Kinetic characterization of inhibition of human thrombin with DNA aptamers by turbidimetric assay.

A sensitive turbidimetric method for detecting fibrin association was used to study the kinetics of fibrinogen hydrolysis with thrombin. The data were complemented by high-performance liquid chromatography (HPLC) measurements of the peptide products, fibrinopeptides released during hydrolysis. Atomic force microscopy (AFM) data showed that the fibril diameter is the main geometric parameter influencing the turbidity. The turbidimetric assay was validated using thrombin with the standard activity. To study thrombin inhibitors, a kinetic model that allows estimating the inhibition constants and the type of inhibition was proposed. The kinetic model was used to study the inhibitory activity of the two DNA aptamers 15-TBA (thrombin-binding aptamer) and 31-TBA, which bind to thrombin exosites. For the first time, 31-TBA was shown to possess the competitive inhibition type, whereas the shortened aptamer 15-TBA has the noncompetitive inhibition type.

Investigation of early stages of fibrin association.

Interactions between fibrinogen molecules proteolytically cleaved with thrombin were investigated using atomic force microscopy (AFM) and dynamic light scattering (DLS). Gradually decreased fibrinogen concentrations were used to study the fibrin network, large separated fibrils, small fibrils in the initial association stages, and protofibrils. In addition, a new type of structure was found in AFM experiments at a low fibrinogen concentration (20 nM): the molecules in these single-stranded associates are arranged in a row, one after the other. The height, diameter, and distance between domains in these single-stranded associates were the same as those in the original fibrinogen molecules. DLS data assumed formation of extended associates in bulk solution at fibrinogen concentration as low as 20 nM.