Inhibition of platelet aggregation by the cAMP-phosphodiesterase inhibitor, cilostamide, may not be associated with activation of cAMP-dependent protein kinase.
We examined the involvement of cAMP-dependent protein kinase (A kinase)2 in the inhibition by cilostamide, a specific inhibitor of the low Km cAMP-phosphodiesterase (PDE), on 9,11-epithio-11,12-methanothromboxane A2 (STA2)-induced platelet aggregation. For comparative purposes, the PGE1 analogue, 17S-20-dimethyl-trans-delta 2-PGE1 (OP-1206) was used. OP-1206 (IC50 = 18 +/- 0.55 nM) and cilostamide (IC50 = 40 +/- 4.5 nM) were both potent inhibitors of the platelet aggregation induced by STA2 (1 microM). OP-1206 and cilostamide dose-dependently inhibited elevations in intracellular free Ca2+ ([Ca2+]i) caused by STA2. OP-1206 caused an almost complete inhibition of Ca2+ mobilization, but cilostamide did not prevent the STA2-induced elevation in [Ca2+]i to the same extent as OP-1206, even at a high concentration (greater than 200 nM). Cilostamide did not increase the cAMP level at concentrations (5-100 nm) which affected STA2-induced aggregation. OP-1206 significantly increased cAMP contents in platelets, and the degree of aggregation inhibition by OP-1206 appears to be related to the size of increase in cAMP. OP-1206 increased phosphorylation of the 50,000 mol. wt vasodilator-stimulated phosphoprotein, at concentrations of 7.9-79 nM, which inhibited aggregation induced by STA2. Cilostamide treatment resulted in a marginal increase in the 50,000 mol. wt phosphorylation at concentrations (10-100 nM) which completely inhibited the STA2-induced aggregation. (8R*, 9S*, 11S*)-(-)-9-Hydroxy-9-n-hexyloxy-8-methyl-2,3,9,10- tetrahydro-8,11-epoxy-1H, 8H, 11H-2, 7b, 11a-triazadibenzo(a,g)-cycloocta(c,d,e)trinden-1-one (KT-5720), a specific inhibitor of A kinase, not only reversed the inhibition by OP-1206 of STA2-induced platelet aggregation, but also inhibited the OP-1206-induced protein phosphorylation. However, the inhibition by cilostamide of STA2-induced aggregation was not prevented by pretreatment with KT-5720. Inhibition of the STA2-induced aggregation by OP-1206 may be associated with cAMP-dependent protein phosphorylation, while cilostamide may have inhibitory effects on STA2-induced platelet activation through mechanisms other than the activation of A kinase.
Nishikawa
M
M
Department of Internal Medicine, Mie University School of Medicine, Japan.
Komada
F
F
Morita
K
K
Deguchi
K
K
Shirakawa
S
S
D015105:3',5'-Cyclic-AMP Phosphodiesterases / Q000037:antagonists & inhibitors; D000527:Alprostadil / Q000031:analogs & derivatives* / Q000494:pharmacology; D001792:Blood Platelets / Q000187:drug effects*; D002118:Calcium / Q000032:analysis / Q000378:metabolism; D002227:Carbazoles; D000242:Cyclic AMP / Q000032:analysis / Q000037:antagonists & inhibitors / Q000378:metabolism; D006801:Humans; D007211:Indoles / Q000494:pharmacology; D010766:Phosphorylation; D010975:Platelet Aggregation Inhibitors / Q000494:pharmacology*; D011494:Protein Kinases / Q000378:metabolism; D011758:Pyrroles / Q000494:pharmacology; D015363:Quinolones / Q000494:pharmacology*; D013928:Thromboxane A2 / Q000031:analogs & derivatives* / Q000494:pharmacology
D002227:Carbazoles; D007211:Indoles; D010975:Platelet Aggregation Inhibitors; D011758:Pyrroles; D015363:Quinolones; C021294:cilostamide; D013928:Thromboxane A2; C057416:KT 5720; C025090:ONO 1206; C037866:STA 2; D000242:Cyclic AMP; D011494:Protein Kinases; D015105:3',5'-Cyclic-AMP Phosphodiesterases; D000527:Alprostadil; D002118:Calcium