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Bioanalytical Sciences

Recombinant Hirudin

Daniel Gygax

School of Life Sciences, Muttenz

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Institute of Chemistry & Bioanalytics 2

Hirudin

- 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 bestimmtenEingriffen einen Blutstau zu lindern, denn in solchen Fllen verursacht diese Methode mit geringerer WahrscheinlichkeitInfektionen als andere Techniken. Auch werden Blutegel erfolgreich gegen GrtelroseHerpes zoster) und Muskelverhrtungensowie zur Schmerzlinderung bei Kniearthrose eingesetzt. Nach Angaben von 2000 werden in Deutschland jhrlich bis400 000 Blutegel Patienten angesetzt; eine kommerzielle Blutegel-Zuchtanlage befindet sich in Biebertal bei Gieen. Blutegelsind in marinen Gewssern weit verbreitet, und in gemigten und tropischen Regionen trifft man sie auch im Swasser und anLand.

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Hirudo medicinalis and Galen

Because of their high capacity for

blood removal, leeches havebeen 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 theMediterranean coast of what is now Turkey), whobecame the most celebrated doctor in the Romanempire. His teachings powerfully influenced thepractice 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 itwas, 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 anNH2-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 nitrogenatom 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 substratespecificities 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 thephysiological 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 similarapparent 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 partis 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 coliof 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 foruse 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 pastorisFermentation System with MethanolSensor

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Improvement of r-hirudin production

In recombinantPichia pastoris fermentation forhirudin production in a 5 l fermenter, a newstrategy was explored to match the shortfermentation time at low NH4+ concentrationwith decreased hirudin degradation at highNH4+ concentration. A combination of a definedmedium containing initial 0.025 m NH4+ with

NH4+

addition up to 0.6 m in the growth phasewas achieved in both the improvement ofhirudin production and the repression ofhirudin degradation. Intact and total hirudinreached 2.63 g l-1 and 4.25 g l-1, respectively.

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Hirudin-thrombin-binding

Hirudin im Komplexmit Thrombin.

Hirudin ist in Form

von "Sticks"

dargestellt, dasThrombinmolekl 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 blockingthe active site of thrombin. This generationof polypeptide inhibitors was discoveredthrough rational design in combination withphagedisplay selection. The figure shows athree-dimensional model of the bivalentpeptide bound to thrombin, suggesting

intricate communications between P3 and P'3residues. The P'3 residue projects its sidechain into contacts with the side chain of theP3 site, which may be the structuralmechanism for enhanced potencies of thenew thrombin inhibitors.

Bivalent peptide inhibitors of thrombin contain twocovalently linked motifs that bind to the catalytic activesite and a protein recognition exosite of thrombin,

respectively.

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Hirudin and hirudin analogues

Lepirudin ([Leu,1 Thr2]-63-

desulfatohirudin; 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 thenaturally occurring drug hirudin (found in

the saliva of the medicinal leechHirudo

medicinalis).

Argatroban ist ein Arzneistoff zur Hemmungder Blutgerinnung. Der synthetische direkteInhibitor des Thrombins ist in Deutschland und

sterreich seit 2005 unter dem NamenArgatra zur Antikoagulation beiErwachsenen mit einer heparininduziertenThrombozytopenie vom Typ II (HIT II)zugelassen, wenn diese einer parenteralenantithrombotischen Therapie bedrfen. Ximelagatran, a direct thrombin inhibitor,

was the first member of this class that canbe taken orally. It acts solely by inhibitingthe actions of thrombin.

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The coagulation cascade

The coagulation cascade of secondary hemostasis has

two pathways, which lead tofibrin formation.

It was previously thought that the coagulationcascade 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 thetissue factorpathway.

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 lowercasea

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 andcommon pathways of coagulation.

The term 'thromboplastin' in this test refers to theformation of a complex from various plasma clottingfactors which then converts prothrombin to thrombinand the subsequent formation of the fibrin clot.

Most laboratories use an automated method for theAPTT in which clot formation is deemed to haveoccurred when the optical density of the mixture hasexceeded a certain threshold (clot formation makesthe mixture more opaque and less light passesthrough).

The clotting time for the APTT lies between 27-35seconds. However, this varies widely betweenlaboratories and is dependent upon a number ofvariables including whether automated or manual, thetype of surface activator and the incubation time.

Patient platelet poor (PPP) plasma is incubated at37C with phospholipid (cephalin) and a contactactivator (e.g. Kaolin) are added followed by thecalcium. Addition of calcium initiates clotting andtiming 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 hufigals HiT II - teilweise auch als HAT II (heparin-associated thrombocytopenia type II) bezeichnet. Eshandelt sich dabei um ein durch Arzneimittelhervorgerufenes immun-vermitteltes Syndrom, das mit

einer Thrombozytopenie und mit thrombotischenEreignissen einhergeht. Die thrombotischen Ereignisseknnen zum Verlust von Extremitten fhren unddarber hinaus lebensbedrohend sein. Eine Heparin-induzierte Thrombozytopenie tritt in bis zu 5 % der Flle

bei Patienten auf, die mit unfraktioniertem Heparinbehandelt werden. Bei der Behandlung mit nieder-molekularem Heparin sind es weniger als 1 %.

Typisches Erscheinungsbild einerheparininduzierten

Hautnekrose, erythematserRandsaum mit deutlich

sichtbarer irregulr begrenzterzentraler 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

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Pharmakokinetische Parameter von Thrombinhemmern

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Monitoring Refludan

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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, well-

tolerated 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, r-

hirudin was rapidly distributed into theextracellular 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-hirudinclearance.

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Pharmacodynamics of hirudin

Activated partial thromboplastintime (aPTT) is a performanceindicator measuring the efficacy ofthe common coagulation pathways.

Dosage is adjusted according toaPTT ratio (patient aPTT at a given

time over aPTT reference value,usually median of laboratory

normal range for aPTT). Target

range for aPTT ratio duringtreatment 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 currentlyno antidote available. Hemofiltration has been reported to be useful in r-

hirudin 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 andbicarbonate substitution fluid. The investigated membranes were high-

flux 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 tostandard formulae.

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Schematic drawing of the in vitro hemofiltration model

Abbreviations are: Cwi, prefilter plasma-water 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 polysulfoneF50 (surface area 1 m2, blood compartment 63 ml, ultrafiltratecompartment 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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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 rapidlywith 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].

The investigated hollow fiber hemofilters were high-flux polysulfoneF50 (surface area 1 m2, blood compartment 63 ml, ultrafiltratecompartment 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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ELISA fr die Bestimmung von r-Hirudin

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