1 Department of Anaesthesiology and Intensive Care Medicine, 2 Department of Clinical Chemistry and 3 Department of Obstetrics and Gynaecology, Helsinki University Hospital, Finland
Corresponding author: PO Box 340 (P-floor), FIN-00029 HUS, Finland. E-mail: edward.munsterhjelm@hus.fi
Accepted for publication: May 4, 2003
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Abstract |
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Methods. Ten healthy male volunteers (2130 yr) were given diclofenac 1.1 mg kg1 alone, a combination of propacetamol 30 mg kg1 (which is hydrolysed to 50% acetaminophen) and diclofenac 1.1 mg kg1 or placebo intravenously in a double blind, crossover study. Platelet function was assessed at 5 min, 90 min and 2224 h by photometric aggregometry, platelet function analyser (PFA-100TM) and by measuring the release of thromboxane B2 (TxB2). Analgesia was assessed with the cold pressor test.
Results. Platelet aggregation induced with arachidonic acid was fully inhibited by both diclofenac alone and the combination at the end of the 30-min drug infusion. Propacetamol augmented the inhibition by diclofenac at 90 min (P=0.014). At 2224 h, platelet function had fully recovered. TxB2 release was inhibited by the combination of propacetamol and diclofenac at 90 min in comparison with diclofenac alone (P=0.027). PFA-100TM detected no difference in platelet function between these two groups. No analgesic effect was detected with the cold pressor test.
Conclusions. The combination of propacetamol and diclofenac inhibits platelet function more than diclofenac alone. This should be considered when assessing the risk of surgical bleeding.
Br J Anaesth 2003; 91: 35762
Keywords: analgesics non-opioid, acetaminophen; analgesics non-opioid, diclofenac; blood, platelet aggregation; non-steroidal anti-inflammatory drugs
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Introduction |
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The mechanism of action of NSAIDs is considered to be inhibition of cyclooxygenase (COX), the key enzyme in prostaglandin formation.4 The inhibition of COX-1 is the basis of their anti-aggregatory effect on platelets, as production of pro-aggregatory thromboxane A2 is blocked.5 The analgesic effect of NSAIDs, on the other hand, is mediated through inhibition of inducible COX-2.5 The analgesic effect of acetaminophen is probably mediated through central COX-inhibition.6 COX-3, a recently characterized variant of the COX enzyme, is mainly expressed in the central nervous system and appears to be more readily inhibited by acetaminophen than are COX-1 and COX-2.7
However, acetaminophen has also peripheral COX-1 inhibiting8 and anti-aggregatory properties.9 We therefore hypothesized that acetaminophen augments the inhibitory effect of NSAIDs on platelet aggregation through increased inhibition of platelet COX-1.
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Methods |
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Experimental procedures
After 3 h of fasting, a 30-min infusion of either diclofenac (Voltaren®, Novartis, Finland) 1.1 mg kg1, a combination of diclofenac 1.1 mg kg1 and propacetamol (Pro-Dafalgan®, Bristol-Myers Squibb, France) 30 mg kg1 or placebo was given intravenously on three different occasions in random order with at least a 1-week interval between the experiments. Propacetamol is a pro-drug that, given intravenously, is rapidly hydrolysed into 50% acetaminophen and 50% diethylglycine by plasma esterases. Diclofenac and propacetamol were diluted in 100 ml or 250 ml of normal saline, respectively. The solutions were blinded, and the code broken when all experiments had been performed. A dorsal vein on the hand was cannulated with a 20-gauge cannula (VenflonTM, Becton Dickinson, UK). During the test the volunteers were resting.
Venous blood samples were drawn from an antecubital vein before the infusion and at 5 min, 90 min and 2224 h thereafter. A 20-gauge needle (PrecisionGlideTM, Becton Dickinson, UK) was used and the samples were collected into polypropylene tubes (Vacuette®, Greiner bio-one, Austria) containing 3.2% buffered citrate, giving a volume ratio of 1:10.
Cold pressor test
Immediately after every blood sample, except that at 22 24 h, a cold pressor test was performed. The volunteer immersed his non-dominant hand, halfway to the elbow, into an ice bath and estimated the pain intensity on a 10 cm visual analogue scale at 30 and 60 s. The volunteer was instructed to withdraw his hand earlier, if the pain became unbearable. There was at least a 60 min interval between the cold pressor test and the next blood sample.
Laboratory tests
Platelet count and haemoglobin concentration were determined with a Sysmex K-1000 blood cell counter (Sysmex Corporation, Kobe, Japan).
Platelet aggregation
Platelet aggregation was measured with a four channel photometric aggregometer (Packs-4, Helena Laboratories, USA) based on the method of Born.10 Platelet-rich plasma (PRP) and platelet-poor plasma (PPP) were prepared by centrifuging at +20°C at 160 g for 9 min and at 3000 g for 5 min, respectively. Platelet count in PRP was adjusted to 300x109 litre1 ± 10% by diluting with autologous PPP. During continuous stirring (1000 r.p.m.) at 37°C, 270 µl PRP was analysed. Aggregation was induced by adding 30 µl of an agonist, adenosine diphosphate (ADP), to a final concentration of 3 µM, arachidonic acid to a final concentration of 1 mM, collagen to a final concentration of 50 µg/ml, and thrombin receptor activating peptide (TRAP, SFLLRN) to a final concentration of 10 µM. The reagents were purchased from Sigma-Aldrich (St Louis, USA) and Bachem (Weil am Rhein, Germany). The concentrations were chosen to ensure a high probability of aggregation based on previous experience. The aggregation was allowed to proceed for 300 s, after which plasma for thromboxane B2 (TxB2) determination was prepared as described earlier.9 The area under the curve of the aggregometry was recorded, and 3000 s% (300 sx10%) was subtracted to compensate for the 10% dilution of PRP when the agonist was added.
Thromboxane B2 concentration
TxB2 is the stable metabolite of thromboxane A2 (TxA2), released during aggregation. The TxB2 concentration in PRP after aggregation induced with ADP or arachidonic acid was determined with a radioimmunoassay as described earlier.11 The assay sensitivity was 80 pg/ml, and the intra-assay coefficient of variation was 17% (n=9).
PFA-100TM here -- KB>TM
With the platelet function analyser (PFA-100TM, Dade Behring, USA) we determined closure times on duplicate samples of 900 µl citrated whole blood. After collection, all samples were incubated at room temperature for 30 min to 2 h. Cartridges containing collagen/epinephrine or collagen/ADP membranes were used. The upper detection limit of the closure time is 300 s. When exceeded, the result was considered 300 s to allow statistical analysis.
All results are expressed as percentage of pre-infusion value.
Statistics
The sample size needed was estimated in advance as described in the statistical literature.12 The study was designed to discover a difference in platelet aggregation between the diclofenac group and the combination group greater than 1 SD, with a power of 80% (-error=5%). The theoretical sample size needed was n=7.85. A difference smaller than 1 SD was considered of minor clinical significance. The difference between the groups was analysed with the Friedman repeated measures analysis of variance on ranks test. When a significant difference was detected, the diclofenac and the combination groups were further compared with the Wilcoxon signed rank test.
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Results |
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Immediate effect of analgesics
Both diclofenac and the combination of diclofenac and propacetamol had an immediate inhibitory effect on platelet aggregation induced with arachidonic acid and ADP (Fig. 1, Table 1). Platelet aggregation induced with TRAP showed results comparable with ADP (data not shown). TxB2 release during aggregation induced with ADP or arachidonic acid was reduced by both diclofenac and the combination (Table 2). The closure time with PFA-100TM was prolonged (Table 3). We did not detect any effect of propacetamol on this immediate inhibition of platelet function by diclofenac with any of the methods used.
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2224 h after infusion
On the next day, the inhibitory effect of both diclofenac and its combination with propacetamol had disappeared. Platelet aggregation, closure time with PFA-100TM and TxB2 release had completely recovered (Fig. 1, Tables 13).
Cold pressor test
Pain induced with the cold pressor test showed a high degree of variation and was not reduced with the analgesics used (Fig. 2).
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Discussion |
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When platelet aggregation was induced with arachidonic acid, the difference between diclofenac only and the combination of propacetamol and diclofenac was significant. Arachidonic acid is the physiologic substrate of COX, the key enzyme in prostaglandin formation. In platelets, COX-1 catalyses the conversion of arachidonic acid to TxA2, which binds to specific G-protein coupled receptors on the surface of the platelet.14 As COX is the rate-limiting step in this reaction, aggregation initiated with arachidonic acid reflects the activity of COX. Traditional NSAIDs inhibit COX by binding to its active site and thereby blocking its catalytic activity.5
When ADP and TRAP were used as agonists the anti-aggregatory effect of diclofenac and its combination with propacetamol were much less pronounced. This supports our hypothesis that the additive effect of propacetamol is mediated through inhibition of platelet COX. Both ADP and TRAP bind directly to receptors on the surface of the platelet. Two ADP-binding receptors, P2Y1 and P2Y12, have been isolated in platelets.15 TRAP activates the thrombin receptor, PAR-1.16 Activation of these receptors initiates intracellular signalling that activates the production of TxA2, although aggregation is not dependent on this pathway.17
The time frame of the additive effect of propacetamol and diclofenac fits well with a COX-mediated mechanism. Immediately after the infusion a high concentration of diclofenac fully inhibits COX and no additional effect of propacetamol could be detected. The anti-aggregatory effect of diclofenac was decreasing at 90 min because of its relatively short half-life in plasma, 1.1 h.18 At this stage the inhibitory effect on platelet aggregation was augmented by propacetamol. The corresponding time frame observed regarding the release of TxB2 further supports a COX-related mechanism. The TxA2, released through platelet COX-1 activity, rapidly decomposes into TxB2. At 90 min the TxB2 release, and therefore COX activity, was significantly inhibited by the presence of propacetamol as compared with diclofenac only. The inhibition of TxB2 release by the combination can be compared with the inhibitory effect of high-dose propacetamol reported earlier.9 The median TxB2 release was 48.4% of pre-infusion value after propacetamol (60 mg kg1) and 34.9% after ketorolac (0.4 mg kg 1) from platelets activated with 8 µM ADP at 90 min after infusion.
A direct additive inhibitory effect on platelet COX by the drug combination is the most likely mechanism, a pharmacokinetic interaction through protein binding or metabolism seems less probable. In contrast to diclofenac, acetaminophen is not highly protein bound at therapeutic concentrations and the drugs have different metabolic pathways in the liver.19
The platelet function analyser PFA-100TM is an in vitro test for primary haemostasis, having fewer sources of errors than conventional Ivys bleeding time.20 The prolongation of collagen/epinephrine closure time demonstrated clearly the immediate inhibitory effect of both diclofenac and the combination of diclofenac and propacetamol. The drugs produced only a minor difference in the collagen/ADP closure time, which is in accordance with results obtained after ingestion of aspirin.21 However, no difference was seen between diclofenac and the combination, possibly because the maximal closure time is restricted to 300 s. In the present study photometric aggregometry was clearly more sensitive than PFA-100TM.
Analgesic effect
In contrast to the clinical observations of a postoperative analgesic effect of both diclofenac1 and acetaminophen2 we detected no analgesic effect of the drugs with the cold pressor test as a noxious stimulus. This experimental pain model has been extensively applied in studies on both pain22 and analgesics.23 In a previous study, acetaminophen showed an analgesic effect in this pain model.24
Although the major mechanism of action of NSAIDs is peripheral, there is evidence in favour of a central mechanism involving spinal COX-2 inhibition.25 This mechanism requires a noxious stimulus of longer duration to allow spinal COX-2 upregulation.25 The lack of an analgesic effect of diclofenac in our short-acting pain model is therefore logical. On the other hand, we did not detect any effect of the addition of acetaminophen either, which was unexpected considering previously published results.24
Clinical implications
The clinical implications of reduced platelet aggregation could be potentially both advantageous and hazardous. Low-dose aspirin has been shown to reduce the incidence of deep-vein thrombosis in patients undergoing surgery for hip fracture, and the incidence of death from pulmonary embolism.26 On the other hand, during the intra- and immediate postoperative period a reduced platelet function may cause bleeding. In a recent systemic review on NSAIDs and bleeding after tonsillectomy, an increased risk for re-operation after use of NSAIDs was found.27 Likewise, pre-treatment with ibuprofen appears to increase perioperative blood loss during total hip replacement28 and low-dose aspirin has the same effect given before transurethral prostatectomy.29
Our results indicate that adding propacetamol to a diclofenac treatment regime can augment the anti-aggregatory effect of this traditional NSAID. This should be taken into consideration when assessing the delicate balance between bleeding and thrombotic complications during and after surgery.
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Acknowledgements |
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References |
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