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PubMed1997

Capillary electrophoresis of r-hirudin and a polyethylene glycol derivative of r-hirudin (PEG-hirudin).

UNLABELLED: Recombinant (r-) hirudins and PEG-hirudin are currently tested for anticoagulant therapy. For their concentration measurement, radioimmunoassay and HPLC methods are available. The separation of r- and PEG-hirudin is currently performed by HPLC. However, the sensitivity of the method is low. Capillary electrophoresis is a rapid, selective technique that requires low sample amounts. Our aim was the development of a capillary electrophoresis method to measure r- and PEG-hirudin. The results are as follows: In a borate solution (0.3% boric acid and 0.4% sodium tetraborate, pH 9.5) r-hirudin was separated from PEG-hirudin in a purified system using a fused silica capillary (50 cm long and 75-micron i.d. and reversed polarity). A neutral capillary with a 20 mM tricine buffer (pH 8.0, field strength 500 V/cm) was also effective in resolving r- from PEG-hirudin. A linear correlation was found between the peak area and the concentration between 20 micrograms/mL and 10 mg/mL for hirudin (r2 = 0.99) and between 1.25 and 10 mg/mL for PEG-hirudin (r2 = 0.99). In human plasma mixtures, r- and PEG-hirudin were completely separated. The linear correlation between the peak area and the concentration was r2 = 0.99. CONCLUSION: In a fused silica capillary, r- and PEG-hirudin are separated in a purified system. Capillary electrophoresis which is performed in a neutral capillary, resolves r- from PEG-hirudin in a purified system, in plasma and in urine. The sensitivities of the methods are comparable. Capillary electrophoresis separates r- from PEG-hirudin and may be applied to biologic systems to measure the concentration and purity of r- and PEG-hirudin.

Electrophoresis, Capillary↗
PubMed1993

Anti-hirudin monoclonal antibodies directed toward discontinuous epitopes of the hirudin amino-terminal and epitopes involving the carboxy-terminal hirudin amino acids.

A panel of eight monoclonal antibodies (MAbs) was obtained against recombinant hirudin variant 2 (rHV2). Specificities of the eight MAbs indicate that four of them recognize C-terminal amino acid residues (Group A) and four are directed against discontinuous epitopes and recognize a determinant (or determinants) within the 43 N-terminal residues (Group B). Using these antibodies recombinant hirudins missing one or more C-terminal amino acids can be distinguished from molecules with an intact C-terminus either in enzyme immunoassays (EIAs) or by immunoaffinity chromatography. A sandwich EIA using the combination of two antibodies, one from each group, can quantitate both recombinant hirudin variant 1 (rHV1) and rHV2 with a detection range from 1 to 10 ng/ml in either buffer or plasma. Using only one MAb a competitive antibody capture EIA can quantitate recombinant or natural hirudin variants 1, 2, and 3 with a detection range from 5 to 100 ng/ml for rHV2 with a lysine in position 47 (rHV2K47). None of the antibodies recognizes hirudin after it is complexed to alpha-thrombin. The ability of any one of these anti-rHV2 antibodies to interfere with hirudin binding to alpha-thrombin as measured by inhibition of thrombin's amidolytic activity correlates with the range of MAb affinity constants (KD = 3.5 x 10(-9) to 1 x 10(-6) M). Incubating hirudin with one antibody from Group A (KD = 3.5 x 10(-8) M) and one from Group B (KD = 6.0 x 10(-9) M) completely blocks the ability of hirudin to bind alpha-thrombin. This MAb panel is thus useful for probing the recombinant C-terminal integrity of hirudin, for sensitive free hirudin quantitations, and the combined use of two MAbs has potential applications as an antidote for hirudin in vivo.

Amino Acid Sequence↗
PubMed1990

Preparation of monoclonal antibodies to hirudin and hirudin peptides. A method for studying the hirudin--thrombin interaction.

A panel of four monoclonal antibodies was obtained against hirudin, a potent and specific inhibitor of thrombin, by immunizing three groups of mice with protein conjugates made of recombinant desulfatohirudin (group I) or two synthetic peptides representing the C-terminal sequences 40-65 (group II) and 52-65 (group III) of hirudin. Only the monoclonal antibody 4049-83-12, obtained from the group I of mice, showed high affinity for hirudin (Kd of 0.6 nM) and in vitro neutralizing properties. The anti-peptide monoclonal antibodies bound hirudin with lower affinity (Kd of 1.5-7 nM) and showed lower neutralizing capacities. An epitope analysis performed by competitive ELISA using various hirudin analogues and by limited proteolysis of the hirudin-antibody complex revealed that the binding domains of all the anti-peptide antibodies were located close to the C-terminus of hirudin, since the bond between Glu-61 and Glu-62 was not cleaved by the V8 staphylococcal protease in the presence of these antibodies. The epitope of the antibody 4049-83-12 was strictly conformation-dependent, it recognized neither S-carboxymethylated hirudin nor any peptides of hirudin. The cleavage of the bond between Glu-43 and Gly-44 by V8 protease, as well as the cleavage of the bond between Lys-47 and Pro-48 by lysyl endopeptidase, was prevented by the binding of the antibody 4049-83-12 to hirudin. The possibility that this epitope overlapped with a region of hirudin involved in the binding to thrombin is discussed.

Animals↗
PubMed1992

Determination of r DNA hirudin and A-human thrombin- hirudin complex in plasma samples: enzyme linked immunosorbent assays for hirudin and complex vs. chromogenic thrombin substrate assay.

rDNA hirudin plasma concentrations in man and rhesus monkeys were determined over a period of 15 and 24 h. The plasma concentration of alpha-human thrombin-hirudin complex was measured after administration of the complex to rhesus monkeys. The complex was also determined after administration of hirudin to man and rhesus monkeys to study a possible formation of a complex with alpha-human thrombin in blood. The determination of hirudin was performed by a sandwich ELISA, using polyclonal and monoclonal antibodies and the chromogenic thrombin substrate assay. The alpha-human thrombin-hirudin complex concentration in the plasma of rhesus monkeys was measured over a period of 48 hours. The results of a sandwich ELISA were compared with those of the chromogenic thrombin substrate assay. A good agreement between the total hirudin concentrations analyzed by the hirudin ELISA and the alpha-human thrombin-hirudin complex ELISA and those of the chromogenic thrombin substrate assay, measuring total hirudin, too, was observed.

Animals↗
PubMed1991

Mechanism of the inhibition of alpha-thrombin by hirudin-derived fragments hirudin(1-47) and hirudin(45-65).

The kinetic mechanism of the inhibition of alpha-thrombin by hirudin was analyzed using the hirudin-derived fragments hirudin(1-47) and hirudin(45-65). Previously, these fragments have been shown to interact with alpha-thrombin at distinct sites inhibiting thrombin-mediated clot formation. Binding to the active site the N-terminal fragment hirudin(1-47) competitively inhibits hydrolysis of the substrates Tos-Gly-Pro-Arg-NH-Mec (Tos, tosyl; NH-Mec, 4-methylcoumaryl-7-amide) and fibrinogen with Ki values of 420 +/- 18 nM and 460 +/- 25 nM, respectively. Interacting with the anion-binding site of alpha-thrombin the C-terminal fragment competitively inhibits the hydrolysis of fibrinogen with a Ki of 760 +/- 40 nM. It was found, however, that this fragment acts as a hyperbolic uncompetitive inhibitor with respect to the hydrolysis of the peptide-NH-Mec substrate. According to the Botts-Morales scheme for enzyme inhibition, the parameters Ki = 710 +/- 38 nM, K'i = 348 +/- 22 nM, as well as alpha = beta = 0.49 of thrombin inhibition by the C-terminal fragment hirudin(45-65), were obtained. The results are discussed in terms of the interaction of hirudin and thrombin.

Amino Acid Sequence↗
PubMed2003

Anti-hirudin antibodies alter pharmacokinetics and pharmacodynamics of recombinant hirudin.

Recombinant hirudin (r-hirudin) is a potent direct thrombin inhibitor with immunogenic properties. Anti-hirudin antibodies (aHAb) are detected in up to 74% of patients treated with r-hirudin for more than 5 days. aHAb may alter the pharmacokinetics and pharmacodynamics of r-hirudin. The effects of aHAb on the pharmacokinetics of r-hirudin were investigated in rats receiving r-hirudin intravenously either without aHAb (controls), 15 min after intravenous administration of non-specific antibodies or aHAb, and after pre-incubation with aHAb. When both were compared to controls and pre-treatment with non-specific antibodies, aHAb significantly altered the pharmacokinetics of r-hirudin with similar effects in both approaches: In the presence of aHAb, the volume of distribution in a steady state and total plasma clearance were diminished, while the half-life of elimination was prolonged. Both the maximum r-hirudin plasma concentration and the area under the curve were increased. In addition, r-hirudin filtration by high-flux hemodialyzer membranes (polysulfone, AN69) was investigated 1) in the absence of aHAb, 2) in the presence of non-specific mouse antibodies, and 3) in the presence of three monoclonal aHAb. In the absence of aHAb, both hemodialyzers allowed for significant r-hirudin filtration. Non-specific mouse antibodies did not markedly affect r-hirudin filtration. By contrast, all three aHAb almost completely hindered r-hirudin filtration. aHAb varied in their capacity to neutralize r-hirudin. In conclusion, aHAb markedly alter the pharmacokinetics of r-hirudin leading to r-hirudin accumulation. In the presence of aHAb, hemofiltration does not allow for rapid reduction of r-hirudin concentration. aHAb are capable of modifying pharmacodynamics of r-hirudin. Close monitoring of aHAb-positive patients treated with r-hirudin is considered mandatory.

Animals↗
PubMed1993

Catabolism of hirudin and thrombin-hirudin complexes in the rat.

The metabolic fate of the anticoagulant protein, hirudin, and its complex with thrombin are presently unknown. Therefore we have labelled hirudin and human thrombin-hirudin complex with the residualizing label dilactitol-125I-tyramine (*I-DLT) in order to identify their tissue sites of catabolism in the rat. The rapid plasma clearance of hirudin after intravenous injection was unaffected by *I-DLT labelling, and by 2 h 6% or less of the injected dose remained in the blood. The majority (80.3 +/- 4.0%, n = 2) of *I-DLT-hirudin radioactivity recovered in tissues was found in kidney, and kidney was also at least 150 times more active in taking up hirudin, on a weight basis, than any other tissue examined (liver, spleen, skin, muscle, intestine, fat, lung). *I-DLT-hirudin which bound to thrombin was isolated by chromatography on concanavalin A-Sepharose; hirudin itself does not bind to concanavalin A. Radioactivity from thrombin-*I-DLT-hirudin was precipitable by anti-thrombin antibody and *I-DLT-thrombin-hirudin was precipitable by anti-hirudin antibody. By 1 h after injection of labelled thrombin-hirudin complexes, the recoveries of radioactivity from hirudin and thrombin in liver were comparable (38.6 +/- 3.0 and 36.4 +/- 4.1%, n = 3), whereas more radioactivity was recovered in kidney from hirudin than from thrombin (27.6 +/- 8.7 compared with 13.6 +/- 4.5%) and less was recovered in lung (0.4 +/- 0.2 compared with 17.7 +/- 2.9%). We conclude that hirudin is catabolized predominantly in kidney, whereas the thrombin-hirudin complex is catabolized by both liver and kidney.

Animals↗
PubMed1994

Displacement of fibrin-bound thrombin by r-hirudin precludes the use of 131I-r-hirudin for detecting pulmonary emboli in the rabbit.

Pulmonary emboli are detectable by filling defects in the pulmonary vasculature upon pulmonary angiography. Emboli derived from venous thrombi are rich in fibrin to which thrombin remains bound. Hirudin, a specific thrombin inhibitor, binds to thrombin to yield a 1:1 stoichiometric complex. We examined whether 131I-recombinant hirudin (r-hirudin) could be used to detect pulmonary emboli in rabbits. Clots were formed by re-calcifying rabbit plasma in vitro, and then injected (0.034 ml) into a femoral vein to lodge in the lungs. 131I-r-hirudin (29 +/- 4 microCi/kg) was injected intravenously but emboli could not be detected by gamma camera in real time. Post-mortem analysis of lung tissue showed that 131I-r-hirudin did not associate with emboli prepared with 125I-fibrin. Because of these findings, we used different techniques to look at the binding of hirudin to plasma clots. Clots formed in vitro were incubated with 131I-r-hirudin in the presence of equimolar amounts of 125I-albumin; specific binding of 131I-r-hirudin was not observed. Experiments with immobilized fibrin(ogen) showed that 125I-r-hirudin did not bind to and remain with fibrin-bound 131I-thrombin but did lead to the inactivation and displacement of up to 70% of bound thrombin as r-hirudin-thrombin complex; residual thrombin bound to fibrin remained active. Thus, released r-hirudin-thrombin complex is probably cleared rapidly from the region of the embolus in vivo; radioiodinated r-hirudin may not, therefore, be useful as a marker for detecting emboli.

Animals↗
PubMed1999

Generation of anti-hirudin antibodies in heparin-induced thrombocytopenic patients treated with r-hirudin.

BACKGROUND: Hirudin is a small protein with strong thrombin inhibition that may be antigenic. The generation and disappearance of anti-hirudin antibodies were investigated in patients with heparin-induced thrombocytopenia who were treated with recombinant hirudin (r-hirudin) for >/=5 days. METHODS AND RESULTS: The IgA, IgE, IgG, and IgM isotypes of anti-hirudin antibodies were determined by ELISA before and after the start of r-hirudin therapy. A total of 56% of patients (13 of 23) developed >/=1 antibody isotype during therapy. No IgE antibodies were generated. IgA, IgG, and IgM antibodies were detected in 30% (7 of 23), 52% (12 of 23), and 17% (4 of 23) of patients, respectively. Four patients generated only IgG, 2 patients developed either IgM or IgG and IgM, 5 patients IgG and IgA, and 2 patients IgG, IgM, and IgA antibodies. IgM antibodies disappeared within 8 days of the cessation of r-hirudin. IgA and IgG antibodies disappeared within 1 year in all but 1 patient. Binding of purified IgG to r-hirudin in IgG antibody-positive patients (n=7) was demonstrated by competitive ELISA for r-hirudin. Of the 7 IgG antibody samples, 1 each neutralized or enhanced the anticoagulant activity of r-hirudin. CONCLUSIONS: R-hirudin may be antigenic in patients with heparin-induced thrombocytopenia. More comprehensive investigations will be required to determine the biological relevance of this and to establish the antibody-generation pattern in other diseases.

Adult↗
PubMed1994

Fibrin-targeted recombinant hirudin inhibits fibrin deposition on experimental clots more efficiently than recombinant hirudin.

BACKGROUND: Although the indirect thrombin inhibitor heparin and the more potent direct inhibitor hirudin are useful in preventing thrombosis, a substantial opportunity remains for improving the thrombus selectivity of thrombin inhibitors. METHODS AND RESULTS: To explore the effect of targeting an antithrombin to the surface of a clot, we covalently linked recombinant hirudin to the Fab' (or IgG) of a monoclonal antibody (59D8) that selectively binds to an epitope on fibrin that becomes exposed only after thrombin cleaves fibrinopeptide B. Antibody-coupled hirudin bound to an immobilized peptide of the fibrin beta-chain amino-terminal sequence and inhibited the peptidolytic activity of thrombin more efficiently than free hirudin. Thrombin inhibition dependent on binding to immobilized fibrin monomer was enhanced 1100-fold (P < .0001). Hirudin-59D8 Fab' was 10 times more effective than hirudin in inhibiting fibrin deposition on experimental clot surfaces in fibrinogen solution (P < .0001) and human plasma (P < .0001). The more effective inhibition of thrombin by the conjugate was supported by significantly diminished concentrations of fibrinopeptide A in the plasma supernatant of the clot (P = .0001). Inhibition of clotting by an uncoupled mixture of hirudin and 59D8 Fab' was indistinguishable from that by hirudin alone, indicating that the conjugate's greater inhibitory activity was due to the covalent linkage between antibody and hirudin. CONCLUSIONS: Fibrin-targeted hirudin (in comparison with unmodified hirudin) significantly reduces fibrin deposition on the surface of experimental clots.

Antibodies, Monoclonal↗
PubMed1999

R-hirudin as anticoagulant in regular hemodialysis therapy: finding of therapeutic R-hirudin blood/plasma concentrations and respective dosages.

Recently heparin-induced thrombocytopenia type II has been diagnosed more frequently and does not exclude hemodialysis patients. Up to now, recombinant hirudin is the only available anticoagulant showing no immunologic cross reactions with heparin. However, the use of r-hirudin in hemodialysis patients with different degrees of residual renal functions is impossible using standard dosages because elimination of r-hirudin varies depending on the degree of residual renal function. Therefore the first study was carried out using consecutive r-hirudin anticoagulated hemodialyses to determine the appropriate dose of r-hirudin. Ten hemodialysis patients with creatinine clearance values ranging between 0 and 13 mL/min/1.73m2 were anticoagulated with r-hirudin. An initial bolus of 0.1 mg/kg bwt before the first hemodialysis, resulted in an average r-hirudin blood concentration of 305 ng/mL at the end of treatment. The dose for each of the following four hemodialyses was adjusted individually to reach the minimum therapeutic r-hirudin blood concentration. At the end of these treatments the mean blood r-hirudin concentration was 422 ng/mL. The necessary mean doses ranged between 0.008 and 0.125 mg/kg bwt correlating to the creatinine clearance values of the patients. All hemodialyses of the study were effective and safe. Bleeding times determined during r-hirudin anticoagulation were significantly lower than control values measured 2 days after a heparin administration. The study proved that r-hirudin may be an efficient and safe heparin alternative as a hemodialysis anticoagulant when the individual's residual renal function is noted for dosage and dose adjustment and is controlled by drug monitoring using the ecarin clotting time.

Adult↗
PubMed1997

Anticoagulation with r-hirudin in regular haemodialysis with heparin-induced thrombocytopenia (HIT II). The first long-term application of r-hirudin in a haemodialysis patient.

A 69-year-old female patient with renal failure developed heparin-induced thrombocytopenia type II (HIT II) two months after starting haemodialysis therapy with heparin as anticoagulant and a 6-week course of thromboembolism prophylaxis with enoxaparin sodium. The platelet count dropped by 50% as compared with initial values and ex vivo platelet aggregation induced by heparin antibodies (HIPA-test) was detected. Haemodialysis therapy was complicated by a massive thrombosis of dialyzer and ensuing repeated interruptions of treatment. After confirmation of the diagnosis of HIT II haemodialysis therapy was continued with hirudin as anticoagulant. Polysulfone dialyzers and an intravenous bolus of 0.14 mg/kg of recombinant hirudin (r-hirudin) achieved efficient haemodialysis therapy of 4.5 hours, with a minimum therapeutic blood level of hirudin of 0.5 micrograms/mL. More than 50 regular haemodialysis with hirudin anticoagulation were performed without additional problems. The ecarin clotting time (ECT) was used as bedside method to monitor blood levels and for dosage adjustments of hirudin. After the 34th haemodialysis, the frequency (previously 3-4 haemodialyses sessions/week) was reduced to 2 sessions/week. The creatinine clearance increased continuously from initially 2.6 to 10.4 ml/min after the 13th week of hirudin-anticoagulated haemodialysis and the platelet count normalized. In conclusion, we report the first long-term administration of r-hirudin to a patient on regular haemodialysis therapy complicated by heparin-induced thrombocytopenia. The use of hirudin as anticoagulant along with dialyzers impermeable to hirudin offers a novel alternative means of anticoagulation and, even in patients with HIT, enables performing an efficient haemodialysis therapy. Hirudin dosage must be individually adjusted by using bedside drug monitoring of plasma concentrations.

Aged↗
PubMed1992

Nuclear magnetic resonance solution structure of hirudin(1-51) and comparison with corresponding three-dimensional structures determined using the complete 65-residue hirudin polypeptide chain.

The three-dimensional structure of the N-terminal 51-residue domain of recombinant hirudin in aqueous solution was determined by 1H nuclear magnetic resonance (NMR) spectroscopy, and the resulting high-quality solution structure was compared with corresponding structures obtained from studies with the intact, 65-residue polypeptide chain of hirudin. On the basis of 580 distance constraints derived from nuclear Overhauser effects and 109 dihedral angle constraints, a group of 20 conformers representing the solution structure of hirudin(1-51) was computed with the program DIANA and energy-minimized with a modified version of the program AMBER. Residues 3 to 30 and 37 to 48 form a well-defined molecular core with two antiparallel beta-sheets composed of residues 14 to 16 and 20 to 22, and 27 to 31 and 36 to 40, and three reverse turns at residues 8 to 11 (type II), 17 to 20 (type II') and 23 to 26 (type II). The average root-mean-square deviation of the individual NMR conformers relative to their mean co-ordinates is 0.38 A for the backbone atoms and 0.77 A for all heavy atoms of these residues. Increased structural disorder was found for the N-terminal dipeptide segment, the loop at residues 31 to 36, and the C-terminal tripeptide segment. The solution structure of hirudin(1-51) has the same molecular architecture as the corresponding polypeptide segment in natural hirudin and recombinant desulfatohirudin. It is also closely similar to the crystal structure of the N-terminal 51-residue segment of hirudin in a hirudin-thrombin complex, with root-mean-square deviations of the crystal structure relative to the mean solution structure of 0.61 A for the backbone atoms and 0.91 A for all heavy atoms of residues 3 to 30 and 37 to 48. Further coincidence is found for the loop formed by residues 31 to 36, which shows increased structural disorder in all available solution structures of hirudin, and of which residues 32 to 35 are not observable in the electron density map of the thrombin complex. Significant local structural differences between hirudin(1-51) in solution and hirudin in the crystalline thrombin complex were identified mainly for the N-terminal tripeptide segment and residues 17 to 21. These are further analyzed in an accompanying paper.

Fibrinolytic Agents↗
PubMed1991

Chemical modifications and amino acid substitutions in recombinant hirudin that increase hirudin-thrombin affinity.

Recombinant hirudin (r-hirudin), unlike the naturally occurring leech protein, lacks a sulfate ester on Tyr-63 which reduces its binding affinity to thrombin by 3-10-fold. We demonstrate that nitration or iodination of Tyr-63 restores hirudin-thrombin affinity to levels similar to or exceeding that of the natural inhibitor. In contrast, nitration of Tyr-3 reduces the affinity of hirudin for thrombin. These chemical modifications results in multiple reaction products that are readily separated by reverse-phase HPLC. The mechanism of the observed changes in thrombin affinity may involve a reduction in the pK of the hydroxyl group of tyrosine due to substitution of the electrophilic iodo or nitro group on the phenyl ring, resulting in an increased negative charge at neutral pH. For Tyr-63, this effect mimics the sulfatotyrosine of natural hirudin, leading to an increased thrombin affinity at the anion-binding exosite. For Tyr-3, the increased polarity may destabilize its interaction within the apolar-binding site of thrombin. Substitution of the highly conserved Tyr-3 residue with Phe or Trp not only enables specific and quantitative chemical modification at Tyr-63 but also independently increases hirudin-thrombin affinity. Kinetic analysis of thrombin inhibition showed that enhanced binding by r-hirudin(nitro-Tyr-63) is due to an increase in the association rate between hirudin and thrombin whereas the reduced binding of r-hirudin(nitro-Tyr-3) results from a large increase in the dissociation rate. These observations indicate that specific segments within both the amino- and carboxy-terminal regions of hirudin interact with thrombin.

DNA Mutational Analysis↗
PubMed1991

Effects of different hirudins and combinations of low doses of hirudin, heparin and acetylsalicylic acid in a rat microcirculatory thrombosis model.

Two recombinant hirudins (r-hirudin), natural hirudin and hirudin in combination with heparin or acetylsalicylic acid (ASA) have been studied in a thrombosis model in which rat mesenteric venules of a diameter of 20-30 microns were injured by well-defined argon laser lesions. In the animal model all hirudins showed significant and dose-dependent antithrombotic effects in doses between 0.05 and 0.1 mg/kg after single intravenous and subcutaneous injections. The antithrombotic effect of single (0.2 mg/kg i.v. or 0.1 mg/kg s.c.) injections lasted longer than 4 h (i.v.) or 6 h (s.c.). Hirudin at a dose of 0.1 mg/kg and heparin at doses of 0.05 mg/kg showed a significant antithrombotic effect 2 h after subcutaneous injection. When heparin and hirudin were injected together at this dosage, the effect of the combination was in the same range as that of unfractionated heparin or hirudin alone. An additive antithrombotic effect was observed if a low dose of r-hirudin (0.1 mg/kg) was combined with a moderate dose of ASA (10 mg/kg).

Animals↗
PubMed1983

Hirudin from leech heads and whole leeches and "pseudo-hirudin" from leech bodies.

Hirudin from whole leeches behaves like hirudin from leech heads and "pseudohirudin" from leech bodies during isolation and purification by means of acetone and ethanol fractionation, affinity chromatography on trypsin-Sepharose, and isoelectric focusing. However, the antithrombin activity of the hirudin fractions obtained after isoelectric focusing was approximately three times lower than that of the corresponding hirudin fractions from the heads. The "pseudohirudin" fractions had practically no antithrombin activity. In hirudin from whole leeches isoleucine and valine were identified as the N-terminal amino acid. Isoleucine was identified as the dominant amino acid in hirudin from leech heads. The dominant N-terminal amino acid in "pseudohirudin" from leech bodies was valine. The data on antithrombin activity and N-terminal amino acids indicate that hirudin from whole leeches contains admixtures of inactive "pseudohirudin". In contrast to hirudin from leech heads, "pseudohirudin" represents associates of di- and three-isomers. The molecular weight of the "pseudohirudin" molecule is 2000 lower than the molecular weight of hirudin, which corresponds to a difference of 20 amino acid residues, calculated from the total number of amino acid residues in the preparations.

Amino Acid Sequence↗
PubMed2004

Hirudin (desulfated, 54-65) contracts canine coronary arteries: extracellular calcium influx mediates hirudin-induced contractions.

OBJECTIVE: Although the anticoagulatory properties of hirudin are well known, its direct vasoactive effects have not been investigated extensively. Hirudin stimulates nitric oxide and prostacyclin production in noncoronary vascular beds, but its actions on coronary arteries are unknown. MATERIALS AND METHODS: Five-millimeter segments of canine left circumflex coronary arteries were obtained for organ chamber experiments. Some segments were denuded of endothelium before study. Segments were exposed to hirudin (10(-10)-10(-6) mol/L) following precontraction with prostaglandin F(2alpha) with or without pretreatment with indomethacin or calcium channel blockers (verapamil and nifedipine). RESULTS: Hirudin stimulated endothelium-independent contraction in coronary arterial segments. Maximum tension (hirudin 10(-6) mol/L) above precontraction baseline was 33.6 +/- 9.0% (n = 10, P < 0.05) for endothelium-intact and 31.8 +/- 11.5% (n = 8, P < 0.05) for endothelium-denuded arterial segments. Differences between endothelium-intact and endothelium-denuded segments were not significant. Contractile responses to hirudin were unaffected by the presence of indomethacin. Pretreatment with either verapamil or nifedipine (10(-4) mol/L) for 1 h attenuated these contractions. The maximal increase in tension above baseline (hirudin 10(-6) mol/L) for verapamil and nifedipine was only 6.2 +/- 12.4 and 3.8 +/- 7.0% (n = 6, P < 0.05 versus endothelium-intact control), respectively. CONCLUSIONS: Hirudin stimulates endothelium-independent contractions of canine coronary arteries in vitro. Pretreatment with calcium channel blockers attenuates this response, suggesting that extracellular influx of calcium has an important mechanistic role in hirudin-mediated coronary artery constriction.

Animals↗