Purification and partial characterization of a novel fibrinogenase from the venom of Deinagkistrodon acutus: inhibition of platelet aggregation

Protein Expr Purif. 2014 Jul:99:99-105. doi: 10.1016/j.pep.2014.04.007. Epub 2014 Apr 20.

Abstract

A novel fibrinogenase, DAnase, was purified from the venom of Deinagkistrodon acutus by a combination of anion and cation exchange chromatography. Unlike other fibrinogenases which are usually single polypeptide chain proteins, the enzyme was a disulfide-linked dimer with an isoelectric point of 6.03 and an apparent molecular weight of 25kDa on SDS-polyacrylamide gel electrophoresis. DAnase showed α-fibrinogenase activity devoid of fibrinolytic activity. It hydrolyzed rapidly the Aα-chain of fibrinogen and followed by the Bβ-chain and did not cleave the γ-chain. It also exhibited arginine esterase activity. The fibrinogenolytic and arginine esterase activities were completely inhibited by phenylmethanesulfonyl fluoride or tris-(2-carboxyethyl)phosphine hydrochloride, but not by EDTA, indicating that DAnase is a serine protease requiring disulfide bridge(s) for its activity. The protease strongly inhibited ADP-induced platelet aggregation in human platelet-rich plasma but was lack of ADPase activity, indicating that its fibrinogenolytic activity is involved in its inhibition of ADP-induced platelet aggregation. DAnase was devoid of hemorrhagic activity and Factor XIII activation activity. DAnase may have a potential clinical application for the therapy of thrombosis disease.

Keywords: DAnase; Deinagkistrodon acutus; Non-hemorrhagic fibrinogenase; Platelet aggregation; Serine protease; Snake venom.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Animals
  • Crotalid Venoms / chemistry
  • Crotalid Venoms / enzymology*
  • Crotalid Venoms / isolation & purification
  • Crotalid Venoms / metabolism
  • Fibrinogen / metabolism
  • Humans
  • Platelet Aggregation / drug effects*
  • Protein Multimerization
  • Serine Endopeptidases / chemistry
  • Serine Endopeptidases / isolation & purification*
  • Serine Endopeptidases / metabolism*
  • Serine Proteases / chemistry
  • Serine Proteases / isolation & purification*
  • Serine Proteases / metabolism*
  • Viperidae

Substances

  • Crotalid Venoms
  • Fibrinogen
  • DAnase alpha-fibrinogenase, Deinagkistrodon acutus
  • Serine Proteases
  • Serine Endopeptidases