X-Ray Structure of an Anticoagulant RNA Aptamer Bound to Factor Xa. Structural Basis for Its Ability to Disrupt Interactions Between Xa and Va within Prothrombinase

Abstract The prothrombinase complex assembles through high affinity interactions between factor Xa and factor Va on the surface of membranes exposing phosphatidylserine. Assembly of prothrombinase allows for the highly accelerated formation of thrombin localized at the injury site. Because the rate of thrombin formation by factor Xa alone is profoundly increased upon its incorporation into prothrombinase, disrupting the proteinase-cofactor interaction could represent an effective therapeutic strategy for the treatment of thrombosis. However, the extended interface between Xa and Va, likely involving shallow surfaces of the interacting proteins, limits the available strategies for the development of ligands that interfere with prothrombinase assembly with high affinity. The RNA aptamer, 11F7t, was selected in an unbiased SELEX screen using human factor Xa as a target and identified as a potent anticoagulant. Detailed functional characterization revealed that 11F7t binds both X and Xa with high affinity (~1 nM) and competes for the interaction between Xa and Va without occluding active site function of the proteinase. Competititive inhibition of the assembly of prothrombinase likely accounts for a major fraction of its anticoagulant effects. However, additional inhibitory effects of 11F7t on coagulation also probably arise from its ability to partially inhibit X activation by the intrinsic Xase and the inhibition of factor VIII activation by Xa. Here we report the crystal structure of 11F7t complexed with desGla-Xa containing the catalytic Ser195 replaced with Ala at 2.5 A resolution and solved by molecular replacement. In the Xa model, the first 30 residues expected in the light chain, 5 residues in the autolysis loop and 7 residues at the C terminus were not defined by electron density. In 11F7t, 25 of 38 nucleotides could be unambiguously modeled. The tertiary fold of 11F7t presents an extended molecular surface for interactions with desGla-Xa and buries 1384 A2 of solvent accessible surface area in the interacting species. The RNA aptamer exclusively binds the proteinase domain with contacts over a broad surface area that includes residues Leu59-Lys62, Val87-Arg93, Phe101, Lys236, Trp237 and Arg240 but not the catalytic site. Some of these residues have been implicated in heparin binding and have also been proposed to participate in Va binding based on modest changes in function upon mutagenesis. Even though 11F7t does not make any contacts in the 165 helix previously proposed to be a key region for the interaction with Va, aptamer binding is sufficient to disrupt the high affinity cofactor-proteinase interaction. The structure also reveals that most of the aptamer–protein contact involves nucleotides that constitute a base-paired stem (formed between G3-C6 and 31G-34C) and loop (C7-G11) of 11F7t with these interactions mostly specified by the nucleotide bases rather than the phosphate backbone which might be expected for a protein surface already implicated in binding a polyanion such as heparin or an acidic peptide from the C terminus of the A2 domain of Va. The regions of desGla-Xa occluded by the aptamer point to an extended surface that plays an important role in mediating interactions with factor Va within prothrombinase and suggests commonalities between this binding mode and the way that factor X might bind to factor VIIIa as a substrate within intrinsic Xase or the way that factor Xa might engage factor VIII as an enzyme. Disclosures No relevant conflicts of interest to declare.