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SYNTHETIC AND NATURAL PEPTIDES AS ANTITHROMBOTIC AGENTS - A VIEW ON THE CURRENT DEVELOPMENT

A. Atanassov1 and B. Tchorbanov2 Thracian University, Faculty of medicine, Department of Biophysics, Stara Zagora, Bulgaria1 Institute of Organic Chemistry with Centre of Phytochemistry, Bulgarian Academy of Sciences, Sofia, Bulgaria2 Correspondence to: B Tchorbanov E-mail: [email protected]

ABSTRACT A view on the current development of synthesis as well as isolation of antithrombotic agents of peptide origin is presented. The structure and action of several anticoagulant peptides/proteins isolated from animal sources during the last 20 years are described. The synthesis of peptide mimetics manifesting even weaker anticoagulant activity is discussed as a main studies purpose. Compounds of both polysaccharides and peptide origin possessing inhibition of platelet aggregation isolated from microbial, animal and plant origin are summarized. Current investigations on a fraction (MW 100-120 kDa) isolated from goat’s rue (Galega officinalis L.) appears to have a polysaccharide nature, including a protein part that is presented with special attention due to the high anti-aggregating activity, especially initiated by adenosine diphosphate, collagen and thrombin.

Keywords: antithrombotic agents, anticoagulants, trombin the pathophysiological mechanisms of anticoagulant and inhibitors, antiaggregants, natural and synthetic peptides, antiaggregation processes has been clearly demonstrated (13) antiaggregants, Galega officinalisL . in numbers of studies published in the last ten years (Fig. 1).

Introduction vascular injury Antithrombotic agents are widely used in the medicine for intrinsic proteases extrinsic proteases treatments of hemostatic impairments such as coronary FIXa/FVIIIa FVIIa/TF angioplasts, coronary thromboembolisms, myocard heart attack, pulmonary embolism, etc. Haemostasis is a key process Factor Xa, Factor Va whose correct functioning is an important defence 1process Ca2+, PL, "prothrombinase" activated in case of injury of the blood system. Coagulation FVIIIa is a defence function of the organism that has to be strictly FVIII FVa regulated. After the bleeding is stopped, a number of limiting self-regulatory mechanisms are initiated. This process is thrombin FV accompanied by a series of enzyme reactions described in 1964 as an enzyme cascade (44). Factor XIIIa activation fibrin formation The living organisms have natural mechanisms to overcome clotting processes, the thrombolytic processes and platelet aggregation other physiological disorders. This is affected via different Fig. 1. Simplified scheme of the coagulation-aggregation system connection substances with defence function called antithrombocytes. They include inhibitors of thrombin, factor Ха, Factor IXa, Thrombin is a serine protease included in the blood factors participating in the extrinsic and intrinsic pathways of coagulation cascade which in the same time also plays a key the coagulation cascade (13). role in the process of thrombin-induced platelet aggregation. In eighties, after disclosing the close relationship between the two The inhibitors of serine proteinases are a large class of processes: aggregation and coagulation, it was revealed that proteins which is divided into families according to their a numbers of natural anticoagulants display antiaggregation structure and mechanism of action. Most inhibitors of properties, due to their ability to inhibit the thrombin (11, 58). coagulation factors of the intrinsic pathway are members of In this global context some previous investigations on the the serpin family. On the other hand, inhibitors of the extrinsic fragment analogues of decorsin, protein with well established pathway such as hirudin and antistasin are classified as antiaggregation properties reveal that it possess anticoagulant members of the family of Kunitz type inhibitors (39). activities too (62, 63, 84). It is well recognized now that the success for the prevention and the treatment of arterial diseases closely relates with Natural inhibitors of serine proteases the necessity for better anti-thrombus therapy to avoid the In the last 20 years a number of proteins and peptides with clots complications. The crucial role of the thrombin in different molecule mass and well established anticoagulation Biotechnol. & Biotechnol. Eq. 23/2009/1 1109 activity have been isolated from the salivary glands of several purified by Jung et al. It is a cystein-rich polypeptide of bloodsucking animals. Many of the strongest anticoagulants 57 amino acid residues with molecular weight of around isolated from bloodsucking animals are found in the saliva of 6 kDa. Guamerin has identical spacing of 10 cysteine different types of leeches. residues like antistasin-type inhibitors, but in the P1 The first systematic investigations date back to the seventies position of the reactive site it has Met instead of Arg (49); of the past century. In 1971 Fritz et al. isolated and characterised • ancylostoma caninum anticoagulant peptide (MW 8.7 bdellin A, an inhibitor of trypsin, plasmin and acrosin, from kDa), a hookworm-derived inhibitor of human coagulation medical leech (42). Later, in 1998, after extensive structural factor Xa, characterised by Cappello et al. Single-stage and kinetic investigations, they assigned it to the family of chromogenic assay reveal that this peptide is a highly antistasine inhibitors of the serine proteinases. Therefore, it potent and specific inhibitor of human coagulation as it was renamed bdellastasin to avoid confusion with Bdellin B, inhibits the specifically free factor Xa (16). which is another trypsin-plasmin inhibitor from the medical The salivary glands of other bloodsucking animals like tics, leech, but of the Kazal type. Bdelastasin does not manifest bats and vampires also contain a large number of anticoagulants inhibitor activity towards Factor Ха, chimotripsin, thrombin, of different types and structure. By the end of the century many tissue or plasma kalikrein, but its Ki values for inhibition of new natural peptides and proteins have been discovered and bovine trypsin are of nanomolar range (64). Steinmetzer et isolated, such as: al. (1999) reported on the highly potent and specific synthetic • therin (MW 5376 Da), specific trypsin inhibitor from the thrombin inhibitors derived from the natural polypeptide rhynchobdellied leech Theromyzon tessulatum, isolated hirudin which they named hirulogs. The C-terminus of the and purified by Chopin et al. It is a tight-binding inhibitor hirulogs is almost identical with C-terminal residues of the of trypsin containing 48 amino acid residues and has no desulfated recombinant hirudin, which bind to the fibrinogen action towards elastase or cathepsin G (25); recognition exo-site of thrombin. This inhibitor segment is connected by a peptide linker to an additional inhibitor moiety, • piguamerin, an antistasin-type protease inhibitor isolated which occupies the active site of thrombin. Similar to hirudin, from a native Korean leech species Hirudo nipponia the hirulogs inhibit thrombin in a bivalent binding mode (80). contains 48 amino acids (MW 5090 Da) showing potent inhibition of plasma and tissue kallikreins, and trypsin Continuing their investigations in this direction in 1993 (54); Strube et al. publish information about isolation, sequence analysis, and cloning of a slow, tight-binding inhibitor with • savignin (MW 12 430 Da), a potent thrombin inhibitor an apparent molecular mass of about 5 kDa, isolated from isolated from the salivary glands of the tick Ornithodoros Haemadipsa sylvestris, an Indian leech of the family of savignyi. Nienaber et al. reveal that it is competitive, Haemadipsidae - Haemadin. It is determined that its inhibitor slow tight-binding inhibitor of α-thrombin. Kinetic activity is thrombin specific since it does not inhibit other investigations show that the inhibitor has a lower affinity proteases like trypsin, chymotripsin, factor Xa, or plasmin. for γ-thrombin. Plasmin, Factor Xa, and trypsin are not Although there is no identity of the sequences between inhibited by savignin (67); haemadin and hirudin it has been established that both • lefaxin (MW 30 kDa), a new factor Xa inhibitor from proteins share common mechanisms for thrombin inhibition Haementeria depressa leech. Nienaber et al. reveal that (82). Seymour et al. report the isolation of ecotin, a potent it can inhibit Factor Xa and thrombin generation in the anticoagulant and reversible tight-binding factor Xa inhibitor. prothrombinase complex (38); They prove that it does not inhibit effectively the human • anophelin, 6.5kDa peptide isolated from salivary gland plasma proteinase, thrombin tissue factor, factor VIIa, factor of Anopheles albimanus. Kinetic analyses by Francischetti XIa, activated protein C, plasmin or tissue plasminogen et al. determine that it is reversible, slow, tight-binding activator (77). The literature data from the middle and the end inhibitor of α-thrombin, displaying a competitive type of of the 90ties of last century is abundant with newly discovered, inhibition (41); isolated and characterized natural polypeptides and proteins • therostasin was described by Chopin et al. (2000) - a novel that are inhibitors of the serine proteinases. For example, the clotting Factor Xa inhibitor from the Rhynchobdellid year 1995 is rich in literature data about the inhibitors of the Leech, Theromyzon tessulatum including 82 amino acid coagulation serine proteinases: residues with 16 cysteines (26). • Draculin (MW 83 kDa), the anticoagulant protein isolated Special attention is devoted in the literature to a protein from the saliva of the vampire bat Desmodus rotundus known as antistasin. In 1988 Nutt et al. reported its isolation from Apitz-Castro et al. It inhibits the activated form of from the salivary glands of the Mexican leech, Haementeria Factor IX and X but does not act on thrombin, trypsin, officinalis as well as its characterisation (69, 70, 71, 85). Kinetic chymotrypsin and does not express fibrinolytic activity (2, studies reveal that antistasin is a potent, slow, tight-binding 10, 40, 41); Factor Xa inhibitor (37, 38, 57, 58). A large part of the natural • guamerin, a new human leukocyte elastase inhibitor from anticoagulant peptides and proteins isolated later, show partial a Korean native leech Hirudo nipponia, extracted and or complete similarity between their active centres and other 1110 Biotechnol. & Biotechnol. Eq. 23/2009/1 parts of their molecules and those of antistasin. Thus it become structures between the isoforms of the antistasin, ghilantens the founder of the largest group of natural anticoagulants - and shortened fragments of TAP realising the above mentioned the antistasin type inhibitors. Two years later, Waxman et al., tripeptide active groups aiming to increase the fragments’ reported the discovery of tick anticoagulant peptide (TAP), a activity. Interesting conclusions have been made regarding the single chain 60 amino acid peptide that was isolated from the structure-activity of these analogues of natural proteins. It is extracts of tick Ornithodoros moubata (89). TAP is a reversible, noticed that a main tendency is that the availability of basic slow, tight-binding inhibitor of Factor Xa with an estimated amino acids and D-amino acids in particular positions of the Ki of 0.5 nM. At the same time Blankenship et al. reported peptide fragments increases the anticoagulant activity. There the isolation and characterisation of ghilantens from the are a lot of data on the structural and kinetic investigations of leech Haementeria ghilianii. They consist of 119 amino acid anticoagulants too (34, 35, 37, 40, 47, 56, 91) as well as several residues and show high homology with natural antistasin (12, publications on in vivo investigation of the anticoagulant 14, 74). Ghilantens are highly specific reversible inhibitors of activity of some analogues (89). Factor Xa, similar to antistasin. Just like antistasin, they form a stoichiometric complex with Factor Xa with dissociation Inhibitors of platelet aggregation constants in the nanomolar range (12, 70, 85). Compounds of both polysaccharides and peptide origin Synthetic inhibitors and serine proteases possessing inhibition of platelet aggregation isolated from microbial, animal and medicinal plants sources are summarized The efforts are focused simultaneously on the isolation of on Table 1 and Table 2. Such compounds of protein and peptide natural anticoagulants and on the synthesis of peptide mimetics origin are isolated from snakes, ticks, leeches, bacterium, etc. possessing similar activity. (Table 1). In the last decade peptide fragments, although manifesting TABLE 1 weaker anticoagulant activity, were reported. They fall under Biological active compounds with inhibition properties against the common name of peptide mimetites because imitating thrombus aggregation isolated from animal sources structure and properties of active proteins. The peptide mimetics as a whole possess many advantages against the Animal source Substance References synthetic substances with non-peptide base, major among them Bothrops atrox batroxostatin peptide (75) being the lack of toxicity, accumulation in the organism and Macrobdella decora decorsin protein (78) easy penetration of the cell membranes. The oldest systematic Human lactotransferrin KRDS peptide (61) data in the literature concerning the anticoagulant structures on Agkistrodon halys snake halysin peptide (79) the basis of amino acids and peptides date back to 1975 when Bajusz et al. reported their work on the tripeptide sequence Trimeresurus flavoviridis triflavin peptide (79) D-Phe-Arg-Pro-H (7). This sequence showed exceptionally Bitis arietans arietin peptide (79) hopeful in vivo results, which led to a number of investigations Haementeria officinalis 16 kDa protein (27) on its modifications (8, 46). About 10 years later a synthesis Bitis gabonica snake gabonin peptide (79) and biological activity of a new tripeptide sequences Tyr-Ile- Ornithodoros moubata protein (89) Arg and Phe-Ile-Arg were described (71). At the same time a study on a new tripeptide with high anticoagulation activity Yersinia pestis toxin protein (21) D-Arg-Gly-Arg was reported (59). Streptococcal sourse exopolysaccharides (50) Many modifications have been made (1, 15, 53) to the snake venom trigramin, rhodostomin (22) obtained peptide sequences with anticoagulant activity mainly cloves oil (77) related to: Crotalus viridis crotavirin (23) • the study of the role of the separate amino acids in them snake venom rhodostomin (23) during the inhibition of the enzymes from the coagulation Trimeresurus tokarensis tokaracetin (24) cascade; Hirudo medicinalis calin (36) • in order to achieve selectivity in regard to one or another serine protease; Vipera lebetina venom lebetins 1 and 2 (9) Not fewer are the data obtained at that time for synthesis of Trimeresurus flavoviridis flavostatin (51) longer structures with similar activity. Brancamp et al. published Cerastes cerastes 40 kDa cerastotin (9) in 1991 their investigations on ghilanten-related inhibitors Clonostachys sp. F5898 clonostatin (24) (14). In a series of publications Danalev et al. (28, 29, 30, 31, Triatoma pallidipennis 17 kDa triabin (43) 32, 33, 45, 87) and Naydenova et al. (65, 66, 67) describe the Agkistrodon rhodostoma rhodostomin (20) synthesis of fragment analogues of the antistasin, ghilantens and TAP by studying the role of the basic and D-amino acids at Austrelaps superbus superbins (83) different positions in the molecule for the anticoagulant activity Ornithodoros savigni apyrase enzyme (9) of the compounds. They also synthesise a series of hybrid Escherichia coli PLATSAK protein (86) Biotechnol. & Biotechnol. Eq. 23/2009/1 1111 Macrobdella decora decorsin (73) It is well established that the inhibition of thrombus Echis carinatus ec-I-PLA2 (52) aggregation by peptides is possible because of some amino The antiplatelet and antithrombotic substances isolated acid sequences (RGD, KRDS, RGDW, RGDS, Pro-X-X-X- from medicinal plants, represent a particular interest. The high Asp-X). They are antagonists of fibrinogen receptors (61). molecular weight biological active compounds which posses’ It is suggested that high molecular polypeptides containing inhibition properties against thrombus formation isolated from the same amino acid sequences can interact with fibrinogen medicinal plants are polysaccharides including protein parts in molecule and to inhibit the thrombus aggregation. All their molecules. Some substances with antiplatelet and anti- inhibitors of thrombus aggregation have a different mechanism aggregating action, isolated from medicinal plants are shown of action but the most perspective are these which could inhibit on Table 2. formation of fibrinogen bridges between aggregating thrombus like the polysaccharide-protein fraction isolated from Galega Usually, the isolated from plants anti-aggregating officinalis L. The current work is focused on the relation polysaccharides and peptides demonstrate a high biological between polysaccharide and protein part of the active fraction activity. It is shown that peptides with given amino acid from Galega officinalis L. sequence (RGD, KRDS , RGDW, RGDS, Pro-X-X-X-Asp-X) appeared as an antagonist against the fibrinigen receptors and Acknowledgements strongly inhibited platelet aggregation (71, 73, 75). The IC of 50 The authors acknowledge financial support for these these peptides is about 10-100 nM. investigations by MES-NFSI-Bulgaria (project TK-X1608). TABLE 2 Biologicaly-active substances inhibiting platelet aggregation isolated from medicinal plants REFERENCES Source Active compound References 1. Angliker H., Stone S., Shaw E. (1990) Peptides, 772- Stichopus japonicus S. mucopolysaccharides (48) 773. Lonicera japonica Thunb. polyphenolic compound (19) 2. 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