Comparison of dose-dependent fibrinogenolytic and BAEE-esterase activities of protease isoenzymes purified from RVV.

<p><a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0086823#pone-0086823-g002" target="_blank">Fig.2</a> (A) shows fibrinogenolytic activity and <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0086823#pone-0086823-g002" target="_blank">Fig 2(B)</a> depcits BAEE-esterase activity of purified protease isoenzymes. The values are mean ± S.D of the three experiments.</p

Activity of protease isoenzymes against various protein and chromogenic substrates.

†<p>Specific activity of partially deglycosylasted protease isoenzymes; ND: not determined.</p>#<p>Incubated for 180 min at 37°C.</p><p>Substrate(s) for ¹trypsin, ^2,3thrombin, ⁴plasmin, ⁵FXa.</p><p>Incubation was carried out with 1% (w/v) protein substrate at pH 8.0, 37°C for 90 min or ^#180 min. For amidolytic activity assay, protease was incubated with 0.2 mM chromogenic substrate for 10 min at 37°C. Values are mean±SD of triplicate determinations. Values in the same row with different subscripts (a–e) are significantly different (P<0.05).</p

The Pro-Coagulant Fibrinogenolytic Serine Protease Isoenzymes Purified from <i>Daboia russelii russelii</i> Venom Coagulate the Blood through Factor V Activation: Role of Glycosylation on Enzymatic Activity

<div><p>Proteases from Russell's viper venom (RVV) induce a variety of toxic effects in victim. Therefore, four new RVV protease isoenzymes of molecular mass 32901.044 Da, 333631.179 Da, 333571.472 Da, and 34594.776 Da, were characterized in this study. The first 10 N-terminal residues of these serine protease isoenzymes showed significant sequence homology with N-terminal sequences of snake venom thrombin-like and factor V-activating serine proteases, which was reconfirmed by peptide mass fingerprinting analysis. These proteases were found to be different from previously reported factor V activators isolated from snake venoms. These proteases showed significantly different fibrinogenolytic, BAEE-esterase and plasma clotting activities but no fibrinolytic, TAME-esterase or amidolytic activity against the chromogenic substrate for trypsin, thrombin, plasmin and factor Xa. Their <i>Km</i> and <i>Vmax</i> values towards fibrinogen were determined in the range of 6.6 to 10.5 µM and 111.0 to 125.5 units/mg protein, respectively. On the basis of fibrinogen degradation pattern, they may be classified as A/B serine proteases isolated from snake venom. These proteases contain ∼42% to 44% of N-linked carbohydrates by mass whereas partially deglycosylated enzymes showed significantly less catalytic activity as compared to native enzymes. <i>In vitro</i> these protease isoenzymes induce blood coagulation through factor V activation, whereas <i>in vivo</i> they provoke dose-dependent defibrinogenation and anticoagulant activity in the mouse model. At a dose of 5 mg/kg, none of these protease isoenzymes were found to be lethal in mice or house geckos, suggesting therapeutic application of these anticoagulant peptides for the prevention of thrombosis.</p></div

Dose-dependent <i>in vivo</i> defibrinogenating activity and <i>in vitro</i> blood clotting activity of RVV protease isoenzymes in mouse model.

<p>The figure (A) shows <i>in vivo</i> defibrinogenating activity and figure (B) shows <i>in vitro</i> clotting blood of control and protease-treated (2 and 4 mg/kg dose) mice after 6 h i.p. injection. The values are mean ± S.D. of triplicate determinations. Significance of difference with respect to control, *p<0.01; ^†p<0.001.</p

Multiple sequence alignment of N-terminal sequence of RVV protease isoenzymes with other known serine proteases isolated from snake venom.

<p>NR: not reported.</p

Effect of inhibitors on fibrinogenolytic, BAEE-esterase and plasma clotting activities of RV-FVP_α.

<p>Values are mean ± SD of triplicate determinations. Significance of difference with respect to control *p<0.01.</p

Kinetics of inhibition of FXa (0.15 µM) and thrombin (0.03 NIH U/ml) by Nk-PLA₂α and Nk-PLA₂β at 37°C, pH 7.4, respectively.

<p>The kinetic parameters (<i>Km</i>, <i>Vmax</i>, and <i>Kcat</i>) values were determined from Michaelis-Menten plot as described in the text. The chromogenic substrates (0.2 mM) used for the amidolytic activity assay of thrombin and FXa were T1637 and F3301, respectively. The values are mean of triplicate determinations and SD was found within 10% of the mean value.</p

Effect of pBPB and monovalent antivenom on catalytic, anticoagulant and thrombin inhibitory activity of Nk-PLA₂α and Nk-PLA₂β.

<p>The activity of enzymes in absence of inhibitor/antivenom was considered as 100% activity and the other values were compared with that. The values are mean ± S. D. of triplicate determinations. ND: not determined; pBPB: p-bromophenacyl bromide. Significance of difference with respect to Nk-PLA₂α;</p>a<p>p<0.01,</p>b<p>P<0.05.</p

A summary of the purification of PLA₂ isoenzymes from the venom of <i>N. kaouthia</i>.

<p>The PLA₂ activity was assayed by egg-yolk phospholipids hydrolysis method using 0.1 M Tris-HCl, pH 8.0 buffer. Data are from a typical experiment.</p>a<p>One unit of PLA₂ activity is defined as decrease of 0.01 absorbance at 740 nm per 10 min at 25°C.</p

Purification and determination of molecular masses of PLA₂ isoenzymes isolated from <i>N. kaouthia</i> venom.

<p>(<b>a</b>) Fractionation of crude <i>N. kaouthia</i> venom (25 mg dry weight) done on a HiPrep CM FF16/10 FPLC cation-exchange column. The fractionation conditions are described in the text. The peak showing major PLA₂ and anticoagulant activity is marked with an arrow. (<b>b</b>) Chromatogram resulting from anion-exchange fractionation of cation-exchange unbound peak [Nk(H)CEXP1] by using a Hiprep DEAE FF16/10 FPLC column. (<b>c</b>) and (<b>d</b>) MALDI-TOF mass spectrum of Nk-PLA₂α [peak Nk(H)AEXP4], and Nk-PLA₂β [peak Nk(H)AEXP5], respectively.</p