*3.1.1. Antiplatelet agents*

The primary response to injury is the activation of circulating platelets, which bind to collagen in the exposed vessel wall and aggregate, arresting bleeding. In addition, thrombin, a multifunctional serine protease, activates platelets by cleaving platelet receptors [24]. Thus, saliva from ticks possess molecules to able to target platelet activation and aggregation in several ways, some of them inhibiting thrombin-induced platelet activation [136], other interfering with the adhesion of platelet to collagen or other ligands [136] or inhibiting the activation of protease-activated receptors (PARs). An example of the first group is the Serpin IRS-2 (*I ricinus* Serpin-2) from *Ixodes ricinus* which inhibits platelet aggregation induced by both thrombin and cathepsin G [133]. Another Serpin, IxscS from *I. scapularis*, was described to inhibit thrombin and to interfere with platelet aggregation induced by thrombin or ADP [137]. Also, in *I. scapularis*, the enzyme apyrase (an adenosine triphosphate (ATP) diphosphohydrolase) degrades active ATP and ADP into non-active AMP [138].

Some molecules can interfere with the adhesion of platelets to collagen, for example, the tick adhesion inhibitor (TAI) from *Ornithodoros moubata* [139, 140]. Other inhibitors act by binding competition through an integrin recognition motif RGD or KGD preventing the binding to fibrinogen or other ligands to platelet receptors such as savignygrin from *O. savignyi* [141]. Variabilin is another anti-platelet RGD-containing peptide from *Dermacentor variabilis* [142]. Some inhibitors identified in *I. pacificus* and *I. scapularis*, known as ixodegrins, display some differences with variabilin by having cysteines flanking the RGD motif, and with savignygrin, which have a non-canonical RGD peptide inserted into a Kunitz fold [127, 143].

Other anti-platelet molecules from ticks were reported: monogrin from *Argas monolakensis* [144], moubatin, a lipocalin derived from *O. moubata* which inhibits collagen-induced platelet aggregation by scavenging thromboxane A2 [139, 140, 144], longicornin, isolated from the salivary gland of *Haemaphysalis longicornis*, which also inhibits collagen-mediated platelet aggregation [145].

### *3.1.2. Tenase complex inhibitors*

**3.1. Components affecting coagulation**

50 Anticoagulant Drugs

TF and FIXa) and/or platelet aggregation [132].

degrades active ATP and ADP into non-active AMP [138].

*savignyi* [135].

*3.1.1. Antiplatelet agents*

Ticks saliva has other strategies besides inhibiting blood coagulation factors, in order to facilitate the feeding. After injury, subendothelial tissue get exposed, activated platelets bind to exposed von Willebrand factor and collagen through its surface receptors and platelets release

there are three major mechanisms that regulate anticoagulation: TFPI, antithrombin III (ATIII), and protein C/thrombomodulin/activated protein C. Until now, there are no description of tick saliva components interfering with or imitating antithrombin, protein S, protein C, heparin, or thrombomodulin [24]. However, many ticks can inhibit thrombin-induced platelet aggregation. On the other hand, anticoagulant molecules from tick saliva also regulate hemostasis by inhibiting blood coagulation factors (FXa or thrombin) or tenase complexes (FVIIa/

Anticoagulants from tick saliva can be classified according with their biochemical characteristics and structure, some of them belonging to the Kunitz-type domain inhibitors and Serpin domain inhibitors [127]. Members of those families can modulate coagulation, inflammation, or vasoconstriction. For example, the Serpin IRS-2 (*I ricinus* Serpin-2) from *I. ricinus* inhibits cathepsin G and chymase, both known as mediators of platelet aggregation and inflammation [133], as well as to mediate vascular permeability [25]. Besides, Kunitz domain inhibitors are widely expressed and characterized as anticoagulants, some of them having just one Kunitz domain being able to inhibit factor Xa [134] or thrombin, such as savigin from *Ornithodoros* 

Depending on the mechanism of action, they can include platelet inhibitors, factor Xa inhibitors and thrombin inhibitors, since they are able to prevent blood clotting and maintain blood incoagulable. Those blood coagulation inhibitors from tick are the major focus of this section.

The primary response to injury is the activation of circulating platelets, which bind to collagen in the exposed vessel wall and aggregate, arresting bleeding. In addition, thrombin, a multifunctional serine protease, activates platelets by cleaving platelet receptors [24]. Thus, saliva from ticks possess molecules to able to target platelet activation and aggregation in several ways, some of them inhibiting thrombin-induced platelet activation [136], other interfering with the adhesion of platelet to collagen or other ligands [136] or inhibiting the activation of protease-activated receptors (PARs). An example of the first group is the Serpin IRS-2 (*I ricinus* Serpin-2) from *Ixodes ricinus* which inhibits platelet aggregation induced by both thrombin and cathepsin G [133]. Another Serpin, IxscS from *I. scapularis*, was described to inhibit thrombin and to interfere with platelet aggregation induced by thrombin or ADP [137]. Also, in *I. scapularis*, the enzyme apyrase (an adenosine triphosphate (ATP) diphosphohydrolase)

Some molecules can interfere with the adhesion of platelets to collagen, for example, the tick adhesion inhibitor (TAI) from *Ornithodoros moubata* [139, 140]. Other inhibitors act by binding competition through an integrin recognition motif RGD or KGD preventing the binding to

). Physiologically,

soluble vasoconstrictor mediators (ADP, serotonin, and thromboxane A2

To target blood coagulation, components from tick saliva have inhibitory activities on the extrinsic tenase complex in blood coagulation [132]. From the studies in *I. scapularis* tick (Acari: Ixodidae) [146], two classes of extrinsic tenase complex inhibitors were identified acting similarly, but not identically, to the physiological inhibitor, tissue factor pathway inhibitor (TFPI) [136]. The first group is represented by ixolaris [147], a 15.7 kDa molecule obtained from the cDNA library of the salivary glands of *I. scapularis* consisting of 140 amino acid residues containing 10 cysteine and two-Kunitz tandem domain which does not bind to FXa active site, in contrast TFPI. It was hypothesized that the second Kunitz domain of ixolaris binds first to FX/FXa (on a heparin binding proexosite/exosite) before binding to the FVIIa-TF complex via the first Kunitz domain. The native inhibitor has a molecular mass of 24 kDa, and both forms are equally effective as anticoagulants. Functionally, the Ixolaris is structurally distinct from human tissue factor pathway inhibitor (TFPI) [146]. The second group is represented by penthalaris [148], a five-Kunitz tandem domain which uses FX or FXa as scaffold to inhibit the FVIIa-TF complex.

#### *3.1.3. Factor Xa inhibitors*

One of the main classes of FXa inhibitors characterized from soft tick saliva is the atypical, non-canonical Kunitz-type inhibitors including the tick anticoagulant peptide (TAP), obtained from the *Ornithodoros moubata* tick [9] and FXa-inhibitor (FXaI) from *O. savignyi* tick [149] (Acari: Argasidae). Both inhibitors possess a single Kunitz domain, in contrast to the tandem Kunitz type thrombin inhibitors. Kinetically, both are slow, tight-binding, competitive inhibitors of FXa. The recombinant (rTAP) TAP has a single-chain acidic polypeptide composed of 60 amino acids including 6 cysteine residues, and is a competitive FXa inhibitor highly selective and reversible. Its molecular weight is 6.8 kDa, pI 4.5 and Ki of 0.588 for the native form, and Ki of 0.18 nM for the recombinant form, expressed in *Saccharomyces cerevisiae* [150–152].

Amblyomin-X is a FXa inhibitor identified molecule in the transcriptomics profile of the salivary glands by Expressed Sequence Tags (ESTs) from the hard tick *Amblyomma cajennense* (currently *Amblyomma sculptum*) [153], containing an unique structure with a N-terminal Kunitz-type domain of 60 amino acids and a C-terminal with 49 amino acids. Amblyomin-X is able to inhibit factor Xa, prothrombinase and tenase activities. As FXa inhibitor, Amblyomin-X acts as a noncompetitive inhibitor (Ki = 3.9 μM) of factor Xa. It is a substrate for plasmin and trypsin, but not for factor Xa and thrombin. The prolongation of PT and aPTT is reversible [154]. Interestingly, several studies pointed out Amblyomin-X as an anti-cancer molecule *in vitro* and *in vivo* [154–161].

*E. coli* as a 20 kDa protein sharing only few similarities with hirudin and more similarity with serine protease inhibitors of the antistasin family. Sculptin is a novel class of competitive, reversible, and specific inhibitor of thrombin because its mechanism of inhibition is slightly different than hirudin. The Ki is comparable with that of hirudin and lower then hirulogs. Interestingly, sculptin phylogenetically diverges from hirudin. Sculptin has not inhibitory activity on FXa, trypsin and plasmin. However, it is degraded by serine proteases including thrombin, thus would not requires antidotes. The sculptin fragments produced by thrombin have not thrombin inhibitory activity, while sculptin fragments produced by FXa can inhibit thrombin independently. Sculptin increases blood coagulation parameter in concentration dependent manner. Sculptin has been filed for patenting in Brazil [171].

Anticoagulants from Hematophagous http://dx.doi.org/10.5772/intechopen.78025 53

From soft ticks, Kunitz-type thrombin inhibitors include ornithodorin from *Ornithodoros moubata* [172], savignin from *Ornithodoros savignyi* [135, 173] and monobin from *Argas monolakensis* [174]. Kinetically, they are slow, tight-binding, competitive inhibitors of thrombin: savignin

As mentioned above, most Ixodidae ticks produce a cement or glue to attach to the host skin to facilitate the penetration of mouthparts for feeding. Interestingly, in *Amblyomma americanum,* the compositions of this cement revealed by the presence of glycine-rich proteins, lipids, and certain carbohydrates, besides serine protease inhibitors and metalloproteases. Some molecules from tick cement were considered promising candidates for an anti-tick vaccine because

Fibrinolytic enzyme with metalloprotease activity has been described in the hard tick *I. scapularis* [176]. On the other hand, an activator of plasminogen, called longistatin from *H. longicornis*, was found to cause hydrolysis of fibrinogen and delay formation of the fibrin clot as comparable to that of tissue-type plasminogen activator (t-PA) [177]. The recombinant form of longistatins is

To conclude, hematophagous animals have evolved effective means of inhibiting thrombosis, thereby facilitating the acquisition and digestion of a blood meal. To date, specific inhibitors of coagulation, platelet function and fibrinolysis regulators have been identified from numerous invertebrate species, mainly leeches, ticks, and mosquitoes, representing an impressive array of convergent functional strategies. These parasites may serve as potentially useful therapeutic agents for the treatment of a variety of conditions associated with activation of thrombosis. A number of anticoagulants and platelet inhibitors from bloodsuckers have been evaluated *in vivo*, with some currently in varying stages of preclinical and clinical development. Because of the unique specificity and potency of anticoagulants from hematophagous, these kinds of products hold great promise for improving the treatment of a variety of human

(Ki = 4.89 pM) [173], and monobin (Ki = 7 pM) [174].

able to inhibit inflammation associated to tick feeding [178].

of their antigenic properties [136, 175].

**3.2. Components affecting fibrinolysis**

illnesses, as heart disease and stroke.

**4. Conclusions**

Other FXa inhibitors were reported in *I. scapularis* belonging to the salivary protein (Salp) family, which specifically inhibits the FXa active site [162]. Other inhibitors act on FXa through binding to prothrombinase complex [163].
