Abstract
Using the three-dimensional structures of thrombin and the leech-derived tryptase inhibitor (LDTI), which does not inhibit thrombin, we were able to construct three LDTI variants inhibiting thrombin. Trimming of the inhibitor reactive site loop to fit thrombin's narrow active site cleft resulted in inhibition constants (Ki) in the 10 nM concentration range; similar values were obtained by the addition of an acidic C-terminal peptide corresponding to hirudin's tail to LDTI. Combination of both modifications is additive, resulting in very strong inhibition of thrombin (Ki in the picomolar range). On the one hand, these results confirm the significance of the restricted active site cleft of thrombin in determining its high cleavage specificity; on the other, they demonstrate that sufficient binding energy at the fibrinogen recognition exosite can force thrombin to accept otherwise unfavorable residues in the active site cleft. The best inhibitor thus obtained is as effective as hirudin in plasma-based clotting assays.
Highlights
The action of thrombin is central to coagulation [1]
We show that trimming the reactive site loop of leech-derived tryptase inhibitor (LDTI) to reduce collisions with residues lining the active site cleft of thrombin can produce an inhibitor with an inhibition constant in the 10 nM range
An inhibitor with similar affinity is obtained upon introduction of a hirudin tail fragment to untrimmed rLDTI, showing that thrombin can accept unfavorable substituents in its active site cleft upon favorable binding at the fibrinogen exosite
Summary
The action of thrombin is central to coagulation [1]. Physiologically, its activity is regulated by serpins such as antithrombin III, heparin cofactor II, and protease nexin I as well as the general proteinase scavenger a2-macroglobulin [2]. Etration into the active site cleft and extensive electrostatic interaction between the acidic carboxyl-terminal “tail” of hirudin with the basic fibrinogen recognition exosite of thrombin. Despite the close structural homology of LDTI to rhodniin [15, 16], the leech-derived inhibitor does not inhibit thrombin. We show that trimming the reactive site loop of LDTI to reduce collisions with residues lining the active site cleft of thrombin can produce an inhibitor with an inhibition constant in the 10 nM range. An inhibitor with similar affinity is obtained upon introduction of a hirudin tail fragment to untrimmed rLDTI, showing that thrombin can accept unfavorable substituents in its active site cleft upon favorable binding at the fibrinogen exosite. A mutant combining both favorable properties inhibits thrombin with a Ki value in the 10 pM range, indicating that the interactions at the active site and exosite are additive
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