The effect of heparin adsorbed on Sepharose-lysine on the inactivation of thrombin by anti-thrombin III [proceedings].

The role of heparin in promoting the reaction between the plasma inhibitor, antithrombin I l l , and several proteolytic enzymes involved in blood coagulation is not clearly understood. Rosenberg and his colleagues (see, for example, Rosenberg & Damus, 1973; Highsmith & Rosenberg, 1974) have claimed that heparin binding modifies the shape of the anti-thrombin I I I molecule, thus exposing a vulnerable arginine residue which reacts with thrombin and other enzymes to create an irreversible anti-thrombin 111-proteinase complex. Some support for this idea came from the kinetic studies by Li e t a / . (1976). An alternative theory by Machovich (1975) proposes that heparin induces a conformational change in the enzyme, in this case thrombin, to encourage interaction with anti-thrombin I l l . The evidence was based in part on the ability of heparin to bind thrombin more avidly than anti-thrombin 111. In an attempt to determine more clearly the mechanism for heparin participation, we studied the thrombinanti-thrombin I 1 1 reaction in the presence of heparin covalently bound to Sepharose, and the results clearly showed that greater inactivation of thrombin and plasmin was obtained if either enzyme was bound to heparin before exposure to anti-thrombin 111 rather than anti-thrombin 111 bound to heparin before exposure to thrombin (Hatton & Regoeczi, 1977). However, it is possible that any chemical modificationof heparin during the preparation of Sepharose-heparin may have influenced the outcome of these inactivation studies. Therefore i t was important to re-examine the enzyme-inhibitor reaction by using heparin ionically bound to Sepharose-lysine. Sepharose-lysine (approx. 250pmoI of lysine per g of dry gel) was prepared by the CNBr-activation method (Cuatrecasas, 1970) and bound lysine determined as described previously (Hatton & Regoeczi, 1974). Small columns (0.6cmx 6.0cm) were poured and equilibrated with Ringer solution/lactate. Each column was loaded with 2mg of heparin (Sigma Chemical Co., St Louis, MO, U.S.A.) in 1 ml of Ringer/lactate. The schemes for loading the enzyme and inhibitor on to the Sepharose-lysine-heparin columns were similar to those described previously (Hatton & Regoeczi, 1977). Bovine thrombin (Parke-Davis Co., Detroit, MI, U.S.A.) was used either in the crude state or