Anticoagulant mechanism and platelet deaggregation property of a non-cytotoxic, acidic phospholipase A2 purified from Indian cobra (Naja naja) venom: inhibition of anticoagulant activity by low molecular weight heparin

Biochimie. 2015 Mar:110:93-106. doi: 10.1016/j.biochi.2014.12.020. Epub 2015 Jan 8.

Abstract

In the present study, anticoagulant and platelet modulating activities of an acidic phospholipase A2 (NnPLA2-I) purified from Indian cobra Naja naja venom was investigated. The NnPLA2-I displayed a mass of 15.2 kDa and 14,186.0 Da when analyzed by SDS-PAGE and MALDI-TOF-MS, respectively. Peptide mass fingerprinting analysis of the NnPLA2-I showed its significant similarity with phospholipase A2 enzymes purified from cobra venom. BLAST analysis of one tryptic peptide sequence of NnPLA2-I demonstrated putative conserved domains of the PLA2-like superfamily. The Km and Vmax values of NnPLA2-I toward hydrolysis of its most preferred substrate-phosphotidylcholine (PC)-were determined to be 0.72 mM and 29.3 μmol min(-1) mg(-1), respectively. The anticoagulant activity of NnPLA2-I was found to be higher than the anticoagulant activity of heparin/AT-III or warfarin. The histidine modifying reagent, monovalent and polyvalent antivenom differentially inhibited the catalytic and anticoagulant activities of NnPLA2-I. Low molecular weight heparin did not inhibit the catalytic and platelet deaggregation activity of NnPLA2-I, albeit its anticoagulant activity was significantly reduced. The NnPLA2-I showed a non-enzymatic, mixed inhibition of thrombin with a Ki value of 9.3 nM. Heparin significantly decreased, with an IC50 value of 15.23 mIU, the thrombin inhibitory activity of NnPLA2-I. The NnPLA2-I uniquely increased the amidolytic activity of FXa without influencing its prothrombin activating property. NnPLA2-I showed dose-dependent deaggregation of platelet rich plasma (PRP) and inhibited the collagen and thrombin-induced aggregation of PRP. However, deaggregation of washed platelets by NnPLA2-I demonstrated in presence of PC or platelet poor plasma. Alkylation of histidine residue of NnPLA2-I resulted in 95% and 21% reduction of its platelet deaggregation and platelet binding properties, respectively. NnPLA2-I did not show cytotoxicity against human glioblastoma U87MG cells, bactericidal or hemolytic activity. The future therapeutic application of NnPLA2-I for treatment and prevention of cardiovascular disorders is therefore suggested.

Keywords: Anticoagulant activity; Heparin neutralization; Naja naja; Phospholipase A(2); Platelet deaggregation; Thrombin inhibition.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Animals
  • Anticoagulants / chemistry
  • Anticoagulants / isolation & purification
  • Anticoagulants / metabolism
  • Anticoagulants / pharmacology*
  • Bacillus subtilis / drug effects
  • Biocatalysis
  • Blood Platelets / drug effects
  • Blood Platelets / physiology
  • Cell Line, Tumor
  • Elapid Venoms / enzymology
  • Elapidae*
  • Factor Xa / metabolism
  • Hemolysis / drug effects
  • Heparin, Low-Molecular-Weight / pharmacology*
  • Humans
  • Phosphatidylcholines / metabolism
  • Phospholipases A2 / chemistry
  • Phospholipases A2 / isolation & purification
  • Phospholipases A2 / metabolism
  • Phospholipases A2 / pharmacology*
  • Platelet Aggregation / drug effects*
  • Structure-Activity Relationship
  • Thrombin / antagonists & inhibitors

Substances

  • Anticoagulants
  • Elapid Venoms
  • Heparin, Low-Molecular-Weight
  • Phosphatidylcholines
  • Phospholipases A2
  • Thrombin
  • Factor Xa