Figure 5.

Heparin is a cofactor for Desmolaris. (A) Inhibition of catalytic activity of FXa. Reactions started with addition of S2222 (250 μM) to a mixture containing FXa (0.5 nM), Desmolaris (25 nM), and heparin (0, 0.1, 0.3, and 1 μg/mL) preincubated for 1 hour. Substrate hydrolysis was followed for 2 hours at 405 nm. (B) The experiment was performed as in (A) but at the indicated concentrations of Desmolaris and heparin. The ratio of Vs/Vo was plotted against Desmolaris concentration and data fitted with the Morrison equation. (C) Determination of the K_i in the presence of heparin. The IC₅₀ value for inhibition of FXa (0.5 nM) by Desmolaris (0-100 nM), in the presence of 1 μg/mL heparin was determined for each S2222 concentration (150, 300, 450, 600, and 750 μM) (not shown). Reactions were started with FXa. The plot of IC₅₀ and S2222 was fitted by linear regression and the K_i was determined by the y intercept. Six experiments were performed, and each data point is the average of duplicate determinations. (D) Inhibition of catalytic activity of FXIa. Reactions started with addition of S2366 (250 μM) to a mixture containing FXIa (0.5 nM), Desmolaris (12.5 nM), and heparin (0, 0.1, 0.3 and 1 μg/mL) preincubated for 1 hour. Substrate hydrolysis was followed for 2 hours at 405 nm. (E) The experiment was performed as in panel D but at the indicated concentrations of Desmolaris and heparin. The ratio of Vs/Vo was plotted against Desmolaris concentration and data fitted with the Morrison equation. (F) Determination of the K_i in the presence of heparin. The IC₅₀ for inhibition of FXIa (0.5 nM) by Desmolaris (0-100 nM), in the presence of 1 μg/mL heparin was determined for each S2366 concentration (150, 300, 450, 600, and 750 μM) (not shown). Reactions were started with FXIa. The plot of IC₅₀ and S2366 was fitted by linear regression and the K_i was determined by the y intercept. Six experiments were performed, and each data point is the average of duplicate determinations. (G) Binding of heparin and dextran sulfate. Desmolaris was immobilized in a CM5 sensor chip followed by injection of DS500K (200 nM), DS50K (200 nM), heparin Grade I (40 μM), or heparan sulfate (8 μM). All analytes were diluted in HEPES-buffered saline Tween (0.05% v/v) (HBS-P). (H) Heparin binding to Desmolaris. Heparin concentrations indicated in the inset: (a) 1.5 μM, (b) 0.75 μM, (c) 0.375 μM, (d) 0.187 μM, (e) 0.093 μM, and (f) 0.0.046 μM were injected over immobilized Desmolaris. The values obtained at equilibrium binding (steady state) were used to determine the KD. A representative experiment is shown. (I) Heparan sulfate binding to Desmolaris. Heparan sulfate concentrations indicated in the inset: (a) 6.25 μM, (b) 3.13 μM, (c) 1.5 μM, (d) 0.75 μM, (e) 0.3 μM, and (f) 0.15 μM were injected over immobilized Desmolaris. The values obtained at equilibrium binding (steady state) were used to determine the KD. A representative experiment is shown. (J) Desmolaris interacts with a heparin-agarose column. Desmolaris (200 μL; 20 μM) was added to the heparin column and equilibrated in TBS, pH 7.4. After washing the column, a gradient (NaCl, 0-2 M) was applied for 40 minutes. The peak corresponding to Desmolaris eluted at ∼1 M NaCl. mAU, milliabsorbance units. Representative experiments are shown.