BIOCHEMICAL-CHARACTERIZATION OF ATROXASE AND NUCLEOTIDE-SEQUENCE ENCODING THE FIBRINOLYTIC ENZYME

Atroxase, isolated from the venom of Crotalus atrox (western diamondba ck rattlesnake), is a non-hemorrhagic protease which has fibrinolytic activity in vitro and in vivo. Fibrin solubilization occurs primarily from the hydrolysis of alpha-polymer and unpolymerized alpha- and beta -chains. The enzyme also cleaves the A alpha-chain of fibrinogen first , followed by the B beta-chain, and shows no effect on the gamma-chain . Although crude venom induces platelet aggregation, atroxase demonstr ated no ability to induce or inhibit aggregation. Intravenous administ ration of atroxase at a dosage of 6.0 mg/kg resulted in thrombolysis w ithin 1 hr followed by recanalization. The primary structure of atroxa se was deduced from the cDNA encoding the atroxase protein. The venom glands of C. atros were used to prepare a cDNA library. Degenerate oli gonucleotides were synthesized based on the partial amino acid sequenc e of atroxase and were used as primers in the polymerase chain reactio n to amplify overlapping cDNA fragments from the C. atrox cDNA library . The resulting cDNA fragments were subcloned, sequenced, and translat ed. The final nucleotide sequence shows high homology to previously de scribed primary structures of non-hemorrhagic fibrinolytic proteases i solated from snake venom. The base sequence of cDNA obtained from colo ny hybridization also showed comparable results to the cDNA fragment a mplified by PCR. Copyright (C) 1996 Elsevier Science Ltd