Characterization of a human coagulation factor Xa-binding site on Viperidae snake venom phospholipases A(2 )by affinity binding studies and molecular bioinformatics

BACKGROUND: The snake venom group IIA secreted phospholipases A(2 )(SVPLA(2)), present in the Viperidae snake family exhibit a wide range of toxic and pharmacological effects. They exert their different functions by catalyzing the hydrolysis of phospholipids (PL) at the membrane/water interface and by highly specific direct binding to: (i) presynaptic membrane-bound or intracellular receptors; (ii) natural PLA(2)-inhibitors from snake serum; and (iii) coagulation factors present in human blood. RESULTS: Using surface plasmon resonance (SPR) protein-protein interaction measurements and an in vitro biological test of inhibition of prothrombinase activity, we identify a number of Viperidae venom SVPLA(2)s that inhibit blood coagulation through direct binding to human blood coagulation factor Xa (FXa) via a non-catalytic, PL-independent mechanism. We classify the SVPLA(2)s in four groups, depending on the strength of their binding. Molecular electrostatic potentials calculated at the surface of 3D homology-modeling models show a correlation with inhibition of prothrombinase activity. In addition, molecular docking simulations between SVPLA(2 )and FXa guided by the experimental data identify the potential FXa binding site on the SVPLA(2)s. This site is composed of the following regions: helices A and B, the Ca(2+ )loop, the helix C-β-wing loop, and the C-terminal fragment. Some of the SVPLA(2 )binding site residues belong also to the interfacial binding site (IBS). The interface in FXa involves both, the light and heavy chains. CONCLUSION: We have experimentally identified several strong FXa-binding SVPLA(2)s that disrupt the function of the coagulation cascade by interacting with FXa by the non-catalytic PL-independent mechanism. By theoretical methods we mapped the interaction sites on both, the SVPLA(2)s and FXa. Our findings may lead to the design of novel, non-competitive FXa inhibitors.