Purification and Characterization of a New Serine Protease (VLCII) Isolated from Vipera lebetina Venom: Its Role in Hemostasis.
Snake venom serine proteinases (SVSPs) affect various physiological functions including blood coagulation, fibrinolysis, and platelet aggregation. Coagulant serine proteinase (VLCII) was purified from Vipera lebetina venom using three chromatographic steps: gel filtration on SephadexG-75, DEAE-Sephadex A-50, and reversed-phase high-performance liquid chromatography (RP-HPLC) on C8 column. VLCII appeared homogenous (60 kDa) when tested on sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE). VLCII as a thrombin-like enzyme was able to hydrolyze Nα-CBZ L-arginine-p-nitroanilide hydrochloride and could be a serine protease because it is inhibited by phenylmethylsulfonyl fluoride. The proteolytic activity of VLCII was not affected by ethylenediaminetetraacetic acid and 1.10-phenanthroline. It showed high coagulant activity against human plasma and cleaved both Aα chain and Bβ chain of bovine fibrinogen. The isolated VLCII displayed proaggregating effect on human platelet in a concentration-dependent manner with an absence of lag time. Clopidogrel P2Y12 adenosine diphosphate (ADP) receptor inhibitor reduced markedly the aggregating effect induced by VLCII than aspirin, indicating the involvement of ADP signaling pathway.
USTHB, Faculty of Biological Sciences, Laboratory of Cellular and Molecular Biology, BP32 El-Alia, Bab Ezzouar, Algiers, Algeria. flaraba@hotmail.com.
Kadi-Saci
KS
Amel
USTHB, Faculty of Biological Sciences, Laboratory of Cellular and Molecular Biology, BP32 El-Alia, Bab Ezzouar, Algiers, Algeria. flaraba@usthb.dz.
Laraba-Djebari
LD
Fatima
D000818:Animals; D002417:Cattle; D002850:Chromatography, Gel; D002851:Chromatography, High Pressure Liquid; D005340:Fibrinogen / Q000378:metabolism; D006801:Humans; D010949:Plasma / Q000378:metabolism; D059748:Proteolysis; D057057:Serine Proteases / Q000302:isolation & purification / Q000378:metabolism*; D014757:Viper Venoms / Q000201:enzymology*
D014757:Viper Venoms; D005340:Fibrinogen; D057057:Serine Proteases
Hemostasis; Mechanism; Snake venom; Thrombin-Like