Summary
The behavior of 125I-labeled recombinant hirudin towards the uninjured and de-endothelialized rabbit
aorta wall has been studied in vitro and in vivo to determine its usefulness as an
indicator of thrombin activity associated with the aorta wall. Thrombin adsorbed to
either sulfopropyl-Sephadex or heparin-Sepharose bound >95% of 125I-r-hirudin and the complex remained bound to the matrix. Binding of 125I-r-hirudin to the exposed aorta subendothelium (intima-media) in vitro was increased
substantially if the tissue was pre-treated with thrombin; the quantity of l25I-r-hirudin bound to the de-endothelialized intima-media (i.e. balloon-injured in
vitro) correlated positively with the quantity of bound 131I-thrombin (p <0.01). Aortas balloon-injured in vivo were measured for thrombin release
from, and binding of 125I-r-hirudin to, the de-endothelialized intimal surface in vitro; 125I-r-hirudin binding correlated with the amount of active thrombin released (p <0.001).
Uptake of 125I-r-hirudin by the aorta wall in vivo was proportional to the uptake of 131I-fibrinogen (as an indicator of thrombin activity) before and after balloon injury.
After 30 min in the circulation, specific 125I-r-hirudin binding to the uninjured and de-endo- thelialized (at 1.5 h after injury)
aorta wall was equivalent to 3.4 (± 2.5) and 25.6 (±18.1) fmol of thrombin/cm2 of intima-media, respectively. Possibly, only hirudin-accessible, glycosaminoglycan-bound
thrombin is measured in this way.
