INHIBITION OF CLOT-BOUND AND FREE (FLUID-PHASE THROMBIN) BY A NOVEL SYNTHETIC THROMBIN INHIBITOR (RO-46-6240), RECOMBINANT HIRUDIN AND HEPARIN IN HUMAN PLASMA

Clot-bound thrombin remains active and is less accessible to heparin-a ntithrombin III than fluid-phase thrombin. To determine whether clot-b ound human thrombin is more susceptible to inactivation by direct thro mbin inhibitors, the activity of a novel synthetic competitive thrombi n inhibitor Ro 46-6240, recombinant hirudin and unfractionated heparin were compared with fluid-phase thrombin and clot-bound thrombin. Fibr inopeptide A generated in human plasma was used as an index of thrombi n activity. Hirudin was the most potent inhibitor of fluid-phase and c lot-bound thrombin. However, Ro 46-6240 inhibited clot-bound thrombin three times more potently than fluid-phase thrombin (IC50 19 vs 56 ng/ ml) while hirudin was two times (IC50 8 us 3 ng/ml) and heparin six ti mes (IC50 1205 vs 200 ng/ml) less active against clot-bound thrombin c ompared with fluid-phase thrombin. The relative selectivity for clot-b ound thrombin is not a unique property of Ro 46-6240 since two other s ynthetic thrombin inhibitors tested inhibited clot-bound thrombin more effectively than fluid-phase thrombin and a third was equally active against both forms of thrombin. In contrast, the affinities of two chr omogenic substrates were similar for both forms of thrombin. This stud y shows that direct thrombin inhibitors inhibit clot-bound thrombin mo re potently than heparin and suggests that an apparent selectivity for clot-bound thrombin can be achieved with some synthetic thrombin inhi bitors. Further studies have to show whether the high potency of these direct thrombin inhibitors translates into antithrombotic efficacy in clinical situations with pre-existing clots.