The anticoagulant effect of Apis mellifera phospholipase A2 is inhibited by CORM-2 via a carbon monoxide-independent mechanism

  • Vance G. NielsenEmail author


Bee venom phospholipase A2 (PLA2) has potential for significant morbidity. Ruthenium (Ru)-based carbon monoxide releasing molecules (CORM) inhibit snake venoms that are anticoagulant and contain PLA2. In addition to modulating heme-bearing proteins with carbon monoxide, these CORM generate reactive Ru species that form adducts with histamine residues resulting in changes in protein function. This study sought to identify anticoagulant properties of bee venom PLA2 via catalysis of plasma phospholipids required for thrombin generation. Another goal was to determine if Ru-based CORM inhibit bee venom PLA2 via carbon monoxide release or via potential binding of reactive Ru species to a key histidine residue in the catalytic site of the enzyme. Anticoagulant activity of bee venom PLA2 was assessed via thrombelastography with normal plasma. Bee venom PLA2 was then exposed to different CORM and a metheme forming agent and anticoagulant activity was reassessed. Using Ru, boron and manganese-based CORM and a metheme forming agent, it was demonstrated that it was unlikely that carbon monoxide interaction with a heme group attached to PLA2 was responsible for inhibition of anticoagulant activity by Ru-based CORM. Exposure of PLA2 to a Ru-based CORM in the presence of histidine-rich human albumin resulted in loss of inhibition of PLA2. Ru-based CORM likely inhibit bee venom PLA2 anticoagulant activity via formation of reactive Ru species that bind to histidine residues of the enzyme.


Anticoagulant Phospholipase A2 Thrombelastography Carbon monoxide releasing molecule Heme Reactive ruthenium species 



This investigation was supported by the Department of Anesthesiology, College of Medicine, at the University of Arizona.

Compliance with ethical standards

Conflict of interest

The author declare that he has no conflict of interest.

Ethical approval

This was an in vitro investigation and did not involve any living subjects.


  1. 1.
    Poinar GO Jr, Danforth BN (2006) A fossil bee from early cretaceous burmese amber. Science 314:614CrossRefGoogle Scholar
  2. 2.
    Schmidt JO (1995) Toxinology of venoms from the honeybee genus Apis. Toxicon 33:917–927CrossRefGoogle Scholar
  3. 3.
    Ownby CL, Powell JR, Jiang MS, Fletcher JE (1997) Melittin and phospholipase A2 from bee (Apis mellifera) venom cause necrosis of murine skeletal muscle in vivo. Toxicon 35:67–80CrossRefGoogle Scholar
  4. 4.
    Petroianu G, Liu J, Helfrich U, Maleck W, Rüfer R (2000) Phospholipase A2-induced coagulation abnormalities after bee sting. Am J Emerg Med 18:22–27CrossRefGoogle Scholar
  5. 5.
    Zolfagharian H, Mohajeri M, Babaie M (2015) Honey bee venom (Apis mellifera) contains anticoagulation factors and increases the blood-clotting time. J Pharmacopuncture 18:7–11CrossRefGoogle Scholar
  6. 6.
    Nielsen VG (2019) Carbon monoxide inhibits the anticoagulant activity of phospholipase A2 purified from Crotalus adamanteus venom. J Thromb Thrombolysis 47:73–79CrossRefGoogle Scholar
  7. 7.
    Nielsen VG (2019) Carbon monoxide inhibits the anticoagulant activity of Mojave rattlesnake venoms type A and B. J Thromb Thrombolysis 48:256–262CrossRefGoogle Scholar
  8. 8.
    Plückthun A, Dennis EA (1985) Activation, aggregation, and product inhibition of cobra venom phospholipase A2 and comparison with other phospholipases. J Biol Chem 260:11099–11106PubMedGoogle Scholar
  9. 9.
    Zambelli VO, Picolo G, Fernandes CAH, Fontes MRM, Cury Y (2017) Secreted phospholipases A2 from animal venoms in pain and analgesia. Toxins (Basel) 9:E406CrossRefGoogle Scholar
  10. 10.
    Nielsen VG, Frank N (2019) The kallikrein-like activity of Heloderma venom is inhibited by carbon monoxide. J Thromb Thrombolysis 47:533–539CrossRefGoogle Scholar
  11. 11.
    Annand RR, Kontoyianni M, Penzotti JE, Dudler T, Lybrand TP, Gelb MH (1996) Active site of bee venom phospholipase A2: the role of histidine-34, aspartate-64 and tyrosine-87. Biochemistry 35:4591–4601CrossRefGoogle Scholar
  12. 12.
    Nielsen VG, Frank N (2018) Differential heme-mediated modulation of Deinagkistrodon, Dispholidus, Protobothrops and Pseudonaja hemotoxic venom activity in human plasma. Biometals 31:951–959CrossRefGoogle Scholar
  13. 13.
    Nielsen VG, Frank N, Afshar S (2019) De novo assessment and review of pan-american pit viper anticoagulant and procoagulant venom activities via kinetomic analyses. Toxins (Basel) 11:E94CrossRefGoogle Scholar
  14. 14.
    Nielsen VG, Sánchez EE, Redford DT (2018) Characterization of the rabbit as an in vitro and in vivo model to assess the effects of fibrinogenolytic activity of snake venom on coagulation. Basic Clin Pharmacol Toxicol 122:157–164CrossRefGoogle Scholar
  15. 15.
    Suntravat M, Langlais PR, Sánchez EE, Nielsen VG (2018) CatroxMP-II: a heme-modulated fibrinogenolytic metalloproteinase isolated from Crotalus atrox venom. Biometals 31:585–593CrossRefGoogle Scholar
  16. 16.
    Gessner G, Sahoo N, Swain SM, Hirth G, Schönherr R, Mede R, Westerhausen M, Brewitz HH, Heimer P, Imhof D, Hoshi T, Heinemann SH (2017) CO-independent modification of K+ channels by tricarbonyldichlororuthenium(II) dimer (CORM-2). Eur J Pharmacol 815:33–41CrossRefGoogle Scholar
  17. 17.
    Meloun B, Morávek L, Kostka V (1975) Complete amino acid sequence of human serum albumin. FEBS Lett 58:134–137CrossRefGoogle Scholar
  18. 18.
    Southam HM, Smith TW, Lyon RL, Liao C, Trevitt CR, Middlemiss LA, Cox FL, Chapman JA, El-Khamisy SF, Hippler M, Williamson MP, Henderson PJF, Poole RK (2018) A thiol-reactive Ru(II) ion, not CO release, underlies the potent antimicrobial and cytotoxic properties of CO-releasing molecule-3. Redox Biol 18:114–123CrossRefGoogle Scholar
  19. 19.
    Motterlini R, Clark JE, Foresti R, Sarathchandra P, Mann BE, Green CJ (2002) Carbon monoxide-releasing molecules: characterization of biochemical and vascular activities. Circ Res 90:E17–E24CrossRefGoogle Scholar
  20. 20.
    Clark JE, Naughton P, Shurey S, Green CJ, Johnson TR, Mann BE, Foresti R, Motterlini R (2003) Cardioprotective actions by a water-soluble carbon monoxide-releasing molecule. Circ Res 93:e2–e8CrossRefGoogle Scholar
  21. 21.
    Nielsen VG, Garza JI (2014) Comparison of the effects of CORM-2, CORM-3 and CORM-A1 on coagulation in human plasma. Blood Coagul Fibrinolysis 25:801–805CrossRefGoogle Scholar
  22. 22.
    Motterlini R, Sawle P, Hammad J, Bains S, Alberto R, Foresti R, Green CJ (2005) CORM-A1: a new pharmacologically active carbon monoxide-releasing molecule. FASEB J 19:284–286CrossRefGoogle Scholar
  23. 23.
    Klein M, Neugebauer U, Schmitt M, Popp J (2016) Elucidation of the CO-release kinetics of CORM-A1 by means of vibrational spectroscopy. ChemPhysChem 17:985–993CrossRefGoogle Scholar
  24. 24.
    Taler G, Eliav U, Navon G (1999) Detection and characterization of boric acid and borate ion binding to cytochrome c using multiple quantum filtered NMR. J Magn Reson 141:228–238CrossRefGoogle Scholar
  25. 25.
    Ali S, Farooqi H, Prasad R, Naime M, Routray I, Yadav S, Ahmad F (2010) Boron stabilizes peroxide mediated changes in the structure of heme proteins. Int J Biol Macromol 47:109–115CrossRefGoogle Scholar
  26. 26.
    Crook SH, Mann BE, Meijer AJ, Adams H, Sawle P, Scapens D, Motterlini R (2011) [Mn(CO)4{S2CNMe(CH2CO2H)}], a new water-soluble CO-releasing molecule. Dalton Trans 40:4230–4235CrossRefGoogle Scholar
  27. 27.
    Fayad-Kobeissi S, Ratovonantenaina J, Dabiré H, Wilson JL, Rodriguez AM, Berdeaux A, Dubois-Randé JL, Mann BE, Motterlini R, Foresti R (2016) Vascular and angiogenic activities of CORM-401, an oxidant-sensitive CO-releasing molecule. Biochem Pharmacol 102:64–77CrossRefGoogle Scholar
  28. 28.
    Nielsen VG, Arkebauer MR, Vosseller K (2011) Redox-based thrombelastographic method to detect carboxyhemefibrinogen-mediated hypercoagulability. Blood Coagul Fibrinolysis 22:657–661CrossRefGoogle Scholar
  29. 29.
    Sears DA (1970) Disposal of plasma heme in normal man and patients with intravascular hemolysis. J Clin Invest 49:5–14CrossRefGoogle Scholar
  30. 30.
    Monzani E, Bonafè B, Fallarini A, Redaelli C, Casella L, Minchiotti L, Galliano M (2011) Enzymatic properties of human hemalbumin. Biochim Biophys Acta 1547:302–312CrossRefGoogle Scholar

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© Springer Science+Business Media, LLC, part of Springer Nature 2019

Authors and Affiliations

  1. 1.Department of AnesthesiologyThe University of Arizona College of MedicineTucsonUSA

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