Aspirin fails to inhibit platelet aggregation in sheep

THROMBOSIS RESEARCH 72; 175-l 82,1993 00443848193 $6.00 + .OOPrinted in the USA. Copyright (c) 1993 Pergamon Press Ltd. All rights reserved.

ASPIRIN FAILS TO INHIBIT PLATELET AGGREGATION IN SHEEP’ Hristos G. Spanos Department of Physiology, University of New England, Armidale N.S.W. 2351 Australia. (Received 27.4.1993; accepted in revised form 22.6.1993 by Editor H.H. Salem) (Received after technical revision by Executive Editorial Office 23.8.1993)

ABSTRACT

This is the first report in which aspirin (ASA) has failed to inhibit aggregation of mammalian platelets. Preincubation of titrated sheep platelet rich plasma with a final concentration of 500 ptM ASA for 3 minutes at 370C did not inhibit aggregation induced by either arachidonic acid (AA; 1.6 mM), ADP (2.5 PM), collagen (5.6 pg/ml) or thrombin (0.04U). Instead, ASA potentiated the aggregation response produced by these agents except AA. Platelet aggregation that was reversible with ADP became irreversible after adding ASA. The inhibitory properties of ASA was confirmed with human platelets, challenged with AA, ADP, adrenalin and collagen. These findings suggest that sheep platelets have an ASA resistant cycle oxygenase and may be able to aggregate by a pathway that is independent of arachidonic acid.

The inhibitory effect of ASA on platelet release and secondary aggregation is well documented for humans (l-2) and other species such as dog (3), rabbit (3,4), rat (5) pig (4) and guinea pig (3). ASA has been reported to inhibit the release reaction (2,4) and subsequent second wave of aggregation induced by ADP (2) adrenalin (6), 5-HT (6) and low doses of thrombin (1) and the single wave of aggregation induced by low doses of collagen (4). Although ingested ASA is rapidly cleared from the bloodstream its inhibitory effect lasts for 3 to 7 days (6) and is dependent upon the presence of the acetyl group (2) as sodium salicylate is not inhibitory (5,6).

Key words: aggregation, aspirin, ADP, collagen, sheep. Corresponding author: H.G. Spanos, Department of Physiology, The University of Melbourne, Parkville, Vie. 3052, Australia. 1 Presented in part to the Australian Physiological and Pharmacological Society, May 1982, (Abstract 36~).

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The mechanism of platelet aggregation involving the arachidonic acid (AA) pathway is well established for humans (7,8) and other species (9) as is the inhibitory effect of ASA on cycle oxygenase (7) and its consequent effect on the platelet release reaction and secondary aggregation (2,6). Various species differences have been reported for platelet aggregation (10-13). Perhaps the most striking is the biphasic aggregation response of titrated human platelets (14). Except for cats and guinea-pigs (12) aggregation responses of titrated PRP from other species such as rabbit, rat, dog, horse, pig, (1 O-13) and sheep (1516) is usually monophasic. Likewise, adrenalin alone does not cause aggregation of mammalian platelets except from humans and some primates (1 O-l 2,15,16). This study reports on another surprising species difference (17), namely the inability of ASA to inhibit aggregation of sheep platelets. The relevance of these findings to patients who do not benefit from ASA prophylaxis, e.g. in the prevention of postoperative thrombosis remains to be established.

MATERIALS AND METHODS

Animalq. Five healthy, 12 month old Merino ewes from the same flock, housed indoors and fed daily on standard stock rations. Blood samples Using the double syringe method, 9 ml blood was drawn from the jugular vein directly into a plastic syringe containing 1 ml 3.8% trisodium citrate, pH 7.4. Platelet rich plasma (PRP) was prepared by centrifuging the 10 ml blood in plastic centrifuge tubes at 4009 for 10 min; which yielded over 95% platelet recovery. The PRP was transferred into a capped plastic tube using siliconised Pasteur pipettes and kept at room temperature for the duration of the experiment (l-2 hr). Platelets were counted on a calibrated Coulter counter (A) and the PRP adjusted to 6-8 x 105 platelets/mm3 with platelet poor plasma (PPP), prepared by centrifuging the remaining blood at 2,000g for 10 min. Platelet Aggregation Studies were carried out on a Zeiss spectrophotometer modified for aggregation studies (16). The recorder was zeroed with the PPP and the full scale deflection set at 0.500 OD units. 0.5 ml PRP was pre-incubated for 10 min at 370C in siliconised glass cuvettes (7.9mm diam). ASA (Drug House of Australia) was prepared fresh before each experiment by dissolving ASA in Ca2+ and Mg2+ free Tyrode solution to a concentration of 5 mM and the pH adjusted to 7.4 with 1M KOH. Further dilutions were made with Ca2+ and Mg2+ free Tyrode as required. 50 pl ASA was added to the preincubated PRP and stirred for three minutes before adding 50 pl of the aggregating agent. Controls had 50 pl of Ca2+ and Mg2+ free Tyrode instead of ASA. Aggregation was allowed to proceed for 6 min following final concentrations of 1.625 mM AA (Sigma # A-0662), 2.5 PM ADP (Calbiochem) or 0.04 U thrombin (Parke Davis) and for 10 min following 50 pl of 5.625 ug/ml collagen (Soluble calfskin :

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Worthington Biochem. Corp.). These concentrations were chosen because they produced aggregation responses that were reversible with ADP and thrombin and delay times of l-5 min with collagen.

Human.

Blood was taken directlv into citrate as described for sheeo, but from the anticubital vein, from 5 human volunteers who had not ingested any medications for the past week. PRP was prepared by centrifuging 10 ml blood at 1509 for 10 min and adjusting to 4 X 105 platelets/mm3 with PPP prepared as above.

enmen& These were carried out separately on PRP from 4 different sheep using a dual channel aggregometer (Payton). Arachidonic acid was prepared by dissolving 50 mg in 1.5 ml hexane, drying with a stream of N2, redissolving in 5 ml 1% sodium carbonate and storing at -200C wrapped in aluminium foil until used. The inhibitory effect of ASA was verified beforehand with human titrated PRP.

RESULTS

ADP.

Figure 1A shows how the normal aggregation responses of sheep platelets increase and become irreversible as the ADP concentration is increased. Figure 1B shows that by increasing collagen concentrations the delay time is decreased. ..

ectof_ Preincubation of sheep PRP with ASA increased the ADP aggregation response from reversible to irreversible (Fig. 2A), decreased the delay time of the collagen response (Fig 2B), and failed to inhibit AA induced aggregation (Fig 2C). Thrombin produced similar responses to ADP (Fig 2D). Significant platelet activation during the preincubation period with 500 pM ASA is evident in some records. Increasing the preincubation period of ASA to 10 min, still had no effect on ADP induced aggregation Table I shows, calculated as

for each animal, the % aggregation

at 6 mins following

ADP,

mm. X 1OQ 1 O.D. of PRP On average platelet aggregation was increased by 33%, 36% and 150% following preincubation with 5, 50, and 500 p.M ASA respectively. Table II shows similar data for delay times following collagen. On average, delay times were decreased by lo%, 30% and 100% respectively by each ASA concentration. These observations are consistent with a general activation of sheep platelets by ASA. Studies The inhibitory effects of the ASA preparation used were confirmed with human platelets for ADP (Fig 3A), collagen (Fig 38) adrenalin (Fig 3C) and AA (Fig 30) induced aggregation.

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B

A - 0.6

-0.0 - 0.6

- 0.4 4

- 0.3

2

. 0.2

. -0.4

2 -4

2.5 pM l.OpY 0.5 PM

* 0.1 -0

-0.3 -0.2 0.1 -0

-

0

’ TIME (min) ’

TIME (min)

lo

FIG. 1.

A: ADP, B: Collagen, dose response curves of sheep platelets. All concentrations in this and subsequent figures are final concentrations in the cuvette. A O.D. = change in optical density of PRP.

??

? ? ? ?

-3

‘.

.‘.’

6

0

TIME (min) C 100%

0.3

%

l-y”,”

0.2

f

s

0.1

bp

0 J,

0%

-3

0

6

3

TIME (mln)

1 C

6

0 TIME (mln)

FIG. 2. Effect of ASA on A: ADP, B: Collagen, C: AA and D: Thrombin induced aggregation of sheep PRP. At -3 min ASA was added at final concentrations of: i 5 pM - -, ii 50 pM - - and iii 500 pM -. Controls (- C) had Ca2+ and Mg2+ free Tyrode instead of ASA. The aggregant was added at time 0.

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TABLE

1

Effects Of ASA On % Aggregation At 6 Minutes Following ADP (2.5 pM)

ASAW4 MEAN SD (n=5)

% AGGREGATION

0

5

50

500

19.8 9.4

26.4 11.7

27.0 14.6

50.0 9.7

TABLE II

Effects Of ASA On Delay Time Following Collagen (5.625 pg)

0

5

50

500

211 7.8

184 68

147 60

0 0

ASA (PM) MEAN SD (n=5)

DELAY TIME (set)

A ,0.3

0

-3

6

TIME

.‘-----------. -3 0

(min)

10

TIME (min)

C WO.4

Adrenalin

d 0.3

-3

0

TIME

,--------,

6

-3

(min)

6

0

TIME (min)

FIG. 3. Effect of ASA on A: ADP, B: Collagen, C: Adrenalin and aggregation of human PRP. ASA was added at -3 concentration of 500 pM - -. Controls (-C) had Ca2+ Tyrode instead of ASA. The aggregant was added at time

D: AA induced min to a final and Mg2+ free 0.

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DISCUSSION

Sheep platelets, like human platelets require stirring and the presence of divalent cations for aggregation. They aggregate with ADP, collagen and thrombin but not adrenalin. The aggregation is monophasic and reversible at lower concentrations of ADP and thrombin. Because they are much smaller than human platelets, sheep PRP requires relatively higher g forces to prepare (16). This is the first report in which ASA has been shown to enhance aggregation of mammalian platelets. In all other species tested so far ASA inhibits platelet aggregation (7). In keeping with this effect, aggregation of human platelets in the present study was inhibited with the same ASA preparation, (refer to Fig. 3). Consistent with the present findings is the reported inability of indomethacin (another non-steroidal anti-inflammatory drug: NSAID) to inhibit sheep platelet aggregation induced by collagen (18). This contrasts it’s potent inhibitory effects on human platelet aggregation even though it can inhibit TXB2 production by sheep platelets with the same potency as in human platelets (18). The mode of action of ASA and other NSAID’s responsible for their anti-inflammatory, antipyretic and analgesic effects is widely accepted to be their inhibition of PG biosynthesis at the cycle oxygenase step. This effect of NSAID’s originally shown for platelets (19) has since been confirmed in many other tissues in man and other species. PG biosynthesis is a universal property of most cells examined and is stimulated when cells are mechanically disrupted or deformed. PG biosynthesis occurs in platelets preceding aggregation and the intermediate products, thromboxanes are powerful platelet aggregants (20). ASA has been shown to irreversibly acetylate the & amino- group of a lysine residue at or near the active site of cycle oxygenase, resulting in the inhibition of the platelet release reaction and secondary aggregation (2). Because platelets lack a nucleus and do not synthesise proteins this effect lasts for the duration of the platelet.life-span in the circulation. Very few studies have investigated the sheep platelet in detail; however PG synthesis by sheep seminal vesicles was among the early studies of PG synthesis by tissues (21). Sheep platelets have also been shown to synthesise PG’s during collagen, but not ADP, induced aggregation (18,22); however the biosynthesis was much less than in humans. The report (21) that ASA inhibits PG biosynthesis by sheep seminal vesicles, in a time-dependent, and concentration-dependent manner, would by analogy, suggest a similar effect on sheep platelets. This has in fact been shown for indomethacin (18). The contrasting observations reported herein for ASA support the view that the AA pathway may not be of major significance in the sheep platelet. The inability of ASA to inhibit AA induced aggregation is quite extraordinary and suggests that sheep platelets, like sheep seminal vesicles (23) have an ASA resistant cycle oxygenase. The possibility of an AA independent aggregation pathway, although not proven by these studies, deserves further investigation. In this regard the sheep platelet could prove to be a valuable model for studying further the various postulated pathways of platelet aggregation (24). The inhibitory effect of ASA on platelet release and aggregation has formed the basis of the rationale of ASA’s prophylactic use against thrombotic occurrences. Unfortunately prophylaxis with ASA has not been universally achieved (2526)

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suggesting that some platelets aggregate independently of ASA inhibition. Whether the findings of the present study can be applied to this human situation needs further investigation as does the potential role of the sheep platelet as a useful model for testing anti-thrombotic drugs for these patients.

Acknowledgments

The author is greatly indebted to Professor H. Salem and the haematology dept. at the Monash University Box Hill hospital for the use of their facilities and reagents and to Dr. M. Brandon of the veterinary sciences dept. at the University of Melbourne for the sheep blood to carry out the arachidonic acid experiments.

REFERENCES

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