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Bioanalytical Sciences

Recombinant Hirudin
Daniel Gygax School of Life Sciences, Muttenz

- Hirudin ist ein Polypeptid aus dem medizinischen Blutegel (Hirudo medicinalis) mit antikoagulatorischen Eigenschaften (Blutgerinnungshemmung). - Die Substanz wurde 1955 erstmals durch Extraktion aus Blutegelkpfen isoliert. - Die Primrstruktur des HV-1 (Hirudin Variant-1) besteht aus 65 Aminosuren. - Die Aminosure Tyr in Position 63 ist natrlicherweise sulfatiert. - Die medizinische Verwendung von Hirudin geschieht entweder durch direktes Ansetzen lebender Egel am Patienten oder mittels von gentechnisch hergestellten Substanzen.
In der modernen Medizin wird kein Aderlass mehr durchgefhrt, aber Blutegel werden weiterhin dazu benutzt, bei bestimmten Eingriffen einen Blutstau zu lindern, denn in solchen Fllen verursacht diese Methode mit geringerer Wahrscheinlichkeit Infektionen als andere Techniken. Auch werden Blutegel erfolgreich gegen Grtelrose Herpes zoster) und Muskelverhrtungen sowie zur Schmerzlinderung bei Kniearthrose eingesetzt. Nach Angaben von 2000 werden in Deutschland jhrlich bis 400 000 Blutegel Patienten angesetzt; eine kommerzielle Blutegel-Zuchtanlage befindet sich in Biebertal bei Gieen. Blutegel sind in marinen Gewssern weit verbreitet, und in gemigten und tropischen Regionen trifft man sie auch im Swasser und an Land.

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Hirudo medicinalis and Galen

Because of their high capacity for blood removal, leeches have been used in medical therapies since ancient times. The Greek scholar Galen mentioned the use of them for venesection in the year 180 and considered this a useful procedure for elective blood removal, if medically desired. Greek physician, born at Pergamus, in Mysia (on the Mediterranean coast of what is now Turkey), who became the most celebrated doctor in the Roman empire. His teachings powerfully influenced the practice of medicine in Europe and the near east for many centuries.
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Galen combined the knowledge and ideas of Herophilus and Erasistratus, the Hippocratic Corpus, and Roman medical writings of his time, in order to create a one-of-a-kind mixture of Platonic, Aristotelian, and Stoic natural philosophy. In his On the Hand, Galen described why the hand was oriented the way it was, why the fingers were positioned where they were, and the space between them (On the Usefulness of the Parts of the Body). Another important contribution of Galen was his physiological system, in which he adopted Plato's tripartite soul theory and correlated it with the three basic physiological functions defined by Erasistratus, resulting in a physiological tripartite organizational framework. In this system, the brain (seat of the soul's rational faculties) was the source of the nerves, accounting for sensation and motor functions; the heart (seat of the passions) was the source of the arteries, conveying life-giving arterial blood (and vital spirit) to all parts of the body; and the liver (seat of desire or appetite) was the source of the veins, nourishing the body with venous blood. These three physiological systems, according to Galen, had interconnections, and were not totally independent.

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The structur of hirudin

The crystallographic structure of a recombinant hirudin-thrombin complex has been solved at 2.3 angstrom (A) resolution. Hirudin consists of an NH2-terminal globular domain and a long (39 A) COOH-terminal extended domain. Residues Ile1 to Tyr3 of hirudin form a parallel beta-strand with Ser214 to Glu217 of thrombin with the nitrogen atom of Ile1 making a hydrogen bond with Ser195 O gamma atom of the catalytic site, but the specificity pocket of thrombin is not involved in the interaction.In all, 27 of the 65 residues of hirudin have contacts less than 4.0 A with thrombin (10 ion pairs and 23 hydrogen bonds). Such abundant interactions may account for the high affinity and specificity of hirudin.

Structure of Hirudin in complex with Thrombin.

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Conversion of r-hirudin by in vitro sulfation

Conversion of recombinant hirudin to the natural form by in vitro tyrosine sulfation. Differential substrate specificities of leech and bovine tyrosylprotein sulfotransferases Hirudin, a tyrosine-sulfated protein secreted by the leech Hirudo medicinalis, is one of the most potent anticoagulants known. The hirudin cDNA has previously been cloned and has been expressed in yeast, but the resulting recombinant protein was found to be produced in the unsulfated form, which is known to have an at least 10 times lower affinity for thrombin than the naturally occurring tyrosine- sulfated hirudin. Here we describe the in vitro tyrosine sulfation of recombinant hirudin by leech and bovine tyrosylprotein sulfotransferase (TPST). With both enzymes, in vitro sulfation of recombinant hirudin occurred at the physiological site (Tyr-63) and rendered the protein biochemically and biologically indistinguishable from natural hirudin. However, leech TPST had an over 20-fold lower apparent Km value for recombinant hirudin than bovine TPST. Further differences in the catalytic properties of leech and bovine TPSTs were observed when synthetic peptides were tested as substrates. Moreover, a synthetic peptide corresponding to the 9 carboxyl-terminal residues of hirudin (which include Tyr-63) was sulfated by leech TPST with a similar apparent Km value as full length hirudin, indicating that structural determinants residing in the immediate vicinity of Tyr-63 are sufficient for sulfation to occur.

J. Biol. Chem., Vol. 265, Issue 16, 9314-9318, Jun, 1990

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Recombinant expression of selectively sulfated proteins in Escherichia coli

Although tyrosine sulfation is a post-translational modification widespread across multicellular eukaryotes, its biological functions remain largely unknown. This is in part is due to the difficulties of synthesizing selectively sulfated proteins. Here we report the selective incorporation of sulfotyrosine into proteins in bacteria by genetically encoding the modified amino acid in response to the amber nonsense codon TAG. Moreover, we show that this strategy enables direct expression in Escherichia coli of sulfo-hirudin, previously inaccessible through recombinant methods. The affinity of sulfo-hirudin toward human thrombin is enhanced more than tenfold over that of desulfo-hirudin, suggesting that sulfo-hirudin may offer clinical advantages for use as an anticoagulant. This general approach to the biosynthesis of sulfated proteins should facilitate further study and application of tyrosine sulfation. Nature Biotechnology - 24, 1436 - 1440 (2006)

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Production of recombinant hirudin

Schematic of Pichia pastoris Fermentation System with Methanol Sensor

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Improvement of r-hirudin production

In recombinant Pichia pastoris fermentation for hirudin production in a 5 l fermenter, a new strategy was explored to match the short fermentation time at low NH4+ concentration with decreased hirudin degradation at high NH4+ concentration. A combination of a defined medium containing initial 0.025 m NH4+ with NH4+ addition up to 0.6 m in the growth phase was achieved in both the improvement of hirudin production and the repression of hirudin degradation. Intact and total hirudin reached 2.63 g l-1 and 4.25 g l-1, respectively.

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Hirudin im Komplex mit Thrombin. Hirudin ist in Form von "Sticks" dargestellt, das Thrombinmolekl als "Ribbon". pdb-Code 4HTC, T.J. Rydel et al., Science V. 249, S. 277 (1990)

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Bivalent binding

Moiety of a new bivalent inhibitor blocking the active site of thrombin. This generation of polypeptide inhibitors was discovered through rational design in combination with phagedisplay selection. The figure shows a three-dimensional model of the bivalent peptide bound to thrombin, suggesting intricate communications between P3 and P'3 residues. The P'3 residue projects its side chain into contacts with the side chain of the P3 site, which may be the structural mechanism for enhanced potencies of the new thrombin inhibitors. Bivalent peptide inhibitors of thrombin contain two covalently linked motifs that bind to the catalytic active site and a protein recognition exosite of thrombin, respectively.

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Hirudin and hirudin analogues

Lepirudin ([Leu,1 Thr2]-63desulfatohirudin; 65 amino acids; molecular weight, 6979.5 Da. Desirudin differs from lepirudin only in the first two N-terminal amino acids (valine-1, valine-2); antithrombin activities are 16 000 and 18 000 antithrombin U/mg, respectively.

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Weitere Thrombinhemmer
Bivalirudin (Angiomax) is a drug that belongs to the anticoagulant class and acts as a direct thrombin inhibitor. Chemically it constitutes a synthetic congener of the naturally occurring drug hirudin (found in the saliva of the medicinal leech Hirudo medicinalis).

Argatroban ist ein Arzneistoff zur Hemmung der Blutgerinnung. Der synthetische direkte Inhibitor des Thrombins ist in Deutschland und sterreich seit 2005 unter dem Namen Argatra zur Antikoagulation bei Erwachsenen mit einer heparininduzierten Thrombozytopenie vom Typ II (HIT II) zugelassen, wenn diese einer parenteralen antithrombotischen Therapie bedrfen.

Ximelagatran, a direct thrombin inhibitor, was the first member of this class that can be taken orally. It acts solely by inhibiting the actions of thrombin. 15

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The coagulation cascade

The coagulation cascade of secondary hemostasis has two pathways, which lead to fibrin formation. It was previously thought that the coagulation cascade consisted of two pathways of equal importance joined to a common pathway. It is now known that the primary pathway for the initiation of blood coagulation is the tissue factor pathway. The pathways are a series of reactions, in which a zymogen (inactive enzyme precursor) of a serine protease and its glycoprotein co-factor are activated to become active components that then catalyze the next reaction in the cascade, ultimately resulting in cross-linked fibrin. Coagulation factors are generally indicated by Roman numerals, with a lowercase a appended to indicate an active form.

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Activated Partial Thromboplastin Time (APTT): PD-parameter

The APTT measures the activity of the intrinsic and common pathways of coagulation. The term 'thromboplastin' in this test refers to the formation of a complex from various plasma clotting factors which then converts prothrombin to thrombin and the subsequent formation of the fibrin clot. Most laboratories use an automated method for the APTT in which clot formation is deemed to have occurred when the optical density of the mixture has exceeded a certain threshold (clot formation makes the mixture more opaque and less light passes through). The clotting time for the APTT lies between 27-35 seconds. However, this varies widely between laboratories and is dependent upon a number of variables including whether automated or manual, the type of surface activator and the incubation time.

Patient platelet poor (PPP) plasma is incubated at 37 C with phospholipid (cephalin) and a contact activator (e.g. Kaolin) are added followed by the calcium. Addition of calcium initiates clotting and timing begins. The APTT is the time taken for a fibrin clot to form.

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Heparin-induzierte Thrombozytopenie

Die Heparin-induzierte Thrombozytopenie wird hufig als HiT II - teilweise auch als HAT II (heparinassociated thrombocytopenia type II) bezeichnet. Es handelt sich dabei um ein durch Arzneimittel hervorgerufenes immun-vermitteltes Syndrom, das mit einer Thrombozytopenie und mit thrombotischen Ereignissen einhergeht. Die thrombotischen Ereignisse knnen zum Verlust von Extremitten fhren und darber hinaus lebensbedrohend sein. Eine Heparininduzierte Thrombozytopenie tritt in bis zu 5 % der Flle bei Patienten auf, die mit unfraktioniertem Heparin behandelt werden. Bei der Behandlung mit niedermolekularem Heparin sind es weniger als 1 %.

Typisches Erscheinungsbild einer heparininduzierten Hautnekrose, erythematser Randsaum mit deutlich sichtbarer irregulr begrenzter zentraler Nekrose.

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Lepirudin bei Heparin-induzierter Thrombozytopenie

- Rekombinantes Hirudin, 65 AS (7.000 D)

- Keine Kreuzreaktivitt mit HIT-Antikrpern - T1/2 1 - 2 h - Renale Elimination (T1/2 52 h bei Krea-Clearance <10ml/min) - Kein Antidot, aber Elimination durch Hmodialyse / -filtration (high-flux Polysulfon Dialysemembranen) -Therapeutischer Bereich: APTT-Ratio 1,5 - 3,0 -Allerg. Reaktionen, AK-Bildung, Anaphylaxie

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Pharmakokinetische Parameter von Thrombinhemmern

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Monitoring Refludan

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Dosing regimen of r-hirudin

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Plasma concentration of r-hirudin

Pharmacological profiling of recombinant hirudin (r-hirudin) has shown that this selective tight-binding thrombin inhibitor is a potent, welltolerated anticoagulant. Clinical pharmacological studies were performed in human volunteers after single and repeated doses of 0.1-0.5 mg/kg. Thrombin time and partial thromboplastin time were prolonged dependent on the r-hirudin level in plasma. Bleeding time was not prolonged. On intravenous injection, rhirudin was rapidly distributed into the extracellular space and eliminated, with a dose-dependent half-life of 1-2 h (first-order kinetics). The high recovery of unchanged r-hirudin in the urine identified renal excretion as the predominant route of r-hirudin clearance. Institute of Chemistry & Bioanalytics 23

Pharmacodynamics of hirudin

Activated partial thromboplastin time (aPTT) is a performance indicator measuring the efficacy of the common coagulation pathways. Dosage is adjusted according to aPTT ratio (patient aPTT at a given time over aPTT reference value, usually median of laboratory normal range for aPTT). Target range for aPTT ratio during treatment should be 1.52.5.

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Hirudin elimination by hemofiltration

Renal function impairment drastically prolongs the elimination half-life time of r-hirudin. In cases of bleeding or overdosage, there is currently no antidote available. Hemofiltration has been reported to be useful in rhirudin elimination. In this study, we determined sieving coefficients (SCs) and drug clearances for two different hemofilters currently used in clinical medicine and intensive care.

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We developed an in vitro postdilution hemofiltration model using 500 ml heparinized (2 IU unfractionated heparin/ml) fresh human blood and bicarbonate substitution fluid. The investigated membranes were highflux polysulfone F50 (1.0 m2, Fresenius) and AN69 Nephral 200 (1.05 m2, Hospal Cobe). After equilibration, a bolus of Lepirudin was injected into the postfilter port to achieve a r-hirudin blood level of approximately 15 g/ml. Serial blood and ultrafiltrate samples were taken for the determination of hirudin levels (chromogenic assay) and control parameters. SC and clearances were calculated according to standard formulae.

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Schematic drawing of the in vitro hemofiltration model

Abbreviations are: Cwi, prefilter plasmawater drug concentration; Cwo, postfilter plasma-water drug concentration; Cu, ultrafiltrate drug concentration; Cw, plasma-water drug concentration; UFR, ultrafiltrate flow rate.

The investigated hollow fiber hemofilters were high-flux polysulfone F50 (surface area 1 m2, blood compartment 63 ml, ultrafiltrate compartment 84 ml; Fresenius Medical Care, Bad Homburg, Germany) and AN69 Nephral 200 (1.05 m2, 64 ml and 41 ml; Hospital Cobe, Lyon, France).

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The investigated hollow fiber hemofilters were high-flux polysulfone F50 (surface area 1 m2, blood compartment 63 ml, ultrafiltrate compartment 84 ml; Fresenius Medical Care, Bad Homburg, Germany) and AN69 Nephral 200 (1.05 m2, 64 ml and 41 ml; Hospital Cobe, Lyon, France).

Time course of the prefilter recombinant (r)-hirudin levels during in vitro hemofiltration. Starting from the same level at t = 0 min, the r-hirudin level (mean SD) declined more rapidly with F50 ( ) compared with Nephral 200 ( ). The differences were statistically significant at time points 5, 10, 15, and 20 minutes (P < 0.05). The control experiments (lines) showed unchanged r-hirudin levels [( ) F50, ( ) Nephral 200].

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ELISA fr die Bestimmung von r-Hirudin

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