Figure 3.

Desmolaris is a slow and tight inhibitor of FXa. (A) Inhibition of the catalytic activity of FXa. Reactions started with addition of S2222 (250 μM) to a mixture containing Desmolaris incubated for 1 hour with FXa (1 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, 2, and 4 nM FXa. 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 FXa produces a straight line typical of tight inhibitors. (D) Slow-type inhibition. Reactions started with addition of FXa (1 nM) to a mixture containing Desmolaris and S2222 (250 μM). Substrate hydrolysis was followed for 2 hours at 405 nm. (E) IC₅₀ determination. FXa was added to Desmolaris in the presence of S2222 (150, 300, 450, 600, 750, and 900 μM). The ratio Vs/Vo obtained (Vmax mode) between 30 and 60 minutes was plotted against S2222 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. (F) Determination of the K_i. Plot of IC₅₀ vs S2222 concentrations was fitted by linear regression and the y intercept equals the K_i. Confidence interval is shown as dotted lines. (G) SPR experiments. Factor Xa at the indicated concentrations was injected over immobilized Desmolaris for 180 seconds. Dissociation of the Desmolaris-FXa complex was monitored for 900 seconds. Representative sensorgrams are shown in black lines; global fitting of the data points using the Langmuir equation is depicted in red lines. (H) Solution binding of Desmolaris to FXa by ITC. (Upper panel) Baseline-adjusted heats per injection of FXa (10 μM) into Desmolaris (1.0 μM). (Lower panel) Molar enthalpies per injection for FXa interaction with Desmolaris. Filled squares, measured enthalpies; solid line, fit of experimental data to a single site-binding model. Thermodynamic parameters: ΔH in kcal/mol, TΔS in kcal/mol, and KD are indicated in the inset. (I) Effects of Desmolaris in the prothrombinase complex. FXa was incubated with Desmolaris (0-30 nM) followed by addition of FVa, phosphatidylcholine/phosphatidylserine, and prothrombin in the presence of Ca²⁺. Aliquots were taken to estimate thrombin generation. Data points are the average for triplicate determination. (J) Cleavage of Desmolaris. Desmolaris (7.5 μM) was incubated with FXa (1.7 μM) for 8 hours. The mixture was loaded in a NuPAGE gel. The bands corresponding to FXa or Desmolaris are indicated. Lane 1, Desmolaris; lane 2, FXa; lane 3, FXa plus Desmolaris. No cleavage is observed.