Variegin, a Novel Fast and Tight Binding Thrombin Inhibitor from the Tropical Bont Tick

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 ϳ 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.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. Within this network of reactions, thrombin plays a central role in maintaining the integrity of hemostasis. Thrombin interacts with most of the zymogens and their cofactors, playing multiple procoagulant and anticoagulant roles in blood coagulation (1, 2). As a procoagulant protease, the first traces of thrombin generated in the initiation phase activate factor V (FV) 2 and factor VIII (FVIII) to provide positive feedback leading to the thrombin burst. Thrombin can also activate factor XI, triggering the intrinsic pathway. Thrombin cleaves fibrinogen to fibrin, forming insoluble clots. Fibrin polymers are further strengthened and stabilized through covalent cross-linking driven by thrombin-activated factor XIII. Thrombin also contributes to the generation of a platelet plug, possibly through two mechanisms: (a) it activates platelets by interacting with protease-activated receptors and glycoprotein V, and (b) it prevents destabilization of the platelet plug, by inactivating ADAMTS13, a disintegrin and metalloprotease with a thrombospondin type 1 motif, that cleaves von Willebrand factor. As an anticoagulant protease, thrombin activates protein C in the presence of the cofactor thrombomodulin.

The Essential Role of Tick Salivary Glands and Saliva in Tick Feeding and Pathogen Transmission

“…Ornithodorin (van de Locht et al, 1996), savignin (Nienaber et al, 1999), and monobin (Mans et al, 2008) from soft ticks are Kunitz-type antithrombins, while IRS-2 and IRIS from I. ricinus (Leboulle et al, 2002; Chmelar et al, 2011), IxscS-1E1 from I. scapularis (Ibelli et al, 2014) and RmS-15 from Rhipicephalus (Boophilus) microplus (Xu et al, 2016) are serpins. Antithrombins belonging to the hirudin-like/madanin/variegin superfamily include madanin 1 and 2 from H. longicornis (Iwanaga et al, 2003), variegin from Amblyomma variegatum (Koh et al, 2007) and hyalomin-1 from Hyalomma marginatum rufipes (Jablonka et al, 2015). The antithrombins microphilin (Ciprandi et al, 2006) and BmAP (Horn et al, 2000) from R.…”

Metalloproteases Affecting Blood Coagulation, Fibrinolysis and Platelet Aggregation from Snake Venoms: Definition and Nomenclature of Interaction Sites

“…In SVMPs there is an exosite C* 241 TRKKHD 246 C* (as numbered in jararhagin) that interacts with human integrin α 2 I-domain [93,94,95]. Because of their importance in determining exquisite selectivity and specificity, the exosites are of immense interest in biomedical research as potential drug targets [153,154,155,156,157,158]. …”

Crystal Structure of Thrombin in Complex with S-Variegin: Insights of a Novel Mechanism of Inhibition and Design of Tunable Thrombin Inhibitors

“…Recently, we described variegin, a novel, fast and tight-binding competitive inhibitor of thrombin (refers to the α form of thrombin unless otherwise stated) isolated from the tropical bont tick, Amblyomma variegatum [16]. Like hirudin/hirulog, variegin targets the thrombin catalytic site and exosite-I.…”