RADIOLABELED R-HIRUDIN AS A MEASURE OF THROMBIN ACTIVITY AT, OR WITHIN, THE RABBIT AORTA WALL IN-VITRO AND IN-VIVO

The behavior of I-125-Iabeled recombinant hirudin towards the uninjure d and de-endothelialized rabbit aorta wall has been studied in vitro a nd in vivo to determine its usefulness as an indicator of thrombin act ivity associated with the aorta wall. Thrombin adsorbed to either sulf opropyl-Sephadex or heparin-Sepharose bound >95% of I-125-r-hirudin an d the complex remained bound to the matrix. Binding of I-125-r-hirudin to the exposed aorta subendothelium (intima-media) in vitro was incre ased substantially if the tissue was pre-treated with thrombin; the qu antity of I-125-r-hirudin bound to the de-endothelialized intima-media (i.e. balloon-injured in vitro) correlated positively with the quanti ty of bound I-131-thrombin (p <0.01). Aortas balloon-injured in vivo w ere measured for thrombin release from, and binding of I-125-r-hirudin to, the de-endothelialized intimal surface in vitro; I-125-r-hirudin binding correlated with the amount of active thrombin released (p <0.0 01). Uptake of I-125-r-hirudin by the aorta wall in vivo was proportio nal to the uptake of I-131-fibrinogen (as an indicator of thrombin act ivity) before and after balloon injury. After 30 min in the circulatio n, specific I-125-r-hirudin binding to the uninjured and de-endothelia lized (at 1.5 h after injury) aorta wall was equivalent to 3.4 (+/- 2. 5) and 25.6 (+/- 18.1) fmol of thrombin/cm(2) of intima-media, respect ively. Possibly, only hirudin-accessible, glycosaminoglycan-bound thro mbin is measured in this way.