*2.1.3. Factor Xa inhibitors*

to its decreased bleeding risk when compared with rHs [62, 63]. Another advantage of bivalirudin was demonstrated in animal studies, where bivalirudin presented a wider therapeutic index than

There are many studies with bivalirudin as an alternative to heparin or the combination of heparin and a GpIIb/IIIa inhibitor in patients with acute coronary syndromes and those undergoing a PCI [65–67]. These trials demonstrated that bivalirudin was not significantly different from other tested inhibitors in relation to reduction in major bleeding; on the other hand, bivalirudin, unlike heparin and GpIIb/IIIa inhibitors, does not cause thrombocytopenia. In this study, it also was demonstrated that bivalirudin reduced cardiac mortality and all-cause mortality among patients undergoing primary PCI for ST-elevation-myocardial infarction in the HORIZONS-AMI trial [66]. Accordingly, bivalirudin (Angiomax, The Medicines Company, Parsippany, NJ, USA) has become one of most widely used antithrombotics in the United States for PCI. Bivalirudin has been further studied in other kind of surgeries, but has not been further developed for these indications. Some examples of clinical studies with bivalirudin were as an alternative to heparin in coronary artery bypass [68, 69] in a dose-finding study for VTE prevention in patients after hip or knee surgery [70] and for the treatment of calf vein thrombosis [71]. Finally, the FDA expanded its approval of bivalirudin to include its use as an alternative to heparin in HIT patients with or without thrombosis undergoing PCI [72].

Besides hirudin, other thrombin inhibitors less studied have been isolated from leeches. Among them are a granuline-similar peptide [73], bufrudin [74], theromin [75], and haemadin [76]. Haemadin and theromin are inhibitors and do not present homology in their sequences with the other inhibitors described up to now in all animal kingdom. Haemadin was isolated from the *Haemadipsa sylvestris*, leech, and it is a 5 kDa peptide with a Ki of 100 fM, kinetically less efficient than hirudin (21 fM) [76, 77]. In addition, in literature, we can find only studies about crystal of haemadin and formation of haemadin-thrombin complex, nothing more besides [78, 79].

Theromin is a potent inhibitor (Ki = 12 fM) which was isolated from the intestines of *Theromyzon tessulatum* leeches [75]. It is homodimer 67 amino acid residues, with 16 cysteines that share 8 disulfide bridges. Just like hirudin, the N-terminal sequence of theromin is highly negatively charged and its C-terminal portion is very compact, due to 10 residues of cysteine present on the sequence. Around 24% of the residues of the molecule be cysteins and this approaches it, in sequence similarity, to protease inhibitors of the antistasin family (more detailed below). Hence, considering the low identity on the general sequence between theromin and the peptides of this family, it is difficult to include theromin as a new member of the mentioned family. However, comparisons of sequences have been made between theromin and four different serine-protease inhibitors isolated from *T. tessulatum* leeches: cytin, therin, therostasin, and tessulin [80–83]. These comparisons revealed that in the case of therostasin [82] and tessulin [83], there was a high degree of sequence identity with

It can also be added that among the leeches from the *Theromyzon* genus, three other thrombin inhibitors were also described [84]. In fact, Merck Company, in 1994, deposited patents for different applications observing three thrombin inhibitors with masses of 3, 9, and 14 kDa

rHs, and an additional advantage of bivalirudin was its lack of immunogenicity [64].

*2.1.2. Other thrombin inhibitors*

44 Anticoagulant Drugs

theromin (70 and 52%, respectively).

While FXa inhibition has emerged as a convenient pathway for management of VTE, currently three FXa inhibitors are available for anticoagulation management—rivaroxaban, apixaban, and edoxaban [85]. New researches about FXa inhibitors of hematophagous animals constantly have been sought.

Antistasin was the first factor Xa inhibitor described that originates from leeches. It is a 15 kDa protein isolated from the salivary glands of the Mexican leech *H. officinalis* [86, 87]. Soon after, a homologous protein, ghilanten, was isolated from the *H. ghiliani* leech [88]. Antistasin features 119 residues of amino acids with the domain I (residues 1–55) being 56% similar to the domain II (residues 56–110). Of the nine residues of the C-terminal (111–119), domain portion four was positively charged [86], and their active site was located on domain I [88–90]. The cDNA of antistasin was cloned [89] and the recombinant protein expressed in system of baculovirus vector in insect cells [90]. Pharmacological studies were carried out, and data showed that the protein remains active after 30 h of injection in animals. Besides this, when tested in different thrombosis models, antistasin proved superior to heparin [91].

Administration of recombinant antistasin in rabbits with atherosclerosis in the femoral artery, as an example, demonstrated reduction of restenosis after balloon angioplasty [91]. Besides this, chimeric peptides corresponding only to domain I were also tested, and it was checked that domains II and III do not feature any intrinsic inhibitory activity over factor Xa, and also do not contribute to activity of domain I [86]. The most powerful synthetic peptide derived from antistasin corresponds to amino acids 27–49, with a disulfide bridge (ATS29–47); this peptide was able to inhibit factor Xa with a Ki of 35 nM. The DRCRVHCP peptide, in micromolar concentrations, featured anticlotting activity and was able to prolong the coagulation time in 50%, when compared with the control [92].
