Effect of phorbol 12-myristate 13-acetate (PMA) on agonist-induced arachidonate release and 5-hydroxytryptamine secretion in human platelets. Dependence of effects on agonist type and time of incubation with PMA

Abstract

We have previously demonstrated synergistic potentiation of secretion by phorbol 12-myristate 13-acetate (PMA) and platelet agonists such as thrombin and the thromboxane mimetic, U46619, with short (less than 2 min) pre-incubations of PMA, despite inhibition of agonist-induced [Ca2+]i mobilization and arachidonate/thromboxane release. In this study, the effect of PMA on 5-hydroxytryptamine secretion in relation to arachidonate/thromboxane B2 release induced by collagen as well as the 'weak agonists', ADP, adrenaline and platelet-activating factor (PAF), was investigated using human platelet-rich plasma. Short incubations (10-30 s) with PMA (400 nM) before agonist addition caused an inhibition (60-100%) of 5-hydroxy[14C]tryptamine secretion and thromboxane B2 formation in response to maximally effective doses of ADP (10 microM), adrenaline (10 microM) and PAF (0.5 microM) but potentiated collagen-induced 5-hydroxy[14C]tryptamine secretion and [3H]arachidonate/thromboxane release. However, a longer pre-incubation with PMA (5 min) caused a significant reduction (20-50%) in the extent of collagen-induced 5-hydroxy[14C]tryptamine secretion and thromboxane B2 formation as seen earlier with thrombin, although collagen-induced [3]arachidonate release was still unaffected. Pretreatment of platelets with the cyclo-oxygenase inhibitor, indomethacin (10 microM), abolished 5-hydroxy[14C]tryptamine secretion in response to the weak agonists and reduced collagen (2.5-10 micrograms/ml) -induced secretion by 50-90%, depending on the collagen concentration. Addition of PMA (400 nM) 10 s before these agonists in indomethacin-treated platelets resulted in synergistic interactions between agonist and PMA leading to enhanced 5-hydroxy[14C]tryptamine secretion, although this was notably less than the synergism observed previously between thrombin and PMA or U46619 and PMA. The results suggest that the effect of short incubations with PMA on 5-hydroxytryptamine secretion induced by 'thromboxane-dependent' agonists, such as those examined in this study, is determined by the effect on agonist-induced thromboxane synthesis. However, when endogenous thromboxane synthesis is blocked, weak agonists as well as collagen can synergize with PMA at potentiating 5-hydroxytryptamine secretion, albeit to a weaker extent than thrombin or U46619. The results also suggest that PMA has differential effects on arachidonate release induced by collagen and thrombin.