Atroxlysin-III, A Metalloproteinase from the Venom of the Peruvian Pit Viper Snake Bothrops atrox (Jergón) Induces Glycoprotein VI Shedding and Impairs Platelet Function

Abstract

Atroxlysin-III (Atr-III) was purified from the venom of Bothrops atrox. This 56-kDa protein bears N-linked glycoconjugates and is a P-III hemorrhagic metalloproteinase. Its cDNA-deduced amino acid sequence reveals a multidomain structure including a proprotein, a metalloproteinase, a disintegrin-like and a cysteine-rich domain. Its identity with bothropasin and jararhagin from Bothrops jararaca is 97% and 95%, respectively. Its enzymatic activity is metal ion-dependent. The divalent cations, Mg2+ and Ca2+, enhance its activity, whereas excess Zn2+ inhibits it. Chemical modification of the Zn2+-complexing histidine residues within the active site by using diethylpyrocarbonate (DEPC) inactivates it. Atr-III degrades plasma fibronectin, type I-collagen, and mainly the α-chains of fibrinogen and fibrin. The von Willebrand factor (vWF) A1-domain, which harbors the binding site for GPIb, is not hydrolyzed. Platelets interact with collagen via receptors for collagen, glycoprotein VI (GPVI), and α2β1 integrin. Neither the α2β1 integrin nor its collagen-binding A-domain is fragmented by Atr-III. In contrast, Atr-III cleaves glycoprotein VI (GPVI) into a soluble ~55-kDa fragment (sGPVI). Thereby, it inhibits aggregation of platelets which had been stimulated by convulxin, a GPVI agonist. Selectively, Atr-III targets GPVI antagonistically and thus contributes to the antithrombotic effect of envenomation by Bothrops atrox.

Pictolysin-III, a Hemorrhagic Type-III Metalloproteinase Isolated from Bothrops pictus (Serpentes: Viperidae) Venom, Reduces Mitochondrial Respiration and Induces Cytokine Secretion in Epithelial and Stromal Cell Lines

“…We regard this fact as an exclusive event of the in vitro condition and not representative of an enzymatic characteristic involved in the biological action of Pic-III. In the presence of Ca 2+ and Mg 2+ , Pic-III significantly increases its proteolytic activity in line with SVMPs [ 30 , 42 , 67 ].…”

“…Total RNA was obtained according to Vivas-Ruiz et al [ 29 ]. The gene of Pic-III was amplified with the kit Master Mix Platinum ® Taq DNA Polymerase (Invitrogen, Carlsbad, CA, USA) according to the manufacturer’s instructions using the external primers MpIIIF and MpIIIR and the internal primers MpIIIFi and MpIIIRi provided by Oliveira et al [ 30 ]. The Pic-III gene expression level was analyzed according to Vivas-Ruiz et al [ 28 ] using Verso 1-Step qPCR ROX Kit (ThermoFisher Scientific, Waltham, MA, USA).…”

“…Proteolytic activities were determined according to Oliveira et al [ 30 ]. Human fibrinogen (H-Fg) was dissolved in 25 mM Tris-HCl, pH 7.4, 154 mM NaCl; samples with 100 µL of H-Fg (2.5 mg/mL) were incubated with 1 µg of Pic-III at 37 °C for 30 min; the reaction was stopped by adding 40 µL of denaturant buffer (SDS, β-mercaptoethanol, glycerol, and bromophenol blue).…”

“…Human blood from two healthy volunteers (age/sex: 22/M and 23/F) was collected in acid–citrate–dextrose (ACD: 78 mM citric acid; 117 mM sodium citrate; 282 mM dextrose) [6:1, ( v / v )], centrifuged at 200× g for 15 min to obtain platelet-rich plasma (PRP). Washed platelets were prepared as previously described by Oliveira et al [ 30 ]. Platelet density was adjusted to 2.5 × 10 5 platelets/µL.…”

Snake Venom Metalloproteinases (SVMPs): A structure-function update

“…There have been several reports of alteration in ligand-receptor interaction; such alteration corresponds to the ectodomain sheddase activities of this domain ( Ho et al, 2002 ; Moura-da-Silva et al, 1996 ; Moura-da-Silva and Baldo, 2012 ). For instance, Oliveira et al (2009) showed how atroxlysin-III from the Peruvian pit viper snake ( Bothrops oligolepis ) induced glycoprotein VI shedding, culminating into platelet dysfunction ( Oliveira et al, 2019 ). Previously, the sheddase activity of atrolysin a, a P-III SVMP from Crotalus atrox venom, was demonstrated against collagen IV and annexin in cultured human fibroblasts ( Pinto et al, 2007 ).…”