Two
DNA aptamers directed against two separate exosites on human
alpha-thrombin were evaluated for
thrombus-imaging potential. Aptamer ODN 1 is directed to the
thrombin substrate binding site (exosite 1). Our finding that ODN 1 competes with
fibrin for binding to exosite 1 on
thrombin suggests that ODN 1 will not be useful for
thrombus imaging. Aptamer ODN 2 is directed against the
thrombin heparin binding site (exosite 2). ODN 2 bound to model thrombi that were formed either by clotting purified
fibrinogen with
thrombin, or by recalcifying citrated plasma. As the
thrombin content of thrombi was increased the rate of ODN 2 uptake into preformed thrombi increased, whereas the rate of release of ODN 2 out of preformed thrombi decreased. This in vitro data suggested that ODN 2 might be useful for
thrombus imaging because it can bind to exosite 2 on
fibrin-bound
thrombin. However, in a rabbit jugular vein model using
thrombus supplemented with human
thrombin, ODN 2 uptake was equal to the
ovalbumin control, and did not reflect
thrombin content. While the in vitro results with ODN 2 were consistent with
thrombus imaging, the rapid clearance of ODN 2 from circulation, combined with slow mass transfer in the clot, seem to work against in vivo
thrombin-dependent imaging or washout analysis.