In vivo behaviour of some antithrombin III-protease complexes

Abstract

Complexes of rabbit, human and rat antithrombin III with rabbit plasmin and complexes of rabbit antithrombin III with rabbit and bovine thrombin were produced and separated from uncomplexed reactants by preparative electrophoresis. After injection in rabbits, the behaviour of these complexes was followed by plasma radioactivity measurements and gel filtration.

All five complex types were unstable in vivo to different extents as evidenced by the divergency of the plasma radioactivity curves representing the inhibitor and protease components respectively. The progressive dissociation of the complexes was confirmed by the changes observed in their elution pattern from a column of Sephadex G-200.

Antithrombin III that had dissociated from a complex ('post-complex antithrombin III') left the circulation at rates which were marginally higher than those for antithrombin III that had not been incubated with protease. Post-complex antithrombin III no longer bound to Sepharose-trypsin and its affinity for Sepharoseheparin was markedly reduced. However, post-complex antithrombin III did react with an antiserum raised against normal antithrombin III.

Post-complex plasmin and, particularly, post-complex thrombin were cleared from circulation considerably more rapidly than post-complex AT III although, from radioactivity measurements, diminishing portions of either enzyme remained detectable in the circulation for approximately 24 h. From chromatographic studies using Sepharose-antithrombin III and -lysine, post-complex plasmin had lost its reactivity with the immobilized inhibitor but had retained its lysine-binding site.

Stability studies in vitro showed that antithrombin III-plasmin complexes, incubated at 37° in the absence of other proteins, slowly dissociated after 6 h. The dissociation was not prevented by the addition of Trasylol after 1 h. In the presence of plasma, the extent of dissociation was reduced, whereas with whole blood it was enhanced.