Dose- and time-dependent antiplatelet effects of aspirin
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© 2006 Schattauer GmbH, Stuttgart
Platelets and Blood Cells
Dose- and time-dependent antiplatelet effects of aspirin
Christina Perneby1, N. Håkan Wallén1,2, Cathy Rooney3, Desmond Fitzgerald4, Paul Hjemdahl1
1
Department of Medicine, Clinical Pharmacology Unit, Karolinska University Hospital (Solna), Stockholm, Sweden; 2Department of Medicine,
Karolinska Institutet at Danderyd's Hospital, Danderyd, Danderyd, Sweden; 3Department of Clinical Pharmacology, Royal College of
Surgeons of Ireland, Dublin, Ireland; 4Molecular Medicine Laboratory, Conway Institute, University College Dublin, Ireland
Summary
Aspirin is widely used, but dosages in different clinical situations pirin. Once daily dosing was associated with considerable re-
and the possible importance of “aspirin resistance” are debated. covery of AA induced platelet aggregation inWB after 24 hours,
We performed an open cross-over study comparing no treat- even after 960 mg aspirin. Collagen induced aggregation in WB
ment (baseline) with three aspirin dosage regimens – 37.5 mg/ with normal extracellular calcium levels (hirudin anticoagulated)
day for 10 days,320 mg/day for 7 days,and,finally,a single 640 mg was inhibitedPerneby, et al.: Dose- and time-dependent aspirin effects
cular disease, and the clinical efficacy of aspirin is reduced or Study design
lost at doses below 75 mg/day (1, 2). A recent retrospective The study was an open cross-over study with measurements at
analysis of two GPIIb/IIIa inhibitor trials suggested that a higher baseline (untreated) and during treatment with three different
dose of aspirin may afford better protection against myocardial dosages of aspirin. Measurements were performed at baseline,
infarction (9), but a similar analysis from the CURE study indi- and after aspirin treatment with 37.5 mg/day for 10 days (half a
cated that ≤100 mg/day was more effective than ≥200 mg/day tablet Trombyl® 75 mg, Pharmacia Sverige AB), and 320 mg/
(10). Prospective, placebo-controlled trials have shown excellent day for 7 days (one tablet Alka Seltzer®, Bayer, Germany). Fin-
protection with 75 mg aspirin daily in patients with stable (11) or ally, they took a single dose of 640 mg aspirin (two tablets of
unstable (12) angina pectoris. Alka Seltzer®) 1–2h after the last 320 mg dose (cumulative dose
The effect of repeated daily low doses of aspirin is cumu- thus 960 mg on the last day). There was a 10–14 day wash-out
lative, and steady state with regard to platelet COX inhibition is period between the low dose and the intermediate/high dose in-
reached within 7 days (8).After a single full dose of aspirin, COX vestigations. The volunteers were instructed to refrain from caf-
activity recovers by some 10% per day, as a function of platelet feine, tobacco or food intake >12 hours before each visit.
turnover (2), which may vary both within and between patients. Blood sampling was performed after 30 min rest in the supine
Phasic increases in thromboxane synthesis were observed in pa- position on all occasions. Between samplings the volunteers
tients with unstable angina, indicating that platelet activation were free to move about, but were instructed not to perform rig-
may occur during spontaneous ischemia (13). Furthermore, dia- orous physical activity. At baseline, the sampling was performed
betes, preeclampsia and advanced coronary artery disease are as- three times during 24 hours (9 AM, 1 PM, and again at 9 AM) be-
sociated with increased platelet turnover (14–16). Thus, the fore the treatment was initiated. Thereafter, the subjects received
requirements for dosages and dosing intervals with aspirin treat- 37.5 mg aspirin daily (low dose) for 10 days, and on the 10th day
ment may differ in different clinical situations. blood sampling was performed 1.5–2 hours (9 AM), ≈6 hours
At low dosages, first pass hepatic metabolism of aspirin li- (1 PM), and 24 hours after the last dose of aspirin. The same pro-
mits the systemic exposure of the patients to COX inhibition, cedure was performed after the last ingested dose of 320 mg as-
thus preserving the vasodilating and antiplatelet effects of en- pirin; i.e. on the 7th day of treatment. However, after the 9 AM
dothelial prostacyclin (17). Higher doses of aspirin may, how- sampling (1.5–2h after drug ingestion) the volunteers took an ad-
ever, attenuate this potentially antithrombotic mechanism (2, ditional single 640 mg aspirin dose to ensure complete platelet
18). Experiments in knock-out mice support an antithrombotic COX-1 inhibition. Sampling was then repeated 4 hours and
role for endogenous prostacyclin (19). The importance of prosta- 24 hours after this high bolus dose.
cyclin in thrombotic processes in humans is not completely Each subject brought his morning urine to the laboratory be-
understood, but several studies have recently demonstrated unfa- fore the first blood sampling and when he returned the next day
vourable cardiovascular outcomes in patients treated with for 24 h sampling. Urine samples were also collected at 1 PM on
COX-2 inhibitors (20), and this may be related to suppression of days of experiment. These 1 PM samples reflect urinary excre-
prostacyclin production (21). tions of analytesPerneby, et al.: Dose- and time-dependent aspirin effects
centrations of 1, 3 or 5 µM and arachidonic acid (AA) dissolved ation and large interindividual variation have to be taken into
in ethanol (Sigma Chemical Co., St. Louis, MO, USA) at final consideration.
concentrations of 0.2, 0.5 or 1 mM. Each experiment included a Urinary 2,3-dinor-6-keto prostaglandin F1α (PGI-M) was de-
test to verify that the final ethanol concentration in the sample termined by LC/MS-MS (Rooney C, Harhen B, Fitzgerald D,
had no effect per se. The amplitude of aggregation was measured Treumann A, manuscript in preparation). Briefly, 2 ml of urine
after 8 minutes. spiked with 4 ng of the internal standard 2,3-dinor-6-keto prostag-
landin F1-D3 (Biomol, PA, USA) was adjusted to pH 3 and centri-
Aggregometry in platelet rich plasma (PRP) fuged at 950 x g for 5 minutes before being applied to Empore
A four-channel platelet aggregation profiler (PAP-4, Bio-Data solid phase extraction disc cartridges (3M, USA) from which the
Corporation, Hatboro, PA, USA) was used to study platelet ag- PGI-M was eluted with 1 ml of 1% methanol in ethyl acetate. Fol-
gregation in PRP. Adenosine diphosphate (ADP; Sigma Chemi- lowing liquid-liquid extraction with 50 mM sodium borate (pH
cal Co.) was diluted in Tris buffer and the EC50 for ADP (i.e. the 8.0) and derivatization with methoxyamine (Sigma, UK), the puri-
concentration required for half-maximal aggregation) was deter- fied and derivatized organic extract was loaded onto an omni-
mined by a dose-response procedure in which the extent of ag- sphere C18 reversed phase column (3 µm x 2.1 x 100 mm, Varian,
gregation after 4 minutes was measured; for details see (24). Pla- UK) and eluted with a linear gradient from 5% to 45% acetonitrile
telet COX inhibiting effects of aspirin treatment were deter- in 10 mM ammonium acetate. The effluent was analyzed by a
mined as the ability of AA (final concentrations 0.5 and 1 mM) triple quadrupole tandem mass spectrometer (API 3000, Applied
to induce aggregation in PRP. Biosystems). The transition of the ion at m/z 370 to 150.2 was
monitored for the analyte and that of the ion at m/z 373 to 150.2
Bleeding time was monitored for the internal standard, using an optimized colli-
Standardized transverse incisions were made on the lateral volar sion energy of 28 V. Concentrations of PGI-M were determined by
side of the forearm, at a constant venous pressure of 40 mmHg, calculating the peak height ratios for sample and internal standard.
using a disposable device (Surgicutt, Ortho Diagnostics, Raritan, Data from two out of nine samples in each of two individuals
NJ, USA). Each incision was made 10 mm distally to the pre- are missing due to technical problems with the analysis. PGI-M
vious one. Total bleeding time was measured by collecting blood data from one individual were completely excluded due to varia-
on filter papers at 15 s intervals, according to a technique pre- bility related to poor compliance with the restrictions of physical
viously shown to reveal aspirin effects in our laboratory (25). activity during the experiment. Thus, the PGI-M data presented
are from 12–14 individuals.
Thromboxane related measurements
Thromboxane B2 (TxB2) is the chemically stable and inactive hy- Platelet function test (PFA-100) and platelet counts
dration product of TxA2, and measurements of TxB2 in serum re- The PFA-100 analyzer (Dade Behring, Germany) was used for
flect platelet COX inhibition. 11-dehydro-TxB2 is the major measurements of platelet-related primary hemostasis capacity of
metabolite of TxB2 which is excreted in man (26). citrate-anticoagulated whole blood under high shear conditions
Urinary 11-dehydro-TxB2 (TxM) was determined by enzyme (29). Closure time (CT) is the time needed to form a platelet plug
immunoassay (Cayman Chemicals, Ann Arbor, MI, USA) using occluding the aperture cut in a collagen/epinephrine coated
a sample work-up procedure previously described and validated membrane. Measurements could only be performed in 6 individ-
by us (27). Two ml of centrifuged urine was diluted 1:2 with uals. Platelets were counted by a semiautomatic cell counter
63 mM ammonium bicarbonate buffer pH 8.6 and was incubated (Cellanalyzer 460, Medonic Solna).
for 3 hours to convert 11-dehydro-TxB2 to its open ring form be-
fore extraction with Bond-elute Certify-II columns (Varian). The Statistics
analyte was eluted with 2% formic acid in methanol. The eluate Data are presented as mean values ± SEM. Normally distributed
was evaporated in a vacuum centrifuge, resuspended in buffer variables were compared with paired t-tests, whereas bleeding
(pH 8.6), and incubated 6 hours before analysis. Urinary TxM is time data were compared with the Wilcoxon test and Spearman´s
expressed in relation to urinary creatinine. Data obtained with rank correlation. Effects of aspirin treatment were analyzed by
this modified assay correlate well with results obtained with GC- 2-factor repeated measures ANOVA:s for overall effects, fol-
MS analysis of 11-dehydro-TxB2 (27). lowed by appropriate after-testing. The software used was Statis-
Serum TxB2 was determined using commercially available tica, version 5.5 for PC. A p valuePerneby, et al.: Dose- and time-dependent aspirin effects Figure 1: Comparison of AA-induced aggregation (final concentrations 0.2, 0.5 and 1.0 mM) in whole blood anticoagulated with hi- rudin after different doses of aspirin. Blood samples were taken 1.5–2, 4–6 or 24 hours after the latest ingested dose. Low dose is 37.5 mg/day for 10 days (filled symbols). High-dose is 320 mg/day for 7 days and a single dose of 640 mg taken immediately after the 1.5–2 hour sample (open symbols). Thus, 6 h is ≈4 h after a cumulative dose of 960 mg aspirin in the high dose condition. 1.5–2 h after the last dose with no significant variation 24 h Aggregometry in platelet rich plasma (PRP) thereafter. The bleeding time was 11.7±1.3 min (p
Perneby, et al.: Dose- and time-dependent aspirin effects
Urinary 2,3-dinor-6-keto prostaglandin F1α (PGI-M)
The urinary PGI-M excretion was 16.5±1.6 and 14.6±1.0 ng/
mmol creatinine in nocturnal and 1 PM samples, respectively,
without treatment. The nocturnal PGI-M excretion decreased by
25±7% (pPerneby, et al.: Dose- and time-dependent aspirin effects
When platelet function is investigated in vitro, the anticoagu- and patient compliance are not known. We found that 960 mg as-
lant used may artifactually modify platelet responses. Sodium ci- pirin resulted in more pronounced inhibition of urinary TxM
trate is the most commonly used anticoagulant, which lowers than 37.5 mg some 5 hours after ingestion, but that this differ-
extracellular calcium and enhances the antiplatelet effects of as- ence had decreased 24 hours after ingestion. Chamorro, et al.
pirin (22–24). We used hirudin (a highly selective thrombin in- showed that non-responders to 300 mg aspirin (defined as pa-
hibitor) to preserve normal ionized calcium levels, and found li- tients with recurrent stroke within 12 months despite aspirin
mited inhibition by aspirin of collagen- and AA-induced platelet treatment) had less effectively inhibited AA dependent platelet
aggregation. Thus, AA-induced platelet aggregation occurred aggregation than patients without events (37). However, a higher
even after 960 mg aspirin if the AA concentration was high dose of aspirin (600 mg/day) resulted in similar inhibition of re-
enough. During low-dose aspirin treatment (37.5 mg/day) pla- sponses to AA in the two groups (37). This is in accordance with
telet aggregation in hirudinized whole blood was virtually unaf- our results showing more effective inhibition of AA-induced pla-
fected when stimulated by 1.0 mM AA. Collagen induced pla- telet aggregation in whole blood with higher doses of aspirin,
telet aggregation in hirudinized whole blood was minimally af- and that aggregation was correlated to serum TxB2 levels at a low
fected by aspirin treatment. aspirin dosage. Thus, the ability of aspirin to block platelet
A problem with in vitro studies of platelet function in PRP is COX-1 (“true aspirin resistance”) should be clearly distin-
that centrifugation of the blood may remove the largest and most guished from treatment failure when discussing “aspirin resis-
active platelets along with the red and white blood cells, which tance” (4), and the time-dependence of inhibitory effects should
are known to influence platelet behaviour (31, 32). Responses to be considered in this context.
antiplatelet agents like aspirin may also be influenced by the re- We found slight reductions of PGI-M excretion after aspirin
moval of other blood cells. For example, the antiplatelet efficacy treatment at all dose levels. After 960 mg aspirin there was a
of low-dose aspirin (50 mg/day) is attenuated by the presence of further reduction of PGI-M excretionPerneby, et al.: Dose- and time-dependent aspirin effects
pirin dose (and the dosing interval) for each patient group, and Acknowledgements
establishing well documented means of monitoring individual The authors are grateful for the expert technical assistance with platelet re-
patients may further improve the secondary prevention of athero- lated parameters by Maud Daleskog, Maj-Christina Johansson and Pia Hil-
thrombotic disease by aspirin treatment. lelsson (Stockholm), and for discussions and advice about prostacyclin
metabolite measurements toAchimTreumann and Brendan Harhen (Dublin).
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