Argatroban (Fig. 2A) is a synthetic arginine-derived direct thrombin inhibitor that exerts a stronger anticoagulant effect compared to heparins and hirudins at equivalent levels. It is hepatically metabolized and predominantly eliminated through the biliary system (7). Dose adjustments are necessary in patients with hepatic, but not renal, impairment. The bioavailability of orally administered argatroban is negligible and therefore, it needs to be administered by infusion. Its half-life is ~30 min. Argatroban can be used as a parenteral anticoagulant for all cases where intravenous administration of heparin is prescribed. It was approved by the Food and Drug Administration (FDA) in 2000 for the treatment of HIT and interventional anticoagulation during percutaneous coronary intervention (PCI). An increased dosage can be used for a coronary artery bypass graft, and a lower dosage can be used for DVT treatment (10). Argatroban passes through endovascular and cellular barriers owing to its low molecular weight. It is therefore effective for the antithrombotic treatment of microvascular disorders (11).

Dabigatran (Fig. 2B) is a synthetic benzamidine-derived direct thrombin inhibitor that rapidly and directly inhibits both free and fibrin-bound thrombin (7). It also exerts an inhibitory effect on thrombin-induced platelet aggregation and prevents the conversion of fibrinogen to fibrin. Dabigatran is metabolized by the glycoprotein system and eliminated through the kidneys (12). However, the absolute bioavailability of dabigatran after oral absorption is very low (6–7%) (13). Dabigatran was the first anticoagulant approved by the FDA for primary prevention of ischemic stroke and systemic thromboembolism in patients without valvular atrial fibrillation (7).

Dabigatran etexilate (Fig. 2C) is an orally administered direct thrombin inhibitor, developed to overcome the limited oral bioavailability of dabigatran. Once absorbed from the gastrointestinal tract, it is rapidly converted to the active form dabigatran. Bioconversion of dabigatran etexilate to dabigatran occurs in enterocytes, hepatocytes and the portal vein. Dabigatran circulates in the blood with a half-life of 12–17 h, which allows oral administration once a day. With a low potential for drug-drug interactions and a predictable anticoagulant effect, dabigatran etexilate can be administered in fixed doses without need for monitoring coagulation (14). In 2008, dabigatran etexilate was approved as a primary preventive agent for venous thromboembolic events (VTEs) in adult patients who underwent elective total hip or total knee replacement surgery in Europe. In October 2010, it was approved by the FDA to reduce the risk of stroke and systemic embolism in patients with non-valvular atrial fibrillation. However, this inhibitor is not currently indicated for treatment of any VTE in the USA (15).

Hirudin is isolated from the salivary glands of medicinal leeches, and has been used as an anticoagulant agent since 1909. Lepirudin and desirudin are two derivatives of hirudin. Lepirudin is composed of 65 amino acids that directly inhibit thrombin by simultaneously binding to its active site and to exosite 1. Lepirudin is intravenously infused and its dosage is dependent on body weight. It is eliminated through the kidneys, which accounts for ~90% of the systemic clearance. Lepirudin is licensed for the treatment of thrombosis complicating HIT. Moreover, unstable angina is an additional syndrome that lepirudin has the potential to treat (16).

Desirudin differs from lepirudin only in the first two N-terminal amino acids. It is also eliminated through the kidneys, accounting for 90% of the systemic clearance. Desirudin reaches maximal plasma concentrations 1–3 h after administration and has a terminal half-life of 2 h. In 2010, desirudin became the only FDA-approved fixed-dose subcutaneously administered DTI for the post-operative prevention of VTE in patients undergoing elective hip replacement surgery (17). Currently, desirudin is under investigation as a potential anticoagulant for HIT patients presenting or not thrombosis (3).

Bivalirudin is a synthetic analog of hirudin. It is a small polypeptide comprising 20 amino acids (D-Phe- Pro-Arg-Pro-Gly-Gly-Gly-Gly-Asn-Gly-Asp-Phe-Glu-Glu-Ile-Pro-Glu-Glu-Tyr-Leu). The N-terminal D-Phe-Pro-Arg-Pro region binds to the active site of thrombin with high affinity, and the C-terminal dodecapeptide Asn-Gly-Asp-Phe-Glu-Glu-Ile-Pro-Glu-Glu-Tyr-Leu binds to exosite 1 of thrombin (18). Bivalirudin exhibits a short half-life (25 min), predominantly non-renal metabolism, and low immunogenicity. It achieves peak plasma concentration within 2 min of intravenous bolus injection. Bivalirudin is primarily used for the treatment of percutaneous transluminal coronary angioplasty, the most frequent type of PCI. It is also indicated for PCI when provisional therapy with the GPIIb/IIIa antagonist is employed, and for patients with HIT or HIT with thrombosis syndrome (HITTS) undergoing PCI (19).

Previous studies have suggested that thrombin inhibitors that contain the tripeptide template D-Phe-Pro-Arg (P3-P2-P1), such as argatroban and dabigatran, are the most effective (23,24). This tripeptide can bind to multiple amino acids at the thrombin active site. It can bind to arginines, especially the positively-charged guanidine group interacting with the Asp-189 residue at the bottom of the S1 pocket. Hydrophobic amino acids, such as proline in the P2 position, can bind to the S2 pocket, and aromatic groups in the P3 position can interact with the lipophilic and aromatic fragments of the S3 pocket (25).

Typically, DTIs possess a P1 group that fills the specific S1 pocket. Generally, to produce potent inhibitors, the P1 ligand features a strongly basic functional group such as guanidine (argatroban), alkylamine, amidine, benzamidine (dabigatran), or 4-aminopyridine (26). Patents WO00/42059 (27), US2007/0249578 A1 (28) and US2010/0087651 Al (29) disclosed a series of compounds in which the P1 position was replaced with amidino, benzamidine, or their analogs. For example, compound 1 (Fig. 3), derived from formula 1 (Fig. 3), has an IC₅₀ of prothrombin time (PT) <0.02 μM, and an IC₅₀ of activated partial thromboplastin time (APTT) <1 μM. Compounds containing strongly basic amines in the P1 position have poor oral absorption properties and poor pharmacokinetics, because the basic amines are protonated at the pH of the intestinal tract.

Strategies to overcome these shortcomings include the use of a prodrug with a masked, weakly basic or non-basic, P1 group, such as dabigatran etexilate (30). N-hydroxylated derivatives such as amidoxime and benzamidoxime, are less basic because of the introduced oxygen atom. They are not protonated under physiological conditions and are expected to have sufficient oral absorption, and therefore, improved bioavailability (31). Patent US2008/0004256 A1 (32) disclosed a series of compounds derived from formula 2 (Fig. 3). Compound 2 (Fig. 3), where the P1 position is replaced by benzamidoxime, is the most bioavailable of these compounds. The inhibition constant (Ki) values for thrombin and trypsin are 0.59 and 32.23 μM, respectively, for this compound. Azoles (26,33) (such as imidazoles, aminothiazoles and N-acetamidoimidazole) and aryl heterocycles (pyridines, pyrazinones, piperidines and pyridinones) are also weakly basic groups. When they are incorporated into the P1 position, the resulting peptides exhibit very good selectivity for thrombin vs. trypsin and lower in vivo toxicity.

A recent study (25) demonstrated that the P1 fragment of the inhibitor does not need to contain a highly basic functional group to efficiently inhibit thrombin. The chlorophenyl fragment in the P1 position can be deeply inserted into the S1 pocket of the thrombin active site (25). In addition, heterocycle-substituted chlorophenyl incorporated into the P1 group was shown to provide more potent inhibitors (34). Patent US8119673 B2 (35) disclosed and described 57 D-Phe-Pro-Arg derivatives with heterocycle-substituted chlorophenyl incorporated into the P1 position, along with their synthetic routes. Compounds 3A and 3B (Fig. 3) were claimed in this patent. Their IC₅₀ values are 4 and 120 nM, respectively, according to a chromogenic robotic assay.

The P2 position of the tripeptide is important, not only in relation to its thrombin inhibitory activity, but also in relation to the oral bioavailability. In previous studies, it was found that using dehydroproline to replace the proline of the tripeptide slightly increases the in vitro potency, in vivo activity, and oral bioavailability (36), replacing it with 4-fluoroproline increased the oral bioavailability (34) and replacing it with a pyrazinone ring increased both activity and oral bioavailability (37). For example, compound 4 (Fig. 3), first described in patent US6455532 B1 (38), is a potentially new thrombin inhibitor with 3-aminopyrazinone in the P2 position. It inhibited thrombin activity with a Ki of 5.2 nM. The 3-aminopyrazinone in the P2 position of compound 3 played a crucial role in thrombin inhibition by forming an hydrogen bond with Gly-216 of thrombin. However, it was less resistant to oxidative metabolism. Researchers from Merck Research Laboratories found that 2-aminopyridine N-oxide in the P2 position confers reduced metabolic liability compared to that of pyrazinones (39).

Derivatives of organoboronic acids exhibit thrombin inhibitory activity. Patent US20070185060 A1 (40) disclosed a series of organoboronic acid compounds such as compound 5 (Fig. 4). This compound has thrombin inhibitory activity with an IC₅₀ <0.1 μm. It has a neutral aminoboronic acid residue, which can bind thrombin S1, linked to a hydrophobic moiety, which can bind thrombin S2 and S3. 1,4-Benzoxazine-3(4H)-one was found to be a suitable scaffold for a thrombin inhibitor. Compound 6 (Fig. 4), which was derived from this scaffold, is a highly potent and selective thrombin inhibitor with Ki of 2.6 nM for thrombin and 112,400-fold selectivity for trypsin. Due to the presence of the benzamidine moiety, it has considerable potential to show improved bioavailability and be developed as an orally active prodrug (41). Compounds derived from formula 3 (Fig. 4) are a series of non-peptide thrombin inhibitors that are described in patent WO2012/174865 A1 (42). These compounds have higher thrombin inhibitory activities compared to dabigatran etexilate. Non-classic tripeptide templates such as Tos-Phe-Arg-OCH₃ and X-D-Arg-D-Phe-OMe can also act as thrombin inhibitors. When X is replaced by myristic acid, the compound (Fig. 4) has high activity in thrombin inhibition (Ki=0.17 μM) and high selectivity towards thrombin in comparison to factor X, plasmin, and trypsin (>600-, 900- and 5,000-fold, respectively) (43).

Aptamers are 15–40 nucleotide, single-stranded DNA or RNA molecules forming three-dimensional structures that bind to their molecular targets with affinity and specificity (44,45). Due to their high binding affinities for target macromolecules, aptamers can act as inhibitors (46). ARC-183, or HD1, is a 15-nucleotide DNA molecule discovered in the 1990s. It is a strong anticoagulant in vitro, and inhibits the thrombin-catalyzed activation of fibrinogen and thrombin-induced platelet aggregation by binding to exosite 1. Despite its high affinity (Kd=30 nM), it has a short half-life (2.5 min). In order to overcome this disadvantage, another DNA aptamer (NU172) was developed. This aptamer binds to thrombin with a Kd of 100 pM and has a half-life of 25 min when tested in pigs. Toggle-25 is a 25-nucleotide RNA aptamer that contains 2′-fluoropyrimidine nucleotides selected to bind exosite 2 of thrombin with high specificity and affinity (Kd=2nM) (47). Other thrombin inhibitors, such as the DNA aptamer RE31 (48), the RNA aptamer HD22 (49), and R9D-14 (50) were also identified. These aptamers possess properties that render them especially suitable for use as thrombin inhibitors: they typically exhibit high affinity and specific binding to the target protein, and demonstrate little to no toxicity or immunogenicity (51).

The widely used in the clinic anticoagulant drugs such as hirudin and its analog lepirudin were isolated from blood-sucking leeches. For these animals to feed on blood from mammalian hosts, they need to prevent local clotting of the host’s blood. These parasites have developed various anti-clotting mechanisms, including the specific inhibition of thrombin (52). In addition to hirudin, which was first described in 1884 (53), other thrombin inhibitors such as haemadin, bufrudin and theromin have been isolated from various species of leeches (54). Haemadin is a slow, tight-binding thrombin inhibitor isolated from the species Haemadipsa sylvestris with an apparent molecular mass of 5 kDa. It exhibits a Ki as low as 100 fM (55). Bufrudin was isolated from the species Hirudinaria manillensis and has 66% sequence homology with hirudin, indicating that it acts as a similar inhibitor (56). Theromin, isolated from the gut leech Theromyzon tessulatum, is the most potent inhibitor (Ki of 12 vs. 21 fM for hirudin). It is a homodimer of 67 amino acid residues with 16 cysteines engaged in eight disulfide bridges (57).

Additional agents targeting thrombin were obtained from various hematophagous animals such as mosquitoes, ticks, and insects. Mosquito saliva carries a high number of factors with anticoagulant activities. Anophelin is a peptide with a molecular weight of 6.5 kDa, isolated from the salivary glands of the mosquito species Anopheles albimanus. It inhibits thrombin with an IC₅₀ of 45 nM and binds to both the active site and exosite 1 (58). An additional peptide of 45 kDa was identified and isolated from the salivary glands of the malarial vector mosquito Anopheles stephensi. It inhibits thrombin only by binding to the active site (59). AaTI is another peptide from the mosquito Aedes aegypti, which inhibits thrombin by binding to exosite 2 (60).

A few potential thrombin inhibitors were also isolated from ticks. Boophilin is a novel 20 kDa thrombin inhibitor isolated from the cattle tick Boophilus microplus. It inhibits thrombin with a Ki of 57 pM by binding to the active site and exosite 1 (61). Boophilin is composed of two canonical Kunitz domains, which inhibit not only thrombin, but also, other serine proteases (62). Microphilin is the smallest salivary thrombin inhibitor peptide known to date, with a molecular weight of 1.7 kDa. It was also isolated from the cattle tick Boophilus microplus. Microphilin inhibits thrombin in a dose-dependent manner with an IC₅₀ of 5.5 μM (63). From the midgut of the parthenogenetic tick species Haemaphysalis longicornis (Ixodidae), another thrombin inhibitor was isolated, known as hemalin. It contains two Kunitz domains and has high homology to boophilin. Hemalin inhibits thrombin in a dose-dependent manner with a Ki of 0.25 μM. It also inhibited both thrombin-induced fibrinogen clotting and thrombin-induced platelet aggregation (64). From the same species, two additional proteins that inhibit thrombin by binding to exosite 1 were identified, madanin 1 and 2 (65).

Other thrombin inhibitors such as triabin, infestin, brasiliensin and dipetalogastin were identified and isolated from blood-sucking insects. Triabin was purified from the saliva of Triatoma pallidipennis (66). Infestin was isolated from midguts of Triatoma infestans (67). Brasiliensin was identified and isolated from Triatoma brasiliensis (68). Dipetalogastin was found and isolated from Dipetalogaster maximus (69).

Besides these blood-sucking animals, other natural sources of compounds targeting thrombin include snake and wasp venom, and skin secretions of toads. TTI-Nh is a new 14.3-kDa protein isolated from venom of the cobra species Naja haje, and consisting of a single polypeptide chain with 14 cysteine residues that form seven intramolecular disulfide bridges. It inhibits thrombin with an IC₅₀ of 73 nM without any cytotoxic side-effects (70). Bothrojaracin is a 27-kDa anticoagulant protein isolated from the venom of the viper Bothrops jararaca. It inhibits thrombin by binding to exosites 1 and 2 (71). Bicolin was purified and characterized from the venom of wasps. It showed inhibitory activity against trypsin and thrombin (72). BMSI-1 is a serine protease inhibitor identified from skin secretions of the toad Bombina microdeladigitora. It inhibits trypsin and thrombin with a Ki of 0.02 and 0.15 μM, respectively. Furthermore, the activity of BMSI-1 was not lost after incubation in boiling water for 10 min, indicating that BMSI-1 is a thermally highly stable inhibitor (73).

Natural flavonoids such as myricetin and quercetin (Fig. 5) were discovered to act as thrombin inhibitors, but with low activity. The IC₅₀ of TT is 0.006 and 0.035 mM for myricetin and quercetin, respectively (74). Glycerolipids, especially the diacylglycerophospholipids isolated from the fungus Stereum hirsutum, can inhibit thrombin by binding to its active site (75). Scutellarin (Fig. 5), the major anti-oxidant in breviscapine extracted from the plant species Erigeron breviscapus, was clinically effective in treating acute cerebral infarction and paralysis induced by cerebrovascular diseases. However, it has certain disadvantages, such as low solubility in water and low bioavailability. Scutellarein (Fig. 5), the hydrolyzed product and metabolic form of scutellarin, was used to design and synthesize derivatives with improved biological activity and water solubility. Many of these derivatives, especially the morpholinyl aminomethylene substituent, showed stronger anticoagulant activity and higher solubility in water (76). A recent study identified thrombin inhibitors isolated from the freshwater cyanobacterium Anabaena compacta. These molecules, known as spumigin J and A (Fig. 5), inhibited thrombin with EC₅₀ values of 4.9 and 2.1 μM, respectively. In addition, the two compounds showed higher inhibitory activity against cathepsin B, with EC₅₀ values of 0.7 and 0.2 μM, respectively (77). Chlorodysinosin A, dysinosin A and oscillarin (Fig. 5), three natural products that belong to the aeruginosin family of serine protease inhibitors, exhibit low inhibitory activity against thrombin in vitro (IC₅₀=5.8, 46 and 28 nM, respectively). These molecules inhibit thrombin owing to their hydrophobic interactions with the S3 site of thrombin (78).