Chemical modification of recombinant hirudin with palmitic acid in mixed aqueous-organic solutions

SummaryThe behavior of 125I-labeled recombinant hirudin towards the uninjured and de-endothelialized rabbit aorta wall has been studied in vitro and in vivo to determine its usefulness as an indicator of thrombin activity associated with the aorta wall. Thrombin adsorbed to either sulfopropyl-Sephadex or heparin-Sepharose bound >95% of 125I-r-hirudin and the complex remained bound to the matrix. Binding of 125I-r-hirudin to the exposed aorta subendothelium (intima-media) in vitro was increased substantially if the tissue was pre-treated with thrombin; the quantity of l25I-r-hirudin bound to the de-endothelialized intima-media (i.e. balloon-injured in vitro) correlated positively with the quantity of bound 131I-thrombin (p <0.01). Aortas balloon-injured in vivo were measured for thrombin release from, and binding of 125I-r-hirudin to, the de-endothelialized intimal surface in vitro; 125I-r-hirudin binding correlated with the amount of active thrombin released (p <0.001). Uptake of 125I-r-hirudin by the aorta wall in vivo was proportional to the uptake of 131I-fibrinogen (as an indicator of thrombin activity) before and after balloon injury. After 30 min in the circulation, specific 125I-r-hirudin binding to the uninjured and de-endo- thelialized (at 1.5 h after injury) aorta wall was equivalent to 3.4 (± 2.5) and 25.6 (±18.1) fmol of thrombin/cm2 of intima-media, respectively. Possibly, only hirudin-accessible, glycosaminoglycan-bound thrombin is measured in this way.

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Combination of Antibiotic Treatment with the Thrombin Inhibitor Recombinant Hirudin for the Therapy of Experimental Klebsiella pneumoniae Sepsis

Thrombosis and Haemostasis ◽

10.1055/s-0038-1642520 ◽

1994 ◽

Vol 71 (06) ◽

pp. 768-772 ◽

Cited By ~ 14

Author(s):

Gerhard Dickneite ◽

Jörg Czech

Keyword(s):

Septic Shock ◽

Klebsiella Pneumoniae ◽

Organ Failure ◽

Therapeutic Effect ◽

Disseminated Intravascular Coagulation ◽

Post Infection ◽

Thrombin Inhibitor ◽

Bolus Administration ◽

Bacterial Burden ◽

Recombinant Hirudin

SummaryRats which were infected with the gramnegative pathogen Klebsiella pneumoniae develop disseminated intravascular coagulation (DIC), multi-organ failure (MOF) and finally die in a septic shock. We investigated the therapeutic effect of antibiotic (tobramycin) treatment combined with the infusion of the highly specific thrombin inhibitor rec. hirudin. Although administration of 2 mg/kg tobramycin alone leads to a decrease of the bacterial burden, DIC could not be prevented. Infusion of rec. hirudin (0.25 mg/kg x h) for 4 h (start of treatment 1 h post infection), in addition to a bolus administration of tobramycin, led to an amelioration of DIC parameters as fibrinogen, thrombin-antithrombin complex (TAT) and platelets. Serum transaminase levels (GOT, GPT) as a marker of MOF were significantly improved by rec. hirudin, the T50 value increased from 17 h in the tobramycin group to 42 h in the tobramycin + rec. hirudin giuup, muilality rates were 90% or 60%, respectively. Combination of heparin (10011/kg x h) and tobramycin was not effective on survival.

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Physico-Chemical Properties of Recombinant Desulphatohirudin

Thrombosis and Haemostasis ◽

10.1055/s-0038-1645073 ◽

1990 ◽

Vol 63 (03) ◽

pp. 499-504 ◽

Cited By ~ 4

Author(s):

A Electricwala ◽

L Irons ◽

R Wait ◽

R J G Carr ◽

R J Ling ◽

...

Keyword(s):

Molecular Weight ◽

Gel Filtration ◽

Chemical Properties ◽

Alpha Helix ◽

Apparent Molecular Weight ◽

Correlation Spectroscopy ◽

Nmr Studies ◽

Recombinant Hirudin ◽

Physico Chemical ◽

Recombinant Molecule

SummaryPhysico-chemical properties of recombinant desulphatohirudin expressed in yeast (CIBA GEIGY code No. CGP 39393) were reinvestigated. As previously reported for natural hirudin, the recombinant molecule exhibited abnormal behaviour by gel filtration with an apparent molecular weight greater than that based on the primary structure. However, molecular weight estimation by SDS gel electrophoresis, FAB-mass spectrometry and Photon Correlation Spectroscopy were in agreement with the theoretical molecular weight, with little suggestion of dimer or aggregate formation. Circular dichroism studies of the recombinant molecule show similar spectra at different pH values but are markedly different from that reported by Konno et al. (13) for a natural hirudin-variant. Our CD studies indicate the presence of about 60% beta sheet and the absence of alpha helix in the secondary structure of recombinant hirudin, in agreement with the conformation determined by NMR studies (17)

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Experimental Studies on a Recombinant Hirudin, CGP 39393

Thrombosis and Haemostasis ◽

10.1055/s-0038-1648151 ◽

1991 ◽

Vol 65 (04) ◽

pp. 355-359 ◽

Cited By ~ 12

Author(s):

E Gray ◽

J Watton ◽

S Cesmeli ◽

T W Barrowcliffe ◽

D P Thomas

Keyword(s):

Thrombin Generation ◽

Bleeding Time ◽

Thrombus Formation ◽

Experimental Studies ◽

Venous Stasis ◽

Antithrombotic Agent ◽

Recombinant Hirudin ◽

Excessive Bleeding ◽

Generation Test

SummaryThe in vitro anticoagulant activities of recombinant desulphatohirudin (r-hirudin) were studied in the activated partial thromboplastin time (APTT) and the thrombin generation test : systems. In the APTT at concentrations below 5 μg/ml, r-hirudin showed a dose-response curye. At concentrations above 5 μg/ml, the plasma became unclottable, but in the thrombin generation test , at least 10 μg/ml of r-hirudin was required for full inhibition of thrombin generation. The antithrombotic effect was assessed using a rabbit venous stasis model; 150 μg/ml r-hirudin completely prevented thrombus formation at 10 and 20 min stasis. At antithrombotic dose, the mean bleeding time ratio measured in a rabbit ear template model, was not prolonged over control values. At higher doses, the bleeding time ratios were higher than those observed for the same dosage of heparin. These data indicate that while r-hirudin is an effective antithrombotic agent, antithrombotic doses have to be carefully titrated to avoid excessive bleeding.

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Monitoring Anticoagulant Therapy by Activated Partial Thromboplastin Time: Hirudin Assessment

Thrombosis and Haemostasis ◽

10.1055/s-0038-1648943 ◽

1994 ◽

Vol 72 (05) ◽

pp. 685-692 ◽

Cited By ~ 43

Author(s):

Michael T Nurmohamed ◽

René J Berckmans ◽

Willy M Morriën-Salomons ◽

Fenny Berends ◽

Daan W Hommes ◽

...

Keyword(s):

Whole Blood ◽

Partial Thromboplastin Time ◽

Ex Vivo ◽

Fold Increase ◽

Heparin Therapy ◽

Activated Partial Thromboplastin Time ◽

Recombinant Hirudin ◽

Interindividual Differences ◽

The Relationship

SummaryBackground. Recombinant hirudin (RH) is a new anticoagulant for prophylaxis and treatment of venous and arterial thrombosis. To which extent the activated partial thromboplastin time (APTT) is suitable for monitoring of RH has not been properly evaluated. Recently, a capillary whole blood device was developed for bed-side monitoring of the APTT and it was demonstrated that this device was suitable to monitor heparin therapy. However, monitoring of RH was not evaluated.Study Objectives. To evaluate in vitro and ex vivo the responsiveness and reproducibility for hirudin monitoring of the whole blood monitor and of plasma APTT assays, which were performed with several reagents and two conventional coagulometers.Results. Large interindividual differences in hirudin responsiveness were noted in both the in vitro and the ex vivo experiments. The relationship between the APTT, expressed as clotting time or ratio of initial and prolonged APTT, and the hirudin concentration was nonlinear. A 1.5-fold increase of the clotting times was obtained at 150-200 ng/ml plasma. However, only a 2-fold increase was obtained at hirudin levels varying from 300 ng to more than 750 ng RH/ml plasma regardless of the assays. The relationship linearized upon logarithmic conversion of the ratio and the hirudin concentration. Disregarding the interindividual differences, and presuming full linearity of the relationship, all combinations were equally responsive to hirudin.Conclusions. All assays were equally responsive to hirudin. Levels up to 300 ng/ml plasma can be reliably estimated with each assay. The manual device may be preferable in situations where rapid availability of test results is necessary.

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Subcutaneous Recombinant Hirudin (HBW 023) Versus Intravenous Sodium Heparin in Treatment of Established Acute Deep Vein Thrombosis of the Legs: a Multicentre Prospective Dose-ranging Randomized Trial

Thrombosis and Haemostasis ◽

10.1055/s-0038-1656063 ◽

1997 ◽

Vol 77 (05) ◽

pp. 0834-0838 ◽

Cited By ~ 59

Author(s):

François Schiele ◽

Folke Lindgaerde ◽

Henry Eriksson ◽

Jean-Pierre Bassand ◽

Anders Wallmark ◽

...

Keyword(s):

Bleeding Complications ◽

Recombinant Hirudin ◽

Serious Adverse Events ◽

Sodium Heparin ◽

Efficacy Analysis ◽

Vein Thrombosis ◽

Dose Ranging ◽

Acute Deep Vein Thrombosis ◽

Deep Vein ◽

Intravenous Sodium

SummaryThe aim of this multicentre, prospective, randomised, dose-ranging study was to compare the safety and efficacy of subcutaneous recombinant hirudin (HBW 023) against intravenous sodium heparin in acute lower limb deep venous thrombosis (DVT). Patients were randomized to treatment with either HBW 023 or heparin for 5 ±1 days. HBW 023 was given according to body-weight in three dose groups. Thromboembolic disease was assessed by phlebography and ventilation/perfusion (V/Q) scanning on Bay 1 and Day 5±1. One hundred and fifty-five patients were enrolled, of these 121 were evaluable for efficacy analysis. Significantly fewer patients on HBW 023 developed new V/Q abnormalities during the treatment period, (p = 0.006). There was no difference between the groups in thrombus extension or regression, major bleeding complications or serious adverse events. There were significantly fewer findings of new V/Q mismatch after treatment with HBW 023, and anticoagulant control was superior in these patients.

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