Calmodulin-independent inhibition of platelet phospholipase A₂ by calmodulin antagonists

We tested the effects of calmodulin, two types of calmodulin antagonists, and various phospholipids on the phospholipase A₂ activities of intact platelets, platelet membranes, and partially purified enzyme preparations. Trifluoperazine, chlorpromazine (phenothiazines) and N-(6-amino-hexyl)-5-chloro-1-naphthalenesulfonamide (W-7), at concentrations which antagonize the effects of calmodulin, significantly inhibited (a) thrombin- and Ca²⁺ ionophore-induced production of arachidonic acid metabolites by suspensions of rabbit platelets and (b) Ca²⁺-induced arachidonic acid release from phospholipids of membrane fractions, but not (c) phospholipase A₂ activity in purified enzyme preparations. The addition of acidic phospholipids, but not calmodulin, stimulated phospholipase A₂ activity in purified enzyme preparations while decreasing its K_m for Ca²⁺. The dose-response and kinetics of inhibition by calmodulin antagonists of acidic phospholipid-activated phospholipase A₂ activity in purified preparations were similar to those of Ca²⁺-induced arachidonic acid release from membrane fractions. Calmodulin antagonists were also found to inhibit Ca²⁺ binding to acidic phospholipids in a similar dose-dependent manner. Our results suggest (a) that the platelet phospholipase A₂ is the key enzyme involved in arachidonic acid mobilization in platelets and is regulated by acidic phospholipids in a Ca²⁺-dependent manner and (b) that calmodulin antagonists inhibit phospholipase A₂ activity via an action on acidic phospholipids.