*2.1.5.2. Disintegrins*

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

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

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

Leech antiplatelet protein (LAPP) is a specific inhibitor by collagen pathway from *Haementeria officinalis* leech salivary glands. It has around 13 kDa and pI 4.0. Recombinant LAPP (rLAPP) is able to inhibit collagen-mediated platelet aggregation under test-tube stirring conditions (IC50 ∼ 60–100 nM) and, also, it is able to block platelet adhesion to soluble collagen under static conditions, a step mediated by integrin α2β1 [96, 97]. There are reports demonstrating that this recombinant prevents integrin α-I domain binding to collagen with IC50 ∼ 125 nM [98]. The platelet adhesion to collagen type I is inhibited by rLAPP at high shear rate (1600 s−1) and this inhibitor is also able to prevent the binding of vWF to collagen type III [99]. In spite of this, rLAPP inhibits platelet deposition to cross sections of human atherosclerotic coronary arteries [99], and studies in baboons proved that rLAPP did not block collagen graft thrombosis, suggesting that inhibition of collagen alone is not enough to prevent thrombosis, possibly because TF exposure plays an important role in the model [100]. The crystal structure of LAPP has been determined and consists of a C-terminal domain which is very compact and a

Calin is isolated from the salivary secretion of the European leech *H. medicinalis,* as well as the rLAPP; it is able to inhibit the vWF-binding and platelet adhesion to collagen both under static and flow conditions [102]. Similarly, Saratin, from *Haementeria ghilianii* leeches, has been described as a platelet aggregation inhibitor that acts on collagen preventing the binding to integrin α2β1 and vWF [103]. The recombinant Saratin was obtained in yeasts (*Hansenula polymorpha*) [104] and it is being commercialized by BioVascular which has developed this product to GMP standards and is evaluating the effects in clinical studies [105]. To date, in the literature, only a few animal studies have been published, where it has been given alone or together with other drugs in glaucoma rabbit models [106, 107]. Saratin, when administrated alone in rat carotid endarterectomy model, significantly decreased platelet adhesion,

important in the progression of, or the response to, various diseases [95].

structure of this molecule still need to be better studied [93].

with 30 kDa and pI of 5.7 [94].

46 Anticoagulant Drugs

*2.1.5. Antiplatelet agents*

*2.1.5.1. Collagen-binding proteins*

disordered N-terminal region [101].

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 ornatin.

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

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].

#### *2.1.6. Regulators of fibrinogenolysis*

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