Abstract
Tick saliva contains potent antihemostatic molecules that help ticks obtain their enormous blood meal during prolonged feeding. We isolated thrombin inhibitors present in the salivary gland extract from partially fed female Amblyomma variegatum, the tropical bont tick, and characterized the most potent, variegin, one of the smallest (32 residues) thrombin inhibitors found in nature. Full-length variegin and two truncated variants were chemically synthesized. Despite its small size and flexible structure, variegin binds thrombin with strong affinity (K(i) approximately 10.4 pM) and high specificity. Results using the truncated variants indicated that the seven residues at the N terminus affected the binding kinetics; when removed, the binding characteristics changed from fast to slow. Further, the thrombin active site binding moiety of variegin is in the region of residues 8-14, and the exosite-I binding moiety is within residues 15-32. Our results show that variegin is structurally and functionally similar to the rationally designed thrombin inhibitor, hirulog. However, compared with hirulog, variegin is a more potent inhibitor, and its inhibitory activity is largely retained after cleavage by thrombin.
Highlights
Blood coagulation is part of the physiological response to vascular injury, in which circulating zymogens of serine proteases are sequentially activated by limited proteolysis leading to the formation of a fibrin clot
The thrombin active site binding moiety of variegin is in the region of residues 8 –14, and the exosite-I binding moiety is within residues 15–32
The results indicate that its N terminus dictates fast binding kinetics, while central residues bind to the active site and the C terminus binds to exosite-I
Summary
Unlike hirudin and bivalirudin, which are bivalent inhibitors (bind to two distinct sites), argatroban is a univalent inhibitor and binds only to the active site [8]. The success of hirudin and bivalirudin demonstrated the feasibility of identifying and developing such anticoagulants. In our search for novel anticoagulants (18 –20), we examined the salivary gland extract (SGE) of the tropical bont tick, Amblyomma variegatum (Acari: Ixodidae). We describe the identification and isolation of a new thrombin inhibitor, variegin. We examined its inhibitory activity, specificity, kinetics, structure-function relationships, and cleavage by thrombin. This 32-residue polypeptide is a potent and specific thrombin inhibitor. The results indicate that its N terminus dictates fast binding kinetics, while central residues bind to the active site and the C terminus binds to exosite-I
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