Phosphorylation of the vasodilator-stimulated phosphoprotein (VASP) by the anti-platelet drug, cilostazol, in platelets.
Vasodilator-stimulated phosphoprotein (VASP) is a regulator of actin dynamics in platelets and a common substrate of both cAMP- and cGMP-dependent protein kinases (PKA and PKG). Elevations of the cAMP and cGMP concentration have been shown to inhibit platelet aggregation. Intracellular levels of cAMP and cGMP are regulated by the synthesizing system of adenylate cyclases, and hydrolysis by cyclic nucleotide phosphodiesterases (PDEs). The present study examined the effect of the anti-platelet drug, cilostazol, which inhibits PDE3 activity, on VASP phosphorylation in platelets. VASP phosphorylation was examined by immunoblotting with an anti-VASP antibody, M4, and an anti-phospho-VASP antibody, 16C2. Cilostazol phosphorylated VASP at both Ser157 and Ser239 in a concentration-dependent manner, but EHNA (PDE2 inhibitor), dipyridamole and zaprinast (PDE5 inhibitors) did not. Forskolin (adenylate cyclase activator) and sodium nitroprusside (SNP, NO donor) resulted in the VASP phosphorylation, with increase in the cAMP and cGMP level, respectively. Cilostazol increased cAMP, but not cGMP levels, in platelets. EHNA, zaprinast and dipyridamole, had no effect on cAMP and cGMP levels. The PKA/PKG inhibitor, H-89, inhibited VASP phosphorylation by cilostazol. These results demonstrated that cilostazol phosphorylates VASP through the PDE3 inhibition, increase of cAMP level, and PKA activation in platelets.
Sudo
Toshiki
T
First Institute of New Drug Research, Otsuka Pharmaceutical Co., Ltd., Tokushima, Japan. t_sudo@research.otsuka.co.jp
Ito
Hideki
H
Kimura
Yukio
Y
D000225:Adenine / Q000031:analogs & derivatives* / Q000494:pharmacology; D000262:Adenylyl Cyclases / Q000097:blood; D001798:Blood Proteins / Q000187:drug effects / Q000378:metabolism; D015815:Cell Adhesion Molecules / Q000097:blood* / Q000187:drug effects; D000077407:Cilostazol; D005576:Colforsin / Q000494:pharmacology; D000242:Cyclic AMP / Q000097:blood; D017868:Cyclic AMP-Dependent Protein Kinases / Q000097:blood; D004789:Enzyme Activation; D004791:Enzyme Inhibitors / Q000494:pharmacology; D006801:Humans; D007546:Isoquinolines / Q000494:pharmacology; D007700:Kinetics; D008840:Microfilament Proteins; D009599:Nitroprusside / Q000494:pharmacology; D010726:Phosphodiesterase Inhibitors / Q000494:pharmacology; D010750:Phosphoproteins / Q000097:blood* / Q000187:drug effects; D010766:Phosphorylation; D010975:Platelet Aggregation Inhibitors / Q000494:pharmacology*; D013449:Sulfonamides; D013777:Tetrazoles / Q000494:pharmacology*; D000098662:Vasodilator-Stimulated Phosphoprotein
D001798:Blood Proteins; D015815:Cell Adhesion Molecules; D004791:Enzyme Inhibitors; D007546:Isoquinolines; D008840:Microfilament Proteins; D010726:Phosphodiesterase Inhibitors; D010750:Phosphoproteins; D010975:Platelet Aggregation Inhibitors; D013449:Sulfonamides; D013777:Tetrazoles; D000098662:Vasodilator-Stimulated Phosphoprotein; D009599:Nitroprusside; D005576:Colforsin; C010192:9-(2-hydroxy-3-nonyl)adenine; D000242:Cyclic AMP; D017868:Cyclic AMP-Dependent Protein Kinases; D000262:Adenylyl Cyclases; D000225:Adenine; C063509:N-(2-(4-bromocinnamylamino)ethyl)-5-isoquinolinesulfonamide; D000077407:Cilostazol