Dose- and time-dependent antiplatelet effects of aspirin Christina Perneby, N. Håkan Wallén, Cathy Rooney, Desmond Fitzgerald, Paul Hjemdahl Published in Thrombosis and Haemostasis2006 Thrombosis and Haemostasis 2006

Aspirin (ASA) has variable antiplatelet activity when assessed by different methods, and the poorly defined phenomenon of “aspirin resistance” is often discussed.Aspirin (ASA) has variable antiplatelet activity when assessed by different methods, and the poorly defined phenomenon of “aspirin resistance” is often discussed. Using various test methods, the range of patients among different studies judged not to have optimal antiplatelet effects during ASA treatment was between 5-57%.Using various test methods, the range of patients among different studies judged not to have optimal antiplatelet effects during ASA treatment was between 5-57%. Among such patients, several authors have reported an increased risk of suffering major cardiovascular events in post hoc analyses.Among such patients, several authors have reported an increased risk of suffering major cardiovascular events in post hoc analyses. However, prospective studies validating the clinical utility of identifying biochemical or functional “aspirin resistance” are lacking.However, prospective studies validating the clinical utility of identifying biochemical or functional “aspirin resistance” are lacking. At low dosages, first pass hepatic metabolism of aspirin limits the systemic exposure of the patients to COX inhibition, thus preserving the vasodilating and antiplatelet effects of endothelial prostacyclin.At low dosages, first pass hepatic metabolism of aspirin limits the systemic exposure of the patients to COX inhibition, thus preserving the vasodilating and antiplatelet effects of endothelial prostacyclin. Higher doses of ASA may attenuate this potentially antithrombotic mechanism.Higher doses of ASA may attenuate this potentially antithrombotic mechanism. Aspirin (ASA) has variable antiplatelet activity when assessed by different methods, and the poorly defined phenomenon of “aspirin resistance” is often discussed.Aspirin (ASA) has variable antiplatelet activity when assessed by different methods, and the poorly defined phenomenon of “aspirin resistance” is often discussed. Using various test methods, the range of patients among different studies judged not to have optimal antiplatelet effects during ASA treatment was between 5-57%.Using various test methods, the range of patients among different studies judged not to have optimal antiplatelet effects during ASA treatment was between 5-57%. Among such patients, several authors have reported an increased risk of suffering major cardiovascular events in post hoc analyses.Among such patients, several authors have reported an increased risk of suffering major cardiovascular events in post hoc analyses. However, prospective studies validating the clinical utility of identifying biochemical or functional “aspirin resistance” are lacking.However, prospective studies validating the clinical utility of identifying biochemical or functional “aspirin resistance” are lacking. At low dosages, first pass hepatic metabolism of aspirin limits the systemic exposure of the patients to COX inhibition, thus preserving the vasodilating and antiplatelet effects of endothelial prostacyclin.At low dosages, first pass hepatic metabolism of aspirin limits the systemic exposure of the patients to COX inhibition, thus preserving the vasodilating and antiplatelet effects of endothelial prostacyclin. Higher doses of ASA may attenuate this potentially antithrombotic mechanism.Higher doses of ASA may attenuate this potentially antithrombotic mechanism. Perneby et al., Thromb Haemost Apr; 95(4): Dose- and time- dependent antiplatelet effects of aspirinStudy Background Dose- and time- dependent antiplatelet effects of aspirin: Study Background

Dose- and time- dependent antiplatelet effects of aspirin: Study Design ASA treatment with 37.5mg/day for 10 days (half tablet of Trombyl® 75mg) Blood Sampling on 10 th day at 1.5-2h (9am),  6h (1pm) and 24 hours after last dose ASA treatment with 37.5mg/day for 10 days (half tablet of Trombyl® 75mg) Blood Sampling on 10 th day at 1.5-2h (9am),  6h (1pm) and 24 hours after last dose ASA treatment with 320 mg/day for 7 days (1 tablet Alka Seltzer ®) Blood Sampling on 10th day at 1.5-2h (9am),  6h (1pm) and 24 hours after last dose ASA treatment with 320 mg/day for 7 days (1 tablet Alka Seltzer ®) Blood Sampling on 10th day at 1.5-2h (9am),  6h (1pm) and 24 hours after last dose Open cross-over study, measurements at baseline and during treatment with 3 different aspirin dosages 15 Males, Ages years, non-smokers, Body Mass Index of 22.9 (range 20-26) Avoidance of antiplatelet drugs for 2 weeks before first visit Open cross-over study, measurements at baseline and during treatment with 3 different aspirin dosages 15 Males, Ages years, non-smokers, Body Mass Index of 22.9 (range 20-26) Avoidance of antiplatelet drugs for 2 weeks before first visit Single dose 640mg ASA (2 tablets Alka Seltzer ®); 1-2h after last 320 mg dose Blood Sampling at 4h and 24h after Single dose 640mg ASA (2 tablets Alka Seltzer ®); 1-2h after last 320 mg dose Blood Sampling at 4h and 24h after Perneby et al., Thromb Haemost Apr; 95(4):652-8.

Dose- and time- dependent antiplatelet effects of aspirin: Methods Whole Blood Aggregometry: Whole Blood Aggregometry: - An impedence aggregometer was used to study platelet aggregation in whole blood (ie, Chrono-log model 570-VS Four Sample, Chrono-log Corp) - Agonists used were collagen type I at final concentrations of 1, 3 or 5 µM and arachidonic acid (AA) dissolved in ethanol at final concentrations of 0.2, 0.5 or 1 mM. Aggregometry in Platelet Rich Plasma (PRP): Aggregometry in Platelet Rich Plasma (PRP): - A four-channel platelet aggregation profiler was used to study platelet aggregation in PRP (ie, PAP-4, Bio-Data Corporation) Perneby et al., Thromb Haemost Apr; 95(4):652-8.

Perneby et al., Thromb Haemost Apr; 95(4): Dose- and time- dependent antiplatelet effects of aspirin: AA-induced aggregation Comparison of AA-induced aggregation (Impedence in Ω ) There was neither a dose- response relationship for platelet aggregation induced by 0.2-1mM AA in whole blood nor a significant diurnal variability without ASA treatment.There was neither a dose- response relationship for platelet aggregation induced by 0.2-1mM AA in whole blood nor a significant diurnal variability without ASA treatment. The 37.5mg ASA suppressed aggregation at low AA concentrations; inhibitory effects of the low-dose ASA were attenuated after 24h compared to 6h (p<0.01).The 37.5mg ASA suppressed aggregation at low AA concentrations; inhibitory effects of the low-dose ASA were attenuated after 24h compared to 6h (p<0.01). After 320 and 960mg of ASA, platelet aggregation was almost completely inhibited (p<0.001 for both) when using 0.2 mM AA, but there was less complete inhibition with higher concentrations of AA, and significant recovery 24h after high-dose ASA.After 320 and 960mg of ASA, platelet aggregation was almost completely inhibited (p<0.001 for both) when using 0.2 mM AA, but there was less complete inhibition with higher concentrations of AA, and significant recovery 24h after high-dose ASA.

All doses of aspirin (37.5mg, 320mg and 960mg) inhibited AA-induced platelet aggregation almost completely (to <1%), indicating good compliance and effective platelet COX inhibition.All doses of aspirin (37.5mg, 320mg and 960mg) inhibited AA-induced platelet aggregation almost completely (to <1%), indicating good compliance and effective platelet COX inhibition. Three individuals had small aggregatory responses (3- 5%) to AA after 37.5mg aspirin, which were abolished after 320 and 960mg.Three individuals had small aggregatory responses (3- 5%) to AA after 37.5mg aspirin, which were abolished after 320 and 960mg. Perneby et al., Thromb Haemost Apr; 95(4): Dose- and time- dependent antiplatelet effects of aspirin: Aggregometry in PRP

Dose- and time- dependent antiplatelet effects of aspirin: Urinary excretion of TxM Perneby et al., Thromb Haemost Apr; 95(4): Urinary excretion of TxM expressed as ng/mmol creatinine after different doses of aspirin. There was no significant diurnal variability of urinary TxM excretion. After 37.5mg ASA, nocturnal TxM excretion was reduced by 74.0±2.0% (p<0.001). After 37.5mg ASA, nocturnal TxM excretion was reduced by 74.0±2.0% (p<0.001). After 320mg ASA the degree of inhibition was 82.0±1.8% (p<0.001 vs. baseline, and 37.5mg). After 320mg ASA the degree of inhibition was 82.0±1.8% (p<0.001 vs. baseline, and 37.5mg). Four hours after 960mg ASA, TxM excretion was reduced by 83.9±2.8% (p<0.001 vs. baseline, and 37.5 mg), but there was significant recovery after <24h to 78.5±1.4% inhibition (TxM excretion increased from 9.4±1.1 to 14.8±1.5 ng/mmol creatinine; p<0.001). Four hours after 960mg ASA, TxM excretion was reduced by 83.9±2.8% (p<0.001 vs. baseline, and 37.5 mg), but there was significant recovery after <24h to 78.5±1.4% inhibition (TxM excretion increased from 9.4±1.1 to 14.8±1.5 ng/mmol creatinine; p<0.001). TxB 2 production in serum was inhibited by 98.5±0.7% (n=8) after 37.5mg ASA, and by 98.9±0.5% (n=9) after 320mg ASA.TxB 2 production in serum was inhibited by 98.5±0.7% (n=8) after 37.5mg ASA, and by 98.9±0.5% (n=9) after 320mg ASA.

Dose- and time- dependent antiplatelet effects of aspirin: Urinary excretion of PGI-M 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. 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. Nocturnal PGI-M excretion decreased by 25±7% (p<0.05) after 37.5mg ASA, and by 28±14% (p=0.066) after 320mg. Nocturnal PGI-M excretion decreased by 25±7% (p<0.05) after 37.5mg ASA, and by 28±14% (p=0.066) after 320mg. PGI-M excretion during the day tended to decrease (by 18±8%; p=0.055) with 37.5mg ASA, and was reduced by 26±10% (p<0.05) 4h after 960mg ASA, but recovered (from 10.6±1.5 to 13.9±2.0 ng/mmol creatinine; p<0.05) in the ensuing nocturnal sample.PGI-M excretion during the day tended to decrease (by 18±8%; p=0.055) with 37.5mg ASA, and was reduced by 26±10% (p<0.05) 4h after 960mg ASA, but recovered (from 10.6±1.5 to 13.9±2.0 ng/mmol creatinine; p<0.05) in the ensuing nocturnal sample. Urinary excretion of PGI-M expressed as ng/mmol creatinine after different doses of aspirin. Perneby et al., Thromb Haemost Apr; 95(4):652-8.

Perneby et al., Thromb Haemost Apr; 95(4): Dose- and time- dependent antiplatelet effects of aspirin: Platelet inhibition by ASA Comparison of platelet inhibition afforded by ASA at different dosages ( mg) using different platelet function assays (% inhibited by ASA) The parameter most sensitive to inhibition by aspirin was AA induced aggregation in PRP, followed by serum TxB 2. The parameter most sensitive to inhibition by aspirin was AA induced aggregation in PRP, followed by serum TxB 2. TxM excretion was less markedly, but dose-dependently reduced by ASA treatment. TxM excretion was less markedly, but dose-dependently reduced by ASA treatment. A dose related but weak inhibition of collagen induced aggregation in hirudinized whole blood was found with the lowest collagen concentration, and some inhibition of PGI-M excretion. A dose related but weak inhibition of collagen induced aggregation in hirudinized whole blood was found with the lowest collagen concentration, and some inhibition of PGI-M excretion. Platelet aggregation in whole blood stimulated by low AA concentrations was correlated to serum TxB 2 levels after 37.5mg ASA (eg, r=0.73, p<0.05, at 0.2 mM AA); correlations disappeared with higher AA concentrations and aspirin dosages.Platelet aggregation in whole blood stimulated by low AA concentrations was correlated to serum TxB 2 levels after 37.5mg ASA (eg, r=0.73, p<0.05, at 0.2 mM AA); correlations disappeared with higher AA concentrations and aspirin dosages.

Dose- and time- dependent antiplatelet effects of aspirin: Discussion Aspirin treatment suppressed TxB ² in serum by more than 98% and abolished AA-induced platelet aggregation in PRP already at a very low dose level (37.5mg daily).Aspirin treatment suppressed TxB ² in serum by more than 98% and abolished AA-induced platelet aggregation in PRP already at a very low dose level (37.5mg daily). Even when using high dosages of ASA, there was significant recovery of platelet function 24 hours after dosing.Even when using high dosages of ASA, there was significant recovery of platelet function 24 hours after dosing. Thus there was incomplete suppression of TxM excretion in urine, and AA-induced platelet aggregation could still occur in whole blood.Thus there was incomplete suppression of TxM excretion in urine, and AA-induced platelet aggregation could still occur in whole blood. Furthermore, treatment with 37.5mg aspirin decreased the sensitivity to stimulation by ADP in platelet rich plasma.Furthermore, treatment with 37.5mg aspirin decreased the sensitivity to stimulation by ADP in platelet rich plasma. In accordance with earlier results, this effect was less pronounced (non-significant) after higher doses of aspirin.In accordance with earlier results, this effect was less pronounced (non-significant) after higher doses of aspirin. Perneby et al., Thromb Haemost Apr; 95(4):652-8.

Dose- and time- dependent antiplatelet effects of aspirin: Discussion cont. The low 37.5mg dose of aspirin, which appears to be subtherapeutic, effectively inhibited platelet-dependent thromboxane formation with very modest effects on aggregation in whole blood.The low 37.5mg dose of aspirin, which appears to be subtherapeutic, effectively inhibited platelet-dependent thromboxane formation with very modest effects on aggregation in whole blood. Nucleated cells are capable of regenerating the COX-1 enzyme after a couple of hours and provide PGH ² to platelets to bypass the inhibition of platelet COX-1.Nucleated cells are capable of regenerating the COX-1 enzyme after a couple of hours and provide PGH ² to platelets to bypass the inhibition of platelet COX-1. These findings of dose- and time-dependent recovery of platelet function within 24h after dosing of aspirin are of relevance for the inability of 100mg ASA every other day to reduce cardiac events in the Women’s Health Study, as such a dose regimen may provide incomplete COX-1 inhibition during at least part of the dosing interval.These findings of dose- and time-dependent recovery of platelet function within 24h after dosing of aspirin are of relevance for the inability of 100mg ASA every other day to reduce cardiac events in the Women’s Health Study, as such a dose regimen may provide incomplete COX-1 inhibition during at least part of the dosing interval. Perneby et al., Thromb Haemost Apr; 95(4):652-8.