**4. Conclusions**

acts as a noncompetitive inhibitor (Ki = 3.9 μM) of factor Xa. It is a substrate for plasmin and trypsin, but not for factor Xa and thrombin. The prolongation of PT and aPTT is reversible [154]. Interestingly, several studies pointed out Amblyomin-X as an anti-cancer molecule *in* 

Other FXa inhibitors were reported in *I. scapularis* belonging to the salivary protein (Salp) family, which specifically inhibits the FXa active site [162]. Other inhibitors act on FXa through

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

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

*vitro* and *in vivo* [154–161].

52 Anticoagulant Drugs

*3.1.4. Thrombin inhibitors*

binding to prothrombinase complex [163].

hemalin from *Haemaphysalis longicornis* [170].

To conclude, hematophagous animals have evolved effective means of inhibiting thrombosis, thereby facilitating the acquisition and digestion of a blood meal. To date, specific inhibitors of coagulation, platelet function and fibrinolysis regulators have been identified from numerous invertebrate species, mainly leeches, ticks, and mosquitoes, representing an impressive array of convergent functional strategies. These parasites may serve as potentially useful therapeutic agents for the treatment of a variety of conditions associated with activation of thrombosis. A number of anticoagulants and platelet inhibitors from bloodsuckers have been evaluated *in vivo*, with some currently in varying stages of preclinical and clinical development. Because of the unique specificity and potency of anticoagulants from hematophagous, these kinds of products hold great promise for improving the treatment of a variety of human illnesses, as heart disease and stroke.
