title: Vasodilator-stimulated phosphoprotein (VASP) phosphorylation assay for platelet response to cilostazol.
abstract: Recent clinical trials have demonstrated that cilostazol, an antiplatelet drug competent to inhibit phosphodiesterase 3, is effective in secondary prevention of atherothrombosis including ischemic stroke, myocardial infarction, and peripheral arterial disease. However, there is no reliable assay for detection of the platelet response to cilostazol. We attempted to establish such an assay for clinical use. Phosphorylation of vasodilator-stimulated phosphoprotein (VASP) subsequent to the pharmacological action of cilostazol was measured by a platelet VASP assay kit that has been widely used for monitoring platelet response to ADP receptor antagonists in clinical settings. We modified the kit and found the optimal conditions for detection of cilostazol-dependent VASP phosphorylation. The assay could detect the in vitro and in vivo platelet responses to cilostazol effectively in the presence of 50 nM PGE1. ROC analysis showed that our assay had 97% sensitivity and 75% specificity when blood drawn between 3 and 9?h after cilostazol ingestion was subjected to the assay. The assay results were verified by immunoblotting specific for VASP phosphorylation. Standard light transmission platelet aggregometry could not detect significant inhibition of agonist-induced platelet aggregation by cilostazol except for the in vitro effect of high concentrations of cilostazol. These results demonstrate that the platelet VASP assay can detect the platelet response to cilostazol with high sensitivity and specificity.
authors.affiliation: Department of Bioregulatory Medicine, Ehime University Graduate School of Medicine, Ehime, Japan.
authors.forename: Jun
authors.initials: J
authors.lastname: Yamanouchi
authors.forename: Takaaki
authors.initials: T
authors.lastname: Hato
authors.forename: Toshiyuki
authors.initials: T
authors.lastname: Niiya
authors.forename: Kou
authors.initials: K
authors.lastname: Nakagawa
authors.forename: Yoshiaki
authors.initials: Y
authors.lastname: Kumon
authors.forename: Hiroshi
authors.initials: H
authors.lastname: Fujiwara
authors.forename: Yoshihiro
authors.initials: Y
authors.lastname: Yakushijin
authors.forename: Masaki
authors.initials: M
authors.lastname: Yasukawa
mesh_terms: D001792:Blood Platelets / Q000187:drug effects* / Q000378:metabolism; D015153:Blotting, Western; D015815:Cell Adhesion Molecules / Q000378:metabolism*; D000077407:Cilostazol; D019411:Clinical Laboratory Techniques / Q000379:methods*; D005434:Flow Cytometry; D006801:Humans; D008840:Microfilament Proteins / Q000378:metabolism*; D010750:Phosphoproteins / Q000378:metabolism*; D010766:Phosphorylation / Q000187:drug effects; D010975:Platelet Aggregation Inhibitors / Q000494:pharmacology; D012372:ROC Curve; D011933:Reagent Kits, Diagnostic; D012680:Sensitivity and Specificity; D013777:Tetrazoles / Q000494:pharmacology*; D000098662:Vasodilator-Stimulated Phosphoprotein
chemical_list: D015815:Cell Adhesion Molecules; D008840:Microfilament Proteins; D010750:Phosphoproteins; D010975:Platelet Aggregation Inhibitors; D011933:Reagent Kits, Diagnostic; D013777:Tetrazoles; D000098662:Vasodilator-Stimulated Phosphoprotein; D000077407:Cilostazol