Clinical Science (1990)79,37-42
37
Effect of aspirin infusions on platelet function in humans K. M. WILSON', D. M. SIEBERT', E. M. DUNCAN2, A. A. SOMOGYI'*3,J. V. LLOYD2 AND F. BOCHNER'.3 'Department of Clinical and Experimental Pharmacology, University of Adelaide, *Divisionof Haematology, Institute of Medical and Veterinary Science and 3Departmentof Clinical Pharmacology, Royal Adelaide Hospital, Adelaide, South Australia, Australia
(Received 9 October 1989/16 January 1990; accepted 13 March 1990)
SUMMARY
INTRODUCTION
1. The inhibitory effects of aspirin on platelet function in vitro have been shown to be both time (over 3 h) and concentration( 1-10 pmol/l) dependent. 2. To determine if these effects occurred in vivo, four volunteers received intravenous infusions on four occasions, to give constant plasma aspirin concentrations of 0, 1, 2 and 4 pmol/l over 3 h. Infusions were performed at intervals of at least 2 weeks. 3. Before and during the infusions, blood was taken for assay of aspirin concentrations, and measurements of platelet aggregation in response to collagen, adenosine 5'pyrophosphate and arachidonate. Thromboxane generation after stimulated platelet aggregation and whole-blood coagulationwas also measured. 4. At each aspirin concentration, both platelet aggregation and thromboxane generation in response to collagen and arachidonate were inhibited progressively over the 3 h infusion period. Greatest inhibition was seen during the 4 pmol/l infusion, which produced maximal or near-maximal inhibition by the third hour. 5. Thromboxane generated during whole-blood coagulation was similarly inhibited in both a time- and concentration-dependent manner throughout all aspirin infusions. 6. The progressive nature of the inhibition of platelet function with these low aspirin concentrations may be due to either slow aspirin transport across the platelet membrane or delayed interaction with cyclo-oxygenase.
Aspirin is a useful agent for prophylaxis against thromboembolic disorders [ 1,2].The doses of aspirin which have been used have ranged from 60 mg to 1.3 g daily. The protective effects are mediated via inhibition of the platelet enzyme cyclo-oxygenase, which is required in the formation of the proaggregatory prostanoid, thromboxane A2 [3, 41. In the endothelium, the formation of the anti-aggregatory prostanoid, prostacyclin, is mediated by cyclo-oxygenase,and is therefore also inhibited by aspirin [5,6]. Burch et al. [7]reported that, in vitro, the inhibitory effects of aspirin were achieved at much lower concentrations in the platelet than in the vessel wall. Accordingly, several dosage regimens have been proposed to optimize the beneficial effects of aspirin (for a review, see Bochner & Lloyd [8]).One such approach has been to use low (100 mg or less)daily doses of aspirin. There has been considerable interest in measuring the concentrations of circulating aspirin and how these may relate to its anti-platelet effects [9-161. In these studies, aspirin was administered orally and plasma concentration versus time profiles and some measurements of platelet function were obtained. In most of these studies, the dose used (and plasma concentrations achieved)were greatly in excess of those now known to be required to produce maximal inhibition of cyclo-oxygenase-dependentplatelet functions. The results of most studies in vitro have indicated an apparent discrepancy between the concentrations (40-500 pmol/l) of aspirin required to inhibit cyclooxygenase in vitro [ 17-19] and those effective in vivo ( < 6 pmol/l) after oral ingestion of aspirin [9-161. Using critical concentrations of stimuli, we have recently demonstrated in vitro that the inhibitory effects of aspirin on platelet aggregation and thromboxane formation were both time and concentration dependent. We observed progressive inhibition by aspirin throughout a 3 h incubation- period at concentrations of 1-10 prnolll [20], confirming similar findings and conclusions to those of Seuter [21],who incubated platelets with aspirin for 20 min.
Key words: aspirin infusions, human platelet aggregation, thromboxane. Abbreviations: ADP, adenosine 5'-pyrophosphate; EC,,, concentration of stimulus producing half the maximal aggregation response; GSE,geometric standard error; PFW, platelet-rich plasma. Correspondence: Ms K. M. Wilson, Department of Clinical and Expefimental pha~acology,University of Adelaide, B~~ 498, Adelaide, South Australia 5000, Australia.
38
K. M. Wilson et al.
These studies in vitro [20, 211 resolved the apparent discrepancy between the concentrations of aspirin required to inhibit platelets in vivo and in vitro. However, more knowledge is needed about the lowest effective concentrations of aspirin required to inhibit platelet cyclooxygenase in vivo. It has been difficult to reliably determine inhibitory concentrations of aspirin in vivo for several reasons. First, assays which measure low plasma concentrations of aspirin (range 0.01-5 pmol/l) have only recently become available [22]. Secondly, plasma aspirin concentrations vary widely with time after oral ingestion [16] and this, together with the irreversible nature of the inhibitory activity of aspirin, has made correlations between plasma aspirin concentrations and platelet inhibitory effects extremely difficult. Other confounding influences include presystemic [9, 10, 12, 16, 231 and possible time-dependent [20,2 11effects of aspirin. The aim of this study was to determine whether platelet functions could be inhibited in vivo in a time- and concentration-dependent fashion, as has been demonstrated in vitro [20, 2 11. To attain these objectives it was necessary to produce a constant plasma aspirin concentration, and this was achieved by intravenous infusion.
infusion pump (Sage Instruments, subsidiary of Orion Research Inc., Cambridge, MA, U.S.A.) by an 18-gauge 1.4 m long sterile connecting tube (Braun, Melsungen, F.R.G.). Sterile aspirin (F.H. Faulding and Co. Limited, Adelaide, Australia) solutions for infusion (10 mg/ml) were prepared by the Royal Adelaide Hospital Pharmacy Department no more than 2 h before administration. These solutions were diluted with 0.9%(w/v) sterile NaCl solution to the appropriate concentration for infusion. There was less than 2% hydrolysis to salicylate over the 3 h infusion period.
METHODS Subjects
Blood sampling protocol
Four healthy subjects (two males, two females) consented to participate after being informed of the study protocol. Their ages ranged from 20 to 48 years, and their weights from 67 to 80 kg. They did not receive any drug known to alter platelet function for 2 weeks before and throughout the study (except for the aspirin administered for study purposes). Each subject was studied on six occasions, with an interval of at least 2 weeks between each occasion. Each subject initially received a rapid intravenous infusion of 50 mg of aspirin over 1 min. Multiple venous blood samples were collected over the next 3.5 h to determine the individual's aspirin pharmacokinetic parameters. On occasions 2-5, each subject received infusions of placebo or aspirin (see the Infusion protocol section). On the sixth occasion, they ingested a solution of 600 mg of aspirin, and after 2 h, venous blood was collected for the measurement of the maximum inhibitory effect of aspirin on thromboxane generation during coagulation of whole blood. This study was approved by the Human Ethics Committee of the Royal Adelaide Hospital and the Committee on the Ethics of Human Experimentation of the University of Adelaide. Infusion protocol Each subject received, in random order, an intravenous infusion containing no aspirin [0.9'/0 (w/v) sterile NaCl solution] or infusions of aspirin to achieve target plasma aspirin steady-state concentrations of 1 ymol/l, 2 pmol/l and 4 pmol/l (see the Design of infusion rates section). The infusions were administered through a forearm vein using a 22-gauge Teflon cannula (Jelco, Criticon Inc., Tampa, FL, U.S.A.) which was connected to a Sage
Design of infusion rates To attain and maintain plasma aspirin concentrations within 10% of the desired steady-state concentration throughout the entire 3 h infusion, the following approach was adopted. The subjects' individual pharmacokinetic parameters (volume of distribution and total body clearance) were first derived from plasma aspirin concentration versus time data after the 50 mg intravenous bolus infusion [24].These values were then used to calculate the required infusion rates for the following infusions [25].
Aspirin concentrations. Venous blood samples ( 5 ml) were collected from an 18-gauge indwelling Teflon cannula (Jelco) placed in the arm contralateral to the infusion arm. The sampling times were 0 and 2 min, one sample between 8 and 16 min, 20, 30 and 45 min and 1, 1.5, 2, 2.5 and 3 h after starting the infusion. Blood was collected into heparinized tubes containing 50 pl of 40 mmol/l physostigmine hemisulphate solution (Sigma Chemical Co., St Louis, MO, U.S.A.) and immediately centrifuged to obtain plasma which was stored at - 70°C until assayed within 1-2 weeks of collection. Whole-blood thromboxane generation. Duplicate 1 ml blood samples were collected into glass tubes at the same times as stated above. After incubation for 1 h at 37"C, the tubes were spun at 1600 g f o r 5 min and 200 pl of serum was transferred into 400 p1 of cold ethanol. These were vortex-mixed and stored at - 70°C for the thromboxane B, assay. Platelet aggregation and thromboxane generation. Before all infusions a 27 ml venous blood sample was mixed with 3 ml of 0.1 mol/l trisodium citrate anticoagulant (pH 6.5). Further 13.5 ml blood samples were collected into 1.5 ml of citrate anticoagulant at 1, 2 and 3 h after initiation of the infusion. For these samples, an initial flush with heparinized saline (10 units/ml; David Bull Laboratories, Melbourne, Australia) was given, and the first 2 ml of withdrawn blood was discarded. Analytical methods Plasma aspirin concentrations. These were measured by high-pressure liquid chromatography as previously described [22]. All samples were assayed in duplicate within 2 weeks of collection. The assay detection limit was 0.01 pmol/l.
39
Aspirin and platelet function Thromboxane. Thromboxane A, was measured as its stable metabolite, thromboxane B,, by radioimmunoassay [26]. Details of the method in our laboratory have been reported previously [27]. The assay detection limit was 0.2 ng/ml corresponding to 0.7 pg/106 platelets in platelet-rich plasma (PRP). For PRP, thromboxane B, was measured 5 min after the addition of arachidonate (1.65 mmol/l), adenosine 5’-pyrophosphate (ADP; 100 pmol/l) or acid-soluble collagen (25 pg/ml) as previously described [27]. Platelet aggregation. This was performed by methods described previously [ 271. Anticoagulated blood was centrifuged at 200 g for 11 min to give PRP. Platelet-poor plasma was prepared by further centrifugation of blood samples at 1600 g for 5 min. PRP platelet counts were standardized where possible to 300 OOO/pl by dilution with platelet-poor plasma. Platelet-free plasma was prepared by centrifugation of platelet-poor plasma (1 ml) at 3000 gfor 2 min. PRP was stored at 37°C in an air-free plastic syringe to control pH (7.4-7.5). For aggregation studies, PRP (240 p l ) was stirred (500 rev./min) for 1 min with Tyrode’s buffer (30 pl) in siliconized cuvettes before stimulus addition (30 p l ) . Five minutes after stimulus addition, percentage platelet aggregation was recorded using a Payton dual-channel aggregometer (model 300B: Payton Associates Ltd, Scarborough, Ontario, Canada). This was calibrated to 100% light transmission for platelet-free plasma and 0%for PRP. Platelet functions were assessed by aggregation responses to the stimuli arachidonate (1.65 mmol/l), acidsoluble collagen (0.05-25 pg/ml) and ADP (1-16 pmol/l). Dose-response curves to collagen and ADP were constructed and EC,, values were determined as the concentration of stimulus required to attain half the maximal aggregation response. For arachidonate, percentage aggregation only was measured. To estimate the maximal inhibitory effects of aspirin, aggregation experiments (performed on blank PRP prepared before infusion) were repeated on PRP which had been incubated with aspirin at a concentration of 1 mmol/l (BDH Chemicals, Sydney, Australia) for 1 min (30 pl in place of Tyrode’s buffer) before addition of stimulus.
concentrations. For the 2 and 4 pmol/l infusions, a similar result was obtained except at 2 min ( - 18.7%) and 2.5 h ( + 11.1%) during the 2 pmol/l infusion, and at 1.5 h ( - 12.0%)during the 4 pmol/l infusion. Platelet aggregation and thromboxane generation Arachidonate. All aspirin infusions produced both concentration- and time-dependent inhibitory effects during the 3 h infusion period (Figs. l a and lb). The 4 pmol/l infusion produced significant ( P < 0.063) inhibition of platelet aggregation responses in 1 h and maximal inhibition was reached in 2 h. Inhibitory effects of 1 and 2 pmol/l infusions continued to increase for up to 3 h, by which time aggregation responses were maximally inhibited by the 2 pmol/l infusion. Maximal inhibition of thromboxane generation was only achieved with the 4 ,umol/l aspirin concentration after 2 h. Collagen. All aspirin infusions produced inhibition in both a concentration- and time-dependent manner (Figs. 2a and 2b). The increasing EC,, values with all aspirin
I
0-1
I
0.0
I
1.0
2.0
3.0
-1
MI
Time (h)
Statistical analysis The Wilcoxon matched pairs signed-rank test was used for statistical analysis to compare the effects of plasma aspirin concentrations against placebo, and the effects of time. With four subjects, the minimum P value for significance was less than 0.063. All data are expressed as means SEM or geometric mean fgeometric standard error ( GSE).
*
RESULTS Plasma aspirin concentrations For the 1 pmol/l infusion the observed individual concentrations were all within f 1 0 % of the targeted
1ooJ
,
0.0
I
1
I
1.0
2.0
3.0
-1
MI
Time (h)
Fig. 1. Effect of infusions on (a) arachidonate (1.65 mmol/l)-stimulated platelet aggregation (arithmetic means fSEM, n = 4) and ( b ) thromboxane generation (geometric mean k GSE, n = 4). Results are shown for the placebo (-) and for 1 pmol/l (-), 2 pmol/l (A-A ) and 4 pmol/l (-) aspirin concentrations. MI (maximal inhibition; m) indicates responses attained when PRP (prepared from blood taken before infusions) was incubated for 1 min with aspirin (1 mmol/l) before the addition of arachidonate.
40
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Effect of aspirin infusions on platelet function in humans K. M. WILSON', D. M. SIEBERT', E. M. DUNCAN2, A. A. SOMOGYI'*3,J. V. LLOYD2 AND F. BOCHNER'.3 'Department of Clinical and Experimental Pharmacology, University of Adelaide, *Divisionof Haematology, Institute of Medical and Veterinary Science and 3Departmentof Clinical Pharmacology, Royal Adelaide Hospital, Adelaide, South Australia, Australia
(Received 9 October 1989/16 January 1990; accepted 13 March 1990)
SUMMARY
INTRODUCTION
1. The inhibitory effects of aspirin on platelet function in vitro have been shown to be both time (over 3 h) and concentration( 1-10 pmol/l) dependent. 2. To determine if these effects occurred in vivo, four volunteers received intravenous infusions on four occasions, to give constant plasma aspirin concentrations of 0, 1, 2 and 4 pmol/l over 3 h. Infusions were performed at intervals of at least 2 weeks. 3. Before and during the infusions, blood was taken for assay of aspirin concentrations, and measurements of platelet aggregation in response to collagen, adenosine 5'pyrophosphate and arachidonate. Thromboxane generation after stimulated platelet aggregation and whole-blood coagulationwas also measured. 4. At each aspirin concentration, both platelet aggregation and thromboxane generation in response to collagen and arachidonate were inhibited progressively over the 3 h infusion period. Greatest inhibition was seen during the 4 pmol/l infusion, which produced maximal or near-maximal inhibition by the third hour. 5. Thromboxane generated during whole-blood coagulation was similarly inhibited in both a time- and concentration-dependent manner throughout all aspirin infusions. 6. The progressive nature of the inhibition of platelet function with these low aspirin concentrations may be due to either slow aspirin transport across the platelet membrane or delayed interaction with cyclo-oxygenase.
Aspirin is a useful agent for prophylaxis against thromboembolic disorders [ 1,2].The doses of aspirin which have been used have ranged from 60 mg to 1.3 g daily. The protective effects are mediated via inhibition of the platelet enzyme cyclo-oxygenase, which is required in the formation of the proaggregatory prostanoid, thromboxane A2 [3, 41. In the endothelium, the formation of the anti-aggregatory prostanoid, prostacyclin, is mediated by cyclo-oxygenase,and is therefore also inhibited by aspirin [5,6]. Burch et al. [7]reported that, in vitro, the inhibitory effects of aspirin were achieved at much lower concentrations in the platelet than in the vessel wall. Accordingly, several dosage regimens have been proposed to optimize the beneficial effects of aspirin (for a review, see Bochner & Lloyd [8]).One such approach has been to use low (100 mg or less)daily doses of aspirin. There has been considerable interest in measuring the concentrations of circulating aspirin and how these may relate to its anti-platelet effects [9-161. In these studies, aspirin was administered orally and plasma concentration versus time profiles and some measurements of platelet function were obtained. In most of these studies, the dose used (and plasma concentrations achieved)were greatly in excess of those now known to be required to produce maximal inhibition of cyclo-oxygenase-dependentplatelet functions. The results of most studies in vitro have indicated an apparent discrepancy between the concentrations (40-500 pmol/l) of aspirin required to inhibit cyclooxygenase in vitro [ 17-19] and those effective in vivo ( < 6 pmol/l) after oral ingestion of aspirin [9-161. Using critical concentrations of stimuli, we have recently demonstrated in vitro that the inhibitory effects of aspirin on platelet aggregation and thromboxane formation were both time and concentration dependent. We observed progressive inhibition by aspirin throughout a 3 h incubation- period at concentrations of 1-10 prnolll [20], confirming similar findings and conclusions to those of Seuter [21],who incubated platelets with aspirin for 20 min.
Key words: aspirin infusions, human platelet aggregation, thromboxane. Abbreviations: ADP, adenosine 5'-pyrophosphate; EC,,, concentration of stimulus producing half the maximal aggregation response; GSE,geometric standard error; PFW, platelet-rich plasma. Correspondence: Ms K. M. Wilson, Department of Clinical and Expefimental pha~acology,University of Adelaide, B~~ 498, Adelaide, South Australia 5000, Australia.
38
K. M. Wilson et al.
These studies in vitro [20, 211 resolved the apparent discrepancy between the concentrations of aspirin required to inhibit platelets in vivo and in vitro. However, more knowledge is needed about the lowest effective concentrations of aspirin required to inhibit platelet cyclooxygenase in vivo. It has been difficult to reliably determine inhibitory concentrations of aspirin in vivo for several reasons. First, assays which measure low plasma concentrations of aspirin (range 0.01-5 pmol/l) have only recently become available [22]. Secondly, plasma aspirin concentrations vary widely with time after oral ingestion [16] and this, together with the irreversible nature of the inhibitory activity of aspirin, has made correlations between plasma aspirin concentrations and platelet inhibitory effects extremely difficult. Other confounding influences include presystemic [9, 10, 12, 16, 231 and possible time-dependent [20,2 11effects of aspirin. The aim of this study was to determine whether platelet functions could be inhibited in vivo in a time- and concentration-dependent fashion, as has been demonstrated in vitro [20, 2 11. To attain these objectives it was necessary to produce a constant plasma aspirin concentration, and this was achieved by intravenous infusion.
infusion pump (Sage Instruments, subsidiary of Orion Research Inc., Cambridge, MA, U.S.A.) by an 18-gauge 1.4 m long sterile connecting tube (Braun, Melsungen, F.R.G.). Sterile aspirin (F.H. Faulding and Co. Limited, Adelaide, Australia) solutions for infusion (10 mg/ml) were prepared by the Royal Adelaide Hospital Pharmacy Department no more than 2 h before administration. These solutions were diluted with 0.9%(w/v) sterile NaCl solution to the appropriate concentration for infusion. There was less than 2% hydrolysis to salicylate over the 3 h infusion period.
METHODS Subjects
Blood sampling protocol
Four healthy subjects (two males, two females) consented to participate after being informed of the study protocol. Their ages ranged from 20 to 48 years, and their weights from 67 to 80 kg. They did not receive any drug known to alter platelet function for 2 weeks before and throughout the study (except for the aspirin administered for study purposes). Each subject was studied on six occasions, with an interval of at least 2 weeks between each occasion. Each subject initially received a rapid intravenous infusion of 50 mg of aspirin over 1 min. Multiple venous blood samples were collected over the next 3.5 h to determine the individual's aspirin pharmacokinetic parameters. On occasions 2-5, each subject received infusions of placebo or aspirin (see the Infusion protocol section). On the sixth occasion, they ingested a solution of 600 mg of aspirin, and after 2 h, venous blood was collected for the measurement of the maximum inhibitory effect of aspirin on thromboxane generation during coagulation of whole blood. This study was approved by the Human Ethics Committee of the Royal Adelaide Hospital and the Committee on the Ethics of Human Experimentation of the University of Adelaide. Infusion protocol Each subject received, in random order, an intravenous infusion containing no aspirin [0.9'/0 (w/v) sterile NaCl solution] or infusions of aspirin to achieve target plasma aspirin steady-state concentrations of 1 ymol/l, 2 pmol/l and 4 pmol/l (see the Design of infusion rates section). The infusions were administered through a forearm vein using a 22-gauge Teflon cannula (Jelco, Criticon Inc., Tampa, FL, U.S.A.) which was connected to a Sage
Design of infusion rates To attain and maintain plasma aspirin concentrations within 10% of the desired steady-state concentration throughout the entire 3 h infusion, the following approach was adopted. The subjects' individual pharmacokinetic parameters (volume of distribution and total body clearance) were first derived from plasma aspirin concentration versus time data after the 50 mg intravenous bolus infusion [24].These values were then used to calculate the required infusion rates for the following infusions [25].
Aspirin concentrations. Venous blood samples ( 5 ml) were collected from an 18-gauge indwelling Teflon cannula (Jelco) placed in the arm contralateral to the infusion arm. The sampling times were 0 and 2 min, one sample between 8 and 16 min, 20, 30 and 45 min and 1, 1.5, 2, 2.5 and 3 h after starting the infusion. Blood was collected into heparinized tubes containing 50 pl of 40 mmol/l physostigmine hemisulphate solution (Sigma Chemical Co., St Louis, MO, U.S.A.) and immediately centrifuged to obtain plasma which was stored at - 70°C until assayed within 1-2 weeks of collection. Whole-blood thromboxane generation. Duplicate 1 ml blood samples were collected into glass tubes at the same times as stated above. After incubation for 1 h at 37"C, the tubes were spun at 1600 g f o r 5 min and 200 pl of serum was transferred into 400 p1 of cold ethanol. These were vortex-mixed and stored at - 70°C for the thromboxane B, assay. Platelet aggregation and thromboxane generation. Before all infusions a 27 ml venous blood sample was mixed with 3 ml of 0.1 mol/l trisodium citrate anticoagulant (pH 6.5). Further 13.5 ml blood samples were collected into 1.5 ml of citrate anticoagulant at 1, 2 and 3 h after initiation of the infusion. For these samples, an initial flush with heparinized saline (10 units/ml; David Bull Laboratories, Melbourne, Australia) was given, and the first 2 ml of withdrawn blood was discarded. Analytical methods Plasma aspirin concentrations. These were measured by high-pressure liquid chromatography as previously described [22]. All samples were assayed in duplicate within 2 weeks of collection. The assay detection limit was 0.01 pmol/l.
39
Aspirin and platelet function Thromboxane. Thromboxane A, was measured as its stable metabolite, thromboxane B,, by radioimmunoassay [26]. Details of the method in our laboratory have been reported previously [27]. The assay detection limit was 0.2 ng/ml corresponding to 0.7 pg/106 platelets in platelet-rich plasma (PRP). For PRP, thromboxane B, was measured 5 min after the addition of arachidonate (1.65 mmol/l), adenosine 5’-pyrophosphate (ADP; 100 pmol/l) or acid-soluble collagen (25 pg/ml) as previously described [27]. Platelet aggregation. This was performed by methods described previously [ 271. Anticoagulated blood was centrifuged at 200 g for 11 min to give PRP. Platelet-poor plasma was prepared by further centrifugation of blood samples at 1600 g for 5 min. PRP platelet counts were standardized where possible to 300 OOO/pl by dilution with platelet-poor plasma. Platelet-free plasma was prepared by centrifugation of platelet-poor plasma (1 ml) at 3000 gfor 2 min. PRP was stored at 37°C in an air-free plastic syringe to control pH (7.4-7.5). For aggregation studies, PRP (240 p l ) was stirred (500 rev./min) for 1 min with Tyrode’s buffer (30 pl) in siliconized cuvettes before stimulus addition (30 p l ) . Five minutes after stimulus addition, percentage platelet aggregation was recorded using a Payton dual-channel aggregometer (model 300B: Payton Associates Ltd, Scarborough, Ontario, Canada). This was calibrated to 100% light transmission for platelet-free plasma and 0%for PRP. Platelet functions were assessed by aggregation responses to the stimuli arachidonate (1.65 mmol/l), acidsoluble collagen (0.05-25 pg/ml) and ADP (1-16 pmol/l). Dose-response curves to collagen and ADP were constructed and EC,, values were determined as the concentration of stimulus required to attain half the maximal aggregation response. For arachidonate, percentage aggregation only was measured. To estimate the maximal inhibitory effects of aspirin, aggregation experiments (performed on blank PRP prepared before infusion) were repeated on PRP which had been incubated with aspirin at a concentration of 1 mmol/l (BDH Chemicals, Sydney, Australia) for 1 min (30 pl in place of Tyrode’s buffer) before addition of stimulus.
concentrations. For the 2 and 4 pmol/l infusions, a similar result was obtained except at 2 min ( - 18.7%) and 2.5 h ( + 11.1%) during the 2 pmol/l infusion, and at 1.5 h ( - 12.0%)during the 4 pmol/l infusion. Platelet aggregation and thromboxane generation Arachidonate. All aspirin infusions produced both concentration- and time-dependent inhibitory effects during the 3 h infusion period (Figs. l a and lb). The 4 pmol/l infusion produced significant ( P < 0.063) inhibition of platelet aggregation responses in 1 h and maximal inhibition was reached in 2 h. Inhibitory effects of 1 and 2 pmol/l infusions continued to increase for up to 3 h, by which time aggregation responses were maximally inhibited by the 2 pmol/l infusion. Maximal inhibition of thromboxane generation was only achieved with the 4 ,umol/l aspirin concentration after 2 h. Collagen. All aspirin infusions produced inhibition in both a concentration- and time-dependent manner (Figs. 2a and 2b). The increasing EC,, values with all aspirin
I
0-1
I
0.0
I
1.0
2.0
3.0
-1
MI
Time (h)
Statistical analysis The Wilcoxon matched pairs signed-rank test was used for statistical analysis to compare the effects of plasma aspirin concentrations against placebo, and the effects of time. With four subjects, the minimum P value for significance was less than 0.063. All data are expressed as means SEM or geometric mean fgeometric standard error ( GSE).
*
RESULTS Plasma aspirin concentrations For the 1 pmol/l infusion the observed individual concentrations were all within f 1 0 % of the targeted
1ooJ
,
0.0
I
1
I
1.0
2.0
3.0
-1
MI
Time (h)
Fig. 1. Effect of infusions on (a) arachidonate (1.65 mmol/l)-stimulated platelet aggregation (arithmetic means fSEM, n = 4) and ( b ) thromboxane generation (geometric mean k GSE, n = 4). Results are shown for the placebo (-) and for 1 pmol/l (-), 2 pmol/l (A-A ) and 4 pmol/l (-) aspirin concentrations. MI (maximal inhibition; m) indicates responses attained when PRP (prepared from blood taken before infusions) was incubated for 1 min with aspirin (1 mmol/l) before the addition of arachidonate.
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