FOREWORD

The clotting enzyme thrombin takes a central position in the blood coagulation system. This is especially evident if one considers that thrombin catalyzes not only the formation of fibrin but also activates clotting factors and blood platelets. Furthermore, it also has direct effects on vascular endothelium and initiates several nonhemostatic cellular events. Therefore, inhibition of thrombin should be very effective in the control of thrombogenesis and various pathophysiological states. Accordingly, thrombin has become a primary target for the development of antithrombotic agents.

Until now, pharmacological control of blood coagulation through thrombin inhibition has been possible only with the aid of the mammalian glycosaminoglycan heparin and derived drugs. However, these drugs may cause certain problems. Apart from the necessity of parenteral administration, heparin exerts its inhibitory effect on the clotting enzymes indirectly via a potentiation of the inhibitory function of antithrombin III.

The resulting pharmacological problem was addressed with the development of thrombin inhibitors different from glycosaminoglycans that block the clotting enzymes directly. We attempted initially to solve this problem by isolation and pharmacological characterization of the specific thrombin inhibitor from medicinal leeches (Hirudo medicinalis), named hirudin. Isolation and sequencing of the inhibitor allowed a molecular cloning of the corresponding cDNA, and synthetic genes coding for the inhibitor could be expressed in Escherichia coli or yeast. Using these methods, the inhibitor was available as a recombinant peptide in adequate amounts sufficient for clinical pharmacological studies. Hirudin served as a standard for designing new inhibitors.

The limitations of antithrombotic therapy with heparins, and the lessons learned from the clinical use of hirudin, have a great impact on the activities in the developments of synthetic low molecular weight antithrombin agents as anticoagulant and potential antithrombotic drugs (as we had pointed out 35 years ago).

Through the mid-1970s, several laboratories synthesized large numbers of thrombin inhibitors. The determination of the crystal structure of thrombin, together with the elucidation of structural features of hirudin, provided a rational basis for the design and synthesis of peptide and nonpeptide thrombin inhibitors. Seen from the vantage point of pharmacological control of coagulation, only a few compounds among the numerous synthetic thrombin inhibitors described have proven to be suitable as anticoagulants.

The development of synthetic thrombin inhibitors is an example of designing protease inhibitors through the mimicry of substrates. Imitating the amino acid sequence around the thrombin scissile bond in fibrinogen, inhibitors were produced that enter into a covalent or noncovalent bond with the active site of the enzyme. The most useful skeleton in designing active site directed thrombin inhibitors was the sequence Phe-Pro-Arg-H, especially if phenylalanine is in the D-configuration. A series of derivatives of the prototype tripeptide aldehyde have been developed as thrombin inhibitors.

One of the building blocks used to develop synthetic inhibitors is arginine with optimal C- and/or N-terminal modifications. Starting from the synthetic thrombin substrate Nα-tosyl-L-arginine methyl ester (TAME), Okamoto and his group have designed competitive inhibitors of considerable potency and selectivity by variation of the carbonyl and Nα-substituents. Argatroban (Tmq-Arg-D-Pip(4-Me)-OH), one of the representatives of the peptidomimetic thrombin inhibitors, has reached the stage of advanced clinical trials and is now available for patient treatment.

A specific thrombin inhibitor may interfere with essential mechanisms of thrombosis. By instantaneous inhibition of the small amount of thrombin generated after activation of the clotting system, the positive feedback on prothrombin activation is prevented and thrombin generation is delayed. Thus, depending on the inhibitor concentration in blood, coagulation is retarded or completely inhibited. Moreover, a thrombin inhibitor not only prevents thrombin-catalyzed hemostatic reactions, including the thrombin-induced platelet aggregation and release reaction, but it also prevents the synthesis and release of some mediators from the vascular endothelium. In addition, thrombin inhibitors interfere with all of thrombin's humoral effects.

Clinicopharmacological studies corroborated the specific pharmacological properties of the site-directed anti-thrombin agents observed in animal experiments. The new thrombin inhibitors are currently undergoing various phases of clinical trials. Moreover, for a broader clinical application of thrombin inhibitors as antithrombotics, the following issues warrant consideration.

Thrombin inhibitors do not find a preformed permanent systemic target in the organism. Instead, their specific binding site is present only when the target enzyme is generated. Therefore, the effect of thrombin inhibitors in vivo depends on their pharmacokinetic behavior, especially the time course of their blood levels. Design strategies for active site-directed inhibitors of thrombin have to focus not only on optimal binding to the target enzyme but also on their pharmacokinetic profile. Synthetic direct thrombin inhibitors represent novel antithrombotic agents in a field where heparin has been the only available drug thus far. Compared with heparins, thrombin inhibitors are advantageous because their effect does not require the presence of endogenous cofactors such as antithrombin III. Furthermore, thrombin inhibitors are not bound or inactivated by platelet factors or other proteins acting as antiheparin substances. They have no direct or immune-mediated platelet activating activity. For the effect in thrombosis, it is of interest that these inhibitors, in contrast to the heparin-antithrombin III complex, are able to block thrombin bound to the fibrin network, whereby the growth of preexistent thrombi can be prevented. The pathophysiological role of thrombin, extending beyond coagulation and platelet activation, suggests that thrombin inhibitors could equally be used as therapeutic tools in areas other than blood coagulation.

All pharmacological aspects speak in favor of the use of the direct thrombin inhibitors as antithrombotic agents, especially in potential indications where thrombin plays a crucial role in the pathogenesis. Demonstrating their ultimate clinical usefulness and advantage, however, represents a substantial challenge.