A prothrombin activator serine protease from the Lonomia obliqua caterpillar venom (Lopap) biochemical characterization.
Lonomia obliqua venom causes a severe consumptive coagulopathy, which can lead to a hemorrhagic syndrome. The crude bristles extract displays a procoagulant activity due to a Factor X and to a prothrombin activating activity. Here, we describe a 69 kDa prothrombin activator serine protease purified from L. obliqua caterpillar bristle extract using gel filtration (Sephadex G 75) and HPLC (C(4) column). The purified protein was able to activate prothrombin in a dose-dependent manner, and calcium ions increased this activity. The prothrombin-derived fluorogenic peptide (Abz-YQTFFNPRTGSQ-EDDnp) had its main cleavage site at the Arg-Thr bond. The kinetic parameters obtained for this substrate were Kmapp of 4.5 microM, kcat of 5.32 s(-1), and a kcat/Kmapp of 1.2 x 10(6) M(-1) s(-1). The prothrombin fragments generated by the purified enzyme corresponded to the molecular masses of prethrombin 2, fragment 1, fragment 2, and thrombin as seen in SDS-PAGE. The thrombin generated was able to clot purified fibrinogen. The partial amino acid sequence of the purified protein, named Lopap (L. obliqua prothrombin activator protease), showed no similarity to any known prothrombin activator.
Reis
C V
CV
Laboratory of Biochemistry and Biophysics, Butantan Institute, Center for Applied Toxinology, CEPID-FAPESP, São Paulo, Brazil.
Portaro
F C
FC
Andrade
S A
SA
Fritzen
M
M
Fernandes
B L
BL
Sampaio
C A
CA
Camargo
A C
AC
Chudzinski-Tavassi
A M
AM
D000595:Amino Acid Sequence; D000818:Animals; D001180:Arthropod Venoms / Q000302:isolation & purification / Q000494:pharmacology; D001665:Binding Sites; D001780:Blood Coagulation Tests; D002118:Calcium / Q000494:pharmacology; D004305:Dose-Response Relationship, Drug; D005170:Factor X / Q000378:metabolism; D005340:Fibrinogen / Q000187:drug effects / Q000378:metabolism; D005456:Fluorescent Dyes / Q000378:metabolism; D006801:Humans; D007700:Kinetics; D007814:Larva; D007915:Lepidoptera; D008969:Molecular Sequence Data; D011516:Prothrombin / Q000378:metabolism*; D012697:Serine Endopeptidases / Q000302:isolation & purification / Q000494:pharmacology*
D001180:Arthropod Venoms; D005456:Fluorescent Dyes; D011516:Prothrombin; D005170:Factor X; D005340:Fibrinogen; C119835:Lonomia obliqua prothrombin activator protease; D012697:Serine Endopeptidases; D002118:Calcium