A View on the Current Development
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REVIEW PB 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