Purification and characterization of a new platelet aggregation inhibitor with dissociative effect on ADP-induced platelet aggregation, from the venom of Protobothrops elegans (Sakishima-habu)

Toxicon. 2009 Jun;53(7-8):706-12. doi: 10.1016/j.toxicon.2009.02.016. Epub 2009 Feb 28.

Abstract

A platelet aggregation inhibitor, named snake venom platelet aggregation dissociator (SV-PAD)-1, with a dissociative reaction of ADP-induced platelet aggregation, was purified from the venom of Protobothrops elegans (Sakishima-habu) by gel-filtration employing Sephadex G-100, and ion-exchange chromatographies using DEAE-Sepharose Fast Flow, CM-Sepharose Fast Flow, and Mono S. By this procedure, about 1.5mg of purified protein was obtained from 1.0g of P. elegans venom. The purified protein showed a single protein band and the molecular weight was about 110kDa on sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) under reducing conditions. The pI of purified protein showed four-bands of 7.7, 7.8, 7.95, and 8.15. This protein strongly inhibited ADP-induced platelet aggregation in rabbit platelet-rich plasma (PRP), and its IC(50) was about 58nM. It inhibited ristocetin-induced platelet aggregation in rabbit PRP (IC(50): 100nM), but hardly blocked collagen-induced platelet aggregation. This protein promptly dissociated platelet aggregation in rabbit PRP stimulated by high-concentration ADP.

MeSH terms

  • Adenosine Diphosphate / antagonists & inhibitors*
  • Adenosine Diphosphate / pharmacology
  • Animals
  • Biotinylation
  • Collagen / antagonists & inhibitors
  • Collagen / pharmacology
  • Crotalid Venoms / chemistry
  • Crotalid Venoms / pharmacology*
  • Dose-Response Relationship, Drug
  • Electrophoresis, Polyacrylamide Gel
  • Enzyme-Linked Immunosorbent Assay
  • Fibrinogen / chemistry
  • In Vitro Techniques
  • Integrin beta3 / metabolism
  • Isoelectric Focusing
  • Molecular Weight
  • Peptides / pharmacology
  • Platelet Aggregation / drug effects*
  • Platelet Aggregation Inhibitors / chemistry*
  • Platelet Aggregation Inhibitors / isolation & purification
  • Platelet Aggregation Inhibitors / pharmacology*
  • Platelet Membrane Glycoprotein IIb / metabolism
  • Rabbits
  • Ristocetin / antagonists & inhibitors
  • Ristocetin / pharmacology
  • Trimeresurus / physiology*

Substances

  • Crotalid Venoms
  • Integrin beta3
  • Peptides
  • Platelet Aggregation Inhibitors
  • Platelet Membrane Glycoprotein IIb
  • elegantin
  • Ristocetin
  • Adenosine Diphosphate
  • Fibrinogen
  • Collagen