Abstract
The inhibition of thrombin is one of the important treatments of pathological blood clot formation. Variegin, isolated from the tropical bont tick, is a novel molecule exhibiting a unique ‘two-modes’ inhibitory property on thrombin active site (competitive before cleavage, noncompetitive after cleavage). For the better understanding of its function, we have determined the crystal structure of the human α-thrombin:synthetic-variegin complex at 2.4 Å resolution. The structure reveals a new mechanism of thrombin inhibition by disrupting the charge relay system. Based on the structure, we have designed 17 variegin variants, differing in potency, kinetics and mechanism of inhibition. The most active variant is about 70 times more potent than the FDA-approved peptidic thrombin inhibitor, hirulog-1/bivalirudin. In vivo antithrombotic effects of the variegin variants correlate well with their in vitro affinities for thrombin. Our results encourage that variegin and the variants show strong potential for the development of tunable anticoagulants.
Highlights
Serine proteinases in the blood coagulation cascade are important molecules in maintaining the integrity of hemostasis
Variegin belongs to a unique class of thrombin inhibitors that have potential as antithrombotic agents [16,17]
Active site inhibitors of thrombin typically target the non-prime subsites, hindering the access of substrates to the catalytic residues [29,31,40]
Summary
Serine proteinases in the blood coagulation cascade are important molecules in maintaining the integrity of hemostasis. The active site contains the classical catalytic triad – His, Asp102 and Ser195 (Figure 1A). Compared to other blood coagulation serine proteinases, thrombin has a prominent active site cleft, which is deep and narrow. Two insertion loops (called the 60-loop with residues Leu59-Asn and the autolysisloop, residues Leu144-Gly150) form the wall of the cleft (Figure 1A–B) [1,2]. The thrombin active site surfaces that interact with substrate residues, at N-terminal to the scissile bond, are described as ‘non-prime subsites’ (S subsites). The surfaces of the active site which are in contact with substrate residues, at C-terminal to the scissile bond, are described as ‘prime subsites’ (S9 subsites) (Figure 1B)
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