Daboxin P, a Major Phospholipase A2 Enzyme from the Indian Daboia russelii russelii Venom Targets Factor X and Factor Xa for Its Anticoagulant Activity

Abstract

In the present study a major protein has been purified from the venom of Indian Daboia russelii russelii using gel filtration, ion exchange and Rp-HPLC techniques. The purified protein, named daboxin P accounts for ~24% of the total protein of the crude venom and has a molecular mass of 13.597 kDa. It exhibits strong anticoagulant and phospholipase A2 activity but is devoid of any cytotoxic effect on the tested normal or cancerous cell lines. Its primary structure was deduced by N-terminal sequencing and chemical cleavage using Edman degradation and tandem mass spectrometry. It is composed of 121 amino acids with 14 cysteine residues and catalytically active His48 -Asp49 pair. The secondary structure of daboxin P constitutes 42.73% of α-helix and 12.36% of β-sheet. It is found to be stable at acidic (pH 3.0) and neutral pH (pH 7.0) and has a Tm value of 71.59 ± 0.46°C. Daboxin P exhibits anticoagulant effect under in-vitro and in-vivo conditions. It does not inhibit the catalytic activity of the serine proteases but inhibits the activation of factor X to factor Xa by the tenase complexes both in the presence and absence of phospholipids. It also inhibits the tenase complexes when active site residue (His48) was alkylated suggesting its non-enzymatic mode of anticoagulant activity. Moreover, it also inhibits prothrombinase complex when pre-incubated with factor Xa prior to factor Va addition. Fluorescence emission spectroscopy and affinity chromatography suggest the probable interaction of daboxin P with factor X and factor Xa. Molecular docking analysis reveals the interaction of the Ca+2 binding loop; helix C; anticoagulant region and C-terminal region of daboxin P with the heavy chain of factor Xa. This is the first report of a phospholipase A2 enzyme from Indian viper venom which targets both factor X and factor Xa for its anticoagulant activity.

Role of crotoxin in coagulation: novel insights into anticoagulant mechanisms and impairment of inflammation-induced coagulation

“…Aside from the prothrombinase complex, we also evaluated the modulation of both intrinsic and extrinsic tenase complexes by CTX, and observed that the toxin inhibited both pathways. Previous reports of this nature have also been published for other svPLA 2 s such as daboxin P, from Daboia russelii snake venom, and 3-finger toxins such as exactin, from Hemachatus haemachatus venom, in which both toxins inhibit extrinsic and intrinsic tenase complexes [33,34]. Moreover, our data shows that CTX is more efficient in inhibiting the intrinsic tenase complex than the extrinsic, similarly to that observed for daboxin P and exactin.…”

Purification and partial characterization of an anticoagulant PLA2 from the venom of Indian Daboia russelii that induces inflammation through upregulation of proinflammatory mediators

“…Alkylation of histidine residue of Rv(i) PLA 2 was carried out using p ‐bromophenacyl bromide (pBpB) . Briefly, 150 μg of Rv(i) PLA 2 was incubated with 30 μg of pBpB (dissolved in 100% ethanol) for 20 h. The same amount of the purified protein in 100% ethanol was considered as the vehicle control.…”

“…Snake venom PLA 2 enzymes play a significant role in inhibiting blood coagulation in envenomated victims. These enzymes interfere with the blood coagulation process either enzymatically by hydrolyzing the phospholipids of platelet membranes and/or nonenzymatically by interacting with the blood clotting factors . In addition to the anticoagulant effect, PLA 2 enzymes also exhibit prominent inflammatory responses in victims both locally and systematically .…”

Expression and characterization of Flavikunin: A Kunitz‐type serine protease inhibitor identified in the venom gland cDNA library of Bungarus flaviceps

“…Coagulation assays were done using platelet‐poor plasma (PPP) prepared from citrated goat blood by centrifugation at 3000 rpm for 20 min at 16°C . A control was run in parallel in each assay where only buffer (20 mM Tris‐Cl, pH 7.4) was used and normal plasma clotting time was recorded using a coagulation analyzer (Tulip Diagnostics, Alto Santa Cruz, Goa, India).…”