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V. Inactivated Thrombin as an Inhibitor of Clot Formation
Page 261
A means to selectively inhibit thrombin's role in coagulation while preserving its anticoagulant functions
involves site-directed mutagenesis of thrombin itself. By introduction of a single mutation, Gibbs et al.
[29] altered thrombin's relative specificity for fibrinogen and protein C. The engineered thrombin's
increased activation of protein C over fibrinogen cleavage offers the possibility of inhibiting clot
formation with a modified human protein, a molecule likely to exhibit few side effects.
VI. The Role of Structural Information
The discovery of thrombin inhibitors has benefited from available protein structural information. Models
of the thrombin overall structure and its active site geometry, constructed from available structures of
related serine proteases [30], aided in the design of the mechanism-based inhibitors such as PPACK [31]
and its boroarginine analog [10]. The unexpected, nonsubstrate binding mode of early thrombin
inhibitors such as NAPAP was revealed by x-ray crystallographic analyses [32]. Iterative structurebased
design methods have been critical in the optimization of bivalent inhibitors and inhibitors directed
at the primary specificity pocket. Structures of inhibitor:thrombin complexes are essential for the
optimization of substitutents forming interactions within the aryl-binding site of the primary specificity
pocket. In some cases (e.g. Table 1), seemingly minor alterations of the inhibitor can result in dramatic
changes in the inhibitor's overall interactions with thrombin [17].
Drug discovery efforts have also been strongly influenced by results of structural studies of thrombin
complexed with effectors and substrate peptides. For example, recently the structures of thrombin
complexed with fibrinopeptide A [33] and human prothrombin fragment F1 [34] have been determined.
In addition to their role in design of high-affinity inhibitors, these structures provide valuable insights
for design of drugs specific for the various subsites and conformational states of thrombin.
VII. Conclusion
Discovery of therapeutically effective thrombin inhibitors involves issues such as affinity and
selectivity, bioavailability, and formulation. In addition to these relatively common concerns, the
complex in vivo mechanisms designed to
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