Dose-Dependent Inhibition of Platelet Function by Acetaminophen in Healthy Volunteers Edward Munsterhjelm, M.D.,* Nina M

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Anesthesiology 2005; 103:712–7 © 2005 American Society of Anesthesiologists, Inc. Lippincott Williams & Wilkins, Inc. Dose-dependent Inhibition of Platelet Function by Acetaminophen in Healthy Volunteers Edward Munsterhjelm, M.D.,* Nina M. Munsterhjelm, B.Pharm.Sc.,† Tomi T. Niemi, M.D., Ph.D.,‡ Olavi Ylikorkala, M.D., Ph.D.,§ Pertti J. Neuvonen, M.D., Ph.D.,࿣ Per H. Rosenberg, M.D., Ph.D.#

Background: Acetaminophen (paracetamol) is widely used Normal platelet function is dependent on the production for postoperative analgesia. Its mechanism of action is inhibi- of proaggregatory thromboxane A2 (TxA2) through tion of prostaglandin synthesis in the central nervous system, COX-1, and acetaminophen has been shown to inhibit and acetaminophen is traditionally not considered to inﬂuence 5 platelet function. The authors studied the dose-dependent inhi- platelet function both in vitro and in high intravenous 6

bition of platelet function by acetaminophen in healthy volun- doses in vivo. However, oral administration of conven- Downloaded from http://pubs.asahq.org/anesthesiology/article-pdf/103/4/712/360846/0000542-200510000-00009.pdf by guest on 29 September 2021 teers. tional doses (approximately 1 g) of acetaminophen does Methods: Thirteen healthy male volunteers (aged 19–26 yr) not alter platelet function.7,8 were given placebo or 15, 22.5, or 30 mg/kg acetaminophen Acetaminophen is widely used for postoperative anal- intravenously in a double-blind, crossover study. Ten and 90 9 10,11 min after infusion, platelet function was assessed by photomet- gesia, although the optimal dose is debatable. In

ric aggregometry and by measuring release of thromboxane B2, pediatric patients, no analgesic ceiling effect was de- analgesia by cold pressor test, and plasma acetaminophen con- tected when acetaminophen was administered rectally centrations by high-performance liquid chromatography. in doses up to 60 mg/kg.12 However, high doses of ␮ Results: When triggered with 500 M arachidonic acid, me- acetaminophen may alter platelet function through pe- dian platelet aggregation (area under the curve) was 25.7, 22.8, -or 3.6 ؋ 103 area units (P < 0.001) 10 min after placebo or ripheral COX-1 inhibition. Because proper platelet func ,4.1 15, 22.5, or 30 mg/kg acetaminophen, respectively. An increas- tion is essential for adequate intraoperative and postop- ing concentration of arachidonic acid attenuated the antiaggre- erative hemostasis, we studied the dose-dependent gatory effect. After 90 min, platelet function was recovering. effect of acetaminophen on platelet function in healthy Release of thromboxane B was also dose-dependently inhib- 2 volunteers. We also measured the analgesic effect of ited by acetaminophen. Although plasma concentration of acetaminophen increased linearly with the dose, no analgesic effect acetaminophen with the cold pressor test as a painful was detected in the cold pressor test. stimulus. Conclusions: Acetaminophen, which is a weak inhibitor of platelet cyclooxygenase 1, has a dose-dependent antiaggregatory effect. This property may become clinically significant Materials and Methods in patients with intrinsic or drug-induced impairment of hemostasis. The protocol was approved by the Ethics Committee for Studies in Healthy Subjects and Primary Care in the CYCLOOXYGENASE (COX), the key enzyme in prosta- Hospital District of Helsinki and Uusimaa (Helsinki, Fin- glandin formation, is an important pharmacologic tar- land) and by the National Agency for Medicines in Fin- get.1 The antithrombotic effect of acetylsalicylic acid is land. Fifteen healthy, nonsmoking men aged between 19 caused by irreversible inhibition of COX-1, constitutively and 26 yr volunteered in this double-blinded, random- expressed in platelets, whereas the analgesic effect of ized, placebo-controlled, crossover study. Written in- nonsteroidal antiinflammatory drugs (NSAIDs) is medi- formed consent was obtained from each subject before ated through inhibition of COX-2, induced during inflam- the study. Normal plasma alanine transaminase and as- mation. The main mechanism of action of acetamino- partate aminotransferase activities were a prerequisite phen is inhibition of prostaglandin synthesis in the for participation. Two volunteers withdrew their con- central nervous system,2 the recently characterized sent before completing the study. The use of acetylsali- COX-3 being a possible target.3 However, acetamino- cylic acid was forbidden for 10 days and that of other phen has also peripheral COX-1 inhibiting properties.4 drugs affecting platelet function was forbidden for 1 week before each experiment.

* Resident, Department of Anesthesiology and Intensive Care Medicine, Hel- Experimental Procedures sinki University Hospital, Helsinki, Finland. † Researcher, ‡ Assistant Professor, Every volunteer participated in four experiments with # Professor, Department of Anesthesiology and Intensive Care Medicine, § Pro- fessor, Department of Obstetrics and Gynecology, ࿣ Professor, Department of at least a 1-week interval. After3hoffasting, 15, 22.5, or Clinical Pharmacology, University of Helsinki, Helsinki, Finland. 30 mg/kg acetaminophen (Perfalgan®; Bristol-Myers Received from the Department of Anesthesiology and Intensive Care Medi- Squibb, New York, NY) or placebo (0.9% NaCl; Braun, cine, University of Helsinki, Helsinki, Finland. Submitted for publication February 15, 2005. Accepted for publication June 17, 2005. Supported by departmental Kronberg, Germany) was given as a 10-min intravenous funding and a grant from the Medical Society of Finland, Helsinki, Finland. infusion through a 20-gauge cannula (Venflon; Becton Presented in part as a poster at the XXth Congress of the International Society on Thrombosis Hemostasis, Sydney, Australia, August 6–12, 2005. Dickinson, Franklin Lakes, NJ) in a dorsal vein of the Address reprint requests to Dr. Munsterhjelm: P.O. Box 340 (P-floor), FIN- hand. The infusions were blinded and administered in 00029 HUS, Finland. Address electronic mail to: edward.munsterhjelm@hus.fi. Individual article reprints may be purchased through the Journal Web site, random order. The code was not broken until all exper- www.anesthesiology.org. iments had been performed.

Anesthesiology, V 103, No 4, Oct 2005 712 ACETAMINOPHEN AND PLATELETS IN HEALTHY VOLUNTEERS 713

Table 1. Plasma Concentration of Acetaminophen (mg/l)

Dose of Acetaminophen

0 mg/kg 15 mg/kg 22.5 mg/kg 30 mg/kg

Time after drug administration 10 min Ͻ 0.1 18.8 (17.0–20.6) 30.2 (27.2–33.3) 39.1 (34.4–43.8) 90 min Ͻ 0.1 10.5 (9.5–11.5) 16.3 (14.6–17.9) 22.2 (20.3–24.2)

Data are reported as mean values (95% conﬁdence intervals). Each volunteer (n ϭ 13) was given placebo and 15, 22.5, and 30 mg/kg acetaminophen. Venous blood samples (approximately 35 ml/sample) stored at Ϫ20°C. Acetaminophen concentration was de-

were drawn from an antecubital vein before infusion and termined using high-performance liquid chromatogra- Downloaded from http://pubs.asahq.org/anesthesiology/article-pdf/103/4/712/360846/0000542-200510000-00009.pdf by guest on 29 September 2021 10 and 90 min after cessation of infusion. The first phy.16 The limit of quantification was 0.1 mg/l, and the sample after infusion was drawn from the contralateral day-to-day coefficients of variation were 7.4% at 16.8 mg/l hand. A 20-gauge needle (PrecisionGlide; Becton Dick- and 4.2% at 36.8 mg/l (n ϭ 6). inson) was used, and samples for aggregation studies were collected into polypropylene tubes (Vacuette; Statistical Analysis Greiner bio-one, Kremsmu¨nster, Austria) containing The sample size needed was estimated in advance as 3.2% buffered citrate, giving a volume ratio of 1:10. described in the statistical literature.17 The study was Samples for acetaminophen concentration measurement designed to discover a difference in platelet aggregation were collected into plastic EDTA tubes (5.9 mg K2EDTA, between each acetaminophen group and placebo VenoSafe; Terumo Europe, Haasrode, Belgium). greater than 1 SD, with a power of 80% (␣ error ϭ 5%, Cold Pressor Test. Immediately after each blood sam- Bonferroni correction was applied). The sample size pling, a cold pressor test was performed. The volunteer needed was n ϭ 11. A difference smaller than 1 SD was immersed his arm, halfway to the elbow, into an ice considered of minor clinical significance. Data distribution bath, and the times elapsed until first sensation of pain was tested with the Kolmogorov-Smirnov test, and non- and sensation of strong pain were recorded. There was parametric statistics were used for nonnormally distributed at least a 50-min interval between the cold pressor test data. Nonnormally distributed data are presented as medi- and the next blood sampling. ans and 25th/75th percentiles; normally distributed data are presented as mean values and 95% confidence intervals. Laboratory Tests The difference between all groups was analyzed with the Platelet Aggregation. Platelet aggregation was mea- Friedman test (repeated-measures analysis of variance on sured with a four-channel photometric aggregometer ranks), and when a significant difference was encountered, (Packs-4; Helena Laboratories, Beaumont, TX) based on 13 each acetaminophen group was further compared with the method of Born. Platelet-rich plasma and platelet- placebo using the Wilcoxon matched pairs signed rank poor plasma were prepared by centrifuging as described sum test and applying the Bonferroni correction. Statistical 14 ␮ previously. Aggregation was induced in 270 l platelet- testing was with SigmaStat for Windows Version 2.03 (SPSS ␮ rich plasma by adding 30 l of one of the following Inc., Chicago, IL). Confidence intervals were calculated triggers: adenosine diphosphate (ADP) to a final concen- using the appropriate t distribution. tration of 1.5 or 3 ␮M; arachidonic acid to a final concentration of 500, 750, or 1,000 ␮M; or epinephrine to a final concentration of 5 ␮M. Reagents were purchased Results from Sigma-Aldrich (St. Louis, MO) and Calbiochem (San Diego, CA). Based on previous experience, these con- Thirteen volunteers completed the study according to centrations are known to cause platelet aggregation. the protocol, all of whom showed normal platelet func- Aggregation was allowed to proceed for 300 s; after that, tion before drug infusion. Plasma acetaminophen con-

plasma for thromboxane B2 (TxB2) determination was centrations increased linearly with dose, and exceeded prepared as described previously.6 The area under the 17.0 mg/l at 10-min sampling after all doses used (table curve of aggregometry was recorded. 1). Ninety minutes after infusion, plasma acetaminophen

Thromboxane B2 Concentration. Thromboxane B2 concentrations decreased, but they remained signiﬁ- is the stable metabolite of TxA2, released during aggre- cantly above 10 mg/l after 22.5 and 30 mg/kg acetamin- gation. TxB2 concentrations in platelet-rich plasma trig- ophen. No acetaminophen was detected before infusion gered with 3 ␮M ADP or 1 mM arachidonic acid were in any of the volunteers. determined with a radioimmunoassay.15 The interassay coefﬁcient of variation was 17.7% (n ϭ 15). Effect of Acetaminophen on Platelets Acetaminophen Concentration. Blood samples Acetaminophen dose-dependently inhibited platelet were centrifuged at 3,000g for 10 min, and plasma was aggregation triggered with arachidonic acid, ADP, or

Anesthesiology, V 103, No 4, Oct 2005 714 MUNSTERHJELM ET AL.

Table 2. Platelet Aggregation Triggered with Arachidonic Acid, Adenosine Diphosphate, or Epinephrine

Dose of Acetaminophen

Aggregation Aggregation Trigger Trigger Administration 0 mg/kg 15 mg/kg 22.5 mg/kg 30 mg/kg P Value (All Groups)

Arachidonic acid 500 ␮M Pre 25.5 (24.5/26.2) 25.4 (24.7/26.1) 24.9 (22.5/26.1) 25.7 (24.4/26.6) 10 min 25.7 (25.3/26.6) 22.8 (3.5/24.1)‡ 4.1 (3.5/4.5)‡ 3.6 (3.4/4.3)‡ Ͻ 0.001 90 min 25.6 (25.0/26.0) 23.9 (3.7/24.9)† 14.7 (3.7/24.7)† 4.8 (3.6/23.4)† 0.007 750 ␮M Pre 26.9 (25.4/27.0) 26.0 (24.9/26.3) 25.9 (25.0/26.4) 26.5 (25.5/27.2) 10 min 26.7 (26.6/27.2) 25.3 (22.0/26.0)‡ 4.1 (2.8/26.0)‡ 16.7 (3.3/25.2)‡ Ͻ 0.001 90 min 25.9 (25.7/26.6) 25.4 (24.4/26.1) 25.6 (23.2/26.6) 24.9 (22.7/26.2) 0.07 Downloaded from http://pubs.asahq.org/anesthesiology/article-pdf/103/4/712/360846/0000542-200510000-00009.pdf by guest on 29 September 2021 1,000 ␮M Pre 26.6 (25.2/27.3) 25.9 (25.3/26.3) 26.0 (25.5/26.7) 26.6 (25.9/27.1) 10 min 26.5 (25.8/26.9) 25.5 (25.1/26.6)* 25.9 (22.6/26.1)† 24.5 (21.8/26.0)‡ Ͻ 0.001 90 min 26.0 (25.7/26.8) 25.8 (23.9/26.8) 25.8 (24.4/26.7) 25.7 (24.4/26.4) 0.49 ADP 1.5 ␮M Pre 15.7 (10.4/22.1) 14.8 (11.1/21.0) 16.0 (12.3/19.0) 16.4 (11.0/23.1) 10 min 14.6 (10.6/20.6) 11.1 (10.2/16.4) 12.2 (8.3/14.9) 11.6 (8.8/14.4)* 0.026 90 min 17.8 (10.2/22.4) 13.5 (10.1/19.1) 13.6 (9.2/19.2) 13.9 (10.7/20.1) 0.49 3.0 ␮M Pre 23.7 (21.7/26.1) 24.1 (20.6/25.1) 23.8 (22.3/24.6) 24.5 (22.7/26.1) 10 min 24.3 (22.2/26.0) 23.5 (20.1/24.6) 22.5 (20.0/23.3) 20.1 (19.0/22.4)† 0.004 90 min 24.4 (22.2/25.2) 23.3 (21.0/24.9) 23.5 (21.0/24.6) 22.4 (19.7/24.7) 0.11 Epinephrine 5 ␮M Pre 21.1 (17.2/22.9) 19.5 (16.4/23.4) 20.4 (16.5/22.7) 20.6 (18.2/23.3) 10 min 19.6 (15.5/21.7) 14.6 (10.6/19.9) 13.9 (10.3/19.6) 12.7 (10.1/16.8)† 0.033 90 min 20.9 (16.4/22.7) 18.3 (16.4/21.5) 20.1 (11.3/21.4) 16.9 (13.6/20.2)* 0.017

Data are aggregation (area under the curve, 103 area units) reported as medians (25th/75th percentiles). Each volunteer (n ϭ 13) was given placebo and 15, 22.5, and 30 mg/kg acetaminophen. Sampling was before drug administration (Pre) and 10 and 90 min after infusion. Statistical tests are the Friedman test (all groups) and Wilcoxon matched pairs signed rank sum test with Bonferroni correction (each acetaminophen dose vs. placebo): * P Ͻ 0.05, † P Ͻ 0.01, ‡ P Ͻ 0.005. ADP ϭ adenosine diphosphate. epinephrine (table 2). Ten minutes after infusion, 15 mg/kg Thromboxane B2 release during aggregation triggered acetaminophen caused a significant inhibition of platelet with arachidonic acid or ADP was dose-dependently aggregation triggered with arachidonic acid. Inhibition inhibited by acetaminophen (table 3). Reduction of TxB2 was most pronounced with 500 ␮M arachidonic acid. An release was significant after all doses of acetaminophen increasing concentration of arachidonic acid counter- at 10 min, and reversibility of the inhibition was evident acted the inhibition; with 1,000 ␮M arachidonic acid, it with both triggers at 90 min. was minimal, although still statistically significant. Aggre- gation triggered with ADP or epinephrine was less sen- sitive to inhibition by acetaminophen; at 10 min, a sig- Cold Pressor Test nificant inhibition was achieved only with 30 mg/kg Pain threshold in the cold pressor test showed a large acetaminophen. The inhibition was reversible; aggrega- variation and was not increased by acetaminophen (fig. tion in response to all triggers was recovering at 90 min 1). Time until sensation of strong pain was also highly after infusion, reflecting the decreasing plasma concen- variable and not significantly influenced by acetamino- tration of acetaminophen in the last sample. phen (data not shown).

Table 3. Thromboxane B2 Release from Activated Platelets

Dose of Acetaminophen

Time after Drug Aggregation Trigger Administration 0 mg/kg 15 mg/kg 22.5 mg/kg 30 mg/kg P Value (All Groups)

1mM arachidonic acid Pre 1,201 (1,111/1,491) 1,227 (1,058/1,406) 1,060 (926/1,496) 1,193 (1,119/1,379) 10 min 1,432 (1,182/1,526) 1,012 (8,20/1,188)‡ 748 (573/979)‡ 923 (739/1,000)‡ Ͻ0.001 90 min 1,262 (1,190/1,458) 1,301 (1,026/1,419) 968 (653/1,222)‡ 1181 (987/1,260)* 0.004 3 ␮M ADP Pre 24.7 (20.9/41.1) 24.9 (13.7/39.5) 21.5 (19.3/36.1) 35.0 (22.2/44.5) 10 min 28.5 (18.1/40.0) 18.5 (9.5/24.2)‡ 10.0 (6.3/20.8)‡ 6.6 (3.2/11.2)‡ Ͻ0.001 90 min 30.8 (20.2/39.7) 22.6 (16.2/29.2)† 15.8 (12.6/26.0)‡ 12.7 (10.0/28.6)* Ͻ0.001

Data are concentrations (␮g/l) reported as medians (25th/75th percentiles). Each volunteer (n ϭ 13) was given placebo and 15, 22.5, and 30 mg/kg acetaminophen. Sampling was before drug administration (Pre) and 10 and 90 min after infusion. Statistical tests are the Friedman test (all groups) and Wilcoxon matched pairs signed rank sum test with Bonferroni correction (each acetaminophen dose vs. placebo): * P Ͻ 0.05, † P Ͻ 0.01, ‡ P Ͻ 0.005. ADP ϭ adenosine diphosphate.

Anesthesiology, V 103, No 4, Oct 2005 ACETAMINOPHEN AND PLATELETS IN HEALTHY VOLUNTEERS 715

acetaminophen was inversely related to the concentration of arachidonic acid used as a trigger. This suggests a competition either between acetaminophen and arachidonic acid for entering the cyclooxygenase reaction or

between acetaminophen and prostaglandin G2 for entering the peroxidase reaction. Previous data from vascular endothelial cells point toward the latter alternative.22 When ADP or epinephrine was used to trigger aggregation, the inhibitory effect of acetaminophen was much less pronounced. In contrast to aggregation induced

with arachidonic acid, no clear difference was detected Downloaded from http://pubs.asahq.org/anesthesiology/article-pdf/103/4/712/360846/0000542-200510000-00009.pdf by guest on 29 September 2021 when the concentration of ADP was increased. This is probably because aggregation triggered with ADP is Fig. 1. Pain threshold with the cold pressor test. The ﬁgure 23 shows all individual measurements before drug administration mainly independent of COX activity and TxA2 release. -ADP and epinephrine bind directly to their own recep (13 ؍ pre) and 10 and 90 min after infusion. Each volunteer (n) was given placebo and 15, 22.5, and 30 mg/kg acetaminophen tors on the surface of the platelet. Two ADP-binding intravenously. P > 0.20 in all groups (Friedman test). 24 receptors, P2Y1 and P2Y12, have been isolated, whereas only one adrenergic receptor, ␣ , seems to be Discussion 2A active in platelets.25 Platelet Function In previous studies, the conclusion was drawn that Traditionally, acetaminophen is considered not to in- acetaminophen does not inhibit platelet function.7,8 Our ﬂuence platelet function in vivo. Studies on conven- results contradict this conclusion. We suggest two main tional doses (approximately 1 g) of oral acetaminophen reasons for this contradiction. First, ADP, epinephrine, have shown negative results.7,8 In the current study, and collagen were used to trigger aggregation in those however, we demonstrate that intravenous acetamino- studies.7,8 When we triggered platelet aggregation with

phen inhibits platelet aggregation and TxB2 release in ADP or epinephrine, only the highest dose of intrave- healthy volunteers in a dose-dependent fashion. We have nous acetaminophen, 30 mg/kg, corresponding to a total previously shown that propacetamol (prodrug of acet- dose of approximately 2 g, caused a signiﬁcant inhibi- aminophen) has a clear inhibitory effect in large doses6 tion. Second, a lower peak plasma acetaminophen con- and that propacetamol augments the antiaggregatory ef- centration is achieved with oral than with intravenous fect of diclofenac.14 administration.26 Because approximately 1 g oral acet- When platelet aggregation was induced with arachi- aminophen was used in the previous studies, no inhibi- donic acid, acetaminophen signiﬁcantly inhibited plate- tion could have been detected under those circum- let function at all doses tested. Arachidonic acid is the stances.

physiologic substrate of COX, the key enzyme in pros- Thromboxane A2, produced in platelets by COX-1 and taglandin formation. The reaction catalyzed by COX pro- thromboxane synthase, is unstable and decomposes rap-

ceeds in two steps: First, arachidonic acid is converted idly into its stable metabolite TxB2. TxB2 release trig- ␮ to prostaglandin G2 in the cyclooxygenase reaction, and gered with arachidonic acid (1 mM) or ADP (3 M) was in the second step, the peroxidase reaction, prostaglan- also dose-dependently inhibited by acetaminophen. Al- 18 din G2 is converted to prostaglandin H2. In platelets, though significant, TxB2 release was considerably less thromboxane synthase further converts prostaglandin inhibited by acetaminophen than by the traditional 14 H2 to TxA2, which initiates aggregation by binding to NSAID diclofenac in our previous study. Under similar specific G protein–coupled receptors on the surface of conditions, median TxB2 release in response to ADP the platelet.19 shortly after an intravenous infusion of diclofenac In contrast to traditional NSAIDs, which inhibit COX (1.1 mg/kg) was only 3.3% of preinfusion release, as by competing with arachidonic acid for entering the compared with 24% after acetaminophen (30 mg/kg) in cyclooxygenase reaction,20 the mechanism by which the current study. Our results in vivo therefore confirm acetaminophen inhibits COX is not known. Acetamino- previous observations in vitro showing that acetaminophen may inhibit either the cyclooxygenase or peroxi- phen is a weaker inhibitor of COX-1 than are conven- dase reaction by competing with the substrate for enter- tional NSAIDs.4,27 ing the reaction or by interfering with the propagation of the reactions in the catalytic center of COX. It has been Analgesic Effect suggested that acetaminophen, like other phenolic com- The plasma concentration of acetaminophen required pounds, can act as a reducing agent and quench a tyrosyl for optimal analgesia is not known. Antipyretic proper- radical necessary for propagation of the cyclooxygenase ties of acetaminophen are evident in the plasma concen- reaction.21 In our study, the antiaggregatory effect of tration range of 10–20 g/l. This concentration or higher

Anesthesiology, V 103, No 4, Oct 2005 716 MUNSTERHJELM ET AL. was observed 10 min after infusion with all doses tested, Large patient studies are needed to determine the clini- but after 90 min, plasma acetaminophen concentration cal impact of acetaminophen-induced inhibition of plate- remained signiﬁcantly above 10 g/l only with doses let function. higher than 15 mg/kg. Optimal analgesia may require higher concentrations than antipyresis in adults,10 but The authors thank Hannele Yki-Ja¨rvinen, M.D., Ph.D. (Professor of Internal 11 Medicine, Helsinki University Hospital, Helsinki, Finland), for the use of her this topic is controversial. When acetaminophen was laboratory facilities; Anna Becker, B.Sci. (Technician, Department of Anesthesi- administrated rectally in children, a linearly increasing ology and Intensive Care Medicine, Helsinki University Hospital), and Miikka Ko¨ykka¨, B.Sci. (Technician, Department of Anesthesiology and Intensive Care morphine-sparing effect was achieved with doses up to Medicine, Helsinki University Hospital), for skilled technical assistance; and our 60 mg/kg.12 Considering that the site of action of acet- volunteers for smooth cooperation. aminophen is mainly in the central nervous system, a

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