**1.2. A little about hirudotherapy**

The number of compounds that have been discovered with anticoagulant potential in leeches and other bloodsuckers has become increasingly larger with the advent of transcriptomic analysis [11–13]. Nevertheless, the use of the animal itself has being also an alternative. Hirudotherapy, after many time, re-emerged in the 1970s. Leeches have been used with success in some kinds of microsurgeries and surgeries, as for example, in repair of lost limbs or still in plastic surgeries, where the animal helps in the blood fluid reestablishment [14, 15]. Hirudotherapy has also been used to treat soft tissue swelling and hematomas in trauma [16]. In the literature, you can find many reviews that report the use of this animal in different applications [14, 16–18]. On the other hand, a study where the use of leeches as an adjunct for the management of venous congestion after reconstructive surgery, realized with 87 patients, demonstrated that the morbidity associated with it should be considered, particularly the need for a blood transfusion [19].

There are even studies that demonstrate the opinion of the population regarding the use of the animal in surgery, of course that is not very comfortable for the patient. However, in general social cognition, the acceptance of hirudotherapy may not be very welcome at first, but provided with proper information and explanations, overall compliance of patients and caregivers can be improved and consequently result in superior outcomes in flap salvage [20].

Actually, no international protocols on leech therapy instructions have been established; some reported that leech application for a week is sufficient to get good results [21, 22]. However, it already was published in the literature, a guide of procedure for the use of leeches in surgical interventions [23].

#### **1.3. Hematophagous animals and role in hemostasis**

In all animals, the circulatory system exists in a perfect balance between coagulation (clot formation) and fibrinolysis (clot dissolution); in other words, to maintain blood in a fluid state, minimizing blood loss when the vascular system is injured. In this mechanism, endothelial cells and platelets are extremely important to form the hemostatic plug helping to arrest bleeding. First, after injury, circulating platelets bind to collagen in the exposed vessel wall and aggregate, and second, the clotting factors are activated in the coagulation cascade, resulting in the formation of a fibrin clot. Thus, hemostasis coagulation reactions and fibrinolysis are crucial for the proper functioning of the hemostatic system [24].

Hematophagous animals such as bloodsuckers (leeches, mosquitoes, and ticks) are rich sources of anticoagulant molecules; among them, clotting inhibitors, fibrinogenolytics, plasminogen activators, and platelet inhibitors, all are present in their fluids and secretions, with roles on physiological processes such as feeding, digestion, self-defense, etc. Bloodsuckers access the blood fluid through the wound made by specialized structures targeting blood coagulation components, specially thrombin, factor Xa and the prothrombinase and tissue factor/FVIIa complexes, as a strategy to maintain blood incoagulable over a relatively long period of time. These new anticoagulants from hematophagous animals have opened new perspectives in the scientific area for basic and applied researches, having applications in the therapeutic area, as anticoagulant medication. Therefore, hematophagous animals have been an excellent target for studies, including pharmacologists [3, 25, 26].
