*2.1.4. Other inhibitors of factor Xa*

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 the prothrombinase complex and Gla-domain less FXa. The authors demonstrated that the inhibition of FXa by vizottin is through binding to the active site rather than an exosite. The structure of this molecule still need to be better studied [93].

intimal hyperplasia, luminal stenosis, and thrombosis. This inhibitor did not increase suture line bleeding or bleeding times, and did not decrease platelet counts. In this study, the authors also have concluded that Saratin may serve as a topical agent to be used for the site-specific

Anticoagulants from Hematophagous http://dx.doi.org/10.5772/intechopen.78025 47

Disintegrins were first discovered in snake venoms where they are very well studied, and were instrumental in our understanding of integrin function and also for the development of antithrombotic drugs [109]. However, this molecule class also has been found in bloodsucker animals. In leeches, there are two more studied molecules with this profile, decorsin and

Decorsin is a 39 amino acids protein purified from *Macrobdella decora* leech salivary glands that acts as an antagonist of glycoprotein GPIIb-IIIa. This disintegrin, like snake family of inhibitors, has six cysteines and an RGD motif near its C-terminus. It completely inhibits platelet aggregation ADP induced at high concentrations (1 μM) and is able to inhibit the interaction of GPIIb-IIIa with fibrinogen in ELISA assays (IC50 ~ 1.5 nM). The secretion of decorsin in the salive of this animal probably is one of its strategy to keep host blood flowing or to keep ingested blood from clotting, as leeches store ingested blood for long periods of time [31]. The structure of decorsin was determined by nuclear magnetic resonance (NMR)

Ornatin is a disintegrin described on *Placobdella ornate* leech that is 40% similar to decorsin. Studies with ornatin demonstrated that it is able to inhibit fibrinogen binding to GPIIb-IIIa (IC50 ~ 5 nM); on the other hand, it inhibits platelet aggregation at higher concentrations (IC50 ~ 300 nM) [110]. Studies with the recombinant protein demonstrated that the native disulfide bonds are required for the optimal GPIIb-IIIa antagonist activity of the ornatin [111].

As described in this chapter, various thrombin inhibitors from hematophagous animals together with other kind of anticoagulant as FXa inhibitor and anti-platelets not only maintain anticoagulant potential of the salivary gland secretions but also play a role of blood preservatives in the gut channel of the bloodsuckers. On the other hand, little is known on the degradation of fibrinogen and fibrin by secretions of bloodsuckers. However, we relate here some data obtained about molecules from some leeches of *Haementeria* genus and from specie

Factor XIIIa promotes the covalent crosslinking of fibrin polymers and incorporation of proteins into the fibrin network and thus the thrombus can be stable and relative resistance to plasmin-mediated degradation. Besides, FXIIIa is involved in other processes such as wound healing and arteriosclerosis. Therefore, selective FXIIIa inhibitors may be a valuable tool for evaluation of the various functions of FXIIIa and their pharmacological control [112]. In this field, a potent FXIIIa inhibitor was found in leeches. Tridegin was discovered in salivary glands of blood-sucking leech, *Haementeria ghilianii*. It is a highly specific inhibitor of factor

and it is interestingly similar to that of hirudin from *Hirudo medicinalis* leech [32].

*Hirudo medicinalis* that act as regulators of fibrinogenolysis and/or fibrinolysis.

inhibition of thrombosis and intimal hyperplasia after vascular manipulation [108].

*2.1.5.2. Disintegrins*

*2.1.6. Regulators of fibrinogenolysis*

*2.1.6.1. FXIIIa inhibitors*

ornatin.

A FXa inhibitor has been described in leech that are proven not part of the antistasin-family, the Lefaxin. This inhibitor was obtained from the salivary glands of the Brazilian leech, *Haementeria depressa*. It is a competitive inhibitor of FXa with a Ki of 3.6 nM, and is able to inhibit the FXa also in the prothrombinase complex with IC50 of 10 nM. It has a simple chain with 30 kDa and pI of 5.7 [94].

Among the FXa inhibitors from leeches, antistasin was the one that came closest to drug development; however, it did not get there. Even if these natural substances, as antistasin, are not being directly used in the human medical clinic, it was through the study of them that synthetic molecules focused on FXa were and are still being designed. This has provided potent and selective tools for evaluating the potential role of FXa in various diseases. In addition, these advances have been instrumental in defining the biology of FXa and have aided in the discovery of specific receptors and intracellular signaling pathways for FXa that may be important in the progression of, or the response to, various diseases [95].
