Structural requirements for preventing the aspirin- and the arachidonate-induced inactivation of platelet cyclo-oxygenase: additional evidence for distinct enzymatic sites.

2-Hydroxybenzoic acid (salicylic acid) prevents the inhibition by aspirin (ASA) of platelet aggregation and of the generation of thromboxane A2 from arachidonic acid (AA). We studied the ability of 2-hydroxybenzoic acid analogues to block ASA and to prevent the platelet desensitization due to a first exposure to AA. Inactivation was prevented when exposure to AA was done in the presence of reversible inhibitors of cyclo-oxygenase. Phenol, methyl salicylate and L8027 were thus strong inhibitors of AA-induced platelet activation and desensitization. The minimal structural requirement for inhibition of thromboxane A2 generation from AA was a phenol group as benzoic acid was fully inactive. 2-Hydroxybenzoic acid, and to some extent 2,6-dihydroxybenzoic acid were effective against ASA, the most active substances being methyl salicylate and L8027. The minimal structural requirement for blocking ASA was that 2-hydroxybenzoic acid, 2-methoxybenzoic acid should be devoid of activity, which highlights the fact that the hydroxyl group must be available. Our work favours the hypothesis that non-steroidal anti-inflammatory drugs react with two sites of cyclo-oxygenase, which were named the supplementary and the catalytic sites. The interaction of 2-hydroxybenzoic acid and of its analogues with the supplementary site is necessary but not sufficient for the efficacy of these compounds as cyclo-oxygenase inhibitors. The intensity of interaction with the supplementary site and the modifications of the catalytic site determine the potency of these compounds as cyclo-oxygenase inhibitors. For preventing ASA inactivation, an interaction with the supplementary sites is always necessary, but furthermore an appropriate group, preferentially in the position ortho to the hydroxyl, is needed.