Isolation of a proteinase with plasminogen-activating activity from Lachesis muta muta (bushmaster) snake venom

A plasminogen activator enzyme (LV-PA) from Lachesis muta muta venom was pu rified to homogeneity using gel filtration and anion exchange chromatograph y, SDS-PAGE under reducing conditions showed a single protein band with an M-r of 33,000 Da, It is an acidic glycoprotein which activates plasminogen to plasmin indirectly, functioning via prior formation of a molecular compl ex, known as plasminogen activator. The purified preparation catalyzes the hydrolysis of several p-nitroanilide peptide substrates containing Lys at t he scissile bond. In contrast, no hydrolysis was detected on the synthetic substrates TAME and BAPNA, which contain arginine. By the use of the plasmi n-specific chromogenic substrate Tos-Gly-Pro-Lys-pNA, the preparation had a plasmin-like activity of 0.68 U/mg, which was 35.8-fold higher than that o f the crude venom from which it was prepared. In vitro, fibrin hydrolysis u sing LV-PA as plasminogen activator displayed more similarity with the effe ct produced by streptokinase (SK), SDS-PAGE (10%) analysis showed a 115-kDa complex formation after incubation of plasminogen with either LV-PA or SK, At a molar ratio of 50:1 (fibrinogen:enzyme), the preparation exhibited we akly fibrinogenolytic activity. However, LV-PA is distinguished from thromb in in that it does not clot fibrinogen. After incubation of LV-PA with plat elet-rich plasma, the enzyme (2 mu M) showed no effect on platelet aggregat ion induced by ADP, epinephrine, or collagen. Comparison of the N-terminal sequence of LV-PA with other snake venom plasminogen activators revealed th at LV-PA exhibits a high degree of sequence identity with the TsVPA from Tr imeresurus stejnegeri (90%) and with the Haly-PA from Agkistrodon halys (85 %). LV-PA also has homology with other snake venom serine proteinases such as the thrombin-like/gyroxin analogue (38%) from bushmaster venom and with other coagulation serine proteases. The proteinase was readily inhibited by treatment with p-nitrophenyl p-guanidinebenzoate, p-aminobenzamidine, and phenylmethanesulfonyl fluoride but was not affected by metal chelators, (C) 2000 Academic Press.