Description of hutmannin-1, a new PIII-metalloprotease from the venom of the neotropical lansberg's hognose viper (Porthidium lansbergii hutmanni) with fibrino (Geno) lytic and haemorrhagic activities

The objective was to characterise hutmannin-1 (hut-1), a new ~ 62 kDa P-III-class metalloprotease from Porthidium lansbergii hutmanni (P.l.h) (Margarita Island, Venezuela). To characterise this protein, the crude venom of P.l.h was fractionated by size exclusion chromatography, anion exchange chromatography and High Performance Liquid Chromatography (HPLC). Hutmannin-1 was identified by MALDI-TOF/TOF mass spectrometry, and the venom was analysed by SDS-PAGE. The lethality, minimum haemorrhagic dose (MHD), effect of temperature on the activity, procoagulant activity on human plasma, and anticoagulant, defibrinating, gelatinolytic fibrinolytic, and fibrinogenolytic and platelet aggregation activities of hut-1 were determined. Antigenic recognition assays were performed on P.l.h crude venom and hut-1 by a venezuelan polyvalent anti-ophidic serum (PAOS) Hut-1 had strong fibrinogenolytic and moderate fibrinolytic activity. These activities and the haemorrhagic activity of hut- 1 were completely inhibited by EDTA. P.l.h crude venom had potent anticoagulant activity on recalcified plasma and inhibited the platelet aggregation induced by thrombin, ADP, collagen and ristocetin. In contrast, the anticoagulant, coagulant and platelet aggregation inhibition of hut-1 were not observed with any of the agonists. This result suggests that other proteins in the crude venom, markedly impact platelet functions and/or coagulation factors. Commercial venezuelan antivenin showed limited ability to neutralise the haemorrhagic activity of hut-1.