Hemostatic and toxinological diversities in venom of Micrurus tener tener, Micrurus fulvius fulvius and Micrurus isozonus coral snakes

Abstract

The coral snake Micrurus tener tener (Mtt) from the Elapidae family inhabits the southwestern United States and produces severe cases of envenomations. Although the majority of Mtt venom components are neurotoxins and phospholipase A₂s, this study demonstrated, by SDS-PAGE and molecular exclusion chromatography (MEC), that these venoms also contain high-molecular-weight proteins between 50 and 150 kDa that target the hemostatic system. The biological aspects of other Micrurus venoms were also studied, such as the LD₅₀s of Micrurus isozonus (from 0.52 to 0.61 mg/kg). A pool from these venoms presented a LD₅₀ of 0.57 mg/kg, Micrurus f. fulvius (Mff) and Mtt had LD₅₀s of 0.32 and 0.78 mg/kg, respectively. These venoms contained fibrino(geno)lytic activity, they inhibited platelet aggregation, as well as factor Xa and/or plasmin-like activities. M. isozonus venoms from different Venezuelan geographical regions inhibited ADP-induced platelet aggregation (from 50 to 68%). Micrurus tener tener venom from the United States was the most active with a 95.2% inhibitory effect. This venom showed thrombin-like activity on fibrinogen and human plasma. Fractions of Mtt showed fibrino(geno)lytic activity and inhibition on plasmin amidolytic activity. Several fractions degraded the fibrinogen Aα chains, and fractions F2 and F7 completely degraded both fibrinogen Aα and Bβ chains. To our knowledge, this is the first report on thrombin-like and fibrino(geno)lytic activity and plasmin or factor Xa inhibitors described in Micrurus venoms. Further purification and characterization of these Micrurus venom components could be of therapeutic use in the treatment of hemostatic disorders.

Fig. 1

An 10–20% SDS-PAGE of M. t. tener (Mtt) crude venom. Lanes: 1) Molecular weight markers; 2) Mtt (50 µg) under non-reducing conditions; 3) Mtt (50 µg) under reducing conditions. The gel was stained with Simply blue.

Fig. 2

Molecular exclusion chromatographic profile of M. t. tener venom. Crude venom (5 mg) was reconstituted in 100 µl of 50 mM ammonium acetate buffer, pH 6.9 and fractionated through a Superdex-200 column (10 × 300 mm) previously equilibrated with the same buffer. The venom was run for 80 min at 0.4 ml/min, and the proteins were detected at 280 nm. The insert shows the fractions F1–F5, which showed the fractions with a high anti ADP-inducer-platelet aggregation and anti-plasmin activities.

Fig. 3

Fibrinogenolytic activity of M. t. tener (Mtt) crude venom. A) A ratio of 100 µg of fibrinogen (Fg):1 µg venom was incubated at 37 °C at different times. Lanes: 1) Molecular weight markers; 2) Fg control (25 µg); 3 – 8) Fg + Mtt at 5 min, 1, 2, 4, 18 and 24 h, respectively; 9) crude venom (15 µg). B) A ratio of 100 µg Fibrinogen (Fg):1 µg venom was incubated in presence of protease inhibitors, for 2 h at 37 °C. Lanes: 1) Molecular weight markers; 2) 25 µg Fg control; 3) Fg + Mtt; 4) Fg + (Mtt + EDTA); 5) Fg + (Mtt + EGTA); 6) Fg + (Mtt + Benzamidine); 7) Fg + (Mtt + Aprotinin); 8) Fg + (Mtt + Iodoacetic acid). An 8% SDS-PAGE under reduced conditions was used and stained with Coomassie blue.

Fig. 4

Fibrinogenolytic activity of M. t. tener chromatographic venom fractions. A) A ratio of 100 µgfibrinogen (Fg):1 µg fraction was incubated for 24 h at 37 °C. A. Lanes: 1) Molecular weight markers; 2 – 11) Fg control at 24 h; 3–11) Fg + fractions F1, F2, F3, F4, F5, F7, F8, and F9, respectively. B) Fibrinolytic activity of fractions in presence of protease inhibitors. Lanes: 1) Molecular weight markers; 2) Fg control at 24 h; 3) Fg + F3; 4) Fg + (F3 + SPI); 5) Fg + (F3 + MPI); 6) Fg + (F3 + CPI); 7) Fg + F7; 8) Fg + (F7 + SPI); 9) Fg+(F7 + MPI); 10) Fg+(F7 + CPI); 11) Fg+(F7+SPI+MPI). An 8% SDS-PAGE was used under reduced conditions and stained with Coomassie blue.

Fig. 5

Fibrinolytic activity on fibrin plates (in presence of plasminogen) observed with M. t. tener (Mtt) crude venom. A) 0.125 nKcat plasmin; B) Mtt (25 µg); C) Mtt (50 µg). The arrows point to the plasmin inhibition halo.

Fig. 6

The effects of M. t. tener crude venom on plasmin fibrinolytic activity evaluated in micro-plates. (PL: plasmin; Mtt: M. t. tener venom; BZ: benzamidine; IA: Iodoacetic acid; EDTA: ethylenediaminetetraacetic acid).