*3.1.4. Thrombin inhibitors*

The main effector blood coagulation factor is thrombin, which is the enzyme involved in the final (common pathway of the blood coagulation, responsible for the conversion of fibrinogen in fibrin and also regulates the activity of other coagulation factor with great specificity. Thrombin is a multifunctional molecule acting in cell signaling, fibrinolysis, and inflammation system [164]. Thrombin has three domains, the active site and two regulator sites, named exosites. Exosite I is the site that links the enzyme with fibrinogen, the platelet receptor and protease activated receptors (PARs), as well as the endothelial receptor, thrombomodulin. Exosite II recognizes glycosaminoglycans such as heparin, platelet receptor GP Ib-IX-V and fibrin (for a recent review on the role of thrombin exosites, see Ref. [165]. Thus, the choice of thrombin as a target for new anticoagulants seems logical, since its inhibition not only attenuates fibrin formation, but also blocks thrombin-mediated feedback amplification of clotting [166].

Kunitz-type thrombin inhibitors from ticks were identified in hard (Ixodidae family) and soft (Argasidae family) ticks, and have differences that place them in two different protein subclasses, based on their sequences, probably as an adaptation of their different blood-feeding behaviors [2]. Avathrin is a recombinant thrombin inhibitor from the salivary glands of the ixodid tick, *Amblyomma variegatum*. It shares 31–34% of identity with variegin. Kinetically, avathrin is a fast, tight binding competitive inhibitor (545 pM) with high affinity for thrombin rather than other serine proteases of the coagulation system. Cristal structure of avathrin and thrombin reveal an interaction through the active site and exosite-I of thrombin. Moreover, cleavage products continue to exert prolonged inhibition in a murine carotid artery thrombosis model [167]. From hard ticks, other thrombin inhibitor was isolated including amblin from *Amblyomma hebraeum* [168], boophilin from the cattle tick *Boophilus microplus* [169], and hemalin from *Haemaphysalis longicornis* [170].

Boophilin has been cloned and overexpressed in *E. coli*, which potently inhibits additional trypsinlike serine proteases, including trypsin and plasmin and displays an apparent molecular mass of ~23 kDa. This inhibitor binds bovine thrombin with tight-binding kinetics, and was determined an apparent Ki of 1.8 nM. The crystal structure of the bovine α-thrombin boophilin complex reveals a non-canonical binding mode to the protease. The N-terminal region of the mature inhibitor binds in a parallel manner across the active site of the protease, while the C-terminal Kunitz domain is negatively charged and docks into the basic exosite I of thrombin [169].

Recently, a new thrombin inhibitor from *Amblyomma sculptum* was identified in the transcriptomics analysis of tick's salivary glands [171]. Scupltin was cloned and expressed in *E. coli* as a 20 kDa protein sharing only few similarities with hirudin and more similarity with serine protease inhibitors of the antistasin family. Sculptin is a novel class of competitive, reversible, and specific inhibitor of thrombin because its mechanism of inhibition is slightly different than hirudin. The Ki is comparable with that of hirudin and lower then hirulogs. Interestingly, sculptin phylogenetically diverges from hirudin. Sculptin has not inhibitory activity on FXa, trypsin and plasmin. However, it is degraded by serine proteases including thrombin, thus would not requires antidotes. The sculptin fragments produced by thrombin have not thrombin inhibitory activity, while sculptin fragments produced by FXa can inhibit thrombin independently. Sculptin increases blood coagulation parameter in concentration dependent manner. Sculptin has been filed for patenting in Brazil [171].

From soft ticks, Kunitz-type thrombin inhibitors include ornithodorin from *Ornithodoros moubata* [172], savignin from *Ornithodoros savignyi* [135, 173] and monobin from *Argas monolakensis* [174]. Kinetically, they are slow, tight-binding, competitive inhibitors of thrombin: savignin (Ki = 4.89 pM) [173], and monobin (Ki = 7 pM) [174].

As mentioned above, most Ixodidae ticks produce a cement or glue to attach to the host skin to facilitate the penetration of mouthparts for feeding. Interestingly, in *Amblyomma americanum,* the compositions of this cement revealed by the presence of glycine-rich proteins, lipids, and certain carbohydrates, besides serine protease inhibitors and metalloproteases. Some molecules from tick cement were considered promising candidates for an anti-tick vaccine because of their antigenic properties [136, 175].
