A New Platelet-Aggregation-Inhibiting Factor Isolated from Bothrops moojeni Snake Venom

Abstract

This work reports the purification and functional characterization of BmooPAi, a platelet-aggregation-inhibiting factor from Bothrops moojeni snake venom. The toxin was purified by a combination of three chromatographic steps (ion-exchange on DEAE-Sephacel, molecular exclusion on Sephadex G-75, and affinity chromatography on HiTrap™ Heparin HP). BmooPAi was found to be a single-chain protein with an apparent molecular mass of 32 kDa on 14% SDS-PAGE, under reducing conditions. Sequencing of BmooPAi by Edman degradation revealed the amino acid sequence LGPDIVPPNELLEVM. The toxin was devoid of proteolytic, haemorrhagic, defibrinating, or coagulant activities and induced no significant oedema or hyperalgesia. BmooPAi showed a rather specific inhibitory effect on ristocetin-induced platelet aggregation in human platelet-rich plasma, whereas it had little or no effect on platelet aggregation induced by collagen and adenosine diphosphate. The results presented in this work suggest that BmooPAi is a toxin comprised of disintegrin-like and cysteine-rich domains, originating from autolysis/proteolysis of PIII SVMPs from B. moojeni snake venom. This toxin may be of medical interest because it is a platelet aggregation inhibitor, which could potentially be developed as a novel therapeutic agent to prevent and/or treat patients with thrombotic disorders.

Figure 1

Purification of BmooPAi from B. moojeni snake venom. (a) Separation on DEAE-Sephacel ion-exchange chromatography: crude venom (200 mg) was applied to the column (2.5 × 20 cm) and elution was carried out at a flow rate of 20 mL/h with ammonium bicarbonate (Ambic) buffer gradients, pH 7.8, from 0.05 M to 0.6 M. Fractions of 3.0 mL/tube were collected and the absorbance was read at 280 nm. (b) Separation on Sephadex G-75 molecular exclusion chromatography: fraction D7 was applied to the column (1.0 × 100 cm) and elution with 0.05 M ammonium bicarbonate was achieved at a flow rate of 20 mL/h. Fractions of 3.0 mL/tube were collected and the absorbance was read at 280 nm. (c) Separation by affinity chromatography on a HiTrap Heparin HP column using the ÄKTApurifier HPLC system: fraction D7S2 was applied to the column (5 × 1 mL), previously equilibrated with 20 mM Tris-HCl buffer (pH 7.0) containing 5 mM calcium chloride. The samples were eluted with an increasing concentration gradient of 20 mM Tris-HCl buffer (pH 7.0) containing 2.0 M sodium chloride, and the absorbance of the fractions was monitored at 280 nm. Fractions of 1.0 mL/tube were collected at a flow rate of 30 mL/h. (d) SDS-PAGE in 14% (w/v) polyacrylamide, Tris-glycine buffer, pH 8.3, and 20 mA. Lanes: 1, standard proteins; 2, reduced crude venom of B. moojeni; 3, reduced BmooPAi; 4, nonreduced BmooPAi. The gel was stained with Coomassie blue R-250. (e) Reverse-phase HPLC on a C2C18 column (4.6 × 100 mm) equilibrated with 0.1% trifluoroacetic acid (TFA) and eluted with a linear concentration gradient from 0 to 100% of solution B (70% acetonitrile in 0.1% TFA).

Figure 2

Sequence alignment of BmooPAi and other metalloproteinases/dis-cys proteins: Moojenin (GI: P0DKR0.1), Bothropasin (GI: 209870468), Jararhagin (GI: P30431.1), Jarastatin (GI: Q0NZX5.1), Jararhagin-C (GI: P30431.1), Leucurolysin-B (GI: P86092.1), Leucurogin (GI: P0DJ87.1), Brevilysin-H6 (GI: P0C7B0.2), AaH-IV (GI: 255917952), Atrolysin-A (GI: Q92043.1), and Salmosin (GI: O93518.1). The nonconserved residues are shown in black frames. The alignment and figure were generated by the MegAlign program from Lasergene (DNAStar Inc., Madison, WI, USA).

Figure 3

Effect of BmooPAi from B. moojeni venom on (a) ristocetin-, (b) ADP-, and (c) collagen-induced platelet aggregation. Aggregation was triggered with the agonists immediately after adding the indicated doses of BmooPAi to citrated human PRP at 37°C. Platelet aggregation was recorded for 10 min in an AggRAM platelet aggregation system with four-channel laser optics (Helena Laboratories, EUA). Results are expressed as an increase in light transmission, where PPP represents the maximum response (100%). Control experiments were performed in the absence of BmooPAi.