13. Development of a clot-targeted anticoagulant

Abstract

The effectiveness of anticoagulant therapy is limited by the inability of many anticoagulant molecules to inhibit completely clot bound thrombin. Despite the development of direct, potent thrombin inhibitors such as hirudin, there remains significant opportunity to improve the treatment efficacy. Research in this area has provided evidence to indicate that the failure of existing anticoagulant therapies is due to the procoagulant nature of the clot itself. Clot-targeted anticoagulants offer the potential to: (1) simplify the administration methods; (2) reduce the quantity required for treatment, thus reducing the potential cost of treatment; (3) increase the local therapeutic concentration and (4) to prevent systemic side-effects. A model system has been developed for preparation of targeted anticoagulants using avidin and biotin for coupling. The Fab fragment of the monoclonal antibody DD3B6/22 which binds to the D-dimer component of cross-linked fibrin was prepared and conjugated to avidin. This conjugate was then reacted with the biotinylated specific active site thrombin inhibitor, biotin-PPACK (D-Phe-Pro-Arg-chloromethyl ketone). This targeted anticoagulant (TAC) molecule retained both the ability to bind D-dimer and inhibit thrombin. The reagent binds clots prepared in vitro, which correlates well with the good binding properties observed in an in vivo imaging trial carried out with DD3B6/22. The inhibition of clot procoagulant activity by the TAC reagent versus conventional inhibitors, biotin-PPACK, PPACK and hirudin was assessed in an in vitro assay developed in this study. Clots were pretreated with inhibitor and then thoroughly washed before the procoagulant activity was assayed. The TAC reagent was able to inhibit 90% of the procoagulant activity whereas the conventional inhibitors were only able to inhibit a maximum of 60%. This novel approach to targeting biological active molecules to the clot surface could be extended to include lytic agents or other anticoagulant molecules.