Desmolaris is a slow and tight inhibitor of FXIa. (A) Inhibition of the catalytic activity of FXIa. Reactions started by addition of S2366 (250 μM) to a mixture containing Desmolaris (0-200 nM) incubated for 1 hour with FXIa (0.5 nM). Substrate hydrolysis was followed for 2 hours at 405 nm. (B) Tight inhibition. Experiments were performed as in (A) but at 0.5, 1, and 2 nM FXIa. The rate of substrate hydrolysis between 30 and 60 minutes (Vmax mode) was transformed as Vs/Vo and plotted against Desmolaris concentration. Data points were fitted with the Morrison equation to calculate the IC₅₀ at each enzyme concentration. (C) Plot of IC₅₀ and FXIa produces a straight line typical of tight inhibitors. (D) Slow-type inhibition. Reactions started with addition of FXIa (0.5 nM) to a mixture containing Desmolaris and S2366 (250 μM). Substrate hydrolysis was followed for 2 hours at 405 nm. (E) IC₅₀ determination. FXIa was added to Desmolaris in the presence of S2366 (150, 300, 450, 600, 750, and 900 μM). The ratio Vs/Vo obtained (Vmax mode) between 30 and 60 minutes was plotted against S2366 concentrations. Data were fitted with the Morrison equation to calculate the IC₅₀ values at each substrate concentration. Six experiments were performed, and each data point is the average of duplicate determinations. Confidence interval is shown as dotted lines. (F) Determination of the K_i: plot of IC₅₀ vs S2366 concentrations was fitted by linear regression and the y intercept equals the K_i. (G) SPR experiments. Factor XIa at the indicated concentrations was injected over immobilized Desmolaris for 180 seconds. Dissociation of the Desmolaris-FXIa complex was monitored for 900 seconds. Representative sensorgrams are shown in black lines; global fitting using the Langmuir equation is depicted in red lines. (H) Equilibrium binding. FXIa was injected for 30 minutes into immobilized Desmolaris, allowing complex formation to reach equilibrium. Representative experiment is illustrated. (I) Steady-state affinity. The resonance values obtained at binding stability (30 minutes of association phase) in panel H were plotted against FXIa concentration, and KD 0.63 ± 0.04 nM was determined. (J) Solution binding of Desmolaris to FXIa by ITC. (Upper panel) Baseline-adjusted heats per injection of FXIa (10 μM) into Desmolaris (1.0 μM). (Lower panel) Molar enthalpies per injection for FXIa interaction with Desmolaris. Filled squares, measured enthalpies; solid line, fit of experimental data to a single site-binding model. Typical experiment is depicted. (K) Desmolaris inhibits FXIa-mediated FIX activation. Controls (lanes 1 and 2): Recombinant FIX (BeneFIX, 1.2 μM) in the presence of phosphate-buffered saline (PBS) (lane 1) or 300 nM Desmolaris (lane 2). Lanes 3 and 4: FXIa (3 nM) was added to FIX in the presence of PBS (lane 3) or Desmolaris (lane 4) and mixture was incubated for 37°C for 60 minutes. Reactions were stopped with reducing Laemmli buffer, and proteins were separated by 4% to 12% SDS-PAGE. The bands correspond to uncleaved FIX, the heavy chain of FIXa (FIXa HC), and the light chain of FIXa (FIXa LC). Typical gel is shown. (L) Cleavage of Desmolaris. Desmolaris (7.5 μM) was incubated with FXIa (1.7 μM) for 8 hours. The mixture was loaded in a NuPAGE gel under reducing conditions. The bands corresponding to FXIa HC, FXIa LC, or Desmolaris are indicated. Lane 1, Desmolaris; lane 2, FXIa plus Desmolaris; lane 3, FXIa. No cleavage is observed.