Abstract
BackgroundThe snake venom group IIA secreted phospholipases A2 (SVPLA2), 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 PLA2-inhibitors from snake serum; and (iii) coagulation factors present in human blood.ResultsUsing 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 SVPLA2s that inhibit blood coagulation through direct binding to human blood coagulation factor Xa (FXa) via a non-catalytic, PL-independent mechanism. We classify the SVPLA2s 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 SVPLA2 and FXa guided by the experimental data identify the potential FXa binding site on the SVPLA2s. This site is composed of the following regions: helices A and B, the Ca2+ loop, the helix C-β-wing loop, and the C-terminal fragment. Some of the SVPLA2 binding site residues belong also to the interfacial binding site (IBS). The interface in FXa involves both, the light and heavy chains.ConclusionWe have experimentally identified several strong FXa-binding SVPLA2s 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 SVPLA2s and FXa. Our findings may lead to the design of novel, non-competitive FXa inhibitors.
Highlights
The snake venom group IIA secreted phospholipases A2 (SVPLA2), present in the Viperidae snake family exhibit a wide range of toxic and pharmacological effects
We have experimentally identified several strong factor Xa (FXa)-binding several toxic group IIA PLA2s (SVPLA2) that disrupt the function of the coagulation cascade by interacting with FXa by the non-catalytic PL-independent mechanism
Identification of SVPLA2s that bind to FXa and inhibit prothrombinase activity As shown in Table 1, the SVPLA2s from Viperidae snake CBc and MtxII interact with FXa with very high affinity
Summary
The snake venom group IIA secreted phospholipases A2 (SVPLA2), 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 PLA2-inhibitors from snake serum; and (iii) coagulation factors present in human blood. The heavy chain of FXa contains the catalytically active serine proteinase domain (254 amino acids, residues 16–269 in chymotrypsinogen numbering system). Residues His, Asp102, and Ser195 (chymotrypsinogen numbering) form a catalytic triad at the active site cleft between the two subdomains[4]. The autolysis loop 149–151 occupies the southern boundary of the active site cleft. Afterwards, thrombin converts fibrinogen into fibrin, consolidating the primary plug
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