title: Purification and characterization of AHPM, a novel non-hemorrhagic P-IIIc metalloproteinase with α-fibrinogenolytic and platelet aggregation-inhibition activities, from Agkistrodon halys pallas venom.
abstract: A novel non-hemorrhagic metalloproteinase, AHPM, was purified from the venom of Agkistrodon halys pallas by a combination of ion-exchange and gel filtration chromatography. AHPM is a dimeric glycoprotein with multiple pIs around pH 7.9 and has a molecular mass of 110 kDa with two blocked N-terminuses. Partial sequence of AHPM obtained by LC-MS/MS analysis together with its dimeric nature reveals that it is a P-IIIc snake venom metalloproteinase composed of metalloproteinase, disintegrin-like and cysteine-rich domains. AHPM has a conserved DECD sequence in the disintegrin-like domain. AHPM hydrolyzes casein and fibrinogen and also dissolves fibrin clots and the proteolytic activity is abolished by EDTA, but not by PMSF, suggesting that it is a metalloproteinase. The protease hydrolyzes rapidly the Aα-chain of fibrinogen followed by the Bβ-chain and does not cleave the γ-chain. AHPM contains endogenous Zn(2+) and Ca(2+) ions at a molar ratio of 1:1.9 and 1:4.2, respectively, and Zn(2+) ions are essential for its proteolytic activity. AHPM inhibits collagen-and ADP-induced platelet aggregation with half maximal inhibitory concentrations of 200 ± 8 nM and 280 ± 10 nM, respectively. EDTA markedly attenuates the inhibition of ADP-induced platelet aggregation by AHPM, indicating that the fibrinogenolytic activity of AHPM is involved in its inhibition of ADP-induced platelet aggregation. AHPM is devoid of hemorrhagic activity when injected (up to 30 μg) subcutaneously into mice. AHPM is so far identified as first non-hemorrhagic P-IIIc SVMP which has both fibrinolytic and platelet aggregation-inhibition activities. The bifunctional enzyme may have a potential clinical application as a thrombolytic agent.
authors.affiliation: Department of Chemistry, University of Science and Technology of China, No. 96, Jinzhai Road, Hefei 230026, PR China.
authors.forename: Jiajia
authors.initials: J
authors.lastname: Song
authors.forename: Xiaolong
authors.initials: X
authors.lastname: Xu
authors.forename: Yan
authors.initials: Y
authors.lastname: Zhang
authors.forename: Mingchun
authors.initials: M
authors.lastname: Guo
authors.forename: Xincheng
authors.initials: X
authors.lastname: Yan
authors.forename: Shasha
authors.initials: S
authors.lastname: Wang
authors.forename: Shang
authors.initials: S
authors.lastname: Gao
mesh_terms: D017836:Agkistrodon; D000595:Amino Acid Sequence; D000818:Animals; D002364:Caseins / Q000378:metabolism; D005340:Fibrinogen / Q000378:metabolism*; D005342:Fibrinolysis / Q000187:drug effects; D005343:Fibrinolytic Agents / Q000737:chemistry / Q000302:isolation & purification / Q000494:pharmacology; D006470:Hemorrhage / Q000139:chemically induced; D045726:Metalloproteases / Q000737:chemistry / Q000302:isolation & purification* / Q000494:pharmacology*; D051379:Mice; D008969:Molecular Sequence Data; D010974:Platelet Aggregation / Q000187:drug effects*; D014757:Viper Venoms / Q000201:enzymology*
chemical_list: D002364:Caseins; D005343:Fibrinolytic Agents; D014757:Viper Venoms; D005340:Fibrinogen; D045726:Metalloproteases