Effects of Recombinant Hirudin (r-Hirudin, HBW 023) on Coagulation and Platelet Activation In Vivo
Comparison With Unfractionated Heparin and a Low-Molecular-Weight Heparin Preparation (Fragmin)
Abstract In a double-blind, randomized, crossover study, we investigated in 15 healthy male volunteers the effects of recombinant (r-) hirudin (HBW 023, 0.35 mg/kg body wt SC), unfractionated heparin (UFH, HeparinNovo; 150 IU/kg body wt SC), and a low-molecular-weight heparin preparation (LMWH, Fragmin; 75 IU/kg body wt SC) on coagulation and platelet activation in vivo by measuring specific coagulation-activation peptides (prothrombin fragment 1+2 [F1+2], thrombin–antithrombin-III complex [TAT], and β-thromboglobulin [β-TG]) in bleeding-time blood (activated state) and venous blood (basal state). In bleeding-time blood, r-hirudin and the heparin preparations significantly inhibited formation of both TAT and F1+2. However, the inhibitory effect of r-hirudin on F1+2 generation was short-lived and weaker compared with that of UFH and LMWH, and the TAT-to-F1+2 ratio was significantly lower after r-hirudin than after UFH or LMWH. Thus, in vivo, when the coagulation system is in an activated state, r-hirudin exerts its anticoagulant effects predominantly by inhibiting thrombin (factor IIa), whereas UFH and LMWH are directed against both factors Xa and IIa. A different mode of action for UFH and LMWH was not detectable. In venous blood, r-hirudin caused a moderate reduction in TAT formation and an increase (at 1 hour) rather than a decrease in F1+2 generation. Formation of TAT and F1+2 was suppressed at various time points following both UFH and LMWH. There was no difference in the TAT-to-F1+2 ratio after r-hirudin and heparin. Thus, a predominant effect of r-hirudin on factor IIa (as found in bleeding-time blood) was not detectable in venous blood. In bleeding-time blood, r-hirudin (but neither UFH nor LMWH) significantly inhibited β-TG release. In contrast, both UFH and LMWH caused an increase in β-TG 10 hours after heparin administration. Our observation of reduced platelet function after r-hirudin compared with delayed platelet activation following UFH and LMWH suggests an advantage of r-hirudin over heparin, especially in those clinical situations (such as arterial thromboembolism) where enhanced platelet activity has been shown to be of particular importance.
- Received January 16, 1995.
- Accepted March 22, 1995.
Natural hirudin produced in the salivary glands of medicinal leeches is the most potent, selective, and specific inhibitor of thrombin. In contrast to unfractionated heparin (UFH) and low-molecular-weight heparin (LMWH), which catalyze inactivation of thrombin by antithrombin-III (AT-III), recombinant (r-) hirudin binds directly to thrombin, thereby forming a complex with thrombin not only at its fibrinogen-binding site but also at its catalytic region, resulting in complete blocking of thrombin. Inactivation of thrombin by r-hirudin not only inhibits fibrin formation but also blocks thrombin-induced feedback activation of the coagulation system, platelet, and physiological anticoagulant pathways, such as protein C/protein S.1 2 Inactivation of thrombin by r-hirudin also results in a dose-dependent prolongation of the activated partial thromboplastin time (APTT) and thrombin time (TT).3 In addition, r-hirudin’s inhibitory effects on thrombin have been confirmed by more specific laboratory assays that show decreases in fibrinopeptide A and thrombin–AT-III complex (TAT) formation, as well as prevention of a thrombin-induced platelet-release reaction in hirudinized plasma.4 5 These unique properties of r-hirudin launched a series of in vivo studies and clinical trials that explored not merely its potential as a novel antithrombotic agent but also—on the basis of the distinct differences in which r-hirudin inhibits thrombin compared with those of heparin—as an alternative for patients who are not suitable candidates for heparin therapy owing to heparin-related complications or other restrictions, such as heparin-induced thrombocytopenia, heparin resistance, or allergy.6 7 8
Despite substantial numbers of in vitro and in vivo studies, profound knowledge of the in vivo interactions of r-hirudin with thrombin with regard to thrombin’s central role in the coagulation system is still lacking. To gain insight into the anticoagulant mechanisms ofr-hirudin in vivo in humans, the effect of r-hirudin on the formation of coagulation-specific peptides generated after activation of the hemostatic system by a standardized injury of the microvasculature (made to determine template bleeding time) was investigated. This method has been shown to represent a suitable approach for analyzing the mechanisms that lead to “plug” formation under conditions similar to in vivo circumstances.9 10 11 12 13 14 TAT formation was measured to assess the extent of AT-III–dependent (in the presence of heparin) and AT-III–independent (in the presence of r-hirudin) blocking of thrombin. Prothrombin activation fragment 1+2 (F1+2), a peptide that is released from prothrombin during its factor Xa–catalyzed conversion to thrombin, was determined to evaluate the inhibition of factor Xa (Xa) either directly by AT-III (in the presence of heparin) or indirectly via inactivation of feedback mechanisms (in the presence of r-hirudin). In addition, β-thromboglobulin (β-TG), a protein that is released from platelet α-granules, was measured to study drug-induced platelet activation in vivo.
The selected dosage of r-hirudin has been shown to substantially affect in vitro clotting assays without causing an increased bleeding risk.15 To further specify the impact of r-hirudin’s effects on the generation of coagulation-activation markers, the effect of r-hirudin was compared with that of UFH and LMWH, which were given at a dosage known to cause comparable inhibition of TAT and F1+2, respectively.16
The results obtained from these investigations, performed after activation of the hemostatic system, were compared with the effects of r-hirudin and both heparin preparations on the generation of coagulation-specific markers in venous blood at a time when the clotting system is inactivated (ie, in a “resting” state) and on conventional in vitro clotting assays, such as APTT and TT.
The study population consisted of 15 healthy white male volunteers (median age, 25 years; range, 21 to 28 years; median weight, 76 kg; range, 62 to 90 kg) who were nonsmokers and drug-free at least 2 weeks before and throughout the study period.
After an overnight fast and a 30-minute rest period, the subjects received a single subcutaneous injection of 0.35 mg/kg body wt of r-hirudin (HBW 023, Behringwerke AG), 150 anti–factor Xa (anti-Xa) IU/kg body wt of a UFH preparation of porcine origin (HeparinNovo, Novo Nordisk), or 75 anti-Xa IU/kg body wt of an LMWH preparation (Fragmin, KabiVitrum). The r-hirudin used in this study resembled the natural hirudin originally described by Markwardt and Walsmann,17 with the exception of the first two amino acids (Leu and Thr) and the absent sulfate residue on Tyr63.
The study was conducted in a randomized, double-blind, three-way crossover design with a washout period of at least 7 days between the three different treatments. The sequence of treatments was determined according to a Latin-square design, and each subject was allocated a random number on the Latin-square table. All treatments were administered by a single subcutaneous injection (injection volume, 1 mL). The treatments, which were indistinguishable from each other, were prepared by a study nurse and were administered by a physician who was blinded to the identity of the treatment. All other investigators involved in the study were not aware of the randomization code, which was broken by the statistician only after all data had been entered into the data base.
The study protocol was approved by the University Ethics Committee, and written, informed consent was obtained from each participant.
Bleeding Time and Blood Sampling From Bleeding-Time Incisions
Template bleeding time was determined according to the original description by Mielke et al,18 and blood sampling from bleeding-time incisions was performed before and 2, 3, 5, and 10 hours after drug administration. After a sphygmomanometer cuff on the upper arm was inflated to 40 mm Hg, two incisions (each 5 mm long and 1 mm deep) parallel to the antecubital crease were made on the lateral volar aspect of the forearm by using a disposable standard device (Simplate II, Organon Teknika). The procedure was carried out by the same investigator each time.
Blood from these incisions was sampled as described before.11 For determination of TAT, F1+2, and β-TG, blood was collected every 20 seconds directly from the edge of the skin wound by using a Gilson pipette and immediately transferred into ice-cooled Eppendorf tubes containing an anticoagulant solution consisting of 3.8% sodium citrate, 100 mmol/L EDTA, 30 μmol/L indomethacin (Sigma Chemical Co), 1000 U/mL aprotinin (Trasylol, Bayer AG), and 1500 U/mL sodium heparin (Immuno AG). Bleeding-time blood was collected in fractions into single tubes over 1-minute periods (1 tube per minute) to yield a total of 4 aliquots representing minutes 1 through 4 for each time point (at 0, 2, 3, 5, and 10 hours). After being mixed, the blood samples were immediately centrifuged at 12 000g for 2 minutes, and the supernatants were frozen and stored at −80°C until assayed.
Collection and Processing of Venous Blood Samples
Venous blood samples were obtained immediately before and 1, 2, 3, 4, 5, 8, and 10 hours after drug administration by sterile puncture of a large antecubital vein with 21-gauge butterfly infusion sets. For measurement of APTT, TT, and anti-Xa levels, blood was drawn into a 1/10 volume of 3.8% sodium citrate. For determination of TAT, F1+2, and β-TG levels, venous blood was collected into ice-cooled plastic tubes containing a 1/10 volume of the anticoagulant mixture described above. Immediately after centrifugation at 2000g for 20 minutes, the sample supernatants were frozen and stored at −80°C until assayed. Determination of APTT, TT, and platelet counts was done on the day of blood sampling.
APTT (Pathromtin, Behringwerke AG), TT (Test-Thrombin, Behringwerke AG; concentration of thrombin in the test mixture, 3 U/mL), anti-Xa units (Coatest LMW Heparin, Chromogenix), and platelet counts were performed by routine laboratory procedures.
TAT and F1+2 were measured by using commercially available assay kits based on the enzyme-linked immunosorbent assay technique (Enzygnost TAT and Enzygnost F1+2, Behringwerke AG). β-TG was measured by use of a commercially available radioimmunoassay (β-TG RIA Kit, Amersham International).
Data Analysis and Statistics
To quantify the generation of TAT, F1+2, and β-TG in the bleeding-time blood, their concentrations were measured in 4 individual aliquots representing minutes 1 through 4 at each time point. The area under the concentration-versus-time curve (minutes 1 through 4) was regarded as the measure of coagulation and platelet-activation marker production in the microcirculation.
To detect a selective inhibitory effect of r-hirudin, UFH, or LMWH on TAT or F1+2 formation, TAT-to-F1+2 ratios were calculated for each minute (minutes 1 through 4) at each time point (hours 0 to 10). The area under the curve was regarded as a measure of the extent of IIa and Xa inhibition by the three treatment regimens at the different time points.
To exclude changes in the platelet-derived parameter β-TG due to variations in platelet count, β-TG concentrations are expressed as nanograms per 105 platelets rather than nanograms per milliliter.
Differences in treatment effects and between various observation time points were assessed by ANOVA for repeated measures. For data description, differences for individual time points were analyzed. To assess whether the data were normally distributed or not, the Shapiro-Wilk test was applied. Depending on whether or not the data were normally distributed, the paired t test or the Wilcoxon signed rank test was used, and α=.05 was considered to be the level of significance.
Anticoagulant Effects of r-Hirudin, UFH, and LMWH
The anticoagulant effects of r-hirudin, UFH, and LMWH were studied directly at the site of plug formation immediately after hemostatic system activation by injury of the microvasculature made to determine template bleeding time.
The amounts of TAT and F1+2 in bleeding-time blood were measured before and 2, 3, 5, and 10 hours after drug administration. At each time point, bleeding-time blood was collected in 1-minute aliquots over a 4-minute period, and the area under the time-versus-concentration curve was regarded as the amount of coagulation and platelet-activation marker production at each time point.
r-Hirudin markedly inhibited the formation of TAT up to 5 hours after injection, with a reduction of 46% at 2 hours (P=.007) and of 40% at 3 hours (P=.003) compared with the pretreatment value (Fig 1⇓).
In contrast to the marked decrease in TAT formation, inhibition of F1+2 generation was seen only 2 hours after r-hirudin (28% reduction compared with baseline, P=.007; Fig 2⇓). The inhibitory effect of r-hirudin on F1+2 generation was not only shorter but also weaker than that on TAT formation.
UFH and LMWH caused similar decreases in TAT formation, with reductions of 31% (P=.02) after UFH and 39% (P=.0001) after LMWH at 2 hours and of 43% (P=.0002) and 40% (P=.0005) at 3 hours, respectively (compared with baseline; Fig 1⇑). Both UFH and LMWH inhibited F1+2 generation, causing significant reductions after 2 hours (29% after UFH, P=.035, and 52% after LMWH, P=.0001), 3 hours (19% after UFH, P=.001, and 52% after LMWH, P=.0001), and 5 hours (38%, P=.002 after LMWH) compared with baseline (Fig 2⇑). At these time points no significant difference between the effects of UFH and LMWH on F1+2 formation was detectable.
To compare the effects of the three substances on TAT and F1+2 formation, the area under the time (0 to 10 hours)–versus-concentration curve was calculated.
There was no significant difference between r-hirudin, UFH, and LMWH in their effects on TAT formation throughout the study period, suggesting a similar extent of IIa inactivation. The inhibitory effect of r-hirudin on F1+2 was weaker compared with those of UFH (P=.017) and LMWH (P=.1). At 3 and 5 hours, F1+2 levels had returned to baseline after r-hirudin, whereas at these time points F1+2 generation was still markedly suppressed by both UFH and LMWH. This reflects inactivation of Xa by both heparin preparations and demonstrates the distinct difference in the mode of action between r-hirudin and the two heparins.
To further characterize different effects of r-hirudin and the two heparin preparations on TAT and F1+2 formation, TAT-to-F1+2 ratios were calculated for each drug at each time point (Fig 3⇓). The TAT-to-F1+2 ratio was significantly lower at 2, 3, and 5 hours following r-hirudin compared with both UFH and LMWH, whereas no difference was found between the two heparin regimens. The difference between r-hirudin on the one hand and the two heparins on the other was mainly due to the pronounced inhibitory effect of r-hirudin on TAT formation in the presence of a comparatively short-lived and minor inhibition of F1+2 generation rather than selective inhibition of F1+2 by UFH or LMWH.
The amounts of TAT and F1+2 were measured in venous blood in the absence of a hemostatic stimulus before and 1, 2, 3, 4, 5, 8, and 10 hours after drug administration.
In accordance with the results obtained with bleeding-time blood, r-hirudin significantly inhibited TAT formation, with reductions of 21% at 1 hour (P=.009), 23% at 2 hours (P=.005), 21% at 3 hours (P=.01), 28% at 4 hours (P=.0007), and 20% at 5 hours (P=.01) compared with baseline (Fig 4⇓). At 8 and 10 hours, TAT levels had returned to pretreatment values. F1+2 levels increased slightly 1 hour after r-hirudin (P=.03) but remained otherwise unchanged throughout the study period compared with pretreatment values (Fig 5⇓).
LMWH inhibited TAT formation after 2, 4, 5, and 8 hours, with reductions of 24% (P=.03), 40% (P=.0013), 24% (P=.02), and 33% (P=.019), respectively, compared with baseline. UFH had no effect on TAT formation throughout the study period (Fig 4⇑). TAT pretreatment levels in the volunteers treated with LMWH were higher (P=.03) than in those given UFH. However, TAT values before LMWH were between 1.2 and 4.5 μg/L and thus, only slightly elevated or within the normal range seen in healthy control subjects. Therefore, we believe that this difference is most likely due to statistical chance.
As depicted in Fig 5⇑, UFH inhibited F1+2 generation at 4 and 8 hours (18% reduction, P=.009, and 15% reduction, P=.01, respectively) as did LMWH at 5, 8, and 10 hours (12% reduction, P=.04; 19% reduction, P=.0004; and 14% reduction, P=.01, respectively).
To compare the three drug regimens, the areas under the time (0 to 10 hours)–versus-concentration curves were calculated. No significant difference between r-hirudin, LMWH, and UFH on either TAT or F1+2 levels was detectable in venous blood.
There was also no difference in the TAT-to-F1+2 ratios between r-hirudin, LMWH, and UFH throughout the study period. Thus, a predominant inhibitory effect of r-hirudin on TAT formation or of LMWH and UFH on F1+2 formation was not detectable in venous blood.
In Vitro Assays
Compared with pretreatment levels, significant prolongation of the APTT and TT was seen throughout the study period following both r-hirudin and UFH, whereas the effect of LMWH was less pronounced (Table⇓). In contrast, a substantial increase in anti-Xa levels was seen after LMWH, whereas the effect of UFH was weaker. As expected, r-hirudin did not affect anti-Xa levels (Table⇓).
Effects of r-Hirudin, UFH, and LMWH on Platelet Function
Neither r-hirudin nor either of the two heparin preparations prolonged the template bleeding time at any time point (data not shown).
r-Hirudin effectively inhibited β-TG release, with a maximum reduction of 27% at 3 hours compared with the pretreatment value (P=.006, Fig 6⇓).
Inhibition of β-TG release was not detectable following UFH and LMWH. At 10 hours, a significant increase in β-TG release was observed after UFH (P=.01) and LMWH (P=.008) compared with baseline. When the area under the time (0 to 10 hours)–versus-concentration curve was calculated for each preparation, β-TG release was lower after r-hirudin compared with those for the two heparin regimens (r-hirudin versus UFH, P=.056; r-hirudin versus LMWH, P=.01).
Neither r-hirudin, UFH, nor LMWH had an effect on β-TG levels in venous blood (data not shown).
Effects of r-Hirudin, UFH, and LMWH on Coagulation Activation
Recently, a series of sensitive assays that measure peptides generated during coagulation have been developed.19 Determination of these coagulation-activation markers, formed at different steps of the coagulation cascade, offers the opportunity to study the extent of coagulation activation and the regulation of thrombin formation under conditions that are closer to the in vivo situation than are conventional in vitro clotting tests. In the present study this technique was applied to investigate the alterations in human hemostasis in the presence of r-hirudin, UFH, and LMWH preparations and to specify their anticoagulant effects at various stages during coagulation by measuring F1+2, which is released immediately after prothrombin cleavage by Xa, and TAT, which is formed after generation of thrombin. Thus, we were able to study the impact of r-hirudin and the two heparin preparations on the coagulation cascade at two different stages, ie, at the IIa and Xa level.
The mechanisms of thrombin formation that continuously occur in vivo and are effectively counterbalanced by various physiological anticoagulant pathways differ widely from those involved in clot formation when the hemostatic system is in an activated state.20 It was, therefore, another objective of this study to investigate the anticoagulant effects of r-hirudin and the two heparins not only in the basal state of hemostasis, ie, in venous blood, but also during the process of thrombin generation and clot formation after activation of the coagulation system. Hemostatic system activation was achieved by applying a standardized injury of the microvasculature made to determine template bleeding time. Subsequently, coagulation-activation markers were measured in blood emerging from this local injury site.
For UFH and LMWH we have recently demonstrated that doses of 150 IU/kg body wt SC and 75 IU/kg body wt SC, respectively, cause comparable inhibition of TAT and F1+2 formation.16 The heparin doses administered in this study were somewhat smaller than those that are normally applied in the treatment of venous thromboembolism.21 22 So far, a comparable dose regimen of r-hirudin has not been established in clinical trials. Thus, a dose of 0.35 mg/kg body wt SC was selected, which has been shown to substantially prolong the APTT and TT without an increased bleeding tendency.15
In the microcirculation, administration of r-hirudin resulted in a significant and long-lasting (up to 5 hours) suppression of TAT generation and in a comparatively short-lived inhibition of F1+2 generation that was seen only 2 hours after drug application. Because r-hirudin has no direct anti-Xa activity, we surmise that at this time point r-hirudin plasma levels were high enough to inactivate thrombin-induced feedback activation of the coagulation system. It has been shown that even small amounts of thrombin that are generated in the basal state of human hemostasis activate VII and XI.23 24 Because there is increasing evidence that both of these clotting factors play a major role during coagulation activation leading to Xa generation,20 25 inactivation of thrombin by r-hirudin would inhibit both pathways, thereby preventing Xa-catalyzed prothrombin activation. The disparate inhibition of the two coagulation-activation markers results from the direct thrombin-blocking properties of r-hirudin that are also seen in vitro and is reflected in a prolongation of the APTT and TT without affecting anti-Xa levels. Most important, these findings support the concept of selective and specific thrombin inactivation as r-hirudin’s major mode of action in vivo.
r-Hirudin’s effects on TAT and F1+2 formation were particularly evident in blood obtained from bleeding-time incisions and were detectable in venous blood to a much lesser extent. Because the amounts of thrombin that are continuously generated in the basal state of the coagulation system are obviously very small, r-hirudin’s inhibitory effects on TAT and F1+2 formation were less pronounced in quantitative terms than they would be in the presence of the large amounts of thrombin that are formed after stimulating hemostasis by injury to the microvasculature. However, the less pronounced differences in TAT and F1+2 levels in venous blood could also be partially due to differences in their half-lives.19 The longer half-life of F1+2 (t1/2=90 minutes) compared with that of TAT (t1/2=15 minutes) could disguise a more distinct and selective inhibition of TAT than F1+2 by r-hirudin.
Administration of r-hirudin was followed by an immediate though short-lived increase in F1+2 levels in venous blood. An observation similar to this apparent paradoxical phenomenon was made by Conway et al,26 who found an increase in F1+2 levels within the first 24 hours after initiation of oral anticoagulant therapy in the venous plasma of two thrombophilic patients without protein C deficiency. The authors explained their findings by postulating an induction of a transient imbalance between the anticoagulant activity of protein C and the levels of other vitamin K–dependent coagulation factors. Because thrombomodulin-bound thrombin is the major activator of protein C, inactivation by r-hirudin could result in diminished activation of protein C,2 which might cause a state of temporary hypercoagulability similar to that seen after initiation of oral anticoagulant treatment.
We16 have recently demonstrated that application of UFH and LMWH results in a similar extent of Xa and IIa inhibition in vivo rather than a predominant suppression of IIa or Xa, respectively, as might be expected from in vitro assays. In the present study, the comparable inhibition of coagulation activation after UFH and LMWH again did not reveal any selective inhibitory effect of one of the two heparin preparations, thereby confirming our recent findings. Both heparin preparations caused similar decreases in TAT and F1+2 for up to 5 hours that were more evident in bleeding-time blood than in venous blood, most likely due to the disparate amounts of thrombin generated in the two systems. Because our assay system is obviously capable of detecting a predominant anticoagulant action against IIa (as is the case for r-hirudin) or Xa, the similar anti-IIa and anti-Xa potencies of UFH and LMWH in vivo further support our concept of a similar mode of action for the two heparin preparations.
Effects of r-Hirudin, UFH, and LMWH on Platelet Activation
r-Hirudin decreased β-TG with a maximum effect at 3 hours, indicating substantial suppression of platelet activation. Thus, the large amounts of thrombin, a potent platelet activator, that are generated after activation of the coagulation system are inhibited by r-hirudin and can no longer contribute to platelet activation. The inhibitory effect of r-hirudin on platelet activation was more pronounced than that of UFH and LMWH. This observation was made in bleeding-time blood, ie, following stimulation of platelet function in the microcirculation. In contrast, neither r-hirudin nor the two heparin preparations caused a substantial decrease in β-TG levels in venous blood. Thus, when the coagulation system is investigated in a resting state, neither r-hirudin nor heparin has a detectable inhibitory effect on platelet activation, most likely because of the small amounts of IIa that are generated under these conditions.
Interestingly, at later time points (5 and 10 hours), administration of UFH and LMWH caused an increase of β-TG levels in bleeding-time blood, indicating enhancement of platelet activation. From our data, it is not clear whether the increase in β-TG levels is the consequence of a direct or an indirect heparin effect. The late onset of platelet activation may be explained by a concentration-dependent phenomenon: at high initial concentrations, heparin’s anti-IIa activity outweighs its intrinsic platelet-activating properties, whereas with subsiding anti-IIa activity, heparin subsequently causes platelet activation. Heparin’s effect on platelet activity has been extensively discussed, and LMWH’s role with regard to its potential lower platelet-activating properties compared with those of UFH is still controversial.27 28
Heparin is widely used as an anticoagulant in clinical situations known to be associated with increases in platelet activity, such as unstable angina or percutaneous transluminal coronary angioplasty. Despite administration of high doses of heparin and concomitant therapy with platelet inhibitors, the efficacy of these anticoagulant regimens in the prevention of arterial vessel occlusion is suboptimal. The limited effect of heparin has been attributed to its inability to inactivate clot-bound thrombin and enhanced binding to acute-phase-reactant plasma proteins.29 In addition to these findings obtained exclusively from in vitro experiments, the results of the present study demonstrate delayed platelet activation following both UFH and LMWH in vivo and suggest an advantage of r-hirudin over heparin, especially in those clinical situations where enhanced platelet activity has been shown to be important. This observation may be relevant in selecting a particular antithrombotic agent for the treatment of patients with arterial thromboembolism (known to be associated with enhanced platelet function) and deserves further investigation.
This study was sponsored in part by Behringwerke AG, Marburg/Lahn, Germany (donation of r-hirudin and some laboratory reagents).
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