The circulation of blood is pivotal for our survival. During injury, the integrity of the system is compromised, and processes such as vasoconstriction, platelet aggregation, blood coagulation and fibrinolysis play a vital role in restoring the balance. Any imbalance in its regulation could either lead to either unclottable blood, resulting in excessive bleeding, or unwanted clot formation, resulting in death and debilitation due to vascular occlusion with the consequence of myocardial infarction, stroke, pulmonary embolism (PE) or deep-vein thrombosis (DVT) Citation[1]. Globally, with changing food habits and lifestyles, atherosclerosis and thromboembolic disorders are taking center stage and unwanted clots have become a leading causes of death. In the USA alone, it is estimated that 2 million people develop DVT each year. DVT progresses to PE in 600,000 of these patients, and the PE is fatal in 200,000 patients/year Citation[2]. Thus, anticoagulants are crucial for the prevention and treatment of thromboembolic disorders. Heparin and coumarin derivatives (vitamin K antagonists) are the cornerstones of anticoagulation therapy. Unfortunately, both classes of drugs have well-documented limitations, such as a narrow therapeutic window and highly variable dose-response. This is further complicated by other factors such as dietary intake Citation[3]. These limitations drive the continual and intense efforts to develop new anticoagulants targeting specific coagulation factors Citation[4].
To search for new lead molecules, we (and) others have been focusing on isolating and characterizing highly specific anticoagulants from blood-sucking (hematophagous) animals. Over the years, a number of exogenous anticoagulant agents have been purified and characterized Citation[5–8]. Based on their mechanism of anticoagulant action, they can be broadly classified as:
• Thrombin inhibitors
• FXa inhibitors
• Extrinsic tenase complex inhibitors
• Intrinsic tenase complex inhibitors
Thrombin inhibitors
The most well-known example of thrombin inhibitor, hirudin, was isolated more than 50 years ago from the medicinal leech Hirudo medicinalisCitation[9]. Since then, several different classes of thrombin inhibitors from hematophagous animals with distinct structural features and mechanisms have been described. Hirudin Citation[9] and variants (eg., bufrudin Citation[10,11]) have 65 residues with a core β-sheeted structure stabilized by three disulfide bridges and an extended, negatively charged C-terminal tail. They bind to the thrombin active site with their compact N terminus and to thrombin exosite-I with their acidic C terminus Citation[12]. Based on these functional segments, a short synthetic thrombin inhibitory peptide hirulog/bivalirudin was designed Citation[13] and is currently a US FDA-approved drug for percutaneous transluminal coronary angioplasty Citation[14]. Variegin, a new class of thrombin inhibitor from hard ticks, is a short and flexible peptide (32 residues) with an acidic C-terminal tail Citation[15]. It binds to both the thrombin active site and exosite-I through its N- and C terminus, respectively. Structurally and functionally, it is similar to hirulog/bivalirudin. Interestingly, hemadin (also isolated from leeches) binds to thrombin exosite-II with its C terminus, in contrast to hirudin, despite their structural similarity Citation[16,17]. Thrombin inhibitors, ornithodorin (from soft ticks) Citation[18] and boophilin (from hard ticks) Citation[19] have two tandem Kunitz domains. Their first domain binds noncanonically to the active site while their second domain binds to exosite-I. A similar two-domain binding mechanism is also observed for Kazal-type inhibitors such as rhodniin (from kissing bugs), but it interacts through a canonical reactive-site loop Citation[20,21]. Triabin (also from kissing bugs) belongs to the lipocalin family of proteins and binds to thrombin exosite-I but not its active site Citation[22]. Similarly, other thrombin inhibitors from hematophagus animals, such as anophelin Citation[23,24], chimadanin Citation[25], madanin Citation[26], NTI-1 Citation[27], microphilins Citation[28], TTI Citation[29] and theromin Citation[30], are all unique molecules with distinct structures and mechanisms.
FXa inhibitors
As with thrombin inhibitors, FXa inhibitors from hematophagous animals also belong to different classes with distinct structural features and mechanisms. One the main classes of FXa inhibitor is TAP Citation[31,32] and FXaI Citation[33,34] isolated from soft ticks. These molecules are single-domain Kunitz-type inhibitors. TAP inserts its N-terminal peptide into the FXa active site in a noncanonical fashion. A secondary epitope makes additional contacts with the FXa autolysis loop and the Na+ binding loop Citation[31,32]. A group of Ascaris-type inhibitors, NAP5 and NAP6 from hookworms, inhibit both the FXa active site (albeit canonically) and the autolysis loop Citation[35,36]. AceAP1 (also from hookworm) is a structurally similar but mechanistically different FXa inhibitor compared with NAP5 and NAP6 Citation[37,38]. AceAP1 needs a different exosite on FXa to which it binds with lower affinity for full inhibition of the FXa active site, thus acting as a partial noncompetitive inhibitor Citation[37,38]. Antistasin-like (antistasin Citation[39–41], ghilanten Citation[42] and therostasin Citation[43], all from leeches) FXa inhibitors belong to a different structural class. They have a unique sequence and cysteine positioning. There are two tandem repetitive domains with five disulfide bonds each, except in therostasin, which retains only the first domain. All of them inhibit FXa with a mechanism similar to that of NAP5. In addition to these specific FXa inhibitors, there are extrinsic tenase complex inhibitors from hematophagous animals that interact with the FXa heparin-binding exosite (see below).
Extrinsic tenase complex inhibitors
There are two main groups of extrinsic tenase complex inhibitors isolated from hematophagous animals. Both classes of inhibitors act through a similar, but not identical, mechanism as the physiological inhibitor, tissue factor pathway inhibitor. The first group of inhibitors was isolated from hard ticks. They have tandem repeats of Kunitz-type inhibitor domains; ixolaris Citation[44] and penthalaris Citation[45] have two and five tandem domains, respectively. It is postulated that the second Kunitz domain of ixolaris Citation[44] first binds to FX/FXa (on a heparin binding proexosite/exosite Citation[46,47]) before binding to the FVIIa–TF complex via its first domain to form a quaternary complex. The second group of inhibitors was isolated from hookworms. They are single-domain Ascaris-type inhibitors, such as NAPc2 (and its isoforms NAPc3 and NAPc4) Citation[48] and AceAP1 Citation[37,38]. NAPc2 binds to the FXa heparin binding exosite with its extended C terminus before binding to the FVIIa–TF complex Citation[49]. On the other hand, AceAP1 uses FXa as a scaffold by binding to both its active site and exosite (yet to be identified) and forms a quaternary complex with the FVIIa–TF complex Citation[37,38].
Intrinsic tenase complex inhibitors
So far, only one intrinsic tenase complex inhibitor, nitrophorin-2 (or prolixin-s) isolated from kissing bugs Citation[50,51], has been characterized. It belongs to the lipocalin protein family and binds specifically to the FIX/FIXa Gla-domain. This binding interferes with FIX activation (by both the FVIIa–TF complex and FXIa) as well as with FIXa activity in the intrinsic tenase complex Citation[50,52].
Molecular diversity in exogenous anticoagulants
Blood-feeding is crucial for the survival of hematophagous organisms and hence they have a number of anticoagulant proteins in their saliva that specifically target blood coagulation proteinases. Thus, hundreds of millions of years of evolution have provided the driving force for the molecular diversity observed in these exogenous anticoagulants. One can observe the functional diversity among closely related proteins as well as the functional convergence among structurally unrelated proteins. In the first case, a set of molecular scaffolds are being used to target various stages of the blood coagulation cascade. In this scenario, the molecular surface is altered through evolution and tailored to recognize distinct serine proteinases. Generally, the same sets of scaffolds are found in closely related species, whereas different sets of scaffolds are found in phylogenetically distant species. For example, exogenous anticoagulants from ticks have Kunitz-type inhibitor scaffolds, while those from hookworms and leeches have Ascaris-type or hirudin-like inhibitor scaffolds, respectively. Thus, potentially new sets of scaffolds are created each time an independent adaptation of blood-feeding behavior occurs. At times, the same domains are duplicated and the proteins containing tandem repeats exhibit distinct/altered specificities Citation[53,54]. When a domain is repeated in tandem, new functions can be gained through mutations. For instance, in soft ticks, FXa inhibitors (TAP Citation[31,32] and FXaI Citation[33,34]) have a single Kunitz domain-inhibiting active site, while thrombin inhibitors (ornithodorin Citation[18], savagnin Citation[55] and monobin Citation[56]) have two domains targeting both active site and exosite-I. Furthermore, in some animals more than one scaffold is utilized to generate distinct anticoagulants during evolution. For example, anticoagulants from kissing bugs have both Kazal-type inhibitors and lipocalin-like scaffolds. Similarly, hirudin-like and antistasin-like scaffolds are widely present in anticoagulants from leeches.
On the other hand, functional convergence by structurally diverse molecules is equally evident. For example, thrombin inhibitors from hematophagous animals belong to at least twelve different classes, while FXa inhibitors belong to six different classes. Despite such diversity in structures, they recognize a limited number of molecular surfaces. For example, thrombin exosite-I and its active sites are targeted by hirudin-like Citation[9–11], variegin Citation[15], Kunitz-type Citation[18,19] and Kazal-type Citation[20,21,53,57] thrombin inhibitors. Structure–function relationships of such functionally convergent anticoagulants will help us to delineate their functional sites and to design novel anticoagulants de novo.
Five-year view
To date, much valuable information has been gained from the study of a small number of exogenous anticoagulants from hematophagous animals. These molecules can either be used as a drug (e.g., hirudin Citation[9]) or provide a template for drug designs (e.g., bivalirudin Citation[13]). In addition, studies on these exogenous anticoagulants often reveal new mechanisms to inhibit coagulation factors. Furthermore, detailed structural analyses of the inhibitor–proteinase complexes often reveal new exosites on coagulation factors that are susceptible to inhibition. Using this information, we can design novel FXa inhibitors with different mechanisms based on competitive Citation[31,32,35,36,39–41], noncompetitive Citation[58,59], partially noncompetitive Citation[37,38] and uncompetitive Citation[60,61] inhibitors. Since blood coagulation factors all belong to serine proteinase with highly similar active sites, high specificity can often be achieved through molecular interactions with distinct exosites Citation[62]. The inhibitors targeting specific exosites will have minimal side effects related to nonspecificity. Bivalirudin is a good, specific thrombin inhibitor designed to target thrombin exosite-I and the active site is based on information derived from the studies on hirudin C-terminal interactions with thrombin Citation[13].
Currently, only a very small number of hematophagous animals (∼50, based on articles listed on the PubMed database) have been studied, also partially for their anticoagulants. In nature, hematophagous animals include an estimated 15,000 species of arthropods (>400 genera) Citation[6] plus a large number of leeches and hookworms. It is postulated that in hematophagous arthopods alone, blood-feeding behavior has evolved independently at least six times Citation[6]. This huge number of species translates into an enormous pool of structurally and functionally diverse exogenous anticoagulants. One of the major limitations in their study is the availability of only small amounts of salivary gland extracts, which leads to difficulties in the identification, isolation and characterization of these interesting proteins. Recent progress in large-scale and sensitive genomic, transcriptomic, structural and proteomic analysis has helped to ease this problem. Thus, discoveries of new exogenous anticoagulants from distinct lineages will be made in the foreseeable future and studies of the existing and newly characterized anticoagulants will provide an ideal, fertile and exciting future for the development of anticoagulant therapies.
Acknowledgement
The authors acknowledge VM Girish for valuable discussions on this work.
Financial & competing interests disclosure
Koh receives support from Graduate Research Scholarship, National University of Singapore. This work is supported by the Academic Research Fund, National University of Singapore. The authors have no other relevant affiliations or financial involvement with any organization or entity with a financial interest in or financial conflict with the subject matter or materials discussed in the manuscript apart from those disclosed.
No writing assistance was utilized in the production of this manuscript.
Related Research Data
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