Requirements for different Ca2+ pools in the activation of rabbit platelets. I. release reaction and protein phosphorylation.

We examined the role of Ca2+, both extracellular and intracellular in origin, in the release reaction and protein phosphorylation in rabbit platelets stimulated with platelet activating factor (acetylglyceryl ether phosphorylcholine), thrombin, or ionophore A23187. In the presence of extracellular Ca2+, 8-(N,N-diethylamino)octyl-3,4,5-trimethoxybenzoate (TMB-8), a putative antagonist of intracellular Ca2+ transport, blocked platelet activating factor-initiated serotonin release at a half-maximal inhibitor concentration of 40 microM, compared to 350 microM for thrombin-induced release and greater than 500 microM, for A23187-induced release. Platelet activating factor-induced phosphorylation of two platelet proteins of Mr = 41,000 (P7P) and 20,000 (P9P) was inhibited by TMB-8, an effect which was additive to that caused by removing extracellular Ca2+. TMB-8 demonstrated only minor to non-existent inhibitory effect on phosphorylation in thrombin- or A23187-stimulated platelets. In contrast to P9P phosphorylation, phosphorylation of P7P caused by platelet activating factor was more dependent on a TMB-8 sensitive step than on the availability of extracellular Ca2+. Experiments with buffers containing fixed concentrations of free Ca2+ revealed that both processes (release and phosphorylation), when stimulated by platelet activating factor and thrombin, had the same threshold requirement (1-3 microM) for extracellular free Ca2+. These studies provide evidence tht stimulation of rabbit platelets by platelet activating factor is more dependent on a TMB-8-sensitive intracellular Ca2+ source than is stimulation caused by thrombin. Furthermore, our data indicate that activation of different intracellular processes involved in platelet secretion (such as P7P and P9P phosphorylation) may require Ca2+ from different pools.