Structure-function relationships and mechanism of anticoagulant phospholipase A2 enzymes from snake venoms

Phospholipase A(2) (PLA(2)) enzymes from snake venom are toxic and induce a wide spectrum of pharmacological effects, despite similarity in primary, secondary and tertiary structures and common catalytic properties. Thus, the structure-function relationships and the mechanism of this group of small proteins are subtle, complex and intriguing challenges. This review, taking the PLA(2) enzymes from spitting cobra (Naja nigricollis) venom as examples, describes the mechanism of anticoagulant effects. The strongly anticoagulant CM-IV inhibits both the extrinsic tenase and prothrombinase complexes, whereas the weakly anticoagulant PLA(2) enzymes (CM-I and CM-II) inhibit only the extrinsic tenase complex. CM-IV binds to factor Xa and interferes in its interaction with factor Va and the formation of prothrombinase complex. In contrast, CM-I and CM-II do not affect the formation of prothrombinase complex. In addition, CM-IV inhibits the extrinsic tenase complex by a combination of enzymatic and nonenzymatic mechanisms, while CM-I and CM-II inhibit by only enzymatic mechanism. These functional differences explain the disparity in the anticoagulant potency of N. nigricollis PLA(2) enzymes. Similarly, human secretory enzyme binds to factor Xa and inhibits the prothrombinase complex. We predicted the anticoagulant region of PLA(2) enzymes using a systematic and direct comparison of amino acid sequences. This region between 54 and 77 residues is basic in the strongly anticoagulant PLA(2) enzymes and neutral or negatively charged in weakly and non-anticoagulant enzymes. The prediction is validated independently by us and others using both site directed mutagenesis and synthetic peptides. Thus, strongly anticoagulant CM-IV binds to factor Xa (its target protein) through the specific anticoagulant site on its surface. In contrast, weakly anticoagulant enzymes, which lack the anticoagulant region fail to bind specifically to the target protein, factor Xa in the coagulation cascade. Thus, these studies strongly support the target model which suggests that protein-protein interaction rather than protein-phospholipid interaction determines the pharmacological specificity of PLA(2) enzymes.