Biochemical characterization of atroxase and nucleotide sequence encoding the fibrinolytic enzyme

Atroxase, isolated from the venom of Crotalus atrox (western diamondback 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 demonstrated no ability to induce or inhibit aggregation. Intravenous administration of atroxase at a dosage of 6.0 mg/kg resulted in thrombolysis within 1 hr followed by recanalization. The primary structure of atroxase was deduced from the cDNA encoding the atroxase protein. The venom glands of C. atrox were used to prepare a cDNA library. Degenerate oligonucleotides were synthesized based on the partial amino acid sequence of atroxase and were used as primers in the polymerase chain reaction to amplify overlapping cDNA fragments from the C. atrox cDNA library. The resulting cDNA fragments were subcloned, sequenced, and translated. The final nucleotide sequence shows high homology to previously described primary structures of non-hemorrhagic fibrinolytic proteases isolated from snake venom. The base sequence of cDNA obtained from colony hybridization also showed comparable results to the cDNA fragment amplified by PCR.