Br. J. clin. Pharmac. (1979), 7, 283-286
EFFECT OF A SINGLE ORAL DOSE OF ASPIRIN ON THE PLATELET AGGREGATION RESPONSE TO ARACHIDONIC ACID A. BYE, YVONNE LEWIS & J. O'GRADY Wellcome Research Laboratories, Beckenham, Kent BR3 3BS
I The platelet aggregation response to arachidonic acid ex vivo was measured in six volunteers daily before and for 10 days after a single oral dose of 600 mg aspirin. 2 Arachidonic acid induced aggregation of platelets from all subjects before aspirin and aggregation ,occurred after an interval which varied inversely with the concentration of arachidonic acid. No aggregation occurred for 4 days after aspirin; a reduced response, compared with pre-aspirin values, was obtained on the 5th, 6th and 8th day. The values on days 7, 9 and 10 were not consistently different from the pre-aspirin values. In 6 subjects 24 h after aspirin ingestion the addition of 10-25% v/v normal platelets restored the aggregation response. 3 It is concluded that aspirin has an effect on the platelet precursors in the marrow in addition to its effect on circulating platelets.
Introduction Aspirin irreversibly acetylates the cyclo-oxygenase enzyme of human platelets (Roth, Stanford & Majerus, 1975; Roth & Majerus, 1975). It thereby inhibits their aggregation response to arachidonic acid. This response depends on conversion of arachidonic acid by cyclo-oxygenase to the prostaglandins PGG2 and PGH2 and thence to thromboxane A2 by thromboxane synthetase. As platelets are capable of only very restricted synthesis of enzyme (Steiner, 1970) the effect of exposure to aspirin persists for the duration of the life of the platelet. The present study was designed to investigate the time course of the effect of a single oral dose of aspirin on the platelet aggregation response to arachidonic acid and to establish whether the platelet survival time could be inferred. Method
Six healthy volunteers, five male, one female, were studied. Their age ranged from 20-35 years, their weight from 64.1-73.2 kg. None had taken any drug for 14 days. Informed consent was obtained. Blood samples (20 ml) were obtained from each subject at the same time on each of three consecutive mornings to provide control values. Each subject then took 600 mg aspirin and blood samples were collected as before over the next 10 days except on Saturdays and Sundays. To cover these omissions aspirin was administered on different days of the week to different subjects and the six subjects took aspirin on two 0306-5251/79/030283-04 $01.00 19
occasions. Blood was delivered into citrated tubes and platelet-rich plasma (PRP) obtained by centrifugation. A platelet count was performed and the platelet aggregation response to arachidonic acid measured within 2 h of blood sampling. Aggregation responses were measured turbidometrically by placing 0.5 ml aliquots of PRP in the cuvette of a Paton dual channel aggregometer. Arachidonic acid was then added to produce concentrations between 100-800 1±g/ml and the interval noted to the onset of aggregation taken as the time for the trace of turbidity to cross the projected baseline before addition of arachidonic acid. When no sign of aggregation appeared within 4 min this interval was expressed as infinity. At concentrations higher than 800 j±g arachidonic acid/ml a fine precipitate occurred instead of the usual flocculent one. This false aggregation set the upper limit of the observations. The concentration of arachidonic acid (tg/ml) was plotted against the reciprocal response interval (s-'). Mean values of the reciprocal response (s-') were calculated for all the platelet samples subjected to a specific concentration of arachidonic acid at a specified interval after aspirin ingestion. In order to stablise the variance of the aggregation times the regression of loge (s.d.) on loge (mean) for the pre-drug aggregation times to arachidonic acid at 150, 200, 250, 300, 400, 500 Lg/ml was calculated. An arbitrary value of 1000 s was assigned to those times recorded as infinity, for the purposes of this analysis. The slope of the regression line was approximately 2, indicating that the reciprocal transformation was appropriate to stablise the variance. The transformao Macmillan Journals Ltd 1979
284
A. BYE, YVONNE LEWIS & J. O'GRADY
tion is fortuitous in that the infinite values transform to zero and may be used without the need to assign arbitrary values subsequently. The mean values for every concentration could then be compared by Duncan's multiple range test with the corresponding initial mean value for every elapsed day after aspirin. Platelet rich plasma from a non-aspirin treated subject was added to PRP obtained from each subject I day after aspirin administration in volumes of 0, 10, 25 and 30% v/v and the aggregation response to arachidonic acid 400 tg/ml measured. A preliminary study showed that a light breakfast did not affect the aggregation response so such a breakfast was allowed before blood sampling.
Table 1
Table 1 summarises the mean reciprocal response times in subjects before aspirin. They increased directly with the concentration of arachidonic acid, up to 500 gg/ml. No aggregation occurred to any concentration of arachidonic acid used on days 1-4 after aspirin. Table 2 summarises the mean reciprocal response times in subjects from day 5 to day 10 after aspirin. On the fifth day aggregation occurred but the mean response times were significantly (P < 0.05) less than before aspirin at all arachidonic acid concentrations except 200 ILg/ml. The mean response times were significantly
Summary of mean reciprocal response times in subjects before aspirin
Reciprocal response time I/time (1) mean s.d. n
Harmonic mean (s)
Table 2
Results
Arachidonic acid 4tgIml) 100 200 300 400 150 250 500 600 0.00094 0.00834 0.01275 0.01603 0.01997 0.02578 0.03244 0.02455 0.00276 0.00655 0.00729 0.00744 0.00818 0.00964 0.009100.00947 17 17 27 26 30 29 25 2 1066
78.45
119.9
62.38
50.08
38.80
30.83
40.72
Summary of mean reciprocal response times (WI) in subjects 5 to 10 days after aspirin.
Arachidonic acid (;Lgml) 150
200
250
300
400
500
d5 0 (4)
d6 0
(6)
d8 0.00154 (5)
d7 pre di1 d9 0.01046 0.00948 0.00834 0.01260
(2)
(4)
(17)
(5)
d8 dlO pre d6 d9 d7 d5 0.00320 0.00640 0.00446 0.01109 0.01630 0.01275 0.01415 (7) (9) (4) (6) (2) (27) (11)
d6 0 (3)
d8 0.00227
d5 0.00424
d7 0.00544
dlO 0.02
(5)
(4)
(2)
(1)
d6 0.00730
d5 0.00804
d8 0.01066
(5)
(8)
(9)
d5 0.01248
d6 0.01545
d8 0.01619
(6)
(9)
(11)
d5 0.01027 (8)
d6 0.01743 (12)
d8 0.01981 (12)
d9 pre 0.01583 0.01603
(8)
(26)
d7 d9 dlO pre 0.01668 0.01756 0.01870 0.01997
(3)
(8)
(11)
(30)
d7 dlO d9 pre 0.01706 0.01785 0.02271 0.02578
(11)
(3)
(12)
(29)
dlO d7 d9 pre 0.02225 0.02738 0.03103 0.03244 (12) (3) (12) (25)
Table 2 shows means (as V/time (s-1)) for each day, with the number of observations in parenthesis. Means joined by a common underlining do not differ at the 5% level.
ASPIRIN AND PLATELET AGGREGATION
(P < 0.05) lower than before aspirin at all concentrations of arachidonic acid on day 6. Mean response time on day 7 were significantly (P
EFFECT OF A SINGLE ORAL DOSE OF ASPIRIN ON THE PLATELET AGGREGATION RESPONSE TO ARACHIDONIC ACID A. BYE, YVONNE LEWIS & J. O'GRADY Wellcome Research Laboratories, Beckenham, Kent BR3 3BS
I The platelet aggregation response to arachidonic acid ex vivo was measured in six volunteers daily before and for 10 days after a single oral dose of 600 mg aspirin. 2 Arachidonic acid induced aggregation of platelets from all subjects before aspirin and aggregation ,occurred after an interval which varied inversely with the concentration of arachidonic acid. No aggregation occurred for 4 days after aspirin; a reduced response, compared with pre-aspirin values, was obtained on the 5th, 6th and 8th day. The values on days 7, 9 and 10 were not consistently different from the pre-aspirin values. In 6 subjects 24 h after aspirin ingestion the addition of 10-25% v/v normal platelets restored the aggregation response. 3 It is concluded that aspirin has an effect on the platelet precursors in the marrow in addition to its effect on circulating platelets.
Introduction Aspirin irreversibly acetylates the cyclo-oxygenase enzyme of human platelets (Roth, Stanford & Majerus, 1975; Roth & Majerus, 1975). It thereby inhibits their aggregation response to arachidonic acid. This response depends on conversion of arachidonic acid by cyclo-oxygenase to the prostaglandins PGG2 and PGH2 and thence to thromboxane A2 by thromboxane synthetase. As platelets are capable of only very restricted synthesis of enzyme (Steiner, 1970) the effect of exposure to aspirin persists for the duration of the life of the platelet. The present study was designed to investigate the time course of the effect of a single oral dose of aspirin on the platelet aggregation response to arachidonic acid and to establish whether the platelet survival time could be inferred. Method
Six healthy volunteers, five male, one female, were studied. Their age ranged from 20-35 years, their weight from 64.1-73.2 kg. None had taken any drug for 14 days. Informed consent was obtained. Blood samples (20 ml) were obtained from each subject at the same time on each of three consecutive mornings to provide control values. Each subject then took 600 mg aspirin and blood samples were collected as before over the next 10 days except on Saturdays and Sundays. To cover these omissions aspirin was administered on different days of the week to different subjects and the six subjects took aspirin on two 0306-5251/79/030283-04 $01.00 19
occasions. Blood was delivered into citrated tubes and platelet-rich plasma (PRP) obtained by centrifugation. A platelet count was performed and the platelet aggregation response to arachidonic acid measured within 2 h of blood sampling. Aggregation responses were measured turbidometrically by placing 0.5 ml aliquots of PRP in the cuvette of a Paton dual channel aggregometer. Arachidonic acid was then added to produce concentrations between 100-800 1±g/ml and the interval noted to the onset of aggregation taken as the time for the trace of turbidity to cross the projected baseline before addition of arachidonic acid. When no sign of aggregation appeared within 4 min this interval was expressed as infinity. At concentrations higher than 800 j±g arachidonic acid/ml a fine precipitate occurred instead of the usual flocculent one. This false aggregation set the upper limit of the observations. The concentration of arachidonic acid (tg/ml) was plotted against the reciprocal response interval (s-'). Mean values of the reciprocal response (s-') were calculated for all the platelet samples subjected to a specific concentration of arachidonic acid at a specified interval after aspirin ingestion. In order to stablise the variance of the aggregation times the regression of loge (s.d.) on loge (mean) for the pre-drug aggregation times to arachidonic acid at 150, 200, 250, 300, 400, 500 Lg/ml was calculated. An arbitrary value of 1000 s was assigned to those times recorded as infinity, for the purposes of this analysis. The slope of the regression line was approximately 2, indicating that the reciprocal transformation was appropriate to stablise the variance. The transformao Macmillan Journals Ltd 1979
284
A. BYE, YVONNE LEWIS & J. O'GRADY
tion is fortuitous in that the infinite values transform to zero and may be used without the need to assign arbitrary values subsequently. The mean values for every concentration could then be compared by Duncan's multiple range test with the corresponding initial mean value for every elapsed day after aspirin. Platelet rich plasma from a non-aspirin treated subject was added to PRP obtained from each subject I day after aspirin administration in volumes of 0, 10, 25 and 30% v/v and the aggregation response to arachidonic acid 400 tg/ml measured. A preliminary study showed that a light breakfast did not affect the aggregation response so such a breakfast was allowed before blood sampling.
Table 1
Table 1 summarises the mean reciprocal response times in subjects before aspirin. They increased directly with the concentration of arachidonic acid, up to 500 gg/ml. No aggregation occurred to any concentration of arachidonic acid used on days 1-4 after aspirin. Table 2 summarises the mean reciprocal response times in subjects from day 5 to day 10 after aspirin. On the fifth day aggregation occurred but the mean response times were significantly (P < 0.05) less than before aspirin at all arachidonic acid concentrations except 200 ILg/ml. The mean response times were significantly
Summary of mean reciprocal response times in subjects before aspirin
Reciprocal response time I/time (1) mean s.d. n
Harmonic mean (s)
Table 2
Results
Arachidonic acid 4tgIml) 100 200 300 400 150 250 500 600 0.00094 0.00834 0.01275 0.01603 0.01997 0.02578 0.03244 0.02455 0.00276 0.00655 0.00729 0.00744 0.00818 0.00964 0.009100.00947 17 17 27 26 30 29 25 2 1066
78.45
119.9
62.38
50.08
38.80
30.83
40.72
Summary of mean reciprocal response times (WI) in subjects 5 to 10 days after aspirin.
Arachidonic acid (;Lgml) 150
200
250
300
400
500
d5 0 (4)
d6 0
(6)
d8 0.00154 (5)
d7 pre di1 d9 0.01046 0.00948 0.00834 0.01260
(2)
(4)
(17)
(5)
d8 dlO pre d6 d9 d7 d5 0.00320 0.00640 0.00446 0.01109 0.01630 0.01275 0.01415 (7) (9) (4) (6) (2) (27) (11)
d6 0 (3)
d8 0.00227
d5 0.00424
d7 0.00544
dlO 0.02
(5)
(4)
(2)
(1)
d6 0.00730
d5 0.00804
d8 0.01066
(5)
(8)
(9)
d5 0.01248
d6 0.01545
d8 0.01619
(6)
(9)
(11)
d5 0.01027 (8)
d6 0.01743 (12)
d8 0.01981 (12)
d9 pre 0.01583 0.01603
(8)
(26)
d7 d9 dlO pre 0.01668 0.01756 0.01870 0.01997
(3)
(8)
(11)
(30)
d7 dlO d9 pre 0.01706 0.01785 0.02271 0.02578
(11)
(3)
(12)
(29)
dlO d7 d9 pre 0.02225 0.02738 0.03103 0.03244 (12) (3) (12) (25)
Table 2 shows means (as V/time (s-1)) for each day, with the number of observations in parenthesis. Means joined by a common underlining do not differ at the 5% level.
ASPIRIN AND PLATELET AGGREGATION
(P < 0.05) lower than before aspirin at all concentrations of arachidonic acid on day 6. Mean response time on day 7 were significantly (P
