*2.1.1. Thrombin inhibitors*

Thrombin is a key enzyme on the pathogenesis of coronary acute thrombosis. Therapies with heparin, an indirect thrombin inhibitor, have been used during the last four decades. Search for new alternatives has demonstrated that the development of direct thrombin inhibitors (DTIs) is a translational success story; an example in which the combination of scientific ingenuity, structure-based design (including leech molecules models), and rigorous clinical trials has created a new class of anticoagulants that has improved patient care [33].

Hirudin was discovered on the salivary glands of the *Hirudo medicinalis* leeches in 1884 [34], and its role as a powerful antithrombotic drug started to be investigated on the 1920s. Markwardt in 1957 started studies with hirudin as a direct agent on the inhibition of thrombin (DTI), and these studies have been progressing significantly [28, 29].

Hirudin is a natural peptide with a simple chain, featuring 65 with three disulfide bridges and one residue of sulfated tyrosine amino acid residues. Part of its N-terminal region is globular and very compact, due to the presence of three disulfide bridges. On the other hand, the C-terminal region is made up of a great number of negatively charged residues [35–38]. More than 100 years after its discovery, the cDNA of hirudin was cloned and the recombinant (rH) obtained in large scale on *Escherichia coli* [39], on *Saccharomyces cerevisiae* [40], and, more recently, on *Acremonium chrysogenum* [41]. Its way of action has been extensively compared to low-molecular-weight heparins. Hirudin is a strict thrombin inhibitor of the "tight binding" type [42], and cofactors are not needed for its activity. Preclinical evaluation and rH clinical selection of analog forms have been improved on the last years [43].

The complex formed between hirudin and thrombin involves the three amino acid residues from the N-terminal region, which link near to the active site, and the C-terminal tail is linked to the fibrinogen-linking site. Crystallographic studies have shown that 10 residues of amino acids of the C-terminal portion (residues 55–65) react with the anion present on the exosite of thrombin, an important region for linking to fibrinogen. The residues 1–48 of the N-terminal portion are also important for the action of hirudin over thrombin; they interact with the enzyme's catalytic site. These types of interaction explain why hirudin links only to thrombin and not to the blood semiproteases [44].

A significant advance was reached with the resolution of the tridimensional structure of hirudin, which allowed for the understanding and development of recombinants equivalent to this protein (rH). The increase of interest on protein inhibitors also was due to studies that demonstrated thrombocytopenia induced by heparin. These new agents produce a direct anticlotting response, having thrombin as target, and they also inhibit the activation of platelets and the increase of thrombin's activity on the coagulation cascade, as thrombin is a multifunctional enzyme responsible for the activation of manly factors, for example, factor V, VIII, and XI [45]. The use of rH has been promising in patients with unstable angina [46].

**2.1. Molecules with activity in the hemostatic system from leeches**

*2.1.1. Thrombin inhibitors*

42 Anticoagulant Drugs

Among different anticlotting molecules from leeches and involved in the coagulation cascade, fibrinolysis, or on the platelet aggregation process, three substances have been the main focus of investigation. They are hirudin (a thrombin inhibitor) [29], antistasin (factor Xa inhibitor) [30], and decorsin (an antagonist of the llb-llla glycoprotein of the platelet membrane) [31]. The amino acid sequences of these substances, together with studies of inhibitory activities from different molecules and designs of the three-dimension structure have been determined, and, then, the structural similarity of these molecules was observed, allowing for the design of a structure motif (L.A.P.: Cys-X6-12-Cys-X-Cys-X3-6-Cys-X3-6-Cys8-14) [32]. However, the mechanisms of action of these inhibitors and important epitopes for the connection to their respective targets are distinct [32], demonstrating the relevance of the many inhibition mechanisms on clotting processes, as well as the evolution of these processes. Many of these substances that come from leeches have been developed by the industry, as targets for different therapies and in different clinical trial stages.

Thrombin is a key enzyme on the pathogenesis of coronary acute thrombosis. Therapies with heparin, an indirect thrombin inhibitor, have been used during the last four decades. Search for new alternatives has demonstrated that the development of direct thrombin inhibitors (DTIs) is a translational success story; an example in which the combination of scientific ingenuity, structure-based design (including leech molecules models), and rigorous clinical trials

Hirudin was discovered on the salivary glands of the *Hirudo medicinalis* leeches in 1884 [34], and its role as a powerful antithrombotic drug started to be investigated on the 1920s. Markwardt in 1957 started studies with hirudin as a direct agent on the inhibition of thrombin

Hirudin is a natural peptide with a simple chain, featuring 65 with three disulfide bridges and one residue of sulfated tyrosine amino acid residues. Part of its N-terminal region is globular and very compact, due to the presence of three disulfide bridges. On the other hand, the C-terminal region is made up of a great number of negatively charged residues [35–38]. More than 100 years after its discovery, the cDNA of hirudin was cloned and the recombinant (rH) obtained in large scale on *Escherichia coli* [39], on *Saccharomyces cerevisiae* [40], and, more recently, on *Acremonium chrysogenum* [41]. Its way of action has been extensively compared to low-molecular-weight heparins. Hirudin is a strict thrombin inhibitor of the "tight binding" type [42], and cofactors are not needed for its activity. Preclinical evaluation and rH clinical

The complex formed between hirudin and thrombin involves the three amino acid residues from the N-terminal region, which link near to the active site, and the C-terminal tail is linked to the fibrinogen-linking site. Crystallographic studies have shown that 10 residues of amino acids of the C-terminal portion (residues 55–65) react with the anion present on the exosite of thrombin, an important region for linking to fibrinogen. The residues 1–48 of the N-terminal portion are also important for the action of hirudin over thrombin; they interact with the enzyme's catalytic site. These types of interaction explain why hirudin links only to thrombin

has created a new class of anticoagulants that has improved patient care [33].

(DTI), and these studies have been progressing significantly [28, 29].

selection of analog forms have been improved on the last years [43].

and not to the blood semiproteases [44].

Lepirudin (Refludan) is an rH, and it was the first direct thrombin inhibitor (DTI) licensed for treatment of thrombosis complicating HIT and associated thromboembolic disease in order to prevent further thromboembolic complications [47]. It is given as an intravenous infusion with or without a bolus, and its dosing is dependent on body weight. It is renally excreted and dose adjustments are required in patients with renal impairment [48]. Significant limitations to its use are its narrow therapeutic window and potential for increased bleeding events [49]. Besides, it is a drug that forms immunogenic complexes and causes a delay in renal excretion causing its accumulation [50, 51]. Therefore, during the treatment, the dose adjustment based on aPTT is recommended. Although not common, anaphylaxis can also occur in patients with hirudin-induced antibodies during the re-exposition to drug [52]. To date, there are no reports of antidotes that reverse these effects of DTIs [53]. There are recent reports that lepirudin has been discontinued from the market [54, 55].

Desirudin (Iprivask) is also an rH, with very similar characteristics as lepirudin. Both rH are structurally identical except for their N-terminus sequences, which are Leu1-Tyr2 in lepirudin and Val1-Val2 in desirudin. It reversibly binds to the active thrombin site of free and clot-associated thrombin. Desirudin is able to inhibit different actions of thrombin as fibrin formation, activation of coagulation factors V, VII, and XIII, and platelet aggregation, resulting in a dose-dependent prolongation of aPTT. It is the only fixed-dose subcutaneously administered DTI approved by FDA for postoperative prevention of VTE in patients undergoing elective hip replacement surgery [56]. Eriksson and collaborators published two clinical studies comparing the efficacy and safety of desirudin (15 mg s.c. twice daily injections) with unfractionated heparin (5000 units s.c. three times daily) and enoxaparin (40 mg s.c. daily), for the prophylaxis of DVT in patients undergoing major orthopedic surgeries. After 8–12 days of treatment, desirudin proved to be superior to both heparin anticoagulants, while showing a similar safety profile [57, 58]. Recently, desirudin was also under investigation as a potential anticoagulant for patients with heparin induced-thrombocytopenia (HIT) with or without thrombosis. Desirudin was also compared with argatroban in PREVENT-HIT study. This is a small, randomized, open-label trial comparing the clinical efficacy, safety, and economic utility of fixed-dose s.c. of drugs. However, just as lepirudin, desirudin is also renally excreted; there is still a risk of accumulation if the renal function is impaired [59].

Bivalirudin, formerly named Hirulog, is not properly a molecule from leech, but is a synthetic peptide (20 amino acids) [60] and bivalent analog of hirudin with a thrombin inhibition activity nearly 800 times weaker than that of hirudin [61]. Unlike the rH, the binding of bivalirudin to thrombin is reversible, and after the binding, the inhibitor is slowly cleaved by thrombin. Then, thrombin activity is only transiently inhibited and its enzymatic activity is restored. This reversible relationship between bivalirudin and thrombin can be seen as a benefit, once may contribute 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 rHs, and an additional advantage of bivalirudin was its lack of immunogenicity [64].

[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

Anticoagulants from Hematophagous http://dx.doi.org/10.5772/intechopen.78025 45

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 con-

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

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

Therostasin is a powerful inhibitor for FXa of the "tight binding" type, isolated from *T. tessulatum*, featuring a Ki of 34 pM [82]. The cDNA (825 bp) encodes 82 amino acids polypeptide (with 16 of them being cysteines) preceded by 19 residues representing the signal peptide. Therefore, just as other inhibitors, therostasin is expressed and kept in cells from the salivary glands of leeches [82]. Vizottin is a FXa inhibitor from the salivary complex of the leech *Haementeria vizottoi*. It has shown anticoagulant effects in human plasma, prolonging the recalcification time in a dose-dependent manner (IC50 40 nM). Vizottin was able to induce blood incoagulability in FX-deficient plasma, whereas in normal and reconstituted plasma, vizottin doubled the prothrombin time at 160 nM. At high concentrations, vizottin inhibited the amidolytic activity of factor VIIa/tissue factor (IC50 96.4 nM). It is a compound which is also able to inhibit FXa in

different thrombosis models, antistasin proved superior to heparin [91].

time in 50%, when compared with the control [92].

*2.1.4. Other inhibitors of factor Xa*

of 0.2 IU for factor Xa inhibition.

*2.1.3. Factor Xa inhibitors*

stantly have been sought.

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].
