Rapid purification and procoagulant and platelet aggregating activities of Rhombeobin: a thrombin-like/gyroxin-like enzyme from Lachesis muta rhombeata snake venom.
We report a rapid purification method using one-step chromatography of SVSP Rhombeobin (LMR-47) from Lachesis muta rhombeata venom and its procoagulant activities and effects on platelet aggregation. The venom was fractionated by a single chromatographic step in RP-HPLC on a C8 Discovery BIO Wide Pore, showing high degree of molecular homogeneity with molecular mass of 47035.49 Da. Rhombeobin showed amidolytic activity upon BA ρ NA, with a broad optimum pH (7-10) and was stable in solution up to 60°C. The amidolytic activity was inhibited by serine proteinase inhibitors and reducing agents, but not chelating agents. Rhombeobin showed high coagulant activity on mice plasma and bovine fibrinogen. The deduced amino acid sequence of Rhombeobin showed homology with other SVSPs, especially with LM-TL (L. m. muta) and Gyroxin (C. d. terrificus). Rhombeobin acts, in vitro, as a strong procoagulant enzyme on mice citrated plasma, shortening the APTT and PT tests in adose-dependent manner. The protein showed, "ex vivo", a strong defibrinogenating effect with 1 µg/animal. Lower doses activated the intrinsic and extrinsic coagulation pathways and impaired the platelet aggregation induced by ADP. Thus, this is the first report of a venom component that produces a venom-induced consumptive coagulopathy (VICC).
Department of Biochemistry, Institute of Biology, University of Campinas (UNICAMP), P.O. Box 6109, 13083-970 Campinas, SP, Brazil.
Frank Denis
FD
Torres-Huaco
Cláudio C
CC
Werneck
Cristina Pontes
CP
Vicente
Talita
T
Vassequi-Silva
Ana Cláudia Coelho
AC
Nery-Diez
Camila B
CB
Mendes
Edson
E
Antunes
Sérgio
S
Marangoni
Daniela C S
DC
Damico
D000595:Amino Acid Sequence; D000818:Animals; D001777:Blood Coagulation / Q000187:drug effects*; D001792:Blood Platelets / Q000187:drug effects*; D002417:Cattle; D002851:Chromatography, High Pressure Liquid; D003435:Crotalid Venoms / Q000737:chemistry / Q000302:isolation & purification* / Q000494:pharmacology*; D005340:Fibrinogen / Q000378:metabolism; D007700:Kinetics; D008297:Male; D051379:Mice; D008969:Molecular Sequence Data; D008970:Molecular Weight; D010314:Partial Thromboplastin Time; D010974:Platelet Aggregation / Q000187:drug effects*; D011517:Prothrombin Time; D057057:Serine Proteases / Q000737:chemistry / Q000302:isolation & purification* / Q000494:pharmacology*; D012910:Snake Venoms / Q000201:enzymology*; D021241:Spectrometry, Mass, Electrospray Ionization; D017819:Viperidae / Q000378:metabolism
D003435:Crotalid Venoms; D012910:Snake Venoms; C017356:gyroxin; D005340:Fibrinogen; D057057:Serine Proteases; C587786:rhombeobin protein, Lachesis muta rhombeata