Quantitative Determination of PEG-Hirudin in Human Plasma Using a Competitive Enzyme-Linked Immunosorbent Assay

Thrombosis Research 99 (2000) 195–202

ORIGINAL ARTICLE

Quantitative Determination of PEG-Hirudin in Human Plasma Using a Competitive Enzyme-Linked Immunosorbent Assay X.H. Song, G. Huhle, L.C. Wang and J. Harenberg First Department of Medicine, Faculty of Clinical Medicine Mannheim, University of Heidelberg, Heidelberg, Germany. (Received 13 December 1999 by Editor S. Schulman; revised/accepted 3 April 2000)

Abstract Polyethylene glycol (PEG) coupled r-hirudin mutein is determined by biological methods—the coagulation system. In the present study, a competitive enzyme-linked immunosorbent assay (ELISA) is described that permits the measurement of PEG r-hirudin. The ELISA system adopts a rabbit IgG antibody to quantitatively detect PEG-hirudin in human plasma. A PEG-hirudin calibration curve ranged from 50 to 7000 ng/mL. The limit of detection was 87 ng/mL. The intraassay coefficients of variation (CV) ranged between 16 and 21%, and interassay CV between 8 and 22% for low and high PEG-hirudin concentrations, respectively. The recovery of the compound in plasma was between 96 and 111.5%. The interindividual differences between 100 and 5000 ng/mL PEG-hirudin were between 12 and 22%. The correlation of the concentration of PEG-hirudin determined with the ELISA and the ecarin clotting time was r ⫽ 0.902. No interactions between unfractionated heparin, Abbreviations: APTT, activated partial thromboplastin time; BSA, bovine serum albumin; DMSO, dimethyl sulfoxide; ECT, ecarin clotting time; ELISA, enzyme-linked immunosorbent assay; PB, phosphate buffer; PBS, phosphate buffer saline; PEG, polyethylenglycol; PBST, PBS with Tween; polyethylene glycol; PT, prothrombin time; TMB, 3,3⬘,5,5⬘-Tetramethylbenzidine. Corresponding author: Job Harenberg, Professor of Medicine, First Department of Medicine, Faculty of Clinical Medicine Mannheim, University of Heidelberg, Theodor-Kutzer-Ufer, DE-68167 Mannheim, Germany. Tel: ⫹49 (621) 383 3378 or 2789; Fax: ⫹49 (621) 383 3808; E-mail: ⬍[email protected]⬎.

low molecular-weight heparin, or phenprocoumon and PEG-hirudin were observed in the ELISA. Deficiencies of thrombin or antithrombin as well as low, normal, and high fibrinogen levels did not interfere with the assay. It is concluded that the ELISA determines the concentration of PEG-hirudin and is not influenced by other major anticoagulants or by plasma levels of some coagulation proteins.  2000 Elsevier Science Ltd. All rights reserved. Key Words: ELISA; PEG-hirudin; Anticoagulants; Antibody; Ecarin clotting time

H

irudin is a 65–66 amino acid polypeptide that was discovered in the saliva of medicinal leeches (Hirudo medicinalis) in the 19th century. Recombinant hirudin (r-hirudin) has been produced in sufficient amounts using the recombinant DNA technique, and is proven as a potent and specific direct thrombin inhibitor [1]. PEG-hirudin is a chemically defined conjugate of a recombinant hirudin (r-hirudin) molecule and two molecules of polyethylene glycol (PEG)-5000. It was developed to prolong the short half-life ⫹ time of r-hirudin, and is undergoing clinical investigation [2,3]. Reliable analytical methods for the determination of PEG-hirudin concentration are essential for the characterization of its pharmacokinetics. Several laboratory methods are available to measure the anticoagulant activity of hirudins.

0049-3848/00 $–see front matter  2000 Elsevier Science Ltd. All rights reserved. PII S0049-3848(00)00246-2

196

X.H. Song et al./Thrombosis Research 99 (2000) 195–202

These assays include clot-based, amidolytic, and physico-chemical techniques. Although hirudin, like heparin, is an effective anticoagulant, the mechanism of action of the two agents is different. Thus, prothrombin time (PT), activated partial thromboplastin time (APTT), and Heptest, do not show adequate responses to low doses of hirudins. APTT is prolonged at hirudin concentrations ranging from 0.1 to 1.0 or 1.5 ␮g/mL, but becomes insensitive at higher concentrations where full anticoagulation is achieved [4]. The sensitivities were increased to a certain extent by dilution of the reagents thrombin, APTT, and Heptest [5]. The amidolytic antifactor IIa assay is based on the inhibition of the cleavage of p-nitroaniline by thrombin in the presence of hirudin [6]. Thrombin generation assays have limited value in monitoring the anticoagulant effect of hirudin because the effect of thrombin inhibition by hirudin on the coagulation feedback mechanism, and thus the effect on thrombin generation, appears to be minimal [7]. The ecarin clotting time (ECT) quantitatively determines hirudin concentrations in blood and biological fluids in a range of 50 to about 5000 ng/mL [8]. The above assay methods are useful for evaluating the biological and pharmacodynamic effects of hirudin. A major breakthrough in the development of laboratory methods to monitor hirudin was the generation of hirudin-specific antisera in animals [4]. This led to the development of enzyme-linked immunosorbent assay (ELISA) methods for r-hirudin [9–11] with high sensitivity and reproducibility. In this article, we report the development and validation of a competitive ELISA for quantitative determination of a modified hirudin bound to polyethylenglycol in human plasma using an antihirudin antibody.

1. Reagents and Materials Microtiter plates (U-form, 96 wells) were purchased from Greiner (Frickenhausen, Germany). PEG-hirudin and rabbit IgG antibody to hirudin purified by protein A affinity chromatography were generously supplied from Knoll AG (Ludwigshafen, Germany). Bovine serum albumin (BSA), Tween-20, biotin-conjugated goat antirabbit antibody, streptavidin–peroxidase complex, and chromogenic substrate TMB (3,3⬘,5,5⬘-Tetrameth-

ylbenzidine), dimethyl sulfoxide (DMSO), 3% hydrogen peroxidase (H2O2) and sulfuric acid (H2SO4) were purchased from Sigma Aldrich (Deisenhofen, Germany). All chemical reagents were of analytical grade. Factor II-deficient plasma (Factor II activity ⬍1% of normal) and antithrombin deficient plasma (AT-concentration ⬍0.1 mg %) were from Behringwerke AG (Marburg, Germany). Human fibrinogen was generously supplied by Chromogenix (Essen, Germany), fibrinogen-deficient plasma (concentration ⬍2 mg/L) by Dr. C.E. Dempfle (Mannheim, Germany), and a plasma sample containing fibrinogen 5.31 g/L from the Clinical Biochemistry Institute of University Hospital Mannheim. Plasma containing fibrinogen 3.44, 2.22, and 1.11 g/L were prepared by dilution of plasma containing fibrinogen 5.31 g/L with fibrinogen-deficient plasma. Fibrinogen (Batch No. 05-1996/ ⫻0498-51, Chromogenix) was added at 8.0 and 10.0 g/L in fibrinogen-deficient plasma. Phenprocoumon (3 mg/tablet) was from Hoffmann–La Roche AG (Grenzach, Germany), heparin (5000 IE/0.5 mL) from B. Braun AG (Melsungen, Germany), dalteparin (antifactor Xa 160 IU/mg) from Pharmacia and Upjohn (Erlangen, Germany). The final concentrations were phenprocoumon 0.3, 1.0, and 3.0 mg/mL; heparin and dalteparin 0.5, 1.0, and 5.0 IU/mL. The plasma samples were spiked with different concentrations of PEG-hirudin, shock frozen, and stored at ⫺80⬚C. Working solutions of the ELISA were prepared according to a practical guide [12], with minor modifications. Phosphate buffer (PB) contained 0.01 M/L, pH 7.2. BPS contained PB with 0.145 M/L NaCl. PBST was prepared by PBS with 0.1% Tween-20. Carbonate/bicarbonate coating buffer contained 0.1 M/L, pH 9.2. Acetic buffer involved 0.1 M/L, pH 4.9. Peroxidase substrate solution was freshly prepared with 10 mL acetic buffer involving TMB 1.88 mg in 100 ␮L DMSO and 3% H2O2 15 ␮L. The standard procedure for a competitive ELISA was performed as reported previously [12]. In brief, 200 ␮L PEG-hirudin per well were incubated overnight at 4⬚C in microtiter plates. After blocking with 300 ␮L/well 0.1% BSA for 1 h at 25⬚C, plates were washed three times with 300 ␮L/ well PBST. Samples (100 ␮L) prediluted 1:20 in PBS, and 100 ␮L rabbit antihirudin antibody were incubated overnight at 4⬚C. The amounts of PEGhirudin used for coating and rabbit IgG antihirudin

X.H. Song et al./Thrombosis Research 99 (2000) 195–202

antibody were optimized by serial dilutions of the antigen and the antibody, i.e., by chessboard titration [13,14]. The wells were washed three times with 300 ␮L/well PBST. Biotin (200 ␮L)-conjugated antirabbit antibody diluted 1:10,000 in 0.1% BSA was added, and incubated for 4 h at 25⬚C, followed by washing three times with PBST. Samples were incubated with 200 ␮L streptavidin peroxidase complex diluted 1:10,000 in 0.1% BSA for 20 min at 25⬚C, and washed three times. Peroxidase substrate solution (200 ␮L) was incubated for 10 min, and the reaction was stopped with 100 ␮L 2 M H2SO4. The absorbance was measured at 450 nm (A450 nm) within 30 min against a blank containing all reagents except the substrate in a microtiter plate reader (MR7000, Dynatec, Denkendorf, Germany). The concentration of PEG-hirudin was calculated from a standard curve obtained with normal pooled plasma spiked with 50 to 7000 ng/ mL PEG-hirudin using the Biolinx program. The multipoint curve was plotted using a logit of concentration vs. A450 nm scale. The assay procedure takes 24 h. The so-called chessboard titration method [13, 14] was used to determine the best combination of the concentrations of PEG-hirudin used for coating and rabbit IgG antihirudin antibody. PEG-hirudin (200 ␮L) (0.5 to 10 ␮g/mL in carbonate buffer 0.1 M, pH 9.2) was incubated overnight at 4⬚C. The wells of the microtiter plates were washed three times with PBST, incubated with 1% BSA for 1 h, washed again, followed by addition 100 ␮L of 0.5 to 5 ␮g/mL rabbit antihirudin antibody, and incubated overnight at 4⬚C. After washing with PBST, biotin-conjugated goat antirabbit antibody (1:10,000), streptavidin–peroxidase complex (1:10, 000), substrate, and sulfuric acid were added, and the results were quantified at 450 nm (see above). The ECT was performed with the ecarin reagent (Pentapharm, Basle, Switzerland) generously supplied by Knoll AG (Ludwigshafen, Germany, normal ⬍55 s) on the KC 10 coagulometer (Amelung, Lemgo, Germany) as described previously [8]. Blood samples (9 vol) were collected by a clean venipuncture from a cubital vein without tourniquet into 10 mL polystyrene tubes containing 1 vol of 3.8% sodium citrate solution, mixed carefully, centrifuged within 30 min at 1800 ⫻ g and 4⬚C for 10 min. Pooled plasma was obtained by mixing equal volumes of plasma samples from 20 healthy donors, who had not taken any medication during

197

Fig. 1. The binding curves obtained by chessboard titration of the ELISA using 0.5 to 10 ␮g/mL PEG-hirudin for coating the microtiter wells and 0.5 to 5.0 ␮g/mL rabbit antihirudin antibody to measure bound PEG-hirudin.

the last 10 d. Aliquots were shock frozen and stored at ⫺80⬚C. A 5-mL plasma sample from each of the 20 healthy volunteers was stored at ⫺80⬚C for evaluation of interindividual variation.

2. Results 2.1. Optimization of Competitive ELISA for PEG-Hirudin The results of the chessboard titration are shown in Figure 1. The coating concentration of PEGhirudin in the competitive ELISA was optimal at 1.0 ␮g/mL. Between 0.5 and 5.0 ␮g/mL, rabbit antihirudin antibody increased the detection of 1 ␮g/ mL bound PEG-hirudin, and was highest at 1.5 ␮g/ mL. As shown in Figure 2, 50 to 7000 ng of PEGhirudin/mL plasma was detected in a linear and dose-dependent manner at a coating concentration of 1.0 ␮g/mL PEG-hirudin and in the presence of 5 ␮g/mL rabbit antihirudin antibody, and of 1:10,000 diluted biotin-labeled goat antirabbit antibody. Pooled plasma containing 50 to 7000 ng PEGhirudin/mL was analyzed in the presence of 1.0 ␮g/ mL PEG-hirudin coated to the surface of the wells

198

X.H. Song et al./Thrombosis Research 99 (2000) 195–202

Fig. 2. PEG-hirudin concentrations in plasma ranging from 50 to 7000 ng/mL were analyzed by the ELISA after coating of the microtiter wells with 1.0, 2.0, and 5.0 ␮g/mL PEG-hirudin. Rabbit antihirudin antibody (5 ␮g/mL) was used for quantification.

of the microtiter plate and 1.0 to 3.0 ␮g/mL goat antirabbit antibody (Figure 3). A concentration of 1.5 ␮g/mL rabbit antihirudin antibody resulted in a linear decrease of the absorption at 450 nm in relation to the log concentration of PEG-hirudin, and was chosen for further experiments. The linearity of the assay was determined in pooled plasma samples spiked with serial PEGhirudin concentrations (0 to 7000 ng/mL). Figure 4 shows an increase of the absorbance (at 450 nm) over time, and reached a plateau for most PEGhirudin concentrations after 30 min. The following

Fig. 3. The dose–response curves of 50 to 7000 ng/mL PEG-hirudin are shown using three different concentrations of the rabbit antihirudin antibody. Microtiter wells were coated with 1 ␮g/mL PEG-hirudin.

conditions were used for further experiments: 1.0 ␮g/mL PEG-hirudin as coating solution, 1.5 ␮g/mL rabbit IgG antihirudin antibody, 1:10,000 diluted biotin-labeled goat antirabbit antibody, and 10-min incubation time of the TMB substrate.

2.2. Validation of the Competitive ELISA Method The limit of detection was defined as the threefold standard deviation of 0 ng/mL PEG-hirudin in nor-

Fig. 4. The absorbance (450 nm) is plotted vs. the incubation time of the enzyme reaction in the presence of 0 to 7000 ng/ mL PEG-hirudin in normal pooled human plasma. Optimized experimental conditions were used for the competitive ELISA.

199

X.H. Song et al./Thrombosis Research 99 (2000) 195–202

Table 1. PEG-hirudin true value (ng/mL) 7000 5000 2000 500 100

Intraassay mean (n⫽10) (ng/mL)

Variation (⫾SD)

CV (%)

Recovery (%)

Interassay mean (n⫽10) (ng/mL)

Variation (⫾SD)

CV (%)

7509.9 5577.5 1975.9 481.8 95.6

1008.1 1084.3 330.0 96.5 20.4

13.4 19.4 16.9 20.0 21.3

107.3 111.5 97.9 96.4 95.6

7517.7 5081.9 1975.3 497.1 114.5

713.9 417.1 437.6 84.8 18.8

9.7 8.1 21.9 18.5 17.2

The added and detected concentrations of PEG-hirudin were determined from 10 samples in one assay (left part) and from 10 assays on different days (right part). Values are given as mean⫾SD. The recovery was determined from the mean of the 10 samples analyzed in one assay. The intraassay and interassay coefficients of variation (CV) of the ELISA are given in % for different concentrations of PEG-hirudin in pooled human plasma.

mal pooled plasma analyzed on 10 different days, resulting in a sensitivity of 87 ng/mL. The mean intraassay (n ⫽ 10) and interassay (n ⫽ 10) coefficients of variation of 100 to 7000 ng/mL PEG-hirudin ranged from 13.4 to 21.3%, and from 8.1 to 21.9%, respectively. The recovery of 100 to 7000 ng/mL PEG-hirudin was between 95.6 and 111.5% (Table 1). Twenty-six blinded plasma samples spiked with different concentrations of PEG-hirudin were analyzed using the competitive ELISA and the ECT. The concentrations of added and measured PEGhirudin concentrations correlated with r ⫽ 0.860 (Figure 5) and with r ⫽ 0.902 when determined by the ECT. Unfractionated heparin and low molecular-

weight heparin (dalteparin) 0.5, 1.0, and 5.0 IU/ mL, and phenprocoumon 0.3, 1.0, and 3.0 mg/mL did not influence the recovery of 500, 2000, or 5000 ng/mL PEG-hirudin/mL plasma (n ⫽ 10) compared to the control (Table 2). Deficiencies of thrombin and antithrombin and fibrinogen concentrations between 1.11 and 10.0 g/L did not influence the determination of PEG-hirudin. The results were expressed as percent of deviations of the added from the measured values of PEG-hirudin (Table 3). The deviations were in the same range as for control samples, and did not show any trend in the anticoagulant supplemented or in the coagulation factor modified samples (Tables 2 and 3).

3. Discussion

Fig. 5. The results of the added vs. the detected plasma concentrations of PEG-hirudin in spiked samples determined by the ELISA.

In the present study we report on the development of a competitive ELISA to determine the concentration of PEG-hirudin in human blood. So far, ELISA methods have been described to quantify recombinant hirudin using polyclonal [10] or monoclonal antibodies [15]. The determination of PEG-hirudin by an immunological method is required to analyze the pharmacokinetics of the compound and to compare these with the pharmacodynamic effects of thrombin inhibition [16]. Recently, the biological effect of antihirudin antibodies in patients with heparin-induced thrombocytopenia during treatment with r-hirudin has been described [17,18]. The development of antibodies to PEGhirudin has not been found during short-term treatment [16] in contrast to hirudin [19]. Using an ELISA for the determination of hirudins the bio-

200

X.H. Song et al./Thrombosis Research 99 (2000) 195–202

Table 2. Deviation of measured from true PEG-hirudin value (%) PEG-hirudin ng/mL 5000 2000 500

Heparin (IU/mL)

Dalteparin (IU/mL)

Phenprocoumon (mg/mL)

Control

0.5

1.0

5.0

0.5

1.0

5.0

0.3

1.0

3.0

11.56 17.06 3.02

20.82 16.07 9.76

30.19 6.82 10.15

14.15 14.60 5.4

26.09 18.26 4.33

10.39 0.14 14.85

22.89 16.14 4.58

19.59 19.62 11.28

4.92 15.46 20.49

15.57 1.53 9.13

The deviation of the measured value of PEG-hirudin in the ELISA from the true value of added PEG-hirudin is given as % in the presence of three concentrations of unfractionated heparin, low molecular-weight heparin dalteparin or phenprocoumon compared with a control sample without these anticoagulants.

logical relevance of antihirudin antibodies in hirudin-treated patients may be identified. r-Hirudin was originally coupled to dextran to prolong the biological half-life [20]. PEG-hirudin is a conjugate of a recombinant hirudin mutein with two molecules of polyethylene glycol of 5000 Dalton each [21]. In contrast to the dextran-coupled r-hirudin, the PEG-hirudin exhibited similar inhibitory activities and selectivities towards thrombin in fluid phase as well as towards clotbound thrombin [22]. The elimination half-life of PEG-hirudin was significantly longer compared to the parent compound [16], and resulted in an improved antithrombotic activity using various models of venous and arterial thrombosis [23,24]. Interestingly, PEG-hirudin effectively reduced plateletdependent thrombus formation without interfering with primary hemostasis [25]. In contrast to r-hirudin, the PEG-hirudin conjugate had a similar safety/efficacy ratio in a cardiopulmonary bypass model, when given as a single bolus in comparison to r-hirudin given as bolus plus a continuous intravenous infusion [26]. The ecarin clotting time (ECT) specifically determines the biological conversion of meizothrom-

bin to thrombin [15]. Fifty to 5000 ng r-hirudin [27] and PEG-hirudin [28] inhibit the ECT in a linear fashion. Therefore, we compared the biological and immunological effect of the PEG-hirudin in spiked plasma samples using the ECT and ELISA. The recovery (r ⫽ 0.860) and correlation (r ⫽ 0.902) of the ELISA with the ECT demonstrates the validity of the assay. Whether the ELISA measures only free, only thrombin-bound PEG-hirudin, or both, remains to be investigated. Large interindividual differences in hirudin responsiveness are a special problem in APTT-based monitoring of hirudin treatment [29]. This becomes particularly important when high levels of anticoagulation are used such as in open-heart surgery [6]. In contrast to the results obtained with the APTT method, the results of competitive ELISA showed an acceptable interindividual variability and a wide range of linearity. PEG-hirudin may also be metabolized in vivo to hirudin or smaller proteins with or without anticoagulant and antigenic properties. Thus, both the original hirudin [18] as well as PEG-hirudin are detected with the ELISA system. Further studies are required for differentiation.

Table 3. Deviation of measured from true PEG-hirudin value (%) PEG-hirudin ng/ml 5000 500

Fibrinogen (g/L) Control

Thrombin

AT III

1.11

2.22

3.44

5.31

8.0

10.0

9.99 17.42

7.36 9.59

32.21 5.04

1.05 7.66

18.77 6.79

1.40 21.58

1.06 10.77

31.74 34.89

18.82 14.87

The deviation of the measured values of PEG-hirudin in the ELISA from the true value of the added compound is given in % in the presence of thrombin, antithrombin, and different fibrinogen concentrations.

X.H. Song et al./Thrombosis Research 99 (2000) 195–202

In many clinical situations switching PEG-hirudin to heparin, low molecular-weight heparin, or oral anticoagulants may be necessary. As demonstrated by our data, concentrations of unfractionated heparin and low molecular-weight heparin up to 5 IU/mL or phenprocoumon up to 3 mg/mL did not affect the determination of PEG-hirudin. Plasma deficiencies of thrombin, antithrombin, or a wide range of fibrinogen concentrations did not influence the results either. Thus, the ELISA method may be of significance for pharmacological studies and of advantage over biological test systems in the presence of one of the analyzed compounds.

9.

10.

11.

12.

References 1. Markwardt F. The development of hirudin as an antithrombotic drug. Thromb Res 1994; 74:1–23. 2. Ru¨bsamen K, Hornberger W, Schweden J, Kurfu¨rst M. Pharmacological characteristics of long acting polyethylene glycol-coupled recombinant hirudin. Thromb Haemost 1991; 65:129. 3. Esslinger H-U, Haas S, Mauer R, Lassmann A, Du¨bbers K, Mu¨ler-Pelzer H. Pharmacodynamic and safety results of PEG-hirudin in healthy volunteers. Thromb Haemost 1997; 77:911–9. 4. Markwardt F, Nowak G, Stu¨rzebecher J, Griessbach U, Walsmann P, Vogel G. Pharmacokinetics and anticoagulant effects of hirudin in man. Thromb Res 1984;52:160–3. 5. Walenga JM, Hoppensteadt D, Kza M, Wallock M, Pifarre R, Fareed J. Laboratory assays for the evaluation of recombinant hirudin. Haemostasis 1991;(Suppl 1):49–63. 6. Griessbach U, Stu¨rzebecher J, Markwardt F. Assay of hirudin in plasma using a chromogenic thrombin substrate. Thromb Res 1985; 37:347–50. 7. Walenga JM, Bakhos M, Messmore HL, Koza M, Wallock M, Orfei E, Fareed J, Pifarre R. Comparison of recombinant hirudin and heparin as an anticoagulant in a cardiopulmonary bypass model. Blood Coagul Fibrinol 1991; 2:105–11. 8. Nowak G, Bucha E. Quantitative determina-

13.

14.

15.

16.

17.

18.

19.

201

tion of hirudin in blood and body fluids. Semin Thromb Haemost 1996;22:197–202. Spinner S, Sto¨ffler G, Fink E. Quantitative enzyme-linked immunosorbent assay (ELISA) for hirudin. J Immunol Methods 1986;87:79–83. Spinner S, Scheffauer F, Maschler R, Sto¨ffler G. A hirudin catching ELISA for quantitating the anticoagulant in the biological fluids. Thromb Res 1988;51:617–25. Iyer L, Adam M, Amiral J, Fareed J, Bermes E Jr. Development and validation of two enzymelinked immunosorbent assay (ELISA) methods for recombinant hirudin. Semin Thromb Hemost 1995;21:184–92. Kemeny DM. A Practical Guide to ELISA. Oxford: Pergamon Press plc; 1991. Voller A, Bidwell DE, Bartlett A. Enzymeimmunoassays in diagnostic medicine; Theory and practice. Bull World Health Org 1976; 53:55–6. Bauch H-J, Ilsemann K, Beeck H, Voss B, Balleisen L. A double antibody sandwich microELISA for measuring human platelet factor 4. Thromb Res 1985;37:573–82. Verstraete M, Nurmohamed M, Kienast J, Siebeck M, Silling-Engelhardt G, Bu¨ller HR, Hoet B, Bichler J, Close P, on behalf of the European Hirudin in Thrombosis Group. Biological effects of recombinant hirudin (CGP 39393) in human volunteers. J Am Coll Cardiol 1993; 22:364–72. Esslinger H-U, Greger G, Lassmann A, Mauer R. General tolerability and effects on clotting parameters after single i.v. and s.c. bolus administration of recombinant hirudin (LU 52369) in man. Thromb Haemost 1991;65:1290. Eichler P, Greinacher A. Anti-hirudin antibodies induced by recombinant hirudin in the treatment of patients with heparin-induced thrombocytopenia. Ann Hematol 1996;72(Suppl 1):20. Song XH, Huhle G, Wang LC, Hoffmann U, Harenberg J. Generation of anti-hirudin antibodies in heparin-induced thrombocytopenia patients treated with r-hirudin. Circulation 1999;101:1528–32. Close P, Bichler J, Kerry R, Ekman S, Bu¨ller HR, Kienast J, Marbet GA, Schramm W, Verstraete M. Weak allergenicity of recombinant hirudin CGP 39393 (Revasc) in immunocompetent volunteers. The European Hirudin in

202

20.

21.

22.

23.

24.

X.H. Song et al./Thrombosis Research 99 (2000) 195–202

Thrombosis Group. Coron Artery Dis 1994; 5:943–9. Markwardt F, Richter M, Walsmann P, Riesener G, Paintz M. Preparation of dextranbound recombinant hirudin and its pharmacokinetic behaviour. Biomed Biochim Acta 1990; 49:1103–8. Ru¨bsamen K, Hornberger W. Prevention of early reocclusion after thrombolysis of copper coil-induced thrombi in the canine carotid artery: Comparison of PEG-hirudin and unfractionated hirudin. Thromb Haemost 1996;76: 105–10. Iorio A, Agnelli G, Leone M, Melelli P, Renga C, Nenci GG. Hirudin and PEG-hirudin inhibit clot-bound thrombin more efficaciously than heparin. Ann Hematol 1993;Suppl 1:1819. Hornberger W, Ru¨bsamen K, Schweden J. Prolonged antithrombotic action of a new polyethylenglycol-coupled hirudin (LU 57291) in two rat thrombosis models. Ann Hematol 1993;Suppl 1:20. Laux V, Schweden J, Maurer R, Hornberger W, Ru¨bsamen K. Antithrombotic effect of a new polyethylenglycol-coupled hirudin (LU 57291) in a venous thrombosis model in the rabbit. Correlation with anti-factor IIa activity in plasma. Ann Hematol 1993;63(Suppl 1):20.

25. Iyer I, Shavit J, Koza M, Calabria R, Moran S, Fareed J. Alteration of pharmacokinetics and pharmacodynamics of recombinant hirudin (rHV 2-lys 47) after repeated intravenous administration in dogs. Thromb Res 1993;69: 59–70. 26. Walenga JM, Terrell MR, Koza MJ, Khenkina Y, Fareed J, Pifarre R. PEG-hiridin as anticoagulant in cardiopulmonary bypass. Thromb Haemost 1995;73:1454. 27. Po¨tzsch B, Madlener K, Seelig C, Riess CF, Greinacher A, Mu¨ller-Berghaus G. The whole blood ecarin clotting time assay allows rapid and accurate monitoring of the anticoagulant response of r-hirudin during cardiopulmonary bypass. Thromb Haemost 1997;77:920–5. 28. Harenberg J, Huhle H, Kessler M, Malsch R, Piazolo L, Du¨bbers K, Esslinger HU: Interaction of combined (overlapping) treatment with PEG-hirudin and heparin in healthy volunteers. Thromb Haemost 1997;(Suppl):PS-2000. 29. Nurmohamed MT, Berckmans RJ, MorrienSalomons WM, Berends F, Hammes WD, Rijmerse JJMM, Sturk A. Monitoring anticoagulant therapy by activated partial thromboplastin time: Hirudin assessment. Thromb Haemost 1994;72:685–92.