Dosage frequency for suppression of platelet function by low dose aspirin therapy

THKOHBOSXS

RESEARCH

27;

99-110,

1982

0049-3838/82/130099-12$03.00/O

Cooyright

Printed in the USA. (c) 1982 Perpamon Press itd. .All rights reserved.

DOSAGE FREQUENCY FOR SUPPRESSION OF ?WTELET BY LOW DOSE ASPIRIN THERAPY

FUNCTIOLI

B.A. Bradlow and N. Chetty, Department of Haematology, School of Pathology of the University of the Witwatersrand and the South African Institute for Medical Research, Johannesburg.

in revised form 22.3.1982. (Received 28.10.1981; Accepted by Editor C.R.M. Prentice. Received in final form by Executive Editorial Office 22.4.1982)

ABSTRACT

A study of platelet aggregation and lMDA production after an oral dose of 300 mg aspirin indicated that partial recovery of platelet function occurred when approximately one third of the circulating _-r. platelets had been replaced by new (uninhibited) platelets. Vitro studies on mixtures of normal and aspirin inhibited platelets indicated partial restoration of platelet aggregation and thromboxane B1 production with as little as 10% of normal platelets in Restoration of full function required a higher some subjects. There was considerable variation proportion of normal platelets. These data suggest that complete suppression between subjects. of platelet functions in all normal subjects requires daily administration of the drug. INTRODUCTION Aspirin (ASA) is one of the agents used in many clinical trials of drugs which aim to inhibit platelet function and prevent thrombosis (1,2,3). The optimum dose of ASA used for the purpose of preventing arterial thrombosis has not been established. The doses used in the clinical trials have varied from 324 to 1500 mg per day given in one to four equal doses. These doses have been questioned (4) and much lower doses have been advocated (5,6,7,5). Higher doses may even be thrombotic in rabbits (9) although not in rats (10). ASA has been shown to inhibit platelet aggregation by acetylation of the enzyme cycle-oxygenase (11,12). This effect persists for the entire life span of the platelet in the circulation. The same enzyme in endothelial cells is also inhibited by ASA but the effect in these cells is of short duration (13). Furthermore, the endothelial cell enzyme was found to be considerably less sensitive to ASA inhibition than the platelet enzyme by

KEY WORDS:

aspirin, dosage, platelet 99

aggregation

The duration of the .ASA effect a-d therefore t:he frequency, of dosage has also not been established. The recovery of cvclo-oxvgenase . activity in circulating platelets follows a time course corresponding approximately 53 the mean life span of platelets (lG,11,17,~8,1,~!. As ne:j platelets enter rhe circulation the proportion of normal to inhibited platelets jiill rise steadily but it is not !
In

hvo

studies

Blood samples were Seven normal subjects were each given 300 mg ASA orally. collected with 1 part 3.8% sodium citrate to 9 parts blood before the aspirin Platelet rich plasma (PR?) was dose, and at intervals for a further 10 days. prepared from the samples and the platelet count of the PRP was adjusted to 250 + Platelet aggregation according to the method of aorn and 20 x 10' per litre. Cross (22) was determined on a Payton aggregometer after stimulation by adenosine disphosphate (ADP) (0.5-10.0 PM) collagen (1.4 tlg/me) adrenalin 1.3-55 UM), arachidonic acid (AA) (0.2-1.0 mM). The concentration of agonist chosen for analysis was that which demonstrated the greatest difference between the pre-treatment aggregability and the maximum effect of the ASA dose. The concentrations used were: ADP 1.6 or 2.1 LX, adrenalin 1.3, 2.6 or 3.9 EM, collagen 1 or 2 ag/mL and arachidonic acid 0.5, 0.6 or 1 &I. For each agonist the height of the maximum aggregation response above the baseline at the chosen concentration was measured in millimeters before and on successive days after the ASA dose. Each post ASA observation was expressed as In the case of ADP only the height of the a percentage of the pre ASA response. secondary *wave was measured. Many of the post ASA responses to ADP showed a When this occurred the response was primary wave followed by disaggregation. recorded as zero. For the other three agonists the .maximu.mresponse z~,hethera single wave or two waves of aggregation was meas,Jred.

Voi.27, No.1

ASPIRIN

SUPPRESSION

OF

?LATEi.ETS

:Ui

Thirteen \yslunteers were given 600 mg and nine 150 ng aspirin arally. a1002 was collected (1 part 3.84 sodilum citrate 50 9 t -arts blood: before and PR? (250 - 20 x 10? per litre) was prepared tvo hoiirs after the aspirin dose. from both snmnlles immediately after collecticE. The ?R? from the first sample time PRP from the second was iept at room temperature for 2 hours by ;;:-.1,-h Aggregation studies .&ere then carried out on both prespec:sen was prepared. The pr+zASA Pi;? was tested for sensitivlt) parations and on mixtures of the two. to each of t‘neaggregating agonists to be test:?dand tine least concentration require_' to qiie maximum aggregation in each case was used for all s)ubsequent The agonists irsed aggregation stcidieson the pre and post ASA PR? mixtures. and the concentrations selected were: AD? fro:.2.1 to 10.6 PM, adrenalin from 1.3 to 5.5 WI, collagen from 1 to 4 ug/me and AA from 0.3 to 0.7 ;?3. The two PRP preparations were mixed to give the follo:
studies

The expected depression of platelet aggregation (Figs. 1 & 2) and fall in MDA production (Table 1) was present at 24 hours after the 300 mg oral dose of ASA. Aggregation in response to AA was reduced to zero and the second wave of aggregation following ADP stimulation disappeared in all cases. The responses to adrenalin and collagen were reduced to very low levels but did not disappear entirely except in two cases in which the collagen response disappeared on day 1 only. Definite evidence of partial recovery from the ASA inhibition was present in all cases on day 4, by day 6 aggregation was above 50% of the base line value in most cases and by day 7 recovery was complete or nearl:r so in all cases except for the response to ADP in three cases. On day 3 there was some evidence of recovery of aggregation responses in only one case but MDA production showed some increase in all cases tested (Table 1).

-cdbpsn ----

ADP

FIG. 1: Effect of 300 mg ASA on aggregation responses to collagen and ADP in seven normal subjects. Aggregation responses calculated as a percentage of the pre ASA response (see methods). Mean + S.D. are shown.

1’1

’ ‘\

/

I

I

/ 1’ / 1’

: I I \

‘\\ ‘\ \

I

I I

/i; I’

I

I

I

5

-e-c

j

AA

3

0

FIG. 2: Effect of 300 mq ASA on aggregation (AA) and adrenalin in seven normal subjects.

responses to arachidonic Mean + S.D. are shown.

acid

vo1.27,

ASPIRIS SUPPRESSION

No.

OF PMTELETS

.ASA ingestion (3133 sg) on I%)A ,rOd.;ctio?. of Results exoressed as percentage of initial x;alue.

ir: seven

Effect

C-OlilntePr3:

7 Days after ASA ingestion 0

1

2

3

4

5

7

. ‘Yean value Standard deviation NO.

of

100

observations

7

51.5 11.7

34 25.0

6

61.2 4. 3 4

3

73.3 18.6 6

81 15.3 5

33 5.6 3

& In four of the subjects studied mean platelet life span was determined In all four subjects platelet several veeks prior to the aspirin experiment. aggregation returned to normal in two to seven days less than tine mean platelet life span (Table 2).

Results obtained from subjects given 150 mg aspirin were not significantly different from those given 600 mg (p> 0.05, Fishers exact probabilities calMixtures of culated from 2 x 2 tables). ~11 the results were therefore pooled. normal and ASA inhibited platelets were tested for aggregation responses. A representative experiment showing the effect of increasing the proportion of normal platelets in the mixture is shown in Fig. 3 for each of the agonists. Fig. 4 shows the percentage of normal platelets required to produce full and partial (defined as more than 75% and 25% of full aggregation respectively) Table 3 shows that the mean values for t‘ne aggregation in 22 normal subjects. percentage of normal platelets required to produce full aggregation in a mixture of normal and ASA treated platelets varied between 25 and 53% for the different agonists. In the case of ADP full aggregation was restored by 10% of normal platelets in 4 cases and 20% in seven cases. Aggregation to adrenalin was restored fully in three cases by 10% of normal platelets and by 20% in three other cases. In most instances the proportion of normal platelets necessary was much higher. TABLE

2

A comparison of mean platelet life span determined by the Indiuml'i labelled platelet method and the time required for aggregation responses to return to normal after ASA (3CC mg) ingestion in four normal subjects.

Subjects

Mean platelet life span (Days)

Days required for aggregation to return to normal after ASA ingestion ADP

1 2 3 4 Mean

10.8 11.0 9.6 10.2 10.4

8 8 5 6 6.75

Adrenalin 5

4 4 6 4.75

j(lLLAGEN

(4&ml)

ARACHIDONIC ACID (ImM)

/

FIG. 3: Aggregation responses in mixtures of normal and ASA inhibited platelet: The figures shown next to each tracing represent the from one normal subject. percentage of normal platelets in the mixture.

100 cn 5

ADP

4RACHIDONIC ACID

I 1

COLLAGEN

ADRENALIN

2 80 z

: ??

em

li

. .

?? e

Tie et,‘,’

. ??

Yi..: . .

.@ie2. . . ?? ***

. .

.:* .

o-

J._

Partial

Partial

Full

i Partial

I

?? J

Full

Partial

Full

FIG. 4: Percentage of normal platelets required to restore platelet filnction in mixtures of normal and ASA inhibited platelets in 22 normal subjects. Full aggregation 0 = subjects given 600 mg ASA A = subjects given 150 mg ASA. was defined as >75% and partial aggregation as >25% of the response of normal (uninhibited) platelets.

ASPIRIN

FU;~

of normal

SiiP?&3SSIO?jOF

?UiiEi.ETS

195

aqgrega:isn

was defined as ::75% and partial as ;-258 of the resoonse (Iuninhibited) platelets. N = nlumbsr of subjects tested. _

?artial restoration of aggregation, was restored by a significantly loi.;er With 10% normal platelets partial restoration proportion of normal platelets. of aggregation was found in 13 cases after stimulation with AD?, 8 cases .with With 20% normal platelets an adrenalin, and 1 case with collagen and AA. additional 5 cases with ADP, 5 with adrenalin, 4 with collagen and 4 with AA achieved partial restoration of function (Fig. 4). Thromboxane production after stimulation with each of four agonists was measured in normal platelets (Table 4) and in mixtures of normal and in'nibited platelets (Fig. 5). With all four of the agonists TXS, production expressed as a percentage of the production in uninhibited platelets (Fig. 5) approximated the percentage of normal platelets in the mixture. The results suggested that normal platelets were not responsible for any production of TXS, in the inhibited platelets.

TABLE

4

L

Aggregating Agent

Concentration*

Thromboxane B, Production p Moles/me of Plasma Mean

Arachidonic acid Collagen ADP Adrenalin

0.5 - 1.0 mM 2-4 Gg/me 2.1 - 10.6 PM 1.3 - 5.5 PM

Results are the mean values derived six subjects.

210.3 11.8 4.2 5.9

from one experiment

S.D. 71.9 3.5 2.1 2.4

on each of

The concentration of aggregating agent used for each reagent was the minimum required to produce maximal aggregation and was determined by experiment for each of the six subjects and for each agonist. The range cf concentrations used is shown.

%

20 %

NORMAL

40

PLATELETS

So

NORMAL

So

%

loo

20

PLATELEI’S

%

NORMAL

40 NORMAL

PLATELETS

60

80

100

PLATELETS

m-8AGGREGATION o-a t

PROOL!CTiON

OF THFIOMEOXANE

ADP RESULTS REFER SECONDARY WAVE

ONLY

- 82 TO THE

HEIGHT

Of

THE

FIG. 5. Aggregation responses and TXB, production in mixtures of normal and ASA inhibited platelets. Means f S.D. derived from 13 normal subjects are shown. All results are calculated as the percentage of the aggregation response and of TXB, production in normal platelets (see methods). DISCUSSION Before discussing the significance of the results obtained in this study The production of MDA after aspirin two technical aspects warrant consideration. ingestion was reduced to an average of 34% of the basal load on the second day. Complete inhibition did not occur suggesting that either the cycle-oxygenase was only partially inhibited or an alternative pathway of MDA production exists The latter explanation is probable or that the assay procedure was not specific. The since it has been shown that lipid peroxides can react with TBA (27,28). second aspect concerns the possibility that the post ASA blood samples contained sufficient ASA to influence aggregation in the mixtures of pre- and post ASA PPP. When post ASA PPP was added to pre-ASA PRP aggregation to all four agonists This experiment is in keeping with used in this study was not inhibited. is pharmaco-kinetic data showing that the half-life of ASA in plasma ir. ViVO It is therefore very unlikely that the mixing experiments 13 to 20 minutes (29). were affected by residual ASA in the post-ASA specimens.

Vo‘

-.-.i,,

SO.1

XS?IRIN SU??XESS:ON 0; ?LAT'ELETS

ij-

-:le =es,lly_;r of +_:qese z_+,,4;,ir*?.l=ye ::;a: 33 :nteractisn between normal an5 ASA treated platelets can occl_irand :hat 5:s may have 3 bearing 3n the frecnenq' 3f .A% 30sage necessary t3 ach;e;*e a prslsnged inhibiti0n 3f platelet function. L__._..._3

T? Y<;',-studies indicate; a return 10 n0rzal aggregation responses several days before all ASA treated platelets were likely ts have disappeared. In four subjects the measured mean platelet s-rT;i-;a1times x2re 2-7 Says 10nger than the The s*zppression 3f times reauired f3r n0rmal aggregation t3 ret'urr.(Tabie 2). aggregati0n res0onses in tiie 25 Y-52: stsdies '&as incom0lete in most 3f the s;ljjects in that although the responses to c0lla;on and AA siere abolished within AZ 72 hours 24 h0urs those t0 AD? and adrenalin %ere red.-.ced 5ut not absent. t'here;v'as verz* little evidence of recovery sf f.unctisn, by 96 hours 2artial recovery was evident and full recovery o:cclurredwithin 5 to S days in all cases. At the tme f~uil recovery was present t:& proportion of new piatelets in the circulation was likely to be 50% or m3re. Partial recoverv occurred sooner at a time when the proportion of new pl.atelets co,ild h3ve been as 10.4 as 35%. \Gne n the pro0ortion of new platelets iias less :han this there was n3 evidence i of flunctisnal recovery alt‘nough the pr06ilctin of YDA ha:d risen frcm its nadir 3 days after ASA ingestion at :;hich time the proporti3n of normal platelets CC:Jld be 303 3r iess. The -'q _I_V~;?J studies showed that in many instances partial restoration of aggregation to acirenalin and ADP occurred with as little as 10% nOrE%i platelets in the mixturn of normal and ASA treated platelets. In all except one case at least 20% normal platelets .&as needed for partial restsrati0n of aggregation ti> collagen and AA. Full restoration cf function required at least 10‘; of normal platelets f0r ADP, 20% for adrenalin, 40% for collagen ani 30% for .A-?.

T'nroinboxan5 . i. B : production did not reach n0rnal values even when the propcrtisn 3f normal platelets ;;as raised t3 50% 0f the mixed olatelets. The normal platelets did not appear to cause TX3> prod~uctinn in the inhibited platelets. The increase in TXB, production as the proportion of normal plateleis in the mixture 'was increased lagged behind the increase in aggregability of the mixed platelets. This would indicate that normal aggregation occurred with considerably less than maximal TXB, production. These results contrast with those 0f Cerskus d: c?. (20) who found that 108 of normal platelets was sufficient t3 restore full aggregation after AA stimulation in a mixture of normal and ASA ?iatelPts. They also reported partial restoration of serotonin release by addition of 104 normal platelets to ASA treated platelets. Their work was carried out with rabbit platelets, and thereobtained fore a species difference might account for the different results '*ie with human cells. The in Vit1-r experiments in this study demonstrate that in most but not all cases a single dose of 300 mg ASA will produce complete suppression of aggregation for 72 hours. In the _r.V~S:T experiments, hodever, there was eviAence AL._.. to show that in some cases 10% of normal platelets could restore some aspects Of platelet func:ion. Twenty-four hours after a dose of ASA it is possible that 103 of normal platelets could have entered the circulation. This would imply that partial recovery of platelet function could 3.-cur as early as 24 hours after a dose. If consistent suppression of aggregatisn is to be achieved in all subjects Support for this view can be this data would argue for a daily dose of ASA. deduced from the observation (30) that platelets derived from donors who had ingested ASA 36 hours before donations were able to correct the bleeding time 3f thrombocytopenic patients although platelets prepared 12 h?3urs after ASA ingestion were ineffective. Considerable variation between ind;viual slubjects in the recovery of plstele: fjunction and in the proportisn of normal olatelets necessary tn restore aggregation responses was observed. Cthers ?1,32,33) have obser;_ed similar

Oheoretical considerations suggest that an i.iealdose of ASA wocld inhibit platelet aggregation Snt not PGl, productian by vascular endotheliui?. The dose used in the present study may be too high since 153 mg of ASA can suppress PGI, production in venous segments (34) although there was ;:ery little suppression of PGL, levels in venous blood after 3 minutes ischae-ia of the forea,rm -with a similar dose (7). in another study (35) a dose of 80 mg inhibited ?Gl: production in venous segments but 40 mg did not. The effect of daily doses of 40 mg may however be cumulative and suppress both PGl, and TXA: production (36). Even doses of 20 mg daily can suppress platelet aggregation (37,38) but the effect of PGl; production after prolonged therapy at this dose has not been assessed. If such low doses are necessary to avoid or ninimise effects on endothelial PGl, production it seems likely that t:heneed for daily adninistration would be even greater than our data suggest in order to achieve consistent suppression of platelet function.

ACKNOWLEDGEMENTS Thanks are due to the South African Medical Research Council, the South African Atomic Energy Board and the South African Institute for Medical Research for their generous financial support and providing facilities. REFERENCES 1.

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G-G.

vo:.;i,

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No.1

SUPPRES3IOS

OF PLATELETS

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J.W. Aspirin

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36.

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PATRIGNANI, P., CATTANI, P., MINUZ, P. and PATRONO, C. Low dose Symposium on Prostaglandins and the aspirin: how low, how often? Universitaire instelling Antwerpen Wilrijk, Cardiovascular system. Belguin, 1980.

38.

BRADLOW,

B.A. and CHETTY, N.

Unpublished

observations

1981.