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

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.