Current status of the anticoagulant hirudin: its biotechnological production and clinical practice

Hirudin is a potent thrombin inhibitor originally derived from the medicinal leech, Hirudo medicinalis. Owing to its high affinity and specificity for thrombin, hirudin has been intensively investigated for research and therapeutic purposes. The investigation of hirudin has contributed greatly to the understanding of the mode of action of thrombin and the clotting system. Hirudin and several hirudin analogues have also been demonstrated to have several advantages as a highly specific anticoagulant over the most widely used drug, heparin. Due to the great demand for hirudin in physicochemical and clinical studies, various recombinant systems have been developed, using bacteria, yeasts, and higher eukaryotes, to obtain the biologically active hirudin in significant quantities. After 10 years of clinical applications, two recombinant hirudins and a hirudin analogue have gained marketing approval from the United States Food and Drug Administration, for several applications. Clinical trials are currently ongoing for other treatments for thrombotic disease. As a consequence, it is conceivable that hirudin may expand its therapeutic utility over heparin in the near future.     

The complete amino acid sequence of a hirudin variant from the leechHirudinaria manillensis

Unlike the European leechHirudo medicinalis, the Asian jawed leechHirudinaria manillensis is specialized for feeding on mammalian blood. In the salivary glands of both these leeches, there is a potent inhibitor of thrombin, called hirudin, which acts as an anticoagulant. We have reported previously the isolation and purification of a variant of hirudin, called bufrudin, from the head portions ofHirudinaria. In the present study, the complete amino acid sequence of bufrudin was determined by automated Edman degradation of peptide fragments generated after cleavage of protein with trypsin or thermolysin. Comparison of the primary structure of bufrudin, with hirudin HV1, show about 70% sequence identity with deletion of two amino acids, but the key amino acids at the C-terminus, involved in the inhibition of thrombin, are conserved. However, similar sequence comparison of bufrudin with hirullin P18, a hirudin variant isolated from the same leech species but from whole leech, instead of heads, reveals even less sequence identity of about 60%. From the amino acid sequence, it is suggested that the conformation of the C-terminal portion of bufrudin may be significantly different from hirullin P18, but similar to hirudin HV1, upon its interaction with thrombin. These results indicate that, as withHirudo leech, various isoforms of hirudin also exist inHirudinaria leech, with a significant change occurring in the structure of the molecule during the evolution of leeches.     

The complete amino acid sequence of a hirudin variant from the leechHirudinaria manillensis

Unlike the European leechHirudo medicinalis, the Asian jawed leechHirudinaria manillensis is specialized for feeding on mammalian blood. In the salivary glands of both these leeches, there is a potent inhibitor of thrombin, called hirudin, which acts as an anticoagulant. We have reported previously the isolation and purification of a variant of hirudin, called bufrudin, from the head portions ofHirudinaria. In the present study, the complete amino acid sequence of bufrudin was determined by automated Edman degradation of peptide fragments generated after cleavage of protein with trypsin or thermolysin. Comparison of the primary structure of bufrudin, with hirudin HV1, show about 70% sequence identity with deletion of two amino acids, but the key amino acids at the C-terminus, involved in the inhibition of thrombin, are conserved. However, similar sequence comparison of bufrudin with hirullin P18, a hirudin variant isolated from the same leech species but from whole leech, instead of heads, reveals even less sequence identity of about 60%. From the amino acid sequence, it is suggested that the conformation of the C-terminal portion of bufrudin may be significantly different from hirullin P18, but similar to hirudin HV1, upon its interaction with thrombin. These results indicate that, as withHirudo leech, various isoforms of hirudin also exist inHirudinaria leech, with a significant change occurring in the structure of the molecule during the evolution of leeches.     

Isolation and characterization of hirudin isoinhibitors and sequence analysis of hirudin PA

A five-step isolation procedure has been developed for the purification of isoforms of hirudin (isohirudins) from whole leeches. The final purification of two thrombin-inhibiting preparations by reversed-phase high-performance liquid chromatography yielded several isohirudins with either N-terminal valine or isoleucine but with identical inhibition characteristics, i.e. specific thrombin inhibiting activities of 680-720 IU/mg and dissociation constants Ki of the thrombin-inhibitor complexes close to 3 X 10(-11) mol/l. The inhibitor with N-terminal isoleucine was designated hirudin PA. This inhibitor contains 66 amino-acid residues and has a molecular mass of 7,087 Da. The complete amino-acid sequence of hirudin PA was established by automated solid-phase Edman degradation of the native and oxidized inhibitor and two of its tryptic fragments. On the basis of the primary structures two types of thrombin inhibitors from the leech can be distinguished, designated hirudin and hirudin PA. The degree of structural homology of both isoinhibitors is approximately 82%; both have a tyrosine-O-sulfate residue near the C-terminus.     

我国产日本医蛭水蛭素的分离和纯化

采用离子交换柱层析与凝胶过滤柱层析的方法对从我国产的日本医蛙［Hirudonipponica（whitman）］活体中提取出的唾液腺分泌物进行分离和纯化,获得了较纯净的水蛭素（Hirudin）。测定结果表明：日本医蛭唾液腺分泌物提取液中水蛭素含量为4AT－U／ml;纯化总得率为15％;纯化后的产物比活力为6708AT－U／mg蛋白。     

Interaction of hirudin with thrombin: identification of a minimal binding domain of hirudin that inhibits clotting activity

Hirudin, isolated from the European leech Hirudo medicinalis, is a potent inhibitor of thrombin, forming an almost irreversible thrombin-hirudin complex. Previously, we have shown that the carboxyl terminus of hirudin (residues 45-65) inhibits clotting activity and without binding to the catalytic site of thrombin. In the present study, a series of peptides corresponding to this carboxyl-terminal region of hirudin have been synthesized, and their anticoagulant activity and binding properties to thrombin were examined. Binding was assessed by their ability to displace 125I-hirudin 45-65 from Sepharose-immobilized thrombin and by isolation of peptide-thrombin complexes. We show that the carboxyl-terminal 10 amino acid residues 56-65 (Phe-Glu-Glu-Ile-Pro-Glu-Glu-Tyr-Leu-Gln) are minimally required for binding to thrombin and inhibition of clotting. Phe-56 was critical for maintaining anticoagulant activity as demonstrated by the loss of activity when Phe-56 was substituted with D-Phe, Glu, or Leu. In addition, we found that the binding of the carboxyl-terminal peptide of hirudin with thrombin was associated with a significant conformational change of thrombin as judged by circular dichroism. This conformational change might be responsible for the loss of clotting activity of thrombin.     

Hirudin-based peptides block the inflammatory effects of thrombin on endothelial cells

Thrombin is a serine protease that plays an essential role in blood coagulation and also induces various responses in endothelial cells. The actions of thrombin on the conversion of fibrinogen to fibrin are inhibited by peptides based on the amino acid sequence of hirudin, a natural anticoagulant from leeches. We show in these studies that the peptides Hir45-64 and sulfated Hir53-64 block the effects of thrombin on endothelial cells. These peptides inhibited, in a concentration-dependent manner, the synthesis of prostaglandin I2 and platelet-activating factor, and the acquisition of an adhesive surface for leukocytes that occur in response to thrombin. These actions of the peptides occurred even though the catalytic site of thrombin was not blocked.     

Secretion of biologically active leech hirudin from baculovirus-infected insect cells.

The thrombin inhibitor, hirudin, from the leech Hirudo Medicinalis, is the most powerful natural anticoagulant known. It has been characterized as a polypeptide of 65 amino acids which exhibits its anticoagulant properties by binding tightly and specifically to alpha-thrombin. The potency and specificity of hirudin have generated interest on its possible use in the treatment or prophylaxis of various thrombotic diseases. We have used the baculovirus expression system to efficiently produce active hirudins in insect cells. The Autographa californica nuclear polyhedrosis virus has proved useful as a helper-independent viral expression vector for high-level production of recombinant proteins in cultured insect cells. Hirudin variants (HV1 and HV2) were produced in infected insect cells as secreted proteins by joining their coding sequences to the leader peptide sequence of the vescicular stomatitis virus G protein. The recombinant products were biologically active and, interestingly, N-terminal sequencing of HV1 revealed that the heterologous leader peptide is correctly removed.     

Deciphering the structural elements of hirudin C-terminal peptide that bind to the fibrinogen recognition site of alpha-thrombin

The C-terminal peptide of a hirudin acts as an anticoagulant by binding specifically to a noncatalytic (fibrinogen recognition) site of thrombin. This binding has been shown to shield five spatially distant lysines of the thrombin B-chain (Lys21, Lys65, Lys77, Lys106, and Lys107). It was also demonstrated that modification of the sequence of the hirudin C-terminal peptide invariably diminished its anticoagulant activity. The major object of this study is to investigate how the decreased activity of the modified hirudin C-terminal peptide is reflected by the change of its binding properties to these five lysines of thrombin. A synthetic peptide representing the last 12 C-terminal amino acids of hirudin (Hir54-65) was (1) truncated from both its N-terminal and its C-terminal ends, or (2) substituted with Gly along residues 57-62, or (3) chemically modified to add (sulfation at Tyr63) or abolish (Asp and Glu modification with carbodiimide/glycinamide) its negatively charged side chains. The binding characteristics of these peptides to thrombin were investigated by chemical methods, and their corresponding anticoagulant activities were studied. Our results demonstrated the following: (1) the anticoagulant activities of hirudin C-terminal peptides were quantitatively related to their abilities to shield the five identified lysines of thrombin. The most potent peptide was sulfated Hir54-65 (S-Hir54-65) with an average binding affinity to the five lysines of 120 nM. A heptapeptide (Hir54-60) also displayed anticoagulant activity and thrombin binding ability at micromolar concentrations. (2) All active hirudin C-terminal peptides regardless of their sizes and potencies were shown to be capable of shielding the five lysines of thrombin.(ABSTRACT TRUNCATED AT 250 WORDS)     

Crystal structure of the human alpha-thrombin-haemadin complex: an exosite II-binding inhibitor

The serine proteinase alpha-thrombin plays a pivotal role in the regulation of blood fluidity, and therefore constitutes a primary target in the treatment of various haemostatic disorders. Haemadin is a slow tight- binding thrombin inhibitor from the land-living leech Haemadipsa sylvestris. Here we present the 3.1 A crystal structure of the human alpha-thrombin- haemadin complex. The N-terminal segment of haemadin binds to the active site of thrombin, forming a parallel beta-strand with residues Ser214-Gly216 of the proteinase. This mode of binding is similar to that observed in another leech-derived inhibitor, hirudin. In contrast to hirudin, however, the markedly acidic C-terminal peptide of haemadin does not bind the fibrinogen-recognition exosite, but interacts with the heparin-binding exosite of thrombin. Thus, haemadin binds to thrombin according to a novel mechanism, despite an overall structural similarity with hirudin. Haemadin inhibits both free and thrombomodulin-bound alpha-thrombin, but not intermediate activation forms such as meizothrombin. This specific anticoagulant ability of haemadin makes it an ideal candidate for an antithrombotic agent, as well as a starting point for the design of novel antithrombotics.     

Antithrombin activity of a peptide corresponding to residues 54-75 of heparin cofactor II

Heparin cofactor II (HCII) is a highly specific serine proteinase inhibitor, which complexes covalently with thrombin in a reaction catalyzed by heparin and other polyanions. The molecular basis for the thrombin specificity may be explained by the identification here of a segment of HCII including residues 54-75 that binds to thrombin. A synthetic peptide, HCII(54-75), based on this segment of HCII, Gly-Glu-Glu-Asp-Asp-Asp-Tyr-Leu-Asp-Leu-Glu- Lys-Ile-Phe-Ala-Glu-Asp-Asp-Asp-Tyr-Ile-Asp inhibited thrombin's cleavage of fibrinogen. Clotting activity of thrombin was inhibited 50% at a concentration of 28 microM. Polyacrylamide gel electrophoresis showed that HCII(54-75) inhibited thrombin's cleavage of both the A alpha and B beta polypeptides in fibrinogen. However, the peptide did not block thrombin's active site, as hydrolysis of chromogenic substrates was not inhibited. HCII(54-75) probably binds to the same site on thrombin as do carboxyl-terminal residues of hirudins, thrombin inhibitors of leeches. HCII(54-75) inhibited binding of thrombin to a synthetic peptide corresponding to residues 54-66 of hirudin PA, but the hirudin peptide was about 30-fold more potent in binding and clotting assays. Both synthetic peptides, as a result of their polyanionic character, might be expected to stimulate the reaction of HCII with thrombin. However, the hirudin-related peptide inhibited this reaction, suggesting that it blocked a site on thrombin required for interaction with HCII. HCII(54-75) had a net stimulatory effect on the thrombin-HCII reaction as a consequence of its lower affinity for thrombin and greater negative charge relative to the hirudin-related peptide. These studies suggest that residues 54-75 of HCII interact with a noncatalytic binding site on thrombin and that this interaction contributes to efficient inhibition of thrombin by HCII.     

Crystal structure of the thrombin-hirudin complex: a novel mode of serine protease inhibition.

Thrombin is a serine protease that plays a central role in blood coagulation. It is inhibited by hirudin, a polypeptide of 65 amino acids, through the formation of a tight, noncovalent complex. Tetragonal crystals of the complex formed between human alpha-thrombin and recombinant hirudin (variant 1) have been grown and the crystal structure of this complex has been determined to a resolution of 2.95 A. This structure shows that hirudin inhibits thrombin by a previously unobserved mechanism. In contrast to other inhibitors of serine proteases, the specificity of hirudin is not due to interaction with the primary specificity pocket of thrombin, but rather through binding at sites both close to and distant from the active site. The carboxyl tail of hirudin (residues 48-65) wraps around thrombin along the putative fibrinogen secondary binding site. This long groove extends from the active site cleft and is flanked by the thrombin loops 35-39 and 70-80. Hirudin makes a number of ionic and hydrophobic interactions with thrombin in this area. Furthermore hirudin binds with its N-terminal three residues Val, Val, Tyr to the thrombin active site cleft. Val1 occupies the position P2 and Tyr3 approximately the position P3 of the synthetic inhibitor D-Phe-Pro-ArgCH2Cl. Thus the hirudin polypeptide chain runs in a direction opposite to that expected for fibrinogen and that observed for the substrate-like inhibitor D-Phe-Pro-ArgCH2Cl.     

Probing the hirudin-thrombin interaction by incorporation of noncoded amino acids and molecular dynamics simulation

Thrombin is a primary target for the development of novel anticoagulants, since it plays two important and opposite roles in hemostasis: procoagulant and anticoagulant. All thrombin functions are influenced by Na+ binding, which triggers the transition of this enzyme from an anticoagulant (slow) form to a procoagulant (fast) form. In previous studies, we have conveniently produced by chemical synthesis analogues of the N-terminal fragment 1-47 of hirudin HM2 containing noncoded amino acids and displaying up to approximately 2700-fold more potent antithrombin activity, comparable to that of full-length hirudin. In the work presented here, we have exploited the versatility of chemical synthesis to probe the structural and energetic properties of the S3 site of thrombin through perturbations introduced in the structure of hirudin fragment 1-47. In particular, we have investigated the effects of systematic replacement of Tyr3 with noncoded amino acids retaining the aromatic nucleus of Tyr, as well as similar hydrophobic and steric properties, but possessing different electronic (e.g., p-fluoro-, p-iodo-, or p-nitro-Phe), charge (p-aminomethyl-Phe), or conformational (homo-Phe) properties. Our results indicate that the affinity of fragment 1-47 for thrombin is proportional to the desolvation free energy change upon complex formation, and is inversely related to the electric dipole moment of the amino acid side chain at position 3 of hirudin. In this study, we have also identified the key features that are responsible for the preferential binding of hirudin to the procoagulant (fast) form of thrombin. Strikingly, shaving at position 3, by Tyr --> Ala exchange, abolishes the differences in the affinity for thrombin allosteric forms, whereas a bulkier side chain (e.g., beta-naphthylalanine) improves binding preferentially to the fast form. These results provide strong, albeit indirect, evidence that the procoagulant (fast) form of thrombin is in a more open and accessible conformation with respect to the less forgiving structure it acquires in the slow form. This view is also supported by the results of molecular dynamics simulations conducted for 18 ns on free thrombin in full explicit water, showing that after approximately 5 ns thrombin undergoes a significant conformational transition, from a more open conformation (which we propose can be related to the fast form) to a more compact and closed one (which we propose can be related to the slow form). This transition mainly involves the Trp148 and Trp60D loop, the S3 site, and the fibrinogen binding site, whereas the S1 site, the Na+-binding site, and the catalytic pocket remain essentially unchanged. In particular, our data indicate that the S3 site of the enzyme is less accessible to water in the putative slow form. This structural picture provides a reasonable molecular explanation for the fact that physiological substrates related to the procoagulant activity of thrombin (fibrinogen, thrombin receptor 1, and factor XIII) orient a bulky side chain into the S3 site of the enzyme. Taken together, our results can have important implications for the design of novel thrombin inhibitors, of practical utility in the treatment of coagulative disorders.     

Anticoagulant activity of synthetic hirudin peptides

Synthetic peptides based on the COOH-terminal 21 residues of hirudin were prepared in order to 1) evaluate the role of this segment in hirudin action toward thrombin, 2) define the shortest peptide derivative with anticoagulant activity, and 3) investigate the role of tyrosine sulfation in the peptides' inhibitory activities. A hirudin derivative of 20 amino acids, Hir45-64 (derived from residues 45-64 of the hirudin polypeptide), was found to effect a dose-dependent increase in the activated partial thromboplastin time (APTT) of normal human plasma but to have no measurable inhibitory activity toward thrombin cleavage of a tripeptidyl p-nitroanilide substrate. Anticoagulant activity in hirudin derivatives was comparable in peptides of 20, 16, and 12 residues truncated from the NH2 terminus. Additional truncated peptides prepared by synthesis and carboxypeptidase treatment reveal that the minimal sequence of a hirudin peptide fragment with maximal anticoagulant activity is contained within the sequence: NH2-Asn-Gly-Asp-Phe-Glu-Glu-Ile-Pro-Glu-Glu-Tyr-Leu-COOH. The 12-residue derivative thus identified was reacted with dicyclohexylcarbodiimide in the presence of sulfuric acid to yield a Tyr-sulfated peptide, S-Hir53-64. By comparison to unsulfated peptide, S-Hir53-64 was found to contain a specific inhibitory activity enhanced by one order of magnitude toward increase in APTT and to effect a dose-dependent increase in thrombin time of normal human plasma to yield a 4-fold increase in thrombin time with 2.5 micrograms/ml peptide using 0.8 units/ml alpha-thrombin. Comparison of S-Hir53-64 to hirudin in thrombin time and APTT assays reveals a 50-fold difference in molar specific activities toward inhibition of thrombin. Comparison of antithrombin activities of S-Hir53-64 using a variety of animal thrombins demonstrates greatest inhibitory activity toward murine, rat, and human enzymes and a 10-fold reduced activity toward bovine thrombin.     

Incorporation of noncoded amino acids into the N-terminal domain 1-47 of hirudin yields a highly potent and selective thrombin inhibitor.

Hirudin is an anticoagulant polypeptide isolated from a medicinal leech that inhibits thrombin with extraordinary potency (Kd = 0.2-1.0 pM) and selectivity. Hirudin is composed of a compact N-terminal region (residues 1-47, cross-linked by three disulfide bridges) that binds to the active site of thrombin, and a flexible C-terminal tail (residues 48-64) that interacts with the exosite I of the enzyme. To minimize the sequence of hirudin able to bind thrombin and also to improve its therapeutic profile, several N-terminal fragments have been prepared as potential anticoagulants. However, the practical use of these fragments has been impaired by their relatively poor affinity for the enzyme, as given by the increased value of the dissociation constant (Kd) of the corresponding thrombin complexes (Kd = 30-400 nM). The aim of the present study is to obtain a derivative of the N-terminal domain 1-47 of hirudin displaying enhanced inhibitory potency for thrombin compared to the natural product. In this view, we have synthesized an analogue of fragment 1-47 of hirudin HM2 in which Val1 has been replaced by tert-butylglycine, Ser2 by Arg, and Tyr3 by beta-naphthylalanine, to give the BugArgNal analogue. The results of chemical and conformational characterization indicate that the synthetic peptide is able to fold efficiently with the correct disulfide topology (Cys6-Cys14, Cys16-Cys28, Cys22-Cys37), while retaining the conformational properties of the natural fragment. Thrombin inhibition data indicate that the effects of amino acid replacements are perfectly additive if compared to the singly substituted analogues (De Filippis V, Quarzago D, Vindigni A, Di Cera E, Fontana A, 1998, Biochemistry 37:13507-13515), yielding a molecule that inhibits the fast or slow form of thrombin by 2,670- and 6,818-fold more effectively than the natural fragment, and that binds exclusively at the active site of the enzyme with an affinity (Kd,fast = 15.4 pM, Kd,slow = 220 pM) comparable to that of full-length hirudin (Kd,fast = 0.2 pM, Kd,slow = 5.5 pM). Moreover, BugArgNal displays absolute selectivity for thrombin over the other physiologically important serine proteases trypsin, plasmin, factor Xa, and tissue plasminogen activator, up to the highest concentration of inhibitor tested (10 microM).     

The structural elements of hirudin which bind to the fibrinogen recognition site of thrombin are exclusively located within its acidic C-terminal tail

Six lysyl residues of human thrombin (LysB21, LysB52, LysB65, LysB106, LysB107 and LysB154) have been previously shown to participate in the binding site of hirudin, a thrombin-specific inhibitor [(1989) J. Biol. Chem. 264, 7141-7146]. In this report, we attempted to delineate the region of hirudin which binds to these basic amino acids of thrombin. Using the N-terminal core domains (r-Hir1-43 and r-Hir1-52) derived from recombinant hirudins and synthetic C-terminal peptides (Hir40-65 and Hir52-65)--all fragments form complexes with thrombin--we are able to demonstrate that the structural elements of hirudin which account for the shielding of these 6 lysyl residues are exclusively located within the acidic C-terminal region. Since hirudin C-terminal peptides were shown to bind to a non-catalytic site of thrombin and inhibit its interaction with fibrinogen [(1987) FEBS Lett. 211, 10-16], our data consequently imply that these 6 lysyl residues are constituents of the fibrinogen recognition site of thrombin.     

The stabilization and release of hirudin from liposomes or lipid-assemblies coated with hydrophobically modified dextran

Hirudin is a 65-amino acid peptide and the most potent and specific known inhibitor of thrombin (K_i=0.2 pM). The short elimination half-life of hirudin from the body (1 hour) necessitates the use of a sustained and controlled delivery system. A proliposome method was used to entrap hirudin in liposomes coated with palmitoyl dextran-coated liposomes and lipid-assemblies. In vitro release studies of hirudin were performed using the lipid systems enclosed in dialysis membranes or deposited in the pores of a vascular graft. The activity of hirudin and released hirudin was measured using a thrombin chromogenic substrate assay.     

Chimeric antithrombin peptide. Characterization of an Arg-Gly-Asp (RGD)- and hirudin carboxyl terminus-containing synthetic peptides

We investigated the properties of an artificial chimeric peptide that contains an Arg-Gly-Asp (RGD)-tripeptide, the versatile cell recognition signal of extracellular matrix protein components, coupled to a carboxyl-terminal fragment of the highly specific alpha-thrombin inhibitor, hirudin (residues 53-64): WGRGDSANGDFEEIPEEYL (RGD-hirudin53-64). Hirudin53-64 and RGD-hirudin53-64 inhibited the fibrinogen clotting activity of alpha-thrombin and prolonged the activated partial thromboplastin time of human plasma. In addition, both peptides afforded total protection to thrombin from trypsionolysis. Neither hirudin53-64 nor RGD-hirudin53-64 dramatically interfered with the thrombin-antithrombin inhibition reaction either in the absence or presence of added heparin. alpha-Thrombin-induced platelet aggregation was effectively inhibited by hirudin53-64 and RGD-hirudin53-64. Unlike hirudin53-64, RGD-hirudin53-64 in solution inhibited integrin-mediated endothelial cell and fibroblast cell attachment to polystyrene wells in the presence of fetal bovine serum. Collectively, our results demonstrate that RGD-hirudin53-64 has anticoagulant/antiplatelet aggregation activity attributable to its hirudin sequence and integrin-directed cell attachment activity due to its RGD site. Our results suggest that this chimeric motif may serve as a prototype for a new class of anticoagulants where an integrin-specific sequence "targets" the peptide to a cell (ultimately through the platelet integrin alpha IIb beta 3) trapped amid a thrombus with ensuing proteinase inhibition.     

Construction and characterization of a novel staphylokinase variant with thrombin-inhibitory activity

Staphylokinase (SAK) is an effective thrombolysis agent for therapy of myocardial infarction. We have constructed a fusion SAK variant (SAK-HV) with a thrombin-binding domain composed of 12 amino acids from hirudin and expressed it in Escherichia coli and purified the resultant protein. SAK-HV maintained fibrinolytic activity similar to SAK and had anticoagulant activity attributable to its hirudin segment. Measurement of thrombin-binding activity in vitro demonstrated that SAK-HV possessed binding activity with thrombin while SAK did not. SAK-HV might thus be a more potent thrombolytic agent with anticoagulation property than SAK.     

Pharmacokinetic studies with recombinant hirudin in dogs

The knowledge of the pharmacokinetics of recombinant hirudin is essential for potential clinical use of this selective thrombin inhibitor. For detailed information about absorption, distribution and elimination, pharmacokinetic studies with recombinant hirudin were carried out in dogs. The plasma concentration time curve after intravenous injection could be best described by an open two-compartment model with first order kinetics. The subcutaneous application of recombinant hirudin produced anticoagulantly effective blood level for a prolonged period of time. Recombinant hirudin is distributed into the extracellular space. This is also found in nephrectomized dogs. Recombinant hirudin is eliminated through the kidneys by glomerular filtration in active form with a half-life of 72 minutes.