The hirudin-like factors HLF3 and HLF4-hidden hirudins of European medicinal leeches.

The hirudin-like factors 3 (HLF3) and 4 (HLF4) belong to a new class of leech-derived factors and are present in specimens of the three European medicinal leeches, Hirudo medicinalis, Hirudo verbana, and Hirudo orientalis, respectively. Here we describe the functional analysis of natural and synthetic variants of HLF3 and HLF4. Whereas the natural variants display only very low or no detectable anti-coagulatory activities, modifications within the N-termini in combination with an exchange of the central globular domain have the potency to greatly enhance the inhibitory effects of respective HLF3 and HLF4 variants on blood coagulation. Our results support previous observations on the crucial importance of all parts (both the N- and C-termini as well as the central globular domains) of hirudin and HLF molecules for thrombin inhibition.

Hirudin and Decorsins of the North American Medicinal Leech Macrobdella decora: Gene Structure Reveals Homology to Hirudins and Hirudin-like Factors of Eurasian Medicinal Leeches.

Blood-sucking leeches, some of which are referred to as medicinal leeches, have caught attention not only because of their medical purposes, but also as study organisms to conduct research within fields as diverse as neurobiology, osmoregulation, ecology, and phylogeny. Of particular interest is the question whether hemophagy in leeches is of single origin or evolved independently several times. A key component in the saliva of hematophagous leeches is hirudin, a strong natural inhibitor of thrombin and hence the blood coagulation cascade. Multiple isoforms of hirudin have been described within and among several leech species and genera, often based on sequence data only. The identification of hirudin-like factors (HLFs) illustrated the necessity to underpin such predictions by functional tests. We overexpressed and purified the hirudin of the North American medicinal leech, Macrobdella decora, and proved its thrombin-inhibiting activity. In addition, analysis of the gene structure of both hirudin and some of the decorsins of M. decora clearly indicated conserved exon and intron positions when compared to genes of hirudins and HLFs of Eurasian medicinal leeches. Our data provide evidence for the incorporation of decorsins into the hirudin superfamily and support the concept of a single origin of blood feeding in jawed leeches.

Primary study on the hypoglycemic mechanism of 5rolGLP-HV in STZ-induced type 2 diabetes mellitus mice.

5rolGLP-HV is a promising dual-function peptide for the treatment of diabetes and thrombosis simultaneously. For investigating the therapeutic mechanism of 5rolGLP-HV for type 2 diabetes mellitus (T2DM), STZ-induced diabetic mice were established and treated with 5rolGLP-HV. The results showed that daily water and food intake, blood glucose, serum and pancreatic insulin levels significantly decreased after 5rolGLP-HV treatment with various oral concentrations, and 16 mg/kg was the optimal dose for controlling diabetes. 5rolGLP-HV treatment decreased the MDA levels and the T-SOD activity in serum and pancreatic of diabetic mice (but not up to significant difference), and significantly increased the expression of signal pathways related genes of rolGLP-1, also the density of insulin expression and the numbers of apoptosis cells in islets of diabetic mice were significantly decreased in comparison to the negative diabetic mice. These effects above may be clarified the hypoglycemic mechanisms of 5rolGLP-HV, and 5rolGLP-HV may be as a potential drug for diabetes in future.

Salivary transcriptome of the North American medicinal leech, Macrobdella decora.

A variety of bioactive proteins from medicinal leeches, like species of Hirudo , have been characterized and evaluated for their potential therapeutic biomedical properties. However, there has not previously been a comprehensive attempt to fully characterize the salivary transcriptome of a medicinal leech that would allow a clearer understanding of the suite of polypeptides employed by these sanguivorous annelids and provide insights regarding their evolutionary origins. An Expressed Sequence Tag (EST) library-based analysis of the salivary transcriptome of the North American medicinal leech, Macrobdella decora, reveals a complex cocktail of anticoagulants and other bioactive secreted proteins not previously known to exist in a single leech. Transcripts were identified that correspond to each of saratin, bdellin, destabilase, hirudin, decorsin, endoglucoronidase, antistatin, and eglin, as well as to other previously uncharacterized predicted serine protease inhibitors, lectoxin-like c-type lectins, ficolin, disintegrins and histidine-rich proteins. This work provides a lens into the richness of bioactive polypeptides that are associated with sanguivory. In the context of a well-characterized molecular phylogeny of leeches, the results allow for preliminary evaluation of the relative evolutionary origins and historical conservation of leech salivary components. The goal of identifying evolutionarily significant residues associated with biomedically significant phenomena implies continued insights from a broader sampling of blood-feeding leech salivary transcriptomes.

The determination of protonation states in proteins.

The protonation states of aspartic acids and glutamic acids as well as histidine are investigated in four X-ray cases: Ni,Ca concanavalin A at 0.94 A, a thrombin-hirugen binary complex at 1.26 A resolution and two thrombin-hirugen-inhibitor ternary complexes at 1.32 and 1.39 A resolution. The truncation of the Ni,Ca concanavalin A data at various test resolutions between 0.94 and 1.50 A provided a test comparator for the ;unknown' thrombin-hirugen carboxylate bond lengths. The protonation states of aspartic acids and glutamic acids can be determined (on the basis of convincing evidence) even to the modest resolution of 1.20 A as exemplified by our X-ray crystal structure refinements of Ni and Mn concanavalin A and also as indicated in the 1.26 A structure of thrombin, both of which are reported here. The protonation-state indication of an Asp or a Glu is valid provided that the following criteria are met (in order of importance). (i) The acidic residue must have a single occupancy. (ii) Anisotropic refinement at a minimum diffraction resolution of 1.20 A (X-ray data-to-parameter ratio of approximately 3.5:1) is required. (iii) Both of the bond lengths must agree with the expectation (i.e. dictionary values), thus allowing some relaxation of the bond-distance standard uncertainties required to approximately 0.025 A for a '3sigma' determination or approximately 0.04 A for a '2sigma' determination, although some variation of the expected bond-distance values must be allowed according to the microenvironment of the hydrogen of interest. (iv) Although the F(o) - F(c) map peaks are most likely to be unreliable at the resolution range around 1.20 A, if admitted as evidence the peak at the hydrogen position must be greater than or equal to 2.5 sigma and in the correct geometry. (v) The atomic B factors need to be less than 10 A(2) for bond-length differentiation; furthermore, the C=O bond can also be expected to be observed with continuous 2F(o) - F(c) electron density and the C-OH bond with discontinuous electron density provided that the atomic B factors are less than approximately 20 A(2) and the contour level is increased. The final decisive option is to carry out more than one experiment, e.g. multiple X-ray crystallography experiments and ideally neutron crystallography. The complementary technique of neutron protein crystallography has provided evidence of the protonation states of histidine and acidic residues in concanavalin A and also the correct orientations of asparagine and glutamine side chains. Again, the truncation of the neutron data at various test resolutions between 2.5 and 3.0 A, even 3.25 and 3.75 A resolution, examines the limits of the neutron probe. These various studies indicate a widening of the scope of both X-ray and neutron probes in certain circumstances to elucidate the protonation states in proteins.

[Construction of phage-displayed library by DNA shuffling and the screening of potent hirudin variants].

AIM: To directed promote the antithrombotic activity of hirudin in vitro with DNA family shuffling method. METHODS: PCR products of HV1, HV2 and HV3 genes of hirudin were combined and digested by DNase I. Then random fragments about 50 bp were purified and reassembled to the same size of hirudin gene by two amplifications of PCR with or without primers. The shuffled hirudin genes were inserted into phagemid pCANTAB5E vector and the hirudin mutants were displayed on the surface of bacteriophage M13. Mutants with increased specific activity of antithrombin were screened by affinity panning with decreased amounts of thrombin. RESULTS: After shuffling and selection, a clone (HV2-N47K) with high antithrombotic activity was obtained. CONCLUSION: Hirudin variants with improved properties could be hopefully aquired by DNA family shuffling and affinity panning.

Proteinase inhibitors from the medicinal leech Hirudo medicinalis.

The medicinal leech Hirudo medicinalis produces various types of proteinase inhibitors: bdellins (inhibitors of trypsin, plasmin, and acrosin), hirustasin (inhibitor of tissue kallikrein, trypsin, alpha-chymotrypsin, and granulocyte cathepsin G), tryptase inhibitor, eglins (inhibitors of alpha-chymotrypsin, subtilisin, and chymasin and the granulocyte proteinases elastase and cathepsin G), inhibitor of factor Xa, hirudin (thrombin inhibitor), inhibitor of carboxypeptidase, and inhibitor of complement component C1s. This review summarizes data on their primary and tertiary structures, action mechanisms, and biological activities.

Electrostatic steering and ionic tethering in the formation of thrombin-hirudin complexes: the role of the thrombin anion-binding exosite-I.

Electrostatic interactions between the thrombin anion-binding exosite-I (ABE-I) and the hirudin C-terminal tail play an important role in the formation of the thrombin-hirudin inhibitor complex and serves as a model for the interactions of thrombin with its many other ligands. The role of each solvent exposed basic residue in ABE-I (Arg(35), Lys(36), Arg(67), Arg(73), Arg(75), Arg(77a), Lys(81), Lys(109), Lys(110), and Lys(149e)) in electrostatic steering and ionic tethering in the formation of thrombin-hirudin inhibitor complexes was explored by site directed mutagenesis. The contribution to the binding energy (deltaG(degrees)b) by each residue varied from 1.9 kJ mol(-)(1) (Lys(110)) to 15.3 kJ mol(-1) (Arg(73)) and were in general agreement to their observed interactions with hirudin residues in the thrombin-hirudin crystal structure [Rydel, T. J., Tulinsky, A., Bode, W., and Huber, R. (1991) J. Mol. Biol. 221, 583-601]. Coupling energies (delta deltaG(degrees) int) were calculated for the major ion-pair interactions involved in ionic tethering using complementary hirudin mutants (h-D55N, h-E57Q, and h-E58Q). Cooperativity was seen for the h-Asp(55)/Arg(73) ion pair (2.4 kJ mol(-1)); however, low coupling energies for h-Asp(55)/Lys(149e) (deltadeltaG(degrees)int 0.6 kJ mol(-1)) and h-Glu(58)/Arg(77a) (deltadeltaG(degrees)int 0.9 kJ mol(-1)) suggest these are not major interactions, as anticipated by the crystal structure. Interestingly, high coupling energies were seen for the intermolecular ion-pair h-Glu(57)/Arg(75) (deltadeltaG(degrees)int 2.3 kJ mol(-1)) and for the solvent bridge h-Glu(57)/Arg(77a) (deltadeltaG(degrees)int 2.7 kJ mol(-1)) indicating that h-Glu(57) interacts directly with both Arg(75) and Arg(77a) in the thrombin-hirudin inhibitor complex. The remaining ABE-I residues that do not form major contacts in tethering the C-terminal tail of hirudin make small but collectively important contributions to the overall positive electrostatic field generated by ABE-I important in electrostatic steering.

Pharmacological effects of a novel recombinant hirudin, CX-397, in vivo and in vitro: comparison with recombinant hirudin variant-1, heparin, and argatroban.

The novel recombinant hirudin analog CX-397 was investigated with respect to its pharmacological activity and antithrombin profiles in vivo and in vitro. In three different types of thrombosis models in rats, including stasis and thrombin-induced venous, glass surface-activated arterio-venous shunt, and ferric chloride-induced arterial thrombosis models, CX-397 and rHV-1 elicited potent antithrombotic effects, where the minimum effective doses of rHV-1 tended to be higher than those of CX-397 in the arterio-venous shunt and arterial thrombosis models. The hemorrhagic risk of CX-397 in template bleeding in rats was not higher than that of rHV-1, indicating that CX-397 is superior to rHV-1 for treating the platelet-dominant type of thrombosis. However, no differences were detected between CX-397 and rHV-1 in their effects on in vitro coagulation times and thrombin-induced platelet aggregation, suggesting the possibility that some unknown mechanisms other than simple thrombin inhibition are also involved in their antithrombotic actions.

Destabilase complexes--natural liposome produced by medicinal leeches Hirudo medicinalis.

Electrophoretic analysis of destabilase preparation demonstrates the presence of protein combinations with MW 12.3, 25 and 50 kD. Fraction (MW 12.3 D) is a monomer of destabilase aggregation having properties of micellar proteins and represents a stable lipid-protein complex, where the role of lipid component is played by the stable analogue of prostacyclin (MW 391 D). The synthesis of a low molecular fraction of destabilase is fulfilled with bacteria--symbiont of leeches Aeromonas hydrophila. When the destabilase (MW 12.3 kD) contacts with blood a process of complexe formation is triggered with hirudin and blood plasma kallikrein inhibitor, forming a stable 'destabilase complex' (DC; MW 25 kD), possessing also a high aggregation capacity. Polymer forms of the destabilase complex form a liposome changing its spatial orientation depending on the nature of the solvent. Such structural organization provides a high stability of DC components and a rapid penetration through cellular membranes (transmembrane transfer) and it also provides prophylactic antithrombotic action in the case of peroral application to animals, due to the blockade of vascular platelets (inhibition of platelet aggregation by prostacyclin analogue) and plasmic (inhibition of thrombin activity and blood plasma kallikrein) links of the hemostasis process. Destabilase fraction with MW 50 kD is a dimer of the destabilase complex. As a result of DC destruction (liposome), hirudin, prostacycline analogue and blood plasma kallikrein inhibitor are released.

[Hirudin: the relative failure of a very promising molecule]. / Hirudine: échec relatif d'une molécule très prometteuse.

Hirudin is a peptide of 65 aminoacids extracted from the leech which very specifically inhibits the action of thrombin. Molecular engineering techniques have made it available for therapeutic usage. Experimental data would suggest that hirudin was the drug of choice for the prevention of coronary thrombosis and recurrences, an indication where heparin is not always effective. Despite well organised studies of tolerance and dosage, three large scale clinical trials have had to be interrupted because of a high incidence of severe bleeding complications. The therapeutic margin of hirudin is therefore as low as that of heparin. The initial results of trials using lower doses have not shown hirudin to be effective than heparin in the prevention of critical events in the long term. The reasons for this relative failure are two-fold: either hirudin and molecules with similar modes of action such as Hirulog are less active in vivo than in vitro, possibly due to the fact that they block the activation of the C protein, a powerful natural antithrombotic mechanism, or, the target of hirudin: thrombin, does not play the fundamental role attributed to it in arterial thrombosis. The good results observed with molecules which inhibit platelet aggregation would suggest a dominant role of the platelets.

Pharmacological properties of hirudin and its derivatives. Potential clinical advantages over heparin.

Hirudin and its derivatives represent the first parenteral anticoagulants introduced since the discovery of heparin in the early 1900s. Hirudin, the naturally occurring anticoagulant of the leech, is a single peptide chain of 65 amino acids with a molecular weight of about 7000. Recombinant technology has developed methods to produce recombinant forms of hirudin (r-hirudin) in sufficient quantities for therapeutic use. Hirudin is a potent thrombin-specific inhibitor that forms equimolar complexes with thrombin. It represents a new anticoagulant agent in a field in which heparin has been the only available drug for many years. In contrast to heparin, hirudin does not require antithrombin III as a cofactor, is not inactivated by antiheparin proteins, has no direct effects on platelets and may also inactivate thrombin bound to clot or the subendothelium. In humans, experience with r-hirudin in preventing or treating venous thromboembolism is very preliminary. However, r-hirudin achieved promising results in patients with unstable angina, or following coronary angioplasty. In patients with acute myocardial infarction, 3 important clinical trials were stopped because of an excess of bleeding complications. At present, the discovery of a r-hirudin regimen that is more efficacious than heparin and at least as safe needs a reappraisal of the drug in further trials.

Hirudins: antithrombin anticoagulants.

OBJECTIVE: To review the chemistry, pharmacology, available clinical data, and adverse effects of the hirudin anticoagulants. DATA SOURCES: A MEDLINE search and a review of recent scientific abstracts was conducted to identify pertinent literature. STUDY SELECTION: Focus was placed on studies conducted in humans. Because hirudin is still an investigational agent, however, relevant animal data, particularly pharmacokinetic studies and studies of preclinical efficacy, were also selected. DATA EXTRACTION: Data from both human and animal studies were evaluated; emphasis was placed on human trials. DATA SYNTHESIS: Hirudin has demonstrated potent anticoagulant effects. Although hirudin could have a significant impact on the therapeutic management of patients requiring anticoagulant therapy, only a limited number of human studies have been published to date. Trials comparing hirudin and heparin in specific patient populations are still ongoing. CONCLUSIONS: Although still in clinical trials, hirudin is a unique agent that may represent a breakthrough in anticoagulant therapy. The specific role that this agent will play in the management of patients has yet to be determined.

Electrostatic interactions in the association of proteins: an analysis of the thrombin-hirudin complex.

The role of electrostatic interactions in stabilization of the thrombin-hirudin complex has been investigated by means of two macroscopic approaches: the modified Tanford-Kirkwood model and the finite-difference method for numerical solution of the Poisson-Boltzmann equations. The electrostatic potentials around the thrombin and hirudin molecules were asymmetric and complementary, and it is suggested that these fields influence the initial orientation in the process of the complex formation. The change of the electrostatic binding energy due to mutation of acidic residues in hirudin has been calculated and compared with experimentally determined changes in binding energy. In general, the change in electrostatic binding energy for a particular mutation calculated by the modified Tanford-Kirkwood approach agreed well with the experimentally observed change. The finite-difference approach tended to overestimate changes in binding energy when the mutated residues were involved in short-range electrostatic interactions. Decreases in binding energy caused by mutations of amino acids that do not make any direct ionic interactions (e.g., Glu 61 and Glu 62 of hirudin) can be explained in terms of the interaction of these charges with the positive electrostatic potential of thrombin. Differences between the calculated and observed changes in binding energy are discussed in terms of the crystal structure of the thrombin-hirudin complex.