Prostacyclin inhibits platelet aggregation induced by phorbol ester or Ca2+ ionophore at steps distal to activation of protein kinase C and Ca2+-dependent protein kinases

Abstract

Suspensions of aspirin-treated, 32P-prelabelled, washed platelets containing ADP scavengers in the buffer were activated with either phorbol 12,13-dibutyrate (PdBu) or the Ca2+ ionophore A23187. High concentrations of PdBu (greater than or equal to 50 nM) induced platelet aggregation and the protein kinase C (PKC)-dependent phosphorylation of proteins with molecular masses of 20 (myosin light chain), 38 and 47 kDa. No increase in cytosolic Ca2+ was observed. Preincubation of platelets with prostacyclin (PGI2) stimulated the phosphorylation of a 50 kDa protein [EC50 (concn. giving half-maximal effect) 0.6 ng of PGI2/ml] and completely abolished platelet aggregation [ID50 (concn. giving 50% inhibition) 0.5 ng of PGI2/ml] induced by PdBu, but had no effect on phosphorylation of the 20, 38 and 47 kDa proteins elicited by PdBu. The Ca2+ ionophore A23187 induced shape change, aggregation, mobilization of Ca2+, rapid phosphorylation of the 20 and 47 kDa proteins and the formation of phosphatidic acid. Preincubation of platelets with PGI2 (500 ng/ml) inhibited platelet aggregation, but not shape change, Ca2+ mobilization or the phosphorylation of the 20 and 47 kDa proteins induced by Ca2+ ionophore A23187. The results indicate that PGI2, through activation of cyclic AMP-dependent kinases, inhibits platelet aggregation at steps distal to protein phosphorylation evoked by protein kinase C and Ca2+-dependent protein kinases.

Translocation-independent activation of protein kinase C by platelet-activating factor, thrombin and prostacyclin. Lack of correlation with polyphosphoinositide hydrolysis in rabbit platelets

“…The observation that phosphorylation of p47 is increased in thrombin-and PAF-treated platelets has been used to argue that agonist-induced PIP2 turnover is coupled to PKC activation (Ieyasu et al, 1982;Sano et al, 1983). However, PGI2 and cAMP-analogue treatments of platelets block PAF-, thrombin-and fluoride-induced p47 phosphorylation (Ieyasu , 1982;Sano et al, 1983;Kroll et al, 1988;Siess & Lapetina, 1989;Lazarowski & Lapetina, 1989), even though PIP2 breakdown in response to thrombin was not sensitive to up to 50 ,ug of PGI2/ml (Fig. 4).…”

“…267 Indeed, recent studies with human platelets (Doni et al, 1988;Kroll et al, 1988;Siess & Lapetina, 1989) …”

“…Phosphorylation-induced retardation of proteins on SDS/PAGE gels has been described previously (Casnellie, 1987). PGI2 treatment leads to the appearance of a 50 kDa phosphoprotein on SDS/PAGE gels of platelet extracts (Siess & Lapetina, 1989). Analysis of the amino acid sequence of the p47 protein reveals the presence of at least five classic consensus sequences for PKA phosphorylation (i.e.…”

“…Via activation of adenylate cyclase, it elevates the level of cyclic AMP (cAMP) in platelets, and this leads to a stimulation of cAMP-dependent protein kinase (PKA) activity. At least two sites of PKA action in the disruption of agonistinduced platelet aggregation have been distinguished, and these are pre-and post-PKC activation (Kroll et al, 1988;Siess & Lapetina, 1989). PGI2 and cAMP-analogue treatment of platelets leads to an inhibition of agonist-stimulated PIP2 breakdown, perhaps via PKA phosphorylation of a putative GTP-binding protein (G-protein) which couples the agonist receptors to phospholipase C (Halenda & Feinstein, 1984;Ieyasu et al, 1982;Doni et al, 1988;Lazarowski & Lapetina, 1989;Yada et al, 1989).…”

Prostacyclin and Sodium Nitroprusside Inhibit the Activity of the Platelet Inositol 1,4,5-Trisphosphate Receptor and Promote Its Phosphorylation

“…In the presence of cAMP, the activation of phospholipase C (and hence the production of InsP 3 and diacylglycerol) by the agonist is depressed, leading to the inhibition of the increment of [Ca 2ϩ ] i and of PKC-dependent phosphorylations (21). The action of cAMP is also on events distal to the activation of phospholipase C since platelet aggregation and secretion induced by the Ca 2ϩ ionophore ionomycin or by phorbol esters are also inhibited by the cyclic nucleotide (22)(23)(24). cAMP also increases the incorporation of diacylglycerol into phosphatidylinositol (25).…”