Characterization of L-amino Acid Oxidase Derived from Crotalus adamanteus Venom: Procoagulant and Anticoagulant Activities.

Vance G Nielsen

doi:10.3390/ijms20194853

Abstract

Snake venom enzymes of the L-amino acid oxidase (LAAO) class are responsible for tissue hemorrhage, edema, and derangement of platelet function. However, what role, if any, these flavoenzymes play in altering plasmatic coagulation have not been well defined. Using coagulation kinetomic analyses (thrombelastograph-based), it was determined that the LAAO derived from Crotalus adamanteus venom displayed a procoagulant activity associated with weak clot strength (no factor XIII activation) similar to thrombin-like enzymes. The procoagulant activity was not modified in the presence of reduced glutathione, demonstrating that the procoagulant activity was likely due to deamination, and not hydrogen peroxide generation by the LAAO. Further, unlike the raw venom of the same species, the purified LAAO was not inhibited by carbon monoxide releasing molecule-2 (CORM-2). Lastly, exposure of the enzyme to phenylmethylsulfonyl fluoride (PMSF) resulted in the LAAO expressing anticoagulant activity, preventing contact activation generated thrombin from forming a clot. In sum, this investigation for the first time characterized the LAAO of a snake venom as both a fibrinogen polymerizing and an anticoagulant enzyme acting via oxidative deamination and not proteolysis as is the case with thrombin-like enzymes (e.g., serine proteases). Using this thrombelastographic approach, future investigation of purified enzymes can define their biochemical nature.

Highlights

Purified, isolated enzymes derived from snake venoms have been extensively investigated to mechanistically explain toxic hypercoagulation or anticoagulation clinically [1]
The effects of the L-amino acid oxidases (LAAO) derived from Agkistrodon halys blomhoffii venom on human coagulation were originally described with concentrations ranging from 3.7 to 379 nM [7], so a similar range of concentrations were used to assess the LAAO activity derived from Crotalus adamanteus venom
The experiments involving carbon monoxide releasing molecule-2 (CORM-2) demonstrated functionally, that the procoagulant effects of LAAO were not affected as was the phospholipase A2 and raw venom activity of Crotalus adamanteus [6,7,8]

Summary

Introduction

Purified, isolated enzymes derived from snake venoms have been extensively investigated to mechanistically explain toxic hypercoagulation or anticoagulation clinically [1] While enzyme classes such as metalloproteinases, serine proteases and phospholipases have had clear mechanisms defined in disturbances in human coagulation [1], other important enzyme classes remain relatively far less studied. The LAAO are dimeric, glycosylated flavoenzymes that catalyze a variety of L-amino acids in the presence of oxygen to yield α-keto acids and hydrogen peroxide (H2O2) [2]. While they are not metalloproteinases, the LAAO activities are modulated by various metal ions and metal chelators. Snake venom LAAO are medically important enzymes that have unusual characteristics that are distinctly different from metalloproteinases and serine proteases

Methods

Results

Conclusion