THERAPY AND PREVENTION
THROMBOSIS
Inhibition of prostacyclin and thromboxane A2
generation by low-dose aspirin at the site of plug
formation
in man in
vivo
P. A. KYRLE, M.D., H. G. EICHLER, M.D., U. JAGER, M.D.,
AND
K. LECHNER, M.D.
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ABSTRACT In a double-blind placebo-controlled crossover study, we investigated in seven healthy
male volunteers the effect of a low-dose aspirin regimen (35 mg acetylsalicylate per day for 7 days) on
the formation of thromboxane A2 (TxA2) and prostacyclin (PG12) in blood emerging from a standardized injury of the microvasculature made to determine skin bleeding time. When subjects were treated
with placebo, there was rapid and substantial generation of TxA2 and PG12 at the site of platelet-vessel
wall interaction within the first 2 min after vascular injury. This was reflected by a greater than 100-fold
and greater than 10-fold increase in thromboxane B2 (TxB2) and 6-keto-prostaglandin F,a (6-ketoPGF,a) in blood obtained from incisions made to determine bleeding time as compared with the
corresponding plasma values. Low-dose aspirin caused a significant inhibition of both TxA2 and PGO2
generation in blood sampled from the skin incisions, represented by a 85% and 92% and 81% and 84%
inhibition of TxB2 and 6-keto-PGFIa, respectively, as compared with controls. We therefore conclude
that (1) rapid activation of both platelet prostaglandin metabolism and vascular PGO2 biosynthesis
occurs at the site of platelet-vessel wall interaction, and (2) low-dose aspirin results in a significant
inhibition of both platelet and vascular cyclooxygenase activity. Thus, our data fail to confirm the
concept of a differential effect of low-dose aspirin on platelet and vascular prostaglandin synthesis in
man in
vivo.
Circulation
75, No.
5,
1025-1029, 1987.
BY ACETYLATING the active site of the enzyme
cyclooxygenase, aspirin blocks the formation of prostaglandin endoperoxides from arachidonic acid.' This
leads to inhibition of the production of thromboxane A2
(TxA2), a potent vasoconstrictor and platelet aggregating agent in platelets,2' 3 and of its counterpart, prostacyclin (PG12), a potent inhibitor of platelet aggregation
and vasodilator in vascular tissue.4 5
It has been suggested that an aspirin regimen that
selectively inhibits TxA2 formation in platelets without
blocking PG02 generation in the vessel wall may improve the efficacy of aspirin as an antithrombotic
agent.6 Whereas single doses of aspirin failed to exhibit a selective reduction of platelet thromboxane
production, it has recently been shown that longterm treatment with very low doses of aspirin substantially inhibits thromboxane B2 (TxB2) serum proFrom the Division of Haematology and Haemostasiology, and the
Division of Clinical Pharnacology, I. Med. Univ. - Klinik, Vienna.
Address for correspondence: Dr. P. A. Kyrle, Division of Haematology and Haemostasiology, I. Med. Univ. - Klinik, Lazarettgasse 14,
A-1090 Wien, Austria.
Received Oct. 9, 1986; revision accepted Jan. 2, 1987.
Vol. 75, No. 5, May 1987
duction without affecting urinary excretion of PG12
metabolites.7' 8
However, one major obstacle in the search for a
solution to the "aspirin dilemma" is that both PG12 and
TxA2 exert their effects on thrombus formation in the
locality where they are generated, i.e., at the site of
platelet-vessel wall interaction rather than in the general circulation. Thus, it is doubtful that PG12 and TxA2
levels in serum, plasma, and urine or the capacity of
isolated vascular tissue to produce PG12 truly reflect
TxA2 and PG12 synthesis at an active site in vivo.
A closer experimental approach in investigating the
mechanisms leading to hemostasis in vivo has recently
been described9l" that consists of the measurement of
mediators of hemostasis in blood emerging from a
standardized injury of the microvasculature made to
determine skin bleeding time.
We have adapted this technique to evaluate the effect of a low-dose aspirin regimen on the formation of
PG12 (measured as 6-keto-prostaglandin Fia [6-ketoPGFIa]) and TxA2 (measured as TxB2) at the site of
platelet-vessel wall interaction in man in vivo.
1025
KYRLE et al.
PGF1,0
Materials and methods
PGE2 less than 0.007, 6,15-dioxo
Study design. The study was conducted as a double-blind,
placebo-controlled, crossover trial. Seven healthy male volunteers, 23 to 35 years old, who had not ingested any drugs for at
least 2 weeks before the study and did not take any during the
study period were investigated. They did not smoke and refrained from coffee consumption throughout the study period.
Each subject received either 35 mg of aspirin (Bayer, Leverkusen, F.R.G.; acetylsalicylate powder) or an oral placebo (starch
powder) dissolved in water daily for 7 days (day 1 to day 7). The
drug was taken by each volunteer at 8.00 P.M. Skin bleeding
time was determined and venous blood and blood obtained from
incisions made to determine bleeding time were sampled 12 hr
after the last dose of aspirin (day 8, 8.00 A.M.). After washout
period of at least 2 weeks, subjects crossed over to the alternate
treatment. The study protocol was approved by the Hospital
Ethics Committee and the volunteers gave written informed
13,14-dihydro-6,15-dioxo-PGF,. less than 0.1%, 15-oxo-E2
less than 0.007%, TxB2 less than 0.007%, 13,14-dihydro-15oxo-PGF2a less than 0.007%, 15-oxo-PGFIa less than 0.007%,
consent.
Bleeding time. The skin bleeding time was determined according to the technique originally described by Mielke et al.'12
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A sphygmomanometer cuff was placed on the upper arm and
inflated to 40 mm Hg. Two incisions, 5 mm long X 1 mm deep,
were placed on the lateral aspect of the volar surface of the
forearm parallel to the antecubital crease with use of a disposable standard device (Simplate II, General Diagnostics, NJ).
The procedure was carried out by the same investigator each
time.
Sampling of blood from incisions made to determine
bleeding time. Blood emerging from the incision made for the
skin bleeding time study was sampled as described recently. '0, l l Briefly, the blood was collcted at 30 sec intervals directly from the edge of the skin wound into calibrated polythene
cannulas (Portex 90 tubing, inner diameter 0.86 mm, outer
diameter 1.27 mm, Portex Ltd, Hythe, U.K.). Nine volumes of
blood were passed into 1 volume of an anticoagulant mixture
consisting of 100 mM ethylene-diamine tetraacetic acid, 60,M
indomethacin (Sigma Chemical Co. Ltd., Poole, U.K.), and
300 mM 6-oxo-prostaglandin El (kindly provided by Dr. John
Westwick, Department of Pharmacology, Royal College of
Surgeons, London, U.K.). After mixing, the tubes were centrifuged at 12,000 g for 1 min. The supematant was removed,
frozen, and stored at - 800 C until assay.
Venous blood. Blood was collected by a clean puncture of a
large antecubital vein without tourniquet by the use of a 19gauge needle. For the measurement of 6-keto-PGF,, and TxB2
in plasma, blood was drawn into 1/10 vol of the anticoagulant
solution described above and immediately centrifuged at 2000 g
for 15 min at 40 C. The supernatants were removed, frozen, and
stored at -80° C until assay. For determination of 6-ketoPGFia and TxB2 serum levels, nonanticoagulated blood was
incubated for 90 min in glass tubes at 370 C. After centrifugation
at 2000 g for 20 min, the supernatants were removed, frozen,
and stored at - 800 C until assay.
Assays. 6-keto-PGFi,a and TxB2 concentrations were measured by the use of radioimmunoassay procedures described in
detail previously.'3' 14 Rabbit anti-TxB2 antiserum and sheep
anti-6-keto-PGFI, antiserum, TxB2, and 6-keto-PGFl, were
provided by Dr. John Westwick, Department of Pharmacology,
Royal College of Surgeons, London, U.K. The rabbit anti-TxB2
antibody has the following cross-reaction at 30% displacement:
TxB2 100%, PGD2 3.5%, PGF2a less than 0.2%, PGE2 less than
0.1%, 6-oxo-PGFi, less than 0.01%, 6,15-dioxo-PGFI0 less
than 0.01%, 15-keto-PGE2 less than 0.001%, 13,14 dihydroPGE2 less than 0.01, and arachidonic acid less than 0.001%.
The sheep anti-6-keto-PGFi,a antibody has the following crossreaction at 10% displacement: 6-keto-PGFI, 100%, PGF2a less
than 0.15%, PGE, less than 0.1%, PGFia less than 0.08%,
1026
less than 0.22%,
and 15-oxo-PGF2a less than 0.007%. [3H]-TxB2 (100 to 140
mCi/mmol) and [3H]-6-keto-PGFia (100 to 140 mCi/ mmol)
were purchased from New England Nuclear, Southhampton,
U.K. The limit of sensitivity of both assays is 20 pg/ml. When
TxB2 or PGI2 concentrations were below the limit of sensitivity
of the assays, 20 pg/ml TxB2 or PGI2 was assumed in order to
carry out statistical evaluation of the data.
Statistical analysis was performed by the use of Wilcoxon's
matched-pairs signed-ranks test.'5
Results
Effect of low-dose aspirin on bleeding time. The mean
bleeding time was 348 + 31 sec when the subjects
were treated with placebo. A statistically significant
prolongation to 410 ± 29 sec was seen after administration of a low-dose aspirin regimen (p < .05).
Effect of low-dose aspirin on 6-keto-PGFia and TXB2
plasma and serum concentrations. 6-keto-PGF,, and
TxB2 plasma levels were below 20 pg/ml after placebo
and after aspirin treatment. 6-keto-PGF,i and TxB2
serum concentrations after aspirin were inhibited by
85% (decrease from 139.4 ± 34.5 pg/ml to less than
20 pg/ml 6-keto-PGF,0, p < .05) and 97.6% (decrease
from 236 ± 43.3 to5.65 ± 1.45 ng/ml TxB2, p < .05)
compared with those in control subjects.
Effect of low-dose aspirin on TxB2 generation in blood
obtained from the incisions made to determine bleeding
time. When subjects were treated with placebo, 2.8 +
1 ng/ml TxB2 was found in blood sampled from the
skin incisions during the first minute (figure 1). A peak
value of 5.3 ± 0.6 ng TxB2/ml was found in samples
from the third minute. Low-dose aspirin treatment
caused a marked reduction in TxB2 generation in blood
obtained throughout the 4 min study period, with a
peak value of 0.62 + 0.25 ng/ml at the fourth minute.
Similarly, aspirin caused a reduction in the mean TxB2
peak values in blood obtained during the bleeding time
studies (from 6.0 ± 0.7 ng/ml in the control subjects
to 0.7 ± 0.3 ng/ml after aspirin, p < .05).
Efect of low-dose aspirin on 6-keto-PGFia generation in
blood obtained from incisions made to determine bleeding
time. When the volunteers were treated with placebo,
6-keto-PGFia in blood sampled from the skin incisions
rose rapidly from 38 ± 6 pg/ml in the first minute to
197 ± 66 pg/ml in the second minute. No further
increase was seen in the third and fourth minute (figure
2). The 6-keto-PGFi, concentrations in blood samples
obtained from the incisions in aspirin-treated volunteers were similar to the values found in the placebo
group in the first minute. No further increase, but
rather a decrease, was found after aspirin treatment
CIRCULATION
THERAPY AND PREVENTION-THROMBOSIS
6
5
4
(N
pp 3-
2
X ~ ~~~~~~~~~
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1
2
mln
'4
3
FIGURE 1. Effect of low-dose aspirin (35 mg dail1 y for 7 days) on the
formation of TxB2 in blood obtained from the incis.,ions made to determine bleeding time. The values are plotted as the n-nean ± SEM from
seven volunteers before aspirin (0) and after (0) a spirin.
.
within the second, third, and fourth mi nutes. Aspirin
caused a reduction in the mean 6-kelto-PGFiat peak
values from 271 + 44 pg/ml in the conitrol subjects to
39 ± 11 pg/ml after drug treatment (F < .05).
nute
A
Discussion
It has recently been shown that the mieasurement of
platelet- and coagulation-derived prodlucts in blood
emerging from a local injury of the mivcrovasculature
induced to determine skin bleeding tinie is a suitable
technique for examining the mechanis ims leading to
primary hemostasis in man in vivo.9-11 VVe have shown
that m'arked platelet activation and conisiderable generation of thrombin occur in the early stages of plug
formation, as indicated by a 50-fold, 3()-fold, and 12fold increase in TxB2, /3-thromboglobul in, and fibrinopeptide A in blood obtained from inciisions made todetermine bleeding time as compared 'with the corresponding plasma values.10' 11 The pres,ent study was
designed to investigate the amount and time course of
the generation of PGI2, a potent antiagg;regatory agent
and vasodilator, at the site of platelet-ve.ssel wall interaction and the effect of a low-dose aspinrn regimen on
this process.
Levels of 6-keto-PGFl, in blood obt;ained from the
Vol. 75, No. 5, May 1987
skin incisions made to determine bleeding time within
4 min after vascular injury were at least 10-fold higher
than plasma concentrations. Since superficial incisions
such as those performed in our experiments mainly
transect the capillary network of the skin and to a lesser
extent vessels of the horizontal subpapillary plexus,
which consist of both arterioles and postcapillary venules, we can assume that 6-keto-PGF1a in blood from
the incision made to determine bleeding time reflects
PGI2 biosynthesis in the microvasculature rather than
in large blood vessels.l6 Evidence has been presented
that PGI2 exhibits its effects on plug formation locally
at the site of vessel injury rather than in the general
circulation.'7 It has also been reported that under resting conditions, levels of 6-keto-PGF,a in plasma are in
the very low picogram range'8 and pulmonary release
of PGI2 is not detectable.'9 Furthermore, inhibition of
PGI2 synthesis does not render intact endothelium adherent for platelets,20 but does increase the number of
platelets that become adherent to subendothelial structures.2' Thus, our observation of a rapid generation of
substantial amounts of PGI2 in the microvasculature
during primary hemostasis are in good agreement with
these reports suggesting that PGJ2 is an important meformation,
of plug
in the vicinity
diator
of not
hemostasis
physiounder
hormone
act as a circulating
does
but
logic conditions in man.
Repetitive administration of a low dose of aspirin
250-
-200E
150-
0
(D
100
-
50
1
2
mln
,
1
3
4
FIGURE 2. Effect of low-dose aspirin (35 mg daily for 7 days) on the
formation of 6-keto-PGFi,, in blood obtained from the incisions made to
determine bleeding time. The values are plotted as the mean ± SEM
from seven volunteers before aspirin (0) and after (0) aspirin.
1027
KYRLE et al.
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(35 mg per day for 7 days) caused significant inhibition
of the generation of TxB2 at the site of plug formation,
as reflected by a 83% to 92% reduction in blood sampled from skin incisions within the first 4 min after
injury of the microvasculature. This is in good agreement with previous reports demonstrating marked reduction of TxB2 generation in vitro by low-dose
aspirin, as indicated by a greater than 90% inhibition
of TxB2 serum production7 and absent or substantially decreased aggregation responses to arachidonate,
ADP, collagen, and epinephrine.22 Thus, blockade of
platelet cyclooxygenase by low-dose aspirin leads to
an impairment of platelet function in vivo, as reflected
by reduction of /3-thromboglobulin and TxB2 in blood
from the incision made to determine bleeding time,10 1 1
significant prolongation of the bleeding time,10 11 22
and an increase in the blood loss from the incision. 10. 1 1
According to the TxA2-PG61 balance theory, an aspirin dosing regimen that blocks platelet thromboxane
production without inhibiting vascular PGI2 synthesis
may increase the efficacy of aspirin as an antithrombotic agent.6 It has been shown that long-term administration of very low doses of aspirin per day results in
cumulative inhibition of TxB2 generation in vitro and
of urinary excretion of 6-keto-TxB2 without significant
changes in urinary 6-keto-PGFl,, and 2,3-dinor-6-ketoPGFl., indexes of renal and systemic PG1, production,
respectively.7' 8 However, significant (>80%) reduction of PG61 biosynthesis was found in vascular tissue
obtained from healthy subjects treated with a similar
aspirin regimen (40 mg of aspirin for 4 days).23 Furthermore, low-dose aspirin (20 mg per day for 1 week)
caused a 50% inhibition of PG12 generation in both
aortic and venous tissue in patients with atherosclerosis.22 Therefore, excretion of urinary PG12 metabolites
and 6-keto-PGFia production by vascular specimens ex
vivo may not necessarily reflect the actual production
rates of PG12 in vivo at the site of platelet-vessel wall
interaction.
In view of these discrepant findings, we have studied the effect of low-dose aspirin on PGJ, biosynthesis
in a recently developed preparation that may be more
relevant to PG61 generation in the microcirculation: the
measurement of 6-keto-PGFIa in blood emerging from
a template incision made to determine bleeding time.
Under these experimental conditions, low-dose aspirin
caused a significant reduction in 6-keto-PGFi, in blood
sampled in this way, as reflected by a greater than 80%
inhibition between the second and fourth minute after
injury. This strongly indicates that repeated doses of
aspirin, even those as low as 35 mg/day, induce significant inhibition of cyclooxygenase, not only in
1028
platelets but also in the microvasculature in vivo. It is
of further interest to investigate whether our observation of a substantial blockade of both platelet and vascular cyclooxygenase by low-dose aspirin holds true
for subjects with an abnormal platelet-vessel wall interaction, such as diabetics and patients with atherosclerosis. This is currently under investigation.
In conclusion, we have shown that rapid and substantial generation of both TxA2 and PG12 occurs at the
site of platelet-vessel wall interaction. Treatment with
low-dose aspirin leads to a significant inhibition of
PG12 and TxA2 formation at the site of plug formation.
Thus, our results fail to confirm the hypothesis of a
differential effect of low-dose aspirin on platelet and
vascular prostaglandin biosynthesis in man in vivo.
References
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CIRCULATION
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Vol. 75, No. 5, May 1987
1029
Inhibition of prostacyclin and thromboxane A2 generation by low-dose aspirin at the site
of plug formation in man in vivo.
P A Kyrle, H G Eichler, U Jäger and K Lechner
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Circulation. 1987;75:1025-1029
doi: 10.1161/01.CIR.75.5.1025
Circulation is published by the American Heart Association, 7272 Greenville Avenue, Dallas, TX 75231
Copyright © 1987 American Heart Association, Inc. All rights reserved.
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