Figure 1: Nature Communications

Fig. 1

From: Engineered factor Xa variants retain procoagulant activity independent of direct factor Xa inhibitors

Fig. 1

The S4 subsite of factor Xa coordinates apixaban binding and inhibition. a The minimal interatomic (MI) distances from the side chains of FXa residues to apixaban were calculated every nanosecond during a 750 ns MD simulation of apixaban-bound human FXa. Box plots (with whiskers from minimum to maximum) display the distribution of the observed MI distances for each of the 15 FXa residues with the shortest side chain to apixaban MI distance (sorted by average). The surface representation (inset) depicts the FXa-bound apixaban (blue) configuration throughout the MD simulation. Color coding (corresponding to box plots): S4 subsite residues Tyr99 (orange), Phe174 (cyan), and Trp215 (yellow), catalytic residue Ser195 (red), and others (gray). b The rate of peptidyl substrate conversion by 5 nM of RVV-X-activated wild-type (gray circles, WT), Tyr99Ala (orange squares, Y99A), Phe174Ala (cyan squares, F174A), or double mutant (black squares, Y99A/F174A) FX was determined with saturating amounts of the cofactor FV-810 (30 nM) and anionic phospholipid vesicles (PCPS, 50 µM) in the absence (V 0) or presence (V i) of increasing apixaban concentrations (0.001–100 µM). The lines were drawn following nonlinear regression analysis of the data sets, and the fitted parameters for IC50 ± 1 standard deviation of the induced fit are shown in the inset. The data are the means of two independent experiments. c The specific extrinsic (PT Activity; filled bars) or intrinsic (APTT Activity; striped bars) clotting activity of wild-type (WT), Tyr99Ala (Y99A), Phe174Ala (F174A), or double mutant (Y99A F174A) FX from conditioned media was determined as described in ‘Methods’ by dividing the PT or APTT clotting activity over the FX antigen concentration. The data represent the average ± 1 standard deviation of three representative high-producing stable cell lines per FX variant