Figure 5.

Molecular interaction of PT with VC1 and sRAGE. A, MST measurements were carried out in 20 mm HEPES, pH 7.1, 100 mm NaCl, 0.05% Tween 20 and standard capillaries with a constant concentration of fluorescently labeled prothrombin (100 nm) mixed with varying amounts of unlabeled VC1-His-Strep (final concentrations from 10.2 nm to 333 μm) or sRAGE (from 8.1 nm to 264 μm). After 30 min incubation at 24 °C, the samples were loaded into Monolith NT.115 capillaries (NanoTemper Technologies) and measurements were performed using the Monolith NT.115 (NanoTemper Technologies) at 20% LED power and medium MST power. The values of the dissociation constant (K_d) derived from the curves for the PT-VC1 interaction (n = 5 independent measurements) and for the PT-sRAGE interaction (n = 4 independent measurements) are indicated in the inset. B, MST measurements with VC1 were performed as in A, but in the presence of increasing concentrations of CaCl₂ or in the presence of fluorescently labeled PT des-Gla instead of PT (n = 3 independent measurements). N/A, not applicable. In A and B, the fraction bound is plotted against the log₁₀ of VC1 or sRAGE molar concentrations. The error bars on individual data points represent the standard deviations among the repeats.