Rapid inactivation of cyclooxygenase activity after stimulation of intact platelets

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Abstract
Trypsin, thrombin and ionophore A23187 activated phospholipid breakdown of [horse] platelets that were labeled with [14C]arachidonate, releasing their cyclooxygenase and lipoxygenase products. Intact platelets could also very effectively directly degrade low concentrations of exogenous, free [14C]arachidonate. Pretreatment of platelets with trypsin, thrombin or ionophore A23187 for a minimum of 30 s led to complete inactivation of cyclooxygenase activity, as demonstrated by subsequent exposure to [14C]arachidonate. Lipoxygenase activity was lost after 5 min. The thrombin-induced inactivation of cyclooxygenase and lipoxygenase was prevented by cyclic[c]AMP (which inhibits the stimulated activity of phospholipase A2), although cAMP did not affect the degradation of exogenous [14C]arachidonate. Exposure of platelets labeled with [14C]arachidonate to unlabeled arachidonate under conditions that led to use of the latter also resulted in a similarly rapid inhibition of cyclooxygenase activity, as determined by subsequent challenge with thrombin. Under these conditions lipoxygenase activity was much less markedly inactivated. The arachidonate-induced inhibition of cyclooxygenase activity was not prevented by cAMP. Trypsin did not induce platelet aggregation, and platelets whose cyclooxygenase activity was inactivated were intact insofar as they were still able to undergo aggregation. Operation in intact platelets of the cyclooxygenase pathway, through use of endogenous or exogenous substrate, leads to a very rapid, irreversible inactivation of this enzyme. The lipoxygenase pathway is also progressively impaired, but much less rapidly than the cyclooxygenase enzyme and much less markedly on use of exogenous compared to endogenous substrate. The possible consequences of these physiological processes of spontaneous inactivation were considered.