1.1. Leeches work with secreted proteins

To date, many scientific studies have shed light on the effect mechanisms of leeches. Although more than 100 particular proteins with different molecular masses are observed in leech secretions, only a few have been identified that have a major active role.16 The effect mechanisms are divided into six types to make them more understandable, but these mechanisms are closely related to each other and should be evaluated as a whole (Table 1). Following a leech bite, it has to establish a sucking pathway (extracellular matrix degradation); inhibit adhesion, aggregation, and coagulation (inhibition of platelet functions, and anticoagulant effect); increase blood flow; protect itself (antimicrobial activity); and avoid detection (analgesic and anti-inflammatory effects).

1.2. Extracellular matrix degradation

Following the bite, leeches immediately release hyaluronidase (27.5 kDa) and collagenase (100 kDa) enzymes to facilitate tissue penetration and spread of their bioactive molecules. These enzymes also support antimicrobial activity.9, 10, 14

1.3. Analgesic and anti-inflammatory effects

It is believed that leeches exert analgesic and anti-inflammatory effects so as to avoid detection by the host while feeding.15 Despite this, it has not been possible to isolate any analgesic molecule acting in this way from leech secretions until now. So, studies have focused on indirect mechanisms to achieve this goal. For example, some studies have indicated that some kininases and antistasin may inhibit the kinin–kallikrein mechanism, which is a major nociceptive route.17 There is more information about the anti-inflammatory effects.

Antistasin was identified from H. officinalis (Mexican medicinal leech) and it serves as a potent factor Xa inhibitor and has an inhibitory effect on the kinin–kallikrein system.17 Factor Xa is a prothrombin activator, and plays a critical role in the common pathway of the coagulation cascade.32 The Kinin–kallikrein system is also connected to the coagulation cascade and has a major role in the inflammatory response.33 Researchers claim that antistasin has both anticoagulant and anti-inflammatory effects, but current studies have often focused on the anticoagulant activity, which seems to be the predominant mechanism of action.34 The Ghilantens were also found in secretions of Haementeria ghilianii (Amazonian Leech) and they show high structural homology with antistasin. There are only few data about their anticoagulant effects, and other possible functions are controversial due to lack of additional studies.18, 19

Leech-derived tryptase inhibitor (LDTI) has three isoforms (a, b, and c) and acts by inhibiting proteolytic enzymes of mast cells. LDTI, a Kazal-type serine protease inhibitor, especially inhibits mast cell tryptase, but also trypsin and chymotrypsin.20 Mast cell tryptases are serine proteases in cell granules and their release causes inflammatory reactions. These effects are strongly related to the kinin–kallikrein system, chemotaxis, leukocyte activation, vasoactive actions, and accordingly, pain-generating interactions. Their levels are correlated with allergic and inflammatory diseases such as anaphylaxis, asthma, and arthritis.35, 36 LDTI is an inhibitor of mast cell tryptase, trypsin, chymotrypsin, thrombin, and plasmin, but inhibitory effects on factor Xa, plasma kallikrein, and neutrophil elastase are controversial.37 Even with inhibition of mast cell tryptase, potential benefits of anti-inflammatory effects can be foreseen. However, recombinant LDTI has shown inconsistent actions in different studies, so it is hard to comment on actual clinical effects of LDTI.37, 38

Eglin C is an inhibitor of human neutrophil elastase and cathepsin G.14 These two enzymes are immune serine proteases in the chymotrypsin family that are stocked in azurophil granules of polymorphonuclear neutrophils and released as a part of the inflammatory response.39, 40 Inhibition by eglin C causes decreasing levels of free oxygen radicals in neutrophils and prevents tissue inflammation and destruction. In test models, eglin C was shown to be a potential therapeutic agent for shock and emphysema.14 Further studies are needed to show other potential effects, but the molecule itself is promising. Other isolated eglins act in similar ways, resulting in anti-inflammatory effects. Another leukocyte elastase inhibitor is cysteine-rich guamerin, which was isolated from H. nipponia (Korean medicinal leech). From the same leech, piguamerin was also isolated and has an inhibitory effect on kallikrein and trypsin. As previously stated, hirustasin (Hirudo antistasin) is a serine protease inhibitor and acts as an inhibitor of kallikrein, trypsin, chymotrypsin, and cathepsin G. It was isolated from H. medicinalis (European medicinal leech) and H. officinalis (Mexican medicinal leech).10, 14 Separately, bdellins and bdellastasin were detected as trypsin, plasmin, and sperm acrozine inhibitors.14, 18 Human neutrophil elastase and cathepsin G have activating effects on factor X (prothrombin activator) and enhancing activity on factor XII and tissue factor, so, as a result, their inhibition by these substances may cause additional anticoagulant outcomes, but this area needs further study.40, 41

Complement component C1 has a critical role in the classic pathway of the complement system.42 In leech secretions, complement C1 inhibitor is a 60- to 70-kDa protein, but the effect mechanism is only partially known.21 This protein might be just one part of the protein pool that inhibits the complement system in many ways. In addition, the original C1 inhibitor in humans suppresses factor XIIa, factor XIa, plasma kallikrein, and thrombin. This substance inhibits both the coagulation cascade and kinin–kallikrein system.43 Currently, there are no data of similar effects of leech C1 inhibitor, but it is possible and needs further study.

The mechanism causing the inhibitory effect on carboxypeptidases (kininase 1) is contentious. The enzymes carboxypeptidase N and M participate in kinin degradation, resulting in agonism of B receptors, which causes a bradykinin-related inflammatory response.33 Inhibition of carboxypeptidases by leech secretions should not affect bradykinin action via B2 (constitutive) receptors, but may prevent B1 (inducible) receptors. Although these two receptors basically work with similar mechanisms, it has been stated that B1 receptors are related to chronic inflammation, whereas B2 receptors are related to acute inflammation. Strong correlations have been found between B1 and inflammatory diseases such as multiple sclerosis, asthma, and rheumatoid arthritis. However, studies have indicated that action of bradykinin is not limited to these receptors, so possible anti-inflammatory effects of carboxypeptidase inhibition are controversial and should be tested separately.44

1.4. Increasing blood flow

Leech feeding and therapeutic effects require increased blood flow. These are achieved mainly by histamine-like molecules that cause vasodilatation and arise via local vascular permeability.7, 10, 14 Acetylcholine is also a component in leech secretions, causing endothelial muscle relaxation and vasodilatation.10, 14, 45

1.5. Inhibition of platelet functions

Destruction of the blood vessel wall for sucking blood causes activation of platelets and the coagulation cascade, which are fatal for the leech. For this reason, leech secretions contain many bioactive molecules to locally inhibit these actions.

In a normal host, wall destruction causes spread and release of collagen particles and they are targets of free vonWillebrand factor (vWF). This complex strongly binds to glycoprotein (GP) Ib on platelets as vWF works like a bridge. With this binding, upregulatory mechanisms occur, especially with the critical role of adenosine diphosphate (ADP), and via GpIIb–IIIa and fibrinogen, platelets bind to each other to make a plug and stop any bleeding. This reaction also starts another chain of releasing substances such as thromboxane A2, platelet activation, and coagulation cascade.42 In leech secretions, various molecules (saratin, calin, decorsin, and apyrase) react against different parts of this chain.9, 10, 14

Saratin, a 12-kDa protein, affects only the initial stage of platelet adhesion, and inhibits collagen–vWF reaction competitively. Some animal studies have indicated promising results with recombinant saratin molecule as a potential local therapeutic agent for antithrombotic therapies and atherosclerosis.22 Other leech-secreted proteins, calin and leech antiplatelet protein, show the same action on platelet adhesion.24 In contrast, decorsin, which is isolated from Macrobdella decora (American medicinal leech), is structurally similar to anticoagulant leech proteins hirudin and antistasin, but functionally it is an efficient GPIIb–IIIa inhibitor and acts potentially against platelet aggregation.24, 25

As previously stated, ADP has a critical role in platelet aggregation by especially activating GPIIb–IIIa receptors and increasing affinity of platelets to vWF.42 The enzyme apyrase converts ADP to adenosine monophosphate and blocks aggregation by indirectly inhibiting these receptor mechanisms. ADP also has strong relations with arachidonic acid, platelet-activating factor, and epinephrine activity, so additionally apyrase indirectly acts in an opposing way to these substances.14 An additional molecule is also described that acts as an inhibitor of platelet-activating factor and thrombin-induced platelet aggregation by suppressing thromboxane production in platelets.46, 47, 48

The enzyme collagenase also destroys collagen particles, which initiates all these adhesion and aggregation reactions, and provides additional supportive action to the inhibitory effects.46

1.6. Anticoagulant effect

Coagulation during feeding is fatal for leeches, so anticoagulant effects are necessary.15 The coagulation cascade is a chain reaction and bioactive molecules in leech secretions have effects at various points. Hirudin and gelin mainly work as thrombin inhibitors, factor Xa inhibitor breaks the chain reaction, and destabilase has a fibrinolytic effect.9, 14 Thrombin has a strong effect on platelet activation and ADP release and so these inhibitors may indirectly have a negative impact on platelet functions.42

Hirudin is a 7.1-kDa protein and irreversibly binds to thrombin, which causes consumption of active thrombin and results in antithrombin activity.7, 15 This substance is the most interesting one and was the subject of many studies. There is a strong consensus about it being a therapeutic alternative to heparin, since it has higher anticoagulant activity and fewer adverse effects.15 Gelin is an eglin analog and a potent thrombin inhibitor. Gelin also shows inhibitory effects on chymotrypsin, cathepsin G, and neutrophil elastase.14

Factor Xa inhibitor breaks the coagulation cascade and has a direct anticoagulant effect. It has a critical role in MLT of osteoarthritis and rheumatoid arthritis.9, 10 In addition, as previously stated, antistasin directly inhibits factor Xa,17 and ghilantens, LDTI, C1 inhibitor, and eglins have possible anticoagulant effects, potentially via direct and/or indirect inhibition of coagulation factors.18, 19, 20, 35, 36, 40, 41, 43

Destabilase is an enzyme with glycosidase activity and shows both antibacterial and fibrinolytic actions.26, 27 This enzyme has various isoforms with different capabilities, and is extracted from different leech species.28 Destabilase has a major degradative action on stabilized fibrin and it should also be evaluated as an anticoagulant agent.26

Recently, novel anticoagulant peptides from different leech species have been identified (new leech protein-1, whitide, and whitmanin). Many other peptides have also been isolated, but their function is unknown at present.29

1.7. Antimicrobial effect

To date, only two main molecules, destabilase and chloromycetin, have been shown to have antimicrobial activity.9, 10, 14 As previously stated, destabilase has β-glycosidase activity, which directly disrupts β1–4 bonds that are important in the peptidoglycan layer in bacterial cell walls.14, 49 It is clear that this action is similar to that of lysozyme (muramidase) that is commonly found in human saliva and lachrymal fluid.50 Other studies have shown that antimicrobial activity does not only depend on glycosidase enzymatic activity, but it also has nonenzymatic components.27 Even the denaturated form destabilase shows a dose-dependent bacteriostatic effect on Staphylococcus aureus, Pseudomonas aeruginosa, and Escherichia coli.51 Chloromycetin is a potent antibiotic found in leech secretions, but unfortunately the data are limited about this molecule. 9 Additionally, theromacin, theromyzin, and peptide B have been isolated as antimicrobial peptides.30, 31