Ixolaris and Factor-X interaction: structural and dynamic investigation by NMR

Coagulation activated by tissue factor (TF) is involved in the maintenance of physiological hemostasis and also in cardiovascular disease and thrombosis. After tissue injury, blood clotting begins with TF exposure, which interacts with Factor VIIa to form the FVIIa/TF complex. This complex will activate Factor X (FX) leading to Factor Xa, culminating in prothrombin's activation into thrombin and subsequent formation of fibrin. Initiation of the blood coagulation cascade by FVIIa/TF is controlled by of tissue factor pathway inhibitor (TFPI), a Kunitz-type inhibitor. Several exogenous coagulation inhibitors from the salivary gland of blood-sucking invertebrates have been characterized, like Ixolaris, a protein with sequence homology to TFPI found in Ixodes scapularis saliva that consists of 2 Kunitz domains (K1 and K2) where K2 is strikingly dynamic and encompasses several residues involved in FX binding (Francischetti et al., 2002). Our research group determined the three-dimensional structure and dynamics of Ixolaris and described the structural basis for recognition of FX, and this was the first report revealing the structure-function relationship of an anticoagulant that targets a zymogen serving as a scaffold for TF inhibition (De Paula et al., 2019). In the current work, we expressed the recombinant 2H, 13C, and 15N isotopic labeled Ixolaris to investigate the formation of the structural details of FX and Ixolaris complex at atomic resolution by Magnetic Nuclear Resonance (NMR). Through NMR TROSY experiments, we determined the relaxation parameters (R1, R2 and HetNOE) and the temperature coefficients for Ixolaris. We then titrated Ixolaris with FX in a 1:1 ratio and mapped the chemical shift perturbation (CSP) caused by the FX/Ixolaris complex formation. Additionally, we measured the dynamics parameters and temperature coefficients of FX/Ixolaris complex at concentrations of 50 and 70 µM. The perdeuteration of Ixolaris improved the characterization of the complex. We will further study the interaction with FVIIa/TF.