A new binding assay of von Willebrand factor and glycoprotein Ib using solid-phase biotinylated platelets.
To obtain compounds that inhibit the interaction of von Willebrand factor (vWF) and glycoprotein (GP) Ib, a novel binding assay was established. The binding of fixed platelets to vWF-R497 mutant was quantified by a solid phase assay. In this assay, fixed platelets bound to the vWF-R497 mutant, carrying the deletion of Glu497-Tyr508 and the missense mutation of Arg545 to Ala, without binding modulators such as ristocetin. The K(d) value of the binding was 2.8 nM, which was consistent with the result from liquid binding assay. The binding was inhibited by aurin tricarboxylic acid (ATA) and an anti GPIb antibody, AK2. Using this binding assay, we screened our library compounds and obtained D74-3736. This compound also inhibited ristocetin-induced platelet aggregation in the human platelet-rich plasma.
R&D Division, Exploratory Research Laboratories II, Daiichi-Sankyo Co. Ltd., 16-13 Kita-Kasai 1-Chome, Edogawa-ku, Tokyo, Japan. hayata.kenji.v6@daiichisankyo.co.jp
Kenji
K
Hayata
Takayuki
T
Nakayama
Tadashi
T
Matsushita
Katsuichi
K
Sakano
D000906:Antibodies; D001312:Aurintricarboxylic Acid / Q000494:pharmacology; D001681:Biological Assay; D019921:Biotinylation; D001792:Blood Platelets / Q000187:drug effects / Q000378:metabolism*; D002460:Cell Line; D004305:Dose-Response Relationship, Drug; D005343:Fibrinolytic Agents / Q000494:pharmacology; D006801:Humans; D009154:Mutation; D010974:Platelet Aggregation / Q000187:drug effects; D010975:Platelet Aggregation Inhibitors / Q000494:pharmacology; D019038:Platelet Glycoprotein GPIb-IX Complex / Q000276:immunology / Q000378:metabolism*; D011485:Protein Binding; D011994:Recombinant Proteins / Q000378:metabolism; D012310:Ristocetin / Q000494:pharmacology; D052616:Solid Phase Extraction; D013997:Time Factors; D014162:Transfection; D014841:von Willebrand Factor / Q000235:genetics / Q000378:metabolism*
D000906:Antibodies; D005343:Fibrinolytic Agents; D010975:Platelet Aggregation Inhibitors; D019038:Platelet Glycoprotein GPIb-IX Complex; D011994:Recombinant Proteins; D014841:von Willebrand Factor; D012310:Ristocetin; D001312:Aurintricarboxylic Acid