Tokaracetin, a new platelet antagonist that binds to platelet glycoprotein ib and inhibits von Willebrand factor-dependent shear-induced platelet aggregation

Abstract

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.

A heat shock‐related protein, p20, plays an inhibitory role in platelet activation

“…In order to further define the inhibitory effect of p20 on platelet aggregation induced by botrocetin, we used fixed platelets [6]. We obtained fixed human platelets as follows: we mixed human PRP with an equal volume of 2.0% paraformaldehyde and incubated the mixture for 30 min at 22°C.…”

“…We investigated the effect of p20 on platelet aggregation induced by thrombin since thrombin is also a potent agonist for platelet aggregation. Washed platelets were prepared as described previously [6]. Human platelets were counted and adjusted to 4×10 8 cells/ml (final concentration).…”

Complete Amino Acid Sequence and Identification of the Platelet Glycoprotein Ib-binding Site of Jararaca GPIb-BP, a Snake Venom Protein Isolated from Bothrops jararaca

“…It is now clear that B. jararaca snake venom contains several proteins with homologous structures but with clearly distinct binding characteristics. The amino-terminal sequences of alboaggregin-B (23), flavocetin-A and -B (7), tokaracetin (8), and agkicetin (4) are highly homologous; the primary structure of jararaca GPIb-BP may reveal a structure typical of these GPIb-binding proteins.…”

Echicetin, a GPIb-binding snake C-type lectin from Echis carinatus, also contains a binding site for IgMκ responsible for platelet agglutination in plasma and inducing signal transduction

“…8 Most of the inhibitory C-type lectins described so far bind to GPIb. These include echicetin, 9 jararaca GPIb-binding protein, 10,11 tokaracetin, 12 CHH-A and -B, 4 and agkicetin. 13 Echicetin, a heterodimeric snake C-type lectin from Echis carinatus, has been shown by several authors to bind specifically to platelet GPIb and to block platelet interactions with von Willebrand factor (vWf) 9 and with thrombin.…”