A new peptide (Ruviprase) purified from the venom of Daboia russelii russelii shows potent anticoagulant activity via non-enzymatic inhibition of thrombin and factor Xa.

Compounds showing dual inhibition of thrombin and factor Xa (FXa) are the subject of great interest owing to their broader specificity for effective anticoagulation therapy against cardiovascular disorders. This is the first report on the functional characterization and assessment of therapeutic potential of a 4423.6 Da inhibitory peptide (Ruviprase) purified from Daboia russelii russelii venom. The secondary structure of Ruviprase is composed of α-helices (61.9%) and random coils (38.1%). The partial N-terminal sequence (E(1)-V(2)-X(3)-W(4)-W(5)-W(6)-A(7)-Q(8)-L(9)-S(10)) of Ruviprase demonstrated significant similarity (80.0%) with an internal sequence of apoptosis-stimulating protein reported from the venom of Ophiophagus hannah and Python bivittatus; albeit Ruviprase did not show sequence similarity with existing thrombin/FXa inhibitors, suggesting its uniqueness. Ruviprase demonstrated a potent in vitro anticoagulant property and inhibited both thrombin and FXa following slow binding kinetics. Ruviprase inhibited thrombin by binding to its active site via an uncompetitive mechanism with a Ki value and dissociation constant (KD) of 0.42 µM and 0.46 µM, respectively. Conversely, Ruviprase demonstrated mixed inhibition (Ki = 0.16 µM) of FXa towards its physiological substrate prothrombin. Furthermore, the biological properties of Ruviprase could not be neutralized by commercial polyvalent or monovalent antivenom. Ruviprase at a dose of 2.0 mg/kg was non-toxic and showed potent in vivo anticoagulant activity after 6 h of intraperitoneal treatment in mice. Because of the potent anticoagulant property as well as non-toxic nature of Ruviprase, the possible application of the peptide as an antithrombotic agent for combating thrombosis-associated ailments appears promising.

Characterization of a pro-angiogenic, novel peptide from Russell's viper (Daboia russelii russelii) venom.

Present report shows for the first time on the induction of in vitro angiogenesis by a 3.9 kDa novel peptide (RVVAP) purified from Russell's viper venom. Secondary structure of RVVAP is made up of 36.8% α-helix, 33.3% ß pleated sheets and 29.9% turns. Optimum angiogenesis and significant elevation in endothelial migration were observed at 50 ng/ml of RVVAP treatment; above this concentration, progressive decrease in wound healing was noted. RVVAP (1.0 µg/ml) was non-cytotoxic to U87-MG, HeLa and HT-29 cells; however, increasing the RVVAP concentration above 500 ng/ml resulted in induction of chromosomal aberrations and delay in cell cycle kinetics of Chinese hamster ovary cells.

Mechanism of in vivo anticoagulant and haemolytic activity by a neutral phospholipase A(2) purified from Daboia russelii russelii venom: correlation with clinical manifestations in Russell's Viper envenomed patients.

A 13.0 kDa neutral phospholipase A2 (NEUPHOLIPASE) purified from venom of Daboia russelii russelii from eastern India was identified by peptide mass fingerprinting analysis. It exerted dose-dependent PLA2, anticoagulant and indirect haemolytic activities. NEUPHOLIPASE showed preferential binding followed by hydrolysis of phosphatidylserine > phosphatidylcholine >> phosphatidylethanolamine. Circular dichroism analysis of NEUPHOLIPASE showed a high content of alpha helix (54.6%) followed by beta-turn (29.7%) in its secondary structure. Gas-chromatographic analysis of plasma from PLA2-treated mice suggested preferential hydrolysis of pro-coagulant phospholipid PS was the primary mechanism to account for in vivo anticoagulant effect of NEUPHOLIPASE. The NEUPHOLIPASE-treated mice blood showed a significant decrease (p < 0.01) in WBC as well as RBC counts with a corresponding decline in Hb content due to indirect damage to erythrocyte membranes by plasma phospholipids hydrolysis products rather than the direct haemolytic activity of PLA2 under study. NEUPHOLIPASE was non-lethal to BALB/c mice, however; it was detrimental to liver of treated-mice. Pathological symptoms observed in NEUPHOLIPASE-treated mice were correlated with the actual clinical manifestations in Russell's Viper envenomed patients from eastern India indicating some contribution of NEUPHOLIPASE in Russell's Viper venom induced toxicity and pathogenesis.

Differential mode of attack on membrane phospholipids by an acidic phospholipase A2 (RVVA-PLA2-I) from Daboia russelli venom.

An acidic phospholipase A2 (RVVA-PLA2-I) purified from Daboia russelli venom demonstrated dose-dependent catalytic, mitochondrial and erythrocyte membrane damaging activities. RVVA-PLA2-I was non-lethal to mice at the tested dose, however, it affected the different organs of mice particularly the liver and cardiac tissues as deduced from the enzymatic activities measured in mice serum after injection of this PLA2 enzyme. RVVA-PLA2-I preferentially hydrolyzed phospholipids (phosphatidylcholine) of erythrocyte membrane compared to the liver mitochondrial membrane. Interestingly, RVVA-PLA2-I failed to hydrolyze membrane phospholipids of HT-29 (colon adenocarcinoma) cells, which contain an abundance of phosphatidylcholine in its outer membrane, within 24h of incubation. The gas-chromatographic (GC) analysis of saturated/unsaturated fatty acids' release patterns from intact mitochondrial and erythrocyte membranes after the addition of RVVA-PLA2-I showed a distinctly different result. The results are certainly a reflection of differences in the outer membrane phospholipid composition of tested membranes owing to which they are hydrolyzed by the venom PLA2s to a different extent. The chemical modification of essential amino acids present in the active site, neutralization study with polyvalent antivenom and heat-inactivation of RVVA-PLA2-I suggested the correlation between catalytic and membrane damaging activities of this PLA2 enzyme. Our study advocates that the presence of a large number of PLA2-sensitive phospholipid domains/composition, rather than only the phosphatidylcholine (PC) content of that particular membrane may determine the extent of membrane damage by a particular venom PLA2 enzyme.

An acidic phospholipase A(2) (RVVA-PLA(2)-I) purified from Daboia russelli venom exerts its anticoagulant activity by enzymatic hydrolysis of plasma phospholipids and by non-enzymatic inhibition of factor Xa in a phospholipids/Ca(2+) independent manner.

A homodimeric acidic PLA(2) (RVVA-PLA(2)-I) of 58.0 kDa molecular weight purified from Russell's viper (Daboia russelli) venom demonstrated dose-dependent catalytic, strong anticoagulant and indirect hemolytic activities and inhibited blood coagulation cascade in both enzymatic and non-enzymatic mechanisms. In in vitro condition, RVVA-PLA(2)-I showed preferential hydrolysis of phosphatidylcholine with a K(m) and V(max) values of 0.65 mM and 28.9 µmol min(-1), respectively. Biochemical study and GC-analysis of plasma phospholipids hydrolysis by PLA(2) revealed that anticoagulant activity of RVVA-PLA(2)-I was partly attributed by the enzymatic hydrolysis of pro-coagulant phospholipids PC, followed by PS. The spectrofluorometric and gel-filtration analyses documented binding of RVVA-PLA(2)-I with activated factor X and PC; however, it does not bind with factor Va, prothrombin and thrombin. Therefore, this anticoagulant PLA(2) inhibits the blood coagulation cascade non-enzymatically by binding with coagulation factor Xa, even in the absence of phospholipids and Ca(2+) and thus slows down the blood coagulation by partially inhibiting the prothrombin activation. Chemical modification of essential amino acids present in the active site, neutralization with Azadirachta indica leaves extract (AIPLAI) and heat-inactivation study reinforce the association of catalytic and anticoagulant activity of RVVA-PLA(2)-I and also throw a light on its non-enzymatic mechanism of anticoagulant action.

Isolation of a snake venom phospholipase A2 (PLA2) inhibitor (AIPLAI) from leaves of Azadirachta indica (Neem): mechanism of PLA2 inhibition by AIPLAI in vitro condition.

A compound (AIPLAI (Azadirachta indica PLA(2) inhibitor)) purified from the methanolic leaf extract of A. indica (Neem) inhibits the cobra and Russell's viper venoms (RVVs) phospholipase A(2) enzymes in a dose-dependent manner. Inhibition of catalytic and tested pharmacological properties of cobra venom (Naja naja and Naja kaouthia) PLA(2) enzymes by AIPLAI is significantly higher (P<0.05) compared to the inhibition of PLA(2) enzymes of crude RVV (Daboia russelli) when tested under the same condition. Kinetic study reveals that in in vitro condition, AIPLAI inhibits the purified N. kaouthia PLA(2) enzymes in a non-competitive manner. The AIPLAI is quite stable at room temperature. The present study shows that AIPLAI holds good promise for the development of novel anti-snake venom drug in future.