A high level of cyclooxygenase-2 inhibitor selectivity is associated with a reduced interference of platelet cyclooxygenase-1 inactivation by aspirin

Abstract

Both nonsteroidal anti-inflammatory drugs, such as ibuprofen, and the prototypical selective cyclooxygenase (Cox)-2 inhibitors DuP-697 and NS-398 block the inhibition of Cox-1 by aspirin in vitro. However, clinical studies have shown that the Cox-2 selective drugs (or coxibs) rofecoxib and etoricoxib, at therapeutic doses, do not interfere with the antiplatelet effect of aspirin, in contrast to ibuprofen. Here, we have evaluated the relative potential of ibuprofen and various coxibs to interfere with the inactivation of Cox-1 by aspirin by using purified enzyme and calcium ionophore-activated human platelets. The irreversible inactivation of Cox-1 by aspirin can be antagonized by ibuprofen and coxibs, albeit with widely different potencies. The rank order of potencies for this process (ibuprofen > celecoxib > valdecoxib > rofecoxib > etoricoxib) parallels that obtained for the inhibition of Cox-1-mediated thromboxane B(2) production by calcium ionophore-stimulated platelets. The antagonism of aspirin therefore likely involves a competition at the enzyme active site. The EC(50) value for the antagonism against 10 microM aspirin for each drug is approximately 10- to 40-fold lower than the corresponding IC(50) value for inhibition of platelet Cox-1 activity, consistent with the much weaker initial binding of aspirin to Cox-1 as compared with arachidonic acid. These results show that a low affinity for Cox-1 and a high degree of Cox-2 selectivity confers a low potential to block aspirin inhibition of platelet Cox-1, consistent with the results of clinical studies.

Figure 1

Antagonism of the aspirin inhibition of oCox-1 by ibuprofen and coxibs. Purified oCox-1 was treated with 200 μM aspirin and 0–100 μM ibuprofen (▿), celecoxib (□), valdecoxib (×), rofecoxib (◊), and etoricoxib (○) for 30 min before the addition of substrate and the determination of the remaining Cox activity. Each point represents the average of duplicate determinations. The complete data were fit to the four-parameter IC₅₀ equation for each inhibitor with the exceptions of ibuprofen and celecoxib, where data from the ranges 0.015 to 3.7 μM and 0.015 to 33.3 μM, respectively, were fit. Higher concentrations of these inhibitors resulted in Cox-1 inhibition from these agents themselves.

Figure 2

Titration and reversibility of inhibition of platelet Cox-1 by ibuprofen, celecoxib, valdecoxib, rofecoxib, etoricoxib, and aspirin. Washed platelets were treated with inhibitor for 25 min before the stimulation of Cox activity with calcium ionophore. After a 10-min reaction, the amount of TXB₂ produced was determined by EIA (○). To determine inhibitor reversibility, platelets were treated with each inhibitor for 25 min, and the platelets were then washed twice with fresh buffer before ionophore stimulation and TXB₂ production as above (□). The data represent the average of titrations from at least three blood donors, except for aspirin (n = 2). The error bars represent the SEM of each value.

Figure 3

Antagonism of the aspirin inhibition of platelet Cox-1 by ibuprofen and coxibs. Platelets were treated with 0–100 μM ibuprofen (▿), celecoxib (□), valdecoxib (×), rofecoxib (◊), and etoricoxib (○) for 5 min before the addition of 10 μM (A) or 100 μM (B) aspirin. After a 20-min incubation, the platelets were washed twice and challenged with calcium ionophore. After 10 min, the reactions were quenched, and the amount of TXB₂ produced was determined by EIA. The data represent the average of (n) titrations from at least three different donors: (A) ibuprofen (10), celecoxib (9), valdecoxib (8), rofecoxib (8), and etoricoxib (6); (B) ibuprofen (11), celecoxib (12), valdecoxib (8), rofecoxib (8), and etoricoxib (6). For celecoxib, only data between 0.005 and 3.7 μM are plotted, as higher concentrations showed inhibition from celecoxib alone.

Figure 4

Effect of ibuprofen on the incorporation of [¹⁴C]acetyl aspirin radioactivity into microsomal platelet proteins. Washed platelets were treated with vehicle (○) or 10 μM ibuprofen (□) for 5 min before the addition of 100 μM [¹⁴C]acetyl aspirin. After 30 min, the cells were washed and lysed, and the microsomal fraction was solubilized with octylglucoside before electrophoresis on denaturing gels. Horizontal fractions were excised and counted in scintillation fluid. Fraction 4 represents the beginning of the separating gel. The mobility of molecular mass markers (in kDa) are shown by arrows. The data shown are representative of multiple results obtained with ibuprofen (n = 2) and vehicle-treated platelets (n = 4).