Requirement for different Ca2+ pools in the activation of rabbit platelets II. Phospholipase activity

The effects of extracellular Ca2+ concentration and the putative antagonist of intracellular Ca2+ movement, 8-(N,N-diethylamino)octyl-3,4,5-trimethoxybenzoate (TMB-8) on platelet phospholipase activity and thromboxane B2 synthesis were examined in rabbit platelets stimulated by platelet activating factor, thrombin and ionophore A23187. TMB-8 markedly inhibited the platelets stimulated by platelet activating factor, thrombin and ionophore A23187. TMB-8 markedly inhibited the platelet activating factor-induced decrease in [14C]arachidonate content in platelet phosphatidylcholine and phosphatidylinositol, while showing minimal effects on thrombin-induced phospholipase activation. A23187 stimulation of these processes was inhibited to an intermediate degree by TMB-8. In contrast, extracellular Ca2+ removal inhibited phospholipase activity to a similar degree with all three stimuli. Moreover, the threshold concentration of extracellular Ca2+ for phospholipase activation, as measured by thromboxane B2 synthesis, was similar for platelet activating factor- and thrombin-stimulated platelets. These data provide evidence that, while platelet activating factor and thrombin may, to some extent, have similar requirements for extracellular Ca2+, they utilize a TMB-8 sensitive step to different degrees during activation of platelet phospholipase.