Fate of Thrombin and Thrombin-Antithrombin-III Complex Adsorbed to a Heparinized Biomaterial: Analysis of the Enzyme-Inhibitor Complexes Displaced by Plasma

Heparin covalently-linked to polyvinyl alcohol (PVA) is a biomaterial which is of potential value as a non-thrombogenic coating. 125I-labelled thrombin adsorbed to heparin-PVA beads was not dislodged by phosphate-buffered saline, pH 7.4, although radioactivity was progressively displaced from the adsorbent by fibrinogen-free human plasma. Analysis by gel filtration and affinity chromatography showed that the released radioactivity was distributed between (thrombin-antithrombin-III) complex (approx. 70%) and, probably, (thrombin-alpha-2-macroglobulin) complex (approx. 30%). Less efficient thrombin displacement was obtained by either bovine serum albumin (5% w/v) or antithrombin-III-free human plasma: in the latter case, the dislodged enzyme was presumably associated with alpha-2-macroglobulin. Purified alpha-2-macroglobulin did not displace thrombin from heparin-PVA. The quantity of thrombin displaced by an alpha-2-macroglobulin-free plasma fraction compared well with fibrinogen-free plasma: The eluted enzyme was largely associated with antithrombin-III. Purified antithrombin-III did not displace thrombin from heparin-PVA despite causing greater than 70% inactivation of the bound enzyme. Subsequent treatment with fibrinogen-free plasma dislodged (thrombin-antithrombin-III) at a similar rate to that of bound thrombin. We conclude that plasma contains a component(s) which displaces (thrombin-antithrombin-III) complex from immobilised heparin: presumably this leaves the heparin sites free for further use in enzyme inactivation.