Tokaracetin, a new platelet antagonist that binds to platelet glycoprotein ib and inhibits von Willebrand factor-dependent shear-induced platelet aggregation.
A new platelet antagonist, tokaracetin, was isolated from the venom of Trimeresurus tokarensis by ion-exchange chromatography, heparin-Sepharose chromatography and hydrophobic HPLC. The purified protein showed an apparent molecular mass on SDS/PAGE of 28.9 kDa under non-reducing conditions. On reduction, 16.1 and 15.4 kDa subunits were observed, suggesting that the molecule is a heterodimer. Tokaracetin inhibited the binding of 125I-labelled bovine von Willebrand factor (vWF) and 125I-labelled human vWF in the presence of botrocetin to fixed human platelets. It did not block ADP-, collagen- or thrombin receptor agonist peptide-induced platelet aggregation in human platelet-rich plasma (PRP), or induce platelet agglutination in PRP. On reduction, tokaracetin lost its inhibitory activity on the agglutination of fixed human platelets by bovine vWF. 125I-Tokaracetin specifically bound to washed human platelets with high affinity (Kd 3.9 +/- 1.4 nM) at 47,440 +/- 2780 binding sites per platelet. Binding of tokaracetin to fixed human platelets was reversible, and was inhibited by monoclonal antibody GUR83-35, which is directed against the N-terminal vWF-binding domain of human glycoprotein Ib (GPIb). Tokaracetin completely inhibited vWF-dependent shear-induced platelet aggregation in PRP at 3 micrograms/ml. The N-terminal amino acid sequences of tokaracetin subunits showed a high degree of identity with those of alboaggregin-B. These results suggest that this new platelet antagonist may be a useful tool in the development of specific inhibitors of the vWF-GPIb interaction.
Kawasaki
T
T
Institute for Drug Discovery Research, Yamanouchi Pharmaceutical Co., Ltd., Ibaraki, Japan.
Taniuchi
Y
Y
Hisamichi
N
N
Fujimura
Y
Y
Suzuki
M
M
Titani
K
K
Sakai
Y
Y
Kaku
S
S
Satoh
N
N
Takenaka
T
T
D000595:Amino Acid Sequence; D000818:Animals; D000911:Antibodies, Monoclonal / Q000276:immunology / Q000378:metabolism / Q000494:pharmacology; D001665:Binding Sites; D001667:Binding, Competitive; D002417:Cattle; D002452:Cell Count / Q000187:drug effects; D002845:Chromatography; D003435:Crotalid Venoms / Q000737:chemistry* / Q000494:pharmacology; D004229:Dithiothreitol / Q000378:metabolism; D006023:Glycoproteins / Q000378:metabolism; D006801:Humans; D008969:Molecular Sequence Data; D010446:Peptide Fragments / Q000737:chemistry* / Q000302:isolation & purification / Q000494:pharmacology; D010974:Platelet Aggregation / Q000187:drug effects; D010975:Platelet Aggregation Inhibitors / Q000737:chemistry / Q000302:isolation & purification / Q000494:pharmacology*; D011485:Protein Binding; D012685:Sepharose / Q000378:metabolism; D017421:Sequence Analysis; D014841:von Willebrand Factor / Q000378:metabolism
D000911:Antibodies, Monoclonal; D003435:Crotalid Venoms; D006023:Glycoproteins; D010446:Peptide Fragments; D010975:Platelet Aggregation Inhibitors; C029748:Trimeresurus venoms; D014841:von Willebrand Factor; C102552:tokaracetin; C030961:botrocetin; D012685:Sepharose; D004229:Dithiothreitol