*2.1.2. Other thrombin inhibitors*

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 theromin (70 and 52%, respectively).

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 [28]. The N-terminal of the 9 kDa inhibitor, EDDNPGPPRACPGE, presented homology with theromin (ECENTECPRACPGE), factor Xa inhibitor (DCENTECPRACPGE) [82], and trypsin inhibitor tessulin (MCENTECPRACPGE) [83]. This 9 kDa inhibitor features a pI of 4.9 and a specific activity at the end of the purification process of 25 IU for inhibition of thrombin and of 0.2 IU for factor Xa inhibition.
