.5+ 2 21.3± 5 11.7+ 2

15 15 30 ± 516.2 ± 2 15 28.3 + 744.7+±14 39 +1 9 38.7±l7.270.5+12 64.2± 7 61.2±1190 +1 6 79 + 14 100

60 +10 48.5±17 63.7 + 12

1 Final concentrations of Paf-acether alone required to induce threshold aggregation vary according to the donor.2 The concentrations of Paf-acether associated with adrenaline or ADP in each case were the same as those that were usedalone, in each individual case.

3ASA = aspirin 0.1 mMValues are means 1 s.d. for n = 4.

0c0

U,.EInc

J4

E

i

a_10-.

0a,a)a0

a)

With ASA329 ± 9

1515

37.3+1 966.2 ± 1276.8± 14

53 +20

b

630 F. FOUQUE & B.B. VARGAFTIG

0 r

20 F00-

c0

en.E_V)c:

J-1

40 F-

60 F-

2 min

ASA

80 L

t

Adrenaline 3 FLMPaf-acether 10 nM

Adrenaline 3 F.MPaf-acether 20 nM

Adrenaline 0.3 FLMPaf-acether 20 nM

Figure 4 Aggregation induced by synergistic action of adrenaline and Paf-acether applied to heparinized PRPat the indicated final concentrations, in presence or in absence of aspirin (0.1 mM; 5 min incubation). Scales as inFigure 1.

arachidonic acid (not shown), but failed to reduceaggregation by Paf-acether alone or associated withadrenaline.

Platelet activation by Paf-acether associated toadrenaline in whole blood

Paf-acether was consistently more active in heparin-ized than in citrated blood, since it required respec-

tively 40 and 200-400nM to trigger aggregation.Adrenaline alone was effective at 100-300nM incitrate, or at 30-100nM in heparin. In both cases

aggregation by adrenaline was suppressed by aspirin4 and 24 h after its oral ingestion at 325 mg (n = 3).As in the case of PRP, the synergism between ad-renaline (30 nM) and Paf-acether (40- 80 nM) in cit-rated blood was not affected consistently by aspirin ateither of the intervals studied. The effects of Paf-acether alone were marginally reduced 4 h after aspi-rin, requiring 400 instead of 200 nM to induce fullaggregation, but within 24 h, 200 nM were sufficientto induce aggregation.

Synergism between Paf-acether and adrenalineapplied to thrombinized platelets

Adrenaline might act synergistically with Paf-acether

through the release of ADP from the platelets. Sincethe ADP scavenging systems are not reliable enoughto rule out the participation of ADP (Kinlough-Rathbone et al., 1976), platelets were exposed toexcess thrombin, recovered and resuspended inheparinized plasma (see Methods). These plateletsfully aggregated in response to Paf-acether or toadrenaline.

Furthermore, as seen in Table 3 the synergisticeffects of their combination persisted, despite theexhaustion of endogenous ADP. Finally, aspirinfailed to prevent aggregation of the degranulatedplatelets induced by Paf-acether (Table 3), or by itsassociation with adrenaline or ADP.

Interference of yohimbine with the activity ofPaf-acether

Yohimbine (5 tiM) reduced the direct effect ofthreshold amounts of Paf-acether tested on citratedor heparinized PRP, inhibition being surmounted byincreasing by 10 fold the concentrations used. Themaximal aggregation induced by the synergism ofadrenaline and Paf-acether was reduced to that in-duced by Paf-acether alone if yohimbine was addedto the PRP 30 s before the aggregating agents.

t t

HUMAN PLATELET AGGREGATION BY Paf-acether 631

100

0

0<C

0

a)

CT)

CD-_n1

50

100

50

003 03 3 30 003 03 3 30Adrenaline (pM)

Figure 5 Comparison between aggregation induced by adrenaline and Paf-acether applied to citrated (a) or toheparinized (b) PRP. Aggregation was measured in presence or in absence of aspirin (0.1 mm incubated for 5 min).Sub-threshold concentrations of Paf-acether were used in each case, in the presence (closed symbols) or in theabsence (open symbols) of the concentrations of adrenaline (0.03 - 30 piM) indicated on the horizontal scale. Symbolsas follows: (A and A) Paf-acether and adrenaline; (E and M), adrenaline alone. Vertical scale: % aggregation.

Discussion

Our results show that the threshold aggregating con-centrations of Paf-acether alone or associated withadrenaline vary according to the anticoagulant used.The lowest threshold was obtained with comrmercialheparin, the highest amounts were needed with cit-rate, whereas intermediate amounts of Paf-acetheraggregated PRP anticoagulated with the low molecu-lar weight heparins. Furthermore, addition of citrateto heparinized PRP increased the aggregatingthresholds, but heparin added to citrated PRP failedto sensitize to Paf-acether. Citrate thus reduces onlyaggregation by Paf-acether alone or associated withadrenaline to a limited extent, heparin failing toreverse this slight inhibitory effect. Since citrate andACD were at two extremes of activity, variations ofthe final free calcium concentrations in PRP alonecannot account for the different platelet sensitivities.At this stage, our results do not indicate which is theanticoagulant likely to introduce the fewest artifactsfor testing Paf-acether, but since the overall resultsobtained with each of them, particularly with respectto the effects of aspirin, were essentially the same inPRP and in whole blood, it is clear that valid conclu-sions can be reached concerning the mode of actionof Paf-acether.

Aggregation by low amounts of Paf-acether or by

its association with low amounts of adrenaline, wasblocked by aspirin, inhibition being surmounted byincreasing either of the agonists, in citrated PRP andmore easily in heparinized PRP. In contrast, secre-tion was always suppressed by aspirin, irrespective ofthe amounts of Paf-acether and of adrenaline used,again dissociating aggregation from the release reac-tion. Surmountability of aggregation by increasingthe concentrations of Paf-acether can be accountedfor by the fact that alone it induces primary non-cyclo-oxygenase-dependent aggregation. Primaryaggregation also accounts for surmountability whenthe concentrations of Paf-acether associated withfixed amounts of adrenaline were increased. In con-trast, it is more difficult to explain why inhibition byaspirin of aggregation by Paf-acether plus adrenalinewas also surmounted when the concentrations of thelatter were increased, since aggregation by ad-renaline alone was inhibited by aspirin through anon-surmountable mechanism. This lack of sur-mountability contrasted with the facility with whichlimited increments of the concentrations of ad-renaline associated with very low concentrations ofPaf-acether surmounted inhibition of cyclo-oxygenase.

Since ADP also acts synergistically with Paf-acether (Kinlough-Rathbone et al., 1976), it seemedreasonable to verify whether the effects of adrenaline

632 F. FOUQUE & B.B. VARGAFTIG

are mediated by the release of ADP from the plateletdense bodies, even though Paf-acether alone or com-bined with adrenaline was a poor secretion inducer.The response obtained by the use of thrombin-treated platelets was clearly negative. Indeed, underconditions where the release reaction was suppressedbecause of granule exhaustion by the pre-exposure tothrombin, the synergism between adrenaline andPaf-acether was maintained. Furthermore, since as-pirin also failed to block the effects of Paf-acetheralone or associated with adrenaline on exhaustedplatelets, it is clear that cyclo-oxygenase products didnot account for the aggregating effects of Paf-acetheralone or associated with adrenaline, when secretionwas prevented. Adrenaline thus exerts at least twodistinct effects on platelets, both initiated by its in-teraction with CX2-adrenoceptors (yohimbine-sensitive) (Grant & Scrutton, 1980; Hsu etal., 1979).A classical effect, amplified by cyclo-oxygenasemetabolites is observed in heparinized and in citratedPRP, since adrenaline-induced aggregation and sec-retion are suppressed by aspirin (Cazenave et al.,1981). Another effect involves the conversion byPaf-acether of the cyclo-oxygenase-dependent ef-fects of adrenaline into cyclo-oxygenase-independent effects. In contrast, since aspirin sup-pressed the aggregation induced by the synergism ofPaf-acether and arachidonic acid, we can concludethat synergism involving the latter is fully explainedby the formation of cyclo-oxygenase metabolites.Rao et al. (1980) showed that adrenaline can cor-

rect the defect in aggregation of aspirin-treatedplatelets stimulated with ADP or with thrombin, butfails to improve the responses to arachidonic acid. Intheir experiments, adrenaline also made the 16 hpost-aspirin platelets (equivalent to our 24 h) sensi-tive to arachidonic acid, in apparent contradiction ofthe persistent inhibition of the direct effects ofarachidonic acid tested on platelets collected frompatients for at least one week after aspirin ingestion.Similar differences between the 4 h and the 24 h

post-aspirin platelets were also observed by us for thesynergism between adrenaline and either Paf-acetheror arachidonic acid. Twenty-four hours after aspiriningestion, approximately 10% of the platelets arenewly formed, and should have functional cyclo-oxygenase capable of synthesizing enough throm-boxane A2 from added arachidonate or on stimula-tion with adrenaline, to act synergistically with Paf-acether and aggregate the remaining 90% aspirin-affected platelets. Indeed, the addition of aspirin, orof the thromboxane/endoperoxide receptor antagon-ist 13-APA resulted in further inhibition of the par-tially recovered aggregation triggered by arachidonicacid associated with adrenaline. In contrast, in noinstance was aggregation due to Paf-acether alone, orby its association with adrenaline, inhibited by addi-tional aspirin or by 13-APA. The synergism partiallyrecovered 24 h after aspirin, involving arachidonicacid, is thus indeed cyclo-oxygenase-dependent. Incontrast, when adrenaline is used, the synergism withPaf-acether is clearly not accounted for by cyclo-oxygenase metabolites. Under different conditions,Kinlough-Rathbone et al. (1976) also observed thatwhenever arachidonic acid participates in synergismwith other agents, aspirin is fully inhibitory.The relatively high amounts of adrenaline needed

to trigger aggregation in PRP, and the effectivenessof aspirin against such aggregation, contrasting withits ineffectiveness against clinically relevant forms ofshock, led to an underestimation of its potentialphysiopathological role when its plasma levels areincreased. Our present results, showing the markedcyclo-oxygenase-independent synergism betweenPaf-acether and adrenaline, both of which may bereleased in blood, should lead to a reappraisal of theimportance of adrenaline-induced platelet activa-tion.

The authors are grateful to Mrs D. Joseph for help duringthe platelet degranulation experiments.

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HUMAN PLATELET AGGREGATION BY Paf-acether 633

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(Received January 12, 1984.Revised June 15, 1984.)