Recombinant Hirudin Research Articles

Display of functional thrombin inhibitor hirudin on the surface of phage M13.

A synthetic gene for hirudin was ligated into phagemid pCANTAB5E. This construct allows production of either soluble hirudin or phage having hirudin displayed on the surface. Similarly, hirudin variants with extensions either at their N- or C-terminus were generated. The genes were expressed in their soluble form in a non-suppressor strain of E. coli. Periplasmatic fractions were evaluated in standard thrombin inhibition assays. Extending hirudin by a single Gln residue at the N-terminus reduces the activity by two orders of magnitude. This suggests that either the terminal amine group makes an important interaction or that steric constraints do not allow additional amino acids here. Only C-terminal extensions maintain most of the thrombin inhibitor activity of r-hirudin. The r-hirudin gene was also expressed on the tips of filamentous phage as a fusion protein with protein III (pIII). The hirudin-pIII fusion protein was detected with anti-hirudin antibody and with anti-E-tag antibody by Western blot analysis. Recombinant phages were shown to bind to immobilized thrombin in a dose-dependent manner. Upon addition of soluble thrombin, recombinant hirudin phages could be eluted specifically. Finally, purified phages carrying displayed r-hirudin were shown to inhibit thrombin in a standard amidolytic assay for thrombin inhibitor activity. These results demonstrate that hirudin can be C-terminally extended without diminishing the antithrombic activity. Beyond that, active hirudin can be displayed on the surface of M13 phage. As a conclusion, applied molecular evolution, i.e. the selection of hirudin-based thrombin inhibitor variants with tailored properties from (partially) randomized peptide pools should now be possible.

Differential effects of anticoagulants on the activation of platelets ex vivo.

Because activation of platelets and of the coagulation system are interdependent mediators of thrombosis, platelet activation was characterized in whole blood in the presence of anticoagulants used to assess platelet function in vitro or as treatment for patients with occlusive arterial disease. Blood was anticoagulated alone or in combination with citrate, ethylenediaminetetraacetatic acid, corn trypsin inhibitor (CTI, an inhibitor of activated factor XII), heparin, enoxaparin, recombinant tick anticoagulant peptide (rTAP), or recombinant hirudin. Platelet activation in response to adenosine diphosphate (ADP) or collagen was detected by assay of P-selectin on the platelet surface delineated by flow cytometry. Although minimal activation was seen without ADP, the fraction of platelets expressing P-selectin in response to ADP was greatest in blood anticoagulated with citrate compared with CTI and all other anticoagulants. ADP-induced platelet activation was greater in blood anticoagulated with heparin compared with an equipotent anti-Xa concentration of enoxaparin. More variable results were seen with collagen, but platelet activation in the presence of citrate was greater than that with CTI. Interpretation of assays of inhibition of platelet activation by potentially therapeutic agents in vitro requires consideration of the effects of anticoagulants used. In addition, anticoagulants other than standard heparin may potentiate efficacy of antiplatelet drugs.

Heparin-induced thrombocytopenia. Pathogenesis, frequency, avoidance and management.

Heparin-induced thrombocytopenia (HIT) is an immunoglobulin-mediated adverse drug reaction associated with a high risk of thrombotic complications. The pathogenic antibody, usually immunoglobulin (Ig) G (HIT-IgG), recognises a multimolecular complex of heparin and platelet factor 4, resulting in platelet activation via platelet Fc receptors. In addition to in vivo platelet activation, it is now recognised that there is a concomitant activation of coagulation, as shown by marked elevations in thrombin-antithrombin complex levels. It is possible that this increased thrombin generation predisposes HIT patients to a newly recognised complication: warfarin-induced venous limb gangrene. This syndrome is characterised clinically by necrosis complicating deep venous thrombosis in the absence of large-vessel arterial occlusion, and appears to result from acquired protein C deficiency during warfarin therapy for deep vein thrombosis and HIT. The recommended treatment for HIT is an agent that reduces thrombin generation, either indirectly via factor Xa inhibition [e.g. danaparoid sodium (a mixture of anticoagulant glycosaminoglycans)] or directly using a specific thrombin inhibitor (e.g. recombinant hirudin; argatroban). HIT is potentially preventable: there is a lower frequency of HIT, associated thrombosis and HIT-IgG seroconversion in patients treated with low-molecular-weight heparins, compared with unfractionated heparin.

Abstracts of Literature

Abstracts of Literature

REEVALUATING THE EFFECTS OF TYROSINE IODINATION OF RECOMBINANT HIRUDIN ON ITS THROMBIN INHIBITION KINETICS

To reevaluate the effects of iodination of hirudin on its thrombin-inhibiting activity, we iodinated a recombinant hirudin analog, CX-397, by a chloramine-T method and isolated the eight kinds of iodinated derivatives by reverse-phase HPLC. Their structure were different combinations of non-, mono-, or di-iodinated residues at Tyr-3 and Tyr-64. Their ability to inhibit α-thrombin were determined and compared with each other. Iodination at Tyr-64 slightly increased the association rate constant ( k on ) 1.26- to 1.79-fold; this may be explained by the increase in the negative charge at C-terminal region of CX-397 caused by the electrophilic ortho substitution of Tyr-64 with iodine. On the contrary, iodination at Tyr-3 caused dual effects on the interaction between CX-397 and α-thrombin; namely, mono-iodination at Tyr-3 caused a small decrease in k off (1.29- to 1.63-fold) than non-iodinated ones, whilst di-iodination at Tyr-3 brought about 3.04- to 3.88-fold increase in k off . Since the N-terminal region of hirudin has been reported to bind with thrombin by hydrophobic interaction, the destabilizing effect of di-iodination at Tyr-3 can be explained by the increase in its polar nature, whereas the opposite effect of mono-iodination can not. These results indicate that it is not appropriate to use a mixture of variably radioiodinated hirudins as a radiotracer. We also provide a simple method to obtain well characterized radioiodinated hirudins. ○ 1997 Elsevier Science Ltd

Influence of Different Anticoagulants on Platelet Aggregation in Whole Blood; A Comparison between Citrate, Low Molecular Mass Heparin and Hirudin

Anticoagulants used for platelet function studies in vitro may affect platelet responsiveness. In the present study we compared the influence of three different anticoagulants, sodium citrate, low molecular mass heparin, and recombinant hirudin, on platelet aggregation in whole blood in vitro, using impedance aggregometry. ADP and collagen induced aggregation was significantly lower in citrated blood compared to hirudin treated blood, reflecting the importance of exctracellular calcium for platelet function. Inhibition of platelet aggregation by aspirin, was more pronounced in citrated blood compared to hirudin treated blood, in agreement with the concept of artifactually enhanced thromboxane generation in media containing low extracellular calcium levels. In blood anticoagulated with low molecular mass heparin, platelet aggregation to collagen tended to be enhanced as compared to hirudin treated blood, whereas platelet responses to ADP at a high concentration were slightly reduced. Of the anticoagulants investigated, the selective thrombin inhibitor hirudin is the most suitable anticoagulant for studies of platelet aggregation in vitro in whole blood. © 1997 Elsevier Science Ltd

HIGH AND CONSTANT PLASMA LEVELS OF TISSUE PLASMINOGEN ACTIVATOR AND PEG-HIRUDIN CAN BE ACHIEVED BY SUBCUTANEOUS DELIVERY

Intramural thrombosis is a consistent finding in the arteries of patients who die following coronary angioplasty. This thrombosis is thought to have a role in restonosis, which is a common complication of coronary angioplasty. It has been hypothesised that antithrombotics such as hirudin or tissue -type plasminogen activator (tPA), may be therapeutically useful following angioplasty. This report describes the bioavailability of both agents following subcutaneous (sc) injection in cholesterol-fed rabbits. Intravenously delivered tPA has a half-life of 3–5 minutes. The half-life of intravenously administered hirudin is less than one hour in many species. In order to prolong the duration of action recombinant hirudin was conjugated to polyethylene glycol (PEG). Polyethylene glycol conjugated recombinant hirudin (PEG-rH) (0.7mg/kg) antigen and activity were measurable after just 1 hr, reaching a maximum (663 and 884 ng/ml respectively) at 12 hours. Significant levels were present in rabbit plasma 24 hours after injection. Subcutaneously delivered recombinant (r-tPA) (1mg/kg) was present in significant amounts 1hr after injection, reaching a maximum (92 IU/ml) at 2 hours. Levels of tPA at 9 hours were approximately 80× normal circulating levels. High and constant levels of functional activity of both PEG-rH and r-tPA in rabbit plasma are achieved by subcutaneous delivery. © 1997 Elsevier Science Ltd

ANTITHROMBOTIC EFFECTS OF NF-6505, A NOVEL ANION-BINDING EXOSITE INHIBITOR

NF-6505, a bi- O-Tyr-sulfated decapeptide, which specifically interacts with the anion-binding exosite of the thrombin molecule, was chemically synthesized and assessed for its antithrombotic effects in vitro and in vivo. The IC 50 value of this peptide on fibrin-clot formation in vitro was about 0.05 μg/ml, which indicated a potency similar to that of a recombinant hirudin. NF-6505 caused a 2-fold prolongation of activated partial thromboplastin time when intravenously administered at 1 mg/kg in rats. In a rat venous thrombosis model, a bolus intravenous administration of this peptide dose-dependently inhibited the thrombus formation with an ED 50 value of 0.03 mg/kg, a value smaller than that of recombinant hirudin (ED 50 = 0.1 mg/kg) or of argatroban (ED 50 = 0.2 mg/kg). These results suggest that NF-6505 is a highly potent and safe agent for the clinical treatment of venous thrombosis diseases. © 1997 Elsevier Science Ltd

Covalent linkage of recombinant hirudin to poly(ethylene terephthalate) (Dacron): creation of a novel antithrombin surface

Thrombus formation and intimal hyperplasia on the surface of implantable biomaterials such as poly(ethylene terepthalate) (Dacron) vascular grafts are major concerns when utilizing these materials in the clinical setting. Thrombin, a pivotal enzyme in the blood coagulation cascade primarily responsible for thrombus formation and smooth muscle cell activation, has been the target of numerous strategies to prevent this phenomenon from occurring. The purpose of this study was to covalently immobilize the potent, specific antithrombin agent recombinant hirudin (rHir) to a modified Dacron surface and characterize the in vitro efficacy of thrombin inhibition by this novel biomaterial surface. Bovine serum albumin (BSA), which was selected as the ‘basecoat’ protein, was reacted with various molar ratios of the cross-linker sulphosuccinimidyl 4-(N-maleimidomethyl) cyclohexane-1-carboxylate (sulpho-SMCC; 1:5-1:50). These BSA-SMCC complexes were then covalently linked to sodium hydroxide-hydrolysed Dacron (HD) segments via the cross-linker 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide hydrochloride (EDC). Covalent linkage of these complexes to HD (HD-BSA-SMCC) was not affected by any of the sulpho-SMCC cross-linker ratios assayed. rHir, which was initially reacted with 2-iminothiolane hydrochloride (Traut's reagent) in order to create sulphydryl groups, was then covalently bound to these HD-BSA-SMCC surfaces (HD-BSA-SMCC-S-rHir). The 1:50 (BSA: sulpho-SMCC) HD-BSA-SMCC-S-rHir segments bound 22-fold more rHir (111 ng per mg Dacron) compared to control segments and also possessed the greatest thrombin inhibition of the segments evaluated using a chromogenic substrate assay for thrombin. Further characterization of the HD-BSA-SMCC-S-rHir segments demonstrated that maximum thrombin inhibition was 20.43 NIHU, 14.6-fold greater inhibition than control segments (1.4 NIHU). Thrombin inhibition results were confirmed by 125L-thrombin binding experiments, which demonstrated that the 1:50 HD-BSA-SMCC-S-rHir segments had significantly greater specific thrombin adhesion compared to control segments. Non-specific 125I-thrombin binding to and release from the 1:50 HD-BSA-SMCC-S-rHir segments was also significantly less than the control segments. Thus, these results demonstrate that rHir can be covalently bound to a clinically utilized biomaterial (Dacron) while still maintaining its ability to bind and inhibit thrombin.

Recombinant hirudin in the prevention of venous thromboembolism in patients undergoing elective hip surgery.

Venous thromboembolism is a major cause of mortality and morbidity in patients undergoing orthopedic surgery. In these patients systemic prophylaxis with anticoagulants appears to be the most effective approach to reduce venous thromboembolic events. Low-molecular-weight heparins (LMWHs) are the prophylactic agents of choice in patients undergoing elective hip replacement. However, their use is still associated with a 15% incidence of deep vein thrombosis (DVT); hence, there is a need for potentially more effective agents. Among these, hirudin and other selective thrombin inhibitors have recently been evaluated. The main pharmacologic properties of recombinant hirudin and the clinical trials carried out with this agent in the prevention of DVT in patients undergoing elective hip replacement are reviewed in this article. Overall, the results of these clinical trials are quite promising; they actually indicate that a b.i.d. subcutaneous injection of 15 or 20 mg of recombinant hirudin is an effective and safe prophylactic approach. Hirudin was more effective than unfractionated heparin, given at a low fixed dose, in two clinical trials and of a LMWH in one clinical trial. However, further studies are required to define the role of recombinant hirudin in the prevention of venous thromboembolism in orthopedic surgery patients.

A comparison of recombinant hirudin with heparin for the treatment of acute coronary syndromes

A comparison of recombinant hirudin with heparin for the treatment of acute coronary syndromes

Biological monitoring of three antithrombotic drugs: single dose therapy

A randomised study using 40 New Zealand White male rabbits was performed to compare the effects of three antithrombotic drugs on eight clinical haemostatic function tests. The animals were divided into four treatment groups. The treatment groups were saline (control), unfractioned heparin (UFH), low-molecular-weight heparin (LMWH), and recombinant hirudin (r-hirudin). Blood samples were collected 2 and 12 h after administration of the drugs. The following tests were performed: bleeding time (BT), platelet count, prothrombin time (PT), activated partial thromboplastin time (APTT), thrombin time (TT), fibrinogen concentration (Fg), antithrombin III (ATIII), and antifactor Xa activity (antiXa activity). Effects attributable to drug treatment for each analyte were determined by comparison with the control group. At 2 h after medication, in the UFH treated group, TT was moderately prolonged (p 0.05) between groups were observed for any of the studied analytes. It might be concluded that the antiXa assay has the potential of being a sensitive screen for heparin therapy and that the absence of changes in the bleeding time, antithrombin III, and antiXa assays —with a markedly prolonged thrombin time - indicates that,in vivo, r-hirudin acts as a specific inhibitor of thrombin.

Hirudin reduced death or MI more than heparin at 48 hours but not at 30 days

Source Citation The Global Use of Strategies to Open Occluded Coronary Arteries (GUSTO) IIb Investigators. A comparison of recombinant hirudin with heparin for the treatment of acute coronary syndr...

Recombinant Hirudin Prevents Postoperative Fibrin Formation after Experimental Cataract Surgery

The authors studied the effect of a direct acting antithrombin agent, desulfatohirudin variant 1 (Revasc, Ciba-Geigy, Ltd., Basel, Switzerland), on postoperative fibrin formation after cataract surgery in rabbits. Phacoemulsification was performed in a masked fashion on 28 eyes of 28 New Zealand white rabbits. Ten control group eyes had lactated Ringer's solution in the infusion and an intracameral injection (approximately 1.5 ml) at the end of the case. Ten group 1 eyes received hirudin 100 micrograms/ml in the infusion and intracameral injection. Eight group 2 eyes had 100 micrograms/ml hirudin in the intracameral injection only. Using slit-lamp examination, all eyes were graded for the amount of fibrin and intraocular hemorrhage in a masked fashion on postoperative day 1. Comparison of the mean postoperative fibrin grade (0-4) in group 1 (mean = 0.3), group 2 (mean = 0.25) and the control group (mean = 3.4) revealed a statistically significant difference between hirudin-treated and control eyes (P = 0.0002 for group 1, P = 0.0005 for group 2). No intraocular hemorrhage was noted in any group. Recombinant hirudin significantly decreases postoperative fibrin formation in a rabbit cataract extraction model. Intracameral injection of hirudin alone appears to be at least as effective as infusion of hirudin throughout the case. With further study, this agent has potential for use in cataract surgery on patients known to be at high risk for postoperative fibrin formation.

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

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. 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.

Kinetic Studies of the Regeneration of Recombinant Hirudin Variant 1 with Oxidized and Reduced Dithiothreitol

The regeneration of native recombinant hirudin variant 1 (rHV1) from the reduced unfolded form to the fully oxidized native state has been carried out with mixtures of oxidized and reduced dithiothreitol at pH 8.3 and 12 degrees C. The regeneration reaction was quenched at various times by the addition of 2-aminoethyl methanethiosulfonate to block unreacted sulfhydryl groups. The quenched protein-folding intermediates were fractionated by both capillary electrophoresis and a combination of anion exchange and reverse phase HPLC and characterized by mass spectrometry, amino acid analysis, and disulfide analysis. These intermediates (before quenching) were found to interconvert rapidly so as to achieve a steady-state distribution early in the regeneration process. The experimental data were fitted to a steady-state kinetic scheme. The analysis reveals that the rate-determining step in the regeneration of rHV1 with oxidized and reduced dithiothreitol involves the oxidation of one or more two-disulfide-containing species, most likely those already containing two native disulfide bonds. This regeneration mechanism is different from one that has been proposed by Chatrenet and Chang [(1993) J. Biol. Chem. 268, 20988]. The differences are discussed, and possible explanations for the differences are presented.

Pharmacodynamic and Safety Results of PEG-Hirudin in Healthy Volunteers

PEG-Hirudin, a chemically defined conjugate of recombinant hirudin and two molecules of polyethylene glycol (PEG)-5000 is a highly selective direct thrombin inhibitor with a significantly longer duration of action than non-conjugated recombinant hirudin permitting once daily subcutaneous administration. A series of placebo-controlled, randomized, Phase I clinical trials were conducted in 75 healthy volunteers to investigate the anticoagulant effects, safety and pharmacodynamics of PEG-Hirudin when administered intravenously as a bolus injection, infusion, and subcutaneously. After single i.v. injections of various doses of PEG-Hirudin (0.03-0.3 mg/kg) dose-dependent increases in anti-IIa activity and APTT were observed. Four hours after injection of 0.3 mg/kg, mean plasma concentration expressed in terms of anti-IIa activity was still 0.89 micrograms/ml, corresponding to a 1.8-fold prolongation of APTT. Continuous intravenous infusions of 0.01 and 0.02 mg/kg/h PEG-Hirudin resulted in maximum anti-IIa activities of 0.42 micrograms/ml and 0.77 micrograms/ml, respectively, at the end of a six-hour infusion period without having reached steady state at this time. After termination of the infusion, anticoagulant activity displayed an immediate exponential decrease. The anticoagulant activities of single subcutaneous doses of 0.05 to 0.6 mg/kg were studied in a further series of investigations and slow increases and prolonged durations of anti-IIa activity and APTT prolongation were found. Repeated, once daily subcutaneous administrations of 0.2 to 0.4 mg/kg for five days resulted in dose-dependent prolongations of APTT and increases in anti-IIa activity without completely reaching steady state conditions. In a further study, 0.3 mg/kg of PEG-Hirudin was given as an i.v. bolus injection followed by three repeated single daily s.c. injections. In this trial, the APTT was shorter than expected from previous studies; therefore, a direct comparison of various APTT reagents was made in the intravenous infusion trial. Of the APTT reagents tested, BioMérieux Silimat and IL-ellagic acid proved to be the most sensitive to PEG-Hirudin. The hirudin derivative PEG-Hirudin was tolerated very well without immuno-allergic side effects. In view of the significantly prolonged anticoagulant efficacy in comparison to non-conjugated r-hirudin, PEG-Hirudin is a promising compound especially for repeated once daily subcutaneous administration.

Effects of Anticoagulants on Thromolytic Process by Recombinant Tissue-type Plasminogen Activator

The aim of this study is to establish a microvascular thrombosis model using a hamster cheek pouch membrane suited to evaluate the thrombolytic process in vivo. Furthermore, the effect of anticoagulants such as unfractionated heparin (UFH), low molecular weight heparin (LMWH), and recombinant hirudin (r-hir) on thrombolysis induced by recombinant tissue plasminogen activator (rt-PA) was investigated in this model.Microvascular thrombus in hamster cheek pouch membrane was produced by the irradiation of filtered light with the intravascular administration of fluorescein sodium. The thrombus, with 99% stenosis of the vascular lumen, was used for the evaluation of thrombolytic process. The thrombolytic process, induced by the administration of rt-PA (36×104IU/kg·hr, 72×104IU/kg·hr, 108×104IU/kg·hr), was monitored through high gain camera connected to microscope and VTR. Then thrombolytic process was assessed by three parameters, % stenosis of vessel, % length of thrombus, and % area of thrombus which were calculated with computed video analyzer. Above mentioned anticoagulants were administered respectively with rt-PA and the thrombolytic process was evaluated by the same method. PT, APTT, fibrinogen, plasminogen, and α2-PI were measured in citrated plasma.The size of the thrombus formed in this study was increased in proportion to the irradiated area by the filtered light. Thrombus size was decreased depending on the dose of rt-PA, however there seemed to be the optimum dose for rt-PA infusion. Compared with UFH and LMWH, r-hir accelerated thrombolysis by rt-PA potently with less decrease of plasma plasminogen level.This microvascular thrombosis model was considered to be excellent for the evaluation of thrombolytic agents in vivo, and the superiority of r-hir compared to heparins in thrombolytic therapy was suggested.

Pharmacokinetics of Recombinant Hirudin in Hemodialyzed End-stage Renal Failure Patients

Recently, hirudin was used for the first time as an anticoagulant during hemodialysis in men. Pharmacokinetic data of this compound in end-stage renal failure are however not available. In this study, the pharmacokinetics of recombinant hirudin (HBW 023) was evaluated in hemodialysis-treated end-stage renal failure patients. HBW 023 was administered as a bolus at the start of a single dialysis (0.02 to 0.08 mg/kg) in 20 patients, and plasma hirudin levels were followed during this and the 5 following dialyses, without additional hirudin administration. The initial dialysis (HD1) was performed with a low flux polysulfone dialyzer, the following dialyses (up to HD6) with a high flux polysulfone dialyzer and regular heparin. Hirudin levels averaged 504.0 +/- 214.0 and 527.7 +/- 217.1 ng/ml in the middle and at the end of HD1, and then gradually decreased to 15.2 +/- 15.2 ng/ml at the end of HD6. Pharmacokinetic data were compared to those obtained in healthy controls (n = 5), receiving the same dose, and reaching the same peak hirudin level. Hirudin half-life was > 30 times longer in hemodialysis patients (51.8 +/- 15.6 vs. 1.7 +/- 1.5 h, p < 0.001), whereas area under the curve was > 60 times higher (34,669 +/- 14,898 vs. 545 +/- 205 ng/ml x h, p < 0.001). Distribution volume was lower in hemodialysis patients (11.0 +/- 3.1 vs. 14.1 +/- 2.01, p < 0.05). Hirudin disappearance rate was the same during high flux polysulfone dialysis as during interdialytic periods. Hirudin removal was markedly higher in those patients still maintaining some residual renal function and parameters of hirudin removal were significantly correlated to residual creatinine clearance. It is concluded that hirudin removal from the body is markedly depressed in hemodialyzed end-stage renal failure patients and that even minor residual renal function may increase this removal rate.

Covalent immobilization of hirudin improves the haemocompatibility of polylactide—polyglycolide in vitro

A biodegradable polymer, poly( d,l-lactide- co-glycolide) RESOMER® RG756, was modified by surface immobilization of recombinant hirudin (r-Hir) with glutaraldehyde as coupling reagent to improve the blood contacting properties of the polymer. The activity of immobilized hirudin on the polymer was estimated by a chromogenic assay to about 2.5 ATU r-Hir cm −2. The improvement of the haemocompatibility of the modified RG756 was evaluated in terms of platelet adhesion/activation, whole blood clotting times and clot formation rate. Fluorescence microscopy revealed that surface modification with r-Hir resulted in decreased platelet adhesion and activation. An ELISA for P-selectin, a marker of platelet activation, was used to confirm this result. Clotting time experiments demonstrated significantly prolonged non-activated partial thromboplastin times, and a decreased clot formation rate of whole blood in contact with r-Hir modified RG756 compared with the plain polymer. Comparison of immobilized r-Hir with bound heparin yielded equivalent improvement of blood-contacting properties of the investigated polymers. These in vitro investigations indicate that the immobilization of r-Hir on RG756 is a useful method to improve the blood contacting properties of polylactides/polyglycolides and other polymers as well.