Desmolaris inhibits kallikrein, but not FXIIa. (A) Inhibition of the catalytic site of Kallikrein. Reactions started with addition of S2302 (250 μM) to a mixture containing Desmolaris incubated for 1 hour with Kallikrein (2 nM). Substrate hydrolysis was followed for 2 hours at 405 nm. (B) The ratio of Vs/Vo was plotted against Desmolaris concentration. (C) SPR experiments. Kallikrein at the indicated concentrations was injected over immobilized Desmolaris for 180 seconds. Dissociation was monitored for 900 seconds. Representative sensorgrams are shown in black lines; global fitting using the Langmuir equation is depicted in red lines. Representative experiment is shown. (D) Reciprocal activation. Factor XII (0.2 nM) was preincubated with Desmolaris (0, 62, 125, 250, 500, and 1000 nM) in 20 mM Tris, 0.15 M NaCl, 0.3% bovine serum albumin, pH 7.4, for 10 minutes at room temperature. Reactions were started by addition of PK (10 nM) and DS 500 (0.2 μg/mL, final concentrations). After 10 minutes, S2302 (250 μM) was used as substrate. Each data point is the mean ± standard error of a triplicate determination. (E) Desmolaris does not inhibit the amidolytic activity of FXIIa. FXIIa (2 nM) was incubated with Desmolaris (50 nM) for 15 minutes, followed by addition of S2302 (250 μM). Substrate hydrolysis was followed for 1 hour at 405 nm. No inhibition was observed. CTI (5 μM) was used as a control inhibitor. (F) Desmolaris does not inhibit FXI activation by FXIIa. FXIIa (25 nM) was incubated with Desmolaris (0, 50, and 250 nM) in Tris-buffered saline for 20 minutes followed by addition of FXI (100 µg/mL). After 8 hours at 37°C, reactions were stopped with reducing SDS sample buffer and samples loaded in 4% to 12% NuPAGE gel. FXIa HC and FXIa LC were visualized in Coomassie Blue-stained gels. CTI (5 μM) was used as control inhibitor. A representative gel is shown. (G) BK formation in plasma. Coagulation activation by the contact pathway was activated by kaolin (to generate BK) in the presence of PBS or 0.6 μM Desmolaris. The mixture was transferred to a chamber containing a guinea-pig ileum, which is responsive to BK. As a control, BK (5 and 20 ng/mL, final concentrations) was added directly to the ileum. (H) Dose-response curve for experiments depicted in panel G. Each data point is the mean ± standard error of a triplicate determination. Confidence interval is shown as dotted lines. IC50 value for inhibition of BK formation is ∼200 nM.
