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

Sharma M, Iyer JK, Shih N, Majumder M, Mattaparthi VS, Mukhopadhyay R, Doley R - PLoS ONE (2016)

Bottom Line: It exhibits strong anticoagulant and phospholipase A2 activity but is devoid of any cytotoxic effect on the tested normal or cancerous cell lines.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.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.

View Article: PubMed Central - PubMed

Affiliation: Department of Molecular Biology and Biotechnology, Tezpur University, Tezpur-784028, Assam, India.

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.

No MeSH data available.


Related in: MedlinePlus

Anticoagulant activities of daboxin P on platelet poor human plasma (PPP).(A): Recalcification time, different concentrations of daboxin P (0.001, 0.01, 0.1) were pre-incubated with 150 μl of plasma at 37°C for 2 min. 100 μl of 50 mM CaCl2 was added to initiate clot formation. (B): Activated partial thromboplastin time, daboxin P (0.01, 0.1 & 1 μM) was pre-incubated with 50 μl of plasma and 50 μl APTT reagent (Liquecelin) for 3 min at 37°C. 50 μl of 25 mM CaCl2 was added to form clot. (C): Prothrombin time, different concentrations of daboxin P (0.01, 0.1 & 1 μM) were pre-incubated with 50 μl of plasma at 37°C for 2 min. 50 μl of PT reagent (Uniplastin) was added to initiate the clot formation. (D): Stypven time, daboxin P (0.01, 0.1 & 1 μM) was pre-incubated with 75 μl plasma for 3 min at 37°C. 75 μl RVV-X (10 ng/ml) was added and incubated for 2 min. 25 mM of CaCl2 was added to initiate clot formation. For all the experiments, the clot formation was monitored using Tulip Coastat-1 coagulo analyser and the time taken for clot formation in the presence of Tris-Cl buffer (20 mM, pH 7.4) was considered as normal clotting time (NCT). The results are mean ± SD of three independent experiments.
© Copyright Policy
Related In: Results  -  Collection

License
getmorefigures.php?uid=PMC4835082&req=5

pone.0153770.g007: Anticoagulant activities of daboxin P on platelet poor human plasma (PPP).(A): Recalcification time, different concentrations of daboxin P (0.001, 0.01, 0.1) were pre-incubated with 150 μl of plasma at 37°C for 2 min. 100 μl of 50 mM CaCl2 was added to initiate clot formation. (B): Activated partial thromboplastin time, daboxin P (0.01, 0.1 & 1 μM) was pre-incubated with 50 μl of plasma and 50 μl APTT reagent (Liquecelin) for 3 min at 37°C. 50 μl of 25 mM CaCl2 was added to form clot. (C): Prothrombin time, different concentrations of daboxin P (0.01, 0.1 & 1 μM) were pre-incubated with 50 μl of plasma at 37°C for 2 min. 50 μl of PT reagent (Uniplastin) was added to initiate the clot formation. (D): Stypven time, daboxin P (0.01, 0.1 & 1 μM) was pre-incubated with 75 μl plasma for 3 min at 37°C. 75 μl RVV-X (10 ng/ml) was added and incubated for 2 min. 25 mM of CaCl2 was added to initiate clot formation. For all the experiments, the clot formation was monitored using Tulip Coastat-1 coagulo analyser and the time taken for clot formation in the presence of Tris-Cl buffer (20 mM, pH 7.4) was considered as normal clotting time (NCT). The results are mean ± SD of three independent experiments.

Mentions: Daboxin P exhibited anticoagulant effect on the various coagulation assays in a dose dependent manner (Fig 7). At a concentration of 0.01 μM, it prolonged the recalcification time beyond 600 s indicating it to be a strong anticoagulant enzyme (Fig 7A). 1 μM of the protein delayed activated partial thromboplastin time up to 130 s but did not exhibit prominent effect on prothrombin time (Fig 7B & 7C). Daboxin P exhibited anticoagulant effect on stypven time in a dose dependent manner displaying its inhibitory effect on FX (Fig 7D). Nevertheless, it did not show any inhibitory effect on thrombin time and was devoid of fibrinogenolytic activity (Figure B i & ii in S1 File). This suggests that daboxin P might target a component/coagulation factor upstream of the common pathway.


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

Sharma M, Iyer JK, Shih N, Majumder M, Mattaparthi VS, Mukhopadhyay R, Doley R - PLoS ONE (2016)

Anticoagulant activities of daboxin P on platelet poor human plasma (PPP).(A): Recalcification time, different concentrations of daboxin P (0.001, 0.01, 0.1) were pre-incubated with 150 μl of plasma at 37°C for 2 min. 100 μl of 50 mM CaCl2 was added to initiate clot formation. (B): Activated partial thromboplastin time, daboxin P (0.01, 0.1 & 1 μM) was pre-incubated with 50 μl of plasma and 50 μl APTT reagent (Liquecelin) for 3 min at 37°C. 50 μl of 25 mM CaCl2 was added to form clot. (C): Prothrombin time, different concentrations of daboxin P (0.01, 0.1 & 1 μM) were pre-incubated with 50 μl of plasma at 37°C for 2 min. 50 μl of PT reagent (Uniplastin) was added to initiate the clot formation. (D): Stypven time, daboxin P (0.01, 0.1 & 1 μM) was pre-incubated with 75 μl plasma for 3 min at 37°C. 75 μl RVV-X (10 ng/ml) was added and incubated for 2 min. 25 mM of CaCl2 was added to initiate clot formation. For all the experiments, the clot formation was monitored using Tulip Coastat-1 coagulo analyser and the time taken for clot formation in the presence of Tris-Cl buffer (20 mM, pH 7.4) was considered as normal clotting time (NCT). The results are mean ± SD of three independent experiments.
© Copyright Policy
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4835082&req=5

pone.0153770.g007: Anticoagulant activities of daboxin P on platelet poor human plasma (PPP).(A): Recalcification time, different concentrations of daboxin P (0.001, 0.01, 0.1) were pre-incubated with 150 μl of plasma at 37°C for 2 min. 100 μl of 50 mM CaCl2 was added to initiate clot formation. (B): Activated partial thromboplastin time, daboxin P (0.01, 0.1 & 1 μM) was pre-incubated with 50 μl of plasma and 50 μl APTT reagent (Liquecelin) for 3 min at 37°C. 50 μl of 25 mM CaCl2 was added to form clot. (C): Prothrombin time, different concentrations of daboxin P (0.01, 0.1 & 1 μM) were pre-incubated with 50 μl of plasma at 37°C for 2 min. 50 μl of PT reagent (Uniplastin) was added to initiate the clot formation. (D): Stypven time, daboxin P (0.01, 0.1 & 1 μM) was pre-incubated with 75 μl plasma for 3 min at 37°C. 75 μl RVV-X (10 ng/ml) was added and incubated for 2 min. 25 mM of CaCl2 was added to initiate clot formation. For all the experiments, the clot formation was monitored using Tulip Coastat-1 coagulo analyser and the time taken for clot formation in the presence of Tris-Cl buffer (20 mM, pH 7.4) was considered as normal clotting time (NCT). The results are mean ± SD of three independent experiments.
Mentions: Daboxin P exhibited anticoagulant effect on the various coagulation assays in a dose dependent manner (Fig 7). At a concentration of 0.01 μM, it prolonged the recalcification time beyond 600 s indicating it to be a strong anticoagulant enzyme (Fig 7A). 1 μM of the protein delayed activated partial thromboplastin time up to 130 s but did not exhibit prominent effect on prothrombin time (Fig 7B & 7C). Daboxin P exhibited anticoagulant effect on stypven time in a dose dependent manner displaying its inhibitory effect on FX (Fig 7D). Nevertheless, it did not show any inhibitory effect on thrombin time and was devoid of fibrinogenolytic activity (Figure B i & ii in S1 File). This suggests that daboxin P might target a component/coagulation factor upstream of the common pathway.

Bottom Line: It exhibits strong anticoagulant and phospholipase A2 activity but is devoid of any cytotoxic effect on the tested normal or cancerous cell lines.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.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.

View Article: PubMed Central - PubMed

Affiliation: Department of Molecular Biology and Biotechnology, Tezpur University, Tezpur-784028, Assam, India.

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.

No MeSH data available.


Related in: MedlinePlus