*2.1.6.2. Fibrino(geno)lytics molecules*

Hementin is responsible for proteolysis of blood fibrinogen with formation of products which block conversion of fibrinogen into fibrin catalyzed by thrombin; this molecule was discovered in salivary gland from *Haementeria ghilianii* [115]. Since fibrinogen is involved in the formation of platelet clot, hementin is able to prevent the platelet aggregation induced by ADP and collagen; on the other hand, it can also induce disaggregation of platelet aggregation induced by ADP, but not collagen [116]. Hementin can lyse fibrin clots; but its fibrinolytic activity is less potent than the fibrinogenolytic one. It does not influence the activity of other plasma proteins [117].

It was also demonstrated that plasma clots formed in the presence of tridegin are more sensitive to lyses by hementin (time required for 50% lysis in the presence and absence of hementin was 16 and about 22 h, respectively) [118]. Study of lysis of clots formed from PRP revealed that in the presence of tridegin the effect of fibrinolytic enzymes was the same as in PPP, whereas lysis of platelet-containing clots occurred slower. Thus, the importance of the platelets in the resistance of plasma clots to fibrinolytic enzymes and also the importance of cross-linking in this process [119].

Considering that both molecules are obtained in the same leech species, it was suggested that hementin and tridegin have a synergic action in feeding process of *Haementeria ghilianii*. They may be considered as promising thrombolytic agents.

Hementerin (HT) is a single-chain 80 kDa, Ca++-dependent metalloproteinase, which specifically degrades fibrin(ogen) through a plasminogen-independent pathway. The amino terminal sequence of 8 residues shows 80% similarity with hementin. However, their activities differ somewhat in terms of kinetics and with regard to the structure of the fibrin(ogen) fragments they may produce. Cleavage by HT of fibrinogen A-alpha, gamma, and B-beta chains, in that order, produces fragments differ from those produced by plasmin. HT was also able to degrade cross-linked fibrin although at a lower rate as compared to fibrinogen. HT is a plasminogenindependent fibrino(geno)lytic metalloproteinase that degrades fibrinogen faster than fibrin, prevents the coagulation and destroys fibrin clots *in vitro* [120]. The action of HT was also studied in different platelet assays and the studies have indicated that HT is an effective inhibitor of human platelet aggregation, presumably through activation of the platelet's nitridergic pathway [121].

Destabilase was discovered in salivary glands from *Hirudo medicinalis* and it was able to hydrolyze the epsilon-(gamma-glutamyl)-lysine bonds as a result of fibrin stabilization by FXIIIa in the presence of calcium ions [122]. It was characterized as a polyfunctional molecule and is a unique representative of invertebrate lysozymes. This molecule combines the properties of endo-s-lysyl-y-glutamyl isopeptidase (D-dimer monomerase), lysozyme, and chitinase and simultaneously is also a non-enzymatic antimicrobial agent. Its ability to hydrolyze endoisopeptide bonds formed by transglutaminases, which are involved in many pathological conditions, including thrombosis, causes this enzyme to become a focus to seek its use in practice [123], on the other hand, none was presented after that.

The substrate of destabilase is the D-D-dimer, a protein of 190 kDa that contains fragments of all three chains of monomer fibrin (alpha, beta, and gamma) and there is a nonlinear dependence of the reaction rate on substrate concentration. The crosslinked fibrin is also a substrate of destabilase, which catalyzes hydrolysis of isopeptide bonds connecting gamma-gamma and alpha-alpha-chains of this protein [124, 125].

Recently, a study demonstrated an optimization procedures related to the expression, isolation, and purification of active destabilase isoforms (mlDL-Ds1, 2, 3) using an *Escherichia coli* expression system, where their muramidase, lytic, isopeptidase and antimicrobial activities were detected and compared. Analyses of the tested activities revealed that all isoforms had almost identical patterns of pH and ionic strength effects. It was determined that three isoforms possessed nonenzymatic antibacterial activity independent of their muramidase activity. It was also demonstrated, for the first time, the fibrinolytic activity of the recombinant destabilase and showed that only intact proteins possessed this activity, suggesting being an enzymatic property [126].
