Antithrombotic Potency of Hirudin Is Increased in Nonhuman Primates by Fibrin Targeting
Background Inhibition of thrombin by either the indirect thrombin inhibitor heparin or by more potent direct thrombin inhibitors such as hirudin reduces thrombus formation after arterial injury. The present study was designed to determine if a fibrin-specific thrombin inhibitor could, by local thrombin inhibition, prevent thrombosis more effectively.
Methods and Results We first studied antithrombotic potency in vitro, comparing fibrin-targeted hirudin (recombinant hirudin covalently linked to the Fab′ fragment of the anti-fibrin monoclonal antibody 59D8) to recombinant hirudin in baboon plasma. Fibrin-targeted hirudin was nine times more effective than recombinant hirudin in inhibiting fibrin deposition on experimental clot surfaces in baboon plasma (P<.01). The potency of fibrin-targeted hirudin was then compared with that of recombinant hirudin in a baboon model of thrombus formation. 111In-labeled platelet deposition was measured in a synthetic graft segment of an extracorporeal arteriovenous shunt in control animals and in animals receiving either fibrin-targeted hirudin or hirudin. In these experiments, fibrin-targeted hirudin was 10-fold more potent than hirudin in inhibiting platelet deposition and thrombus formation (P<.05).
Conclusions These data indicate that targeting a thrombin inhibitor such as hirudin to an epitope present in thrombi results in increased antithrombotic potency.
Thrombin is the most potent physiological stimulator of platelet activation, and it plays a pivotal role in generating fibrin. As such, thrombin has a central role in the development of arterial clots. Inhibition of thrombin by either the indirect thrombin inhibitor heparin or by more potent direct thrombin inhibitors such as hirudin reduces thrombus formation after arterial injury in animal models1 2 3 and in humans with unstable coronary syndromes.4 5 Heparin, which inhibits thrombin indirectly by facilitating the action of antithrombin III, has been shown to have a limited antagonistic effect on thrombin, being unable to inhibit thrombin that is bound to fibrin. Recently, hirudin has been shown to be more effective than heparin in preventing platelet-dependent arterial thrombosis and rethrombosis after reperfusion in animal models.6 7 However, clinical trials with hirudin have been less successful. GUSTO IIa,8 TIMI 9,9 and HIT-III10 were halted and then continued at lower doses of hirudin because the initial dosages of hirudin chosen (on the basis of antithrombotic potency) caused excessive bleeding. In the GUSTO IIa study, a substantial reduction in hirudin dose (to <50% of the initial dose) resulted in diminished bleeding but yielded an antithrombotic efficacy only equal to that of heparin. Fibrin targeting was a strategy adopted to augment the safety and efficacy of using hirudin by increasing only local concentrations of hirudin.11
The fact that both approaches require systemic thrombin inhibition to inhibit thrombus formation provides an opportunity for improvement. The present study was designed to determine if a fibrin-specific thrombin inhibitor could, through local thrombin inhibition, prevent thrombosis in vivo at lower antithrombotic doses than are required of r-hirudin. The basis for these experiments was our previous observations that anti-fibrin antibody–plasminogen activator hybrids constructed by chemical or recombinant methods demonstrated enhanced thrombolytic potency and specificity in vitro and in vivo12 13 14 15 16 17 and that fibrin-targeted hirudin was more efficient than hirudin in vitro.11
To generate a fibrin-specific thrombin inhibitor (fibrin-targeted hirudin), we covalently linked r-hirudin to the Fab′ fragment of a monoclonal antibody (59D8) that selectively binds to an epitope on fibrin that is exposed only after thrombin cleavage of fibrinogen. The present study was designed to determine if this fibrin-specific thrombin inhibitor could, by local thrombin inhibition, prevent thrombosis in baboon plasma and ultimately in vivo more effectively than r-hirudin.
r-Hirudin (r-hirudin LU 52369, with a specific activity of 17 000 antithrombin units [ATU] per milligram) was a gift from Knoll AG (Ludwigshafen, Germany). Synthetic peptides Bb (15-21) (β-peptide) and Bb (15-21)-Cys were prepared at the Emory Microchemical Facility. Chromogenic substrate S-2238 was purchased from Helena Laboratories. SPDP was purchased from Pierce Chemical, and 125I-labeled fibrinogen (IBRIN) was purchased from Amersham. Other chemicals were purchased from Sigma Chemical Co.
Preparation of Fibrin-Targeted Hirudin (Hirudin-59D8 Fab′ Conjugate)
Fibrin-targeted hirudin was prepared and characterized as previously described.11 In brief, the (Fab′)2 of anti-fibrin antibody 59D817 was obtained by pepsin cleavage and was purified by affinity chromatography on a β-peptide–Sepharose column.14 59D8 Fab′ was obtained by reducing 59D8 (Fab′)2 at room temperature for 18 hours in 1 mmol/L 2-mercaptoethylamine, 1 mmol/L EDTA, and 10 mmol/L sodium arsenate, followed by addition of Ellman's reagent to a final concentration of 5 mmol/L. 59D8 Fab′ was purified by gel filtration on a Sephadex G-25 column. 59D8 Fab′ was thiolated by 2-mercaptoethylamine and mixed with SPDP-modified hirudin. After 16 hours, the reaction was stopped with excess iodoacetamide, and the hirudin–59D8 Fab′ conjugate was purified by affinity chromatography on a β-peptide–Sepharose column. For the in vivo experiments described herein, a single aliquot from a preparation of fibrin-targeted hirudin (with specific activity of 2300 ATU/mg) was used.
Measurement of Antithrombin Activity by r-Hirudin and Fibrin-Targeted Hirudin
Thrombin activity can be quantified by cleavage of the chromogenic substrate S-2238. The antithrombin activity of a preparation of either r-hirudin or fibrin-targeted hirudin can then be determined by the degree of inhibition of S-2238 cleavage. Because the in vitro and in vivo assays described herein require precise quantification of antithrombin activity, the thrombin inhibitory activity of preparations of r-hirudin and fibrin-targeted hirudin was quantified in the S-2238 assay and compared with a standard curve. In this assay, 20 μL thrombin solution (2.5 U/mL water) was added to 100 μL sample r-hirudin (or fibrin-targeted hirudin) in assay buffer (20 mmol/L sodium dihydrogen carbonate, 0.15 mol/L NaCl, and 0.1% BSA, pH 7.4). Then 50 μL chromogenic substrate S-2238 (0.833 mg/mL) was added. After exactly 5 minutes of incubation, the reaction was stopped by adding 50 μL 20% acetic acid. Absorbance was measured at 405 nm and compared with that of the thrombin standard. The assay was linear for hirudin concentrations in the range of 0 to 0.6 U/mL. Samples with higher activity were diluted so that they could be measured within this range.
Inhibition of Thrombus Growth in Baboon Plasma In Vitro
This assay was designed to compare the effectiveness of r-hirudin and fibrin-targeted hirudin in inhibiting fibrin deposition on the surface of a plasma clot incubated with platelet-free baboon plasma before beginning in vivo comparison of r-hirudin and fibrin-targeted hirudin. Plasma was obtained from three to four baboons and was pooled, separated into aliquots, and frozen for future use. Before each experiment, plasma was centrifuged at 30 000 rpm for 60 minutes in a Beckman SW 40 rotor to obtain platelet-free plasma (<1000 platelets/mL). Immediately before each experiment, the antithrombin activities of r-hirudin and fibrin-targeted hirudin preparations in the S-2238 assay were determined and appropriate dilutions were made such that the antithrombin activities of r-hirudin and fibrin-targeted hirudin were identical for each experimental point. 125I-labeled, platelet-free plasma clots were made by adding 125I-labeled human fibrinogen (20 000 cpm/mL plasma), 0.5 mol/L CaCl2 (0.05 mol/L final concentration), and thrombin (0.5 NIH units/mL plasma) to aliquots of baboon plasma. Immediately after the addition of thrombin, the solution was drawn into silicone elastomer tubing (4-mm ID) and allowed to clot for 2 hours at 37°C. The tubing was then cut into 1.8-cm sections to yield clots of ≈0.2 mL. The clots were removed from the tubing, and each was placed in a plastic vial and washed three times with 2 mL of 0.15 mol/L NaCl. 125I radioactivity was measured in a gamma counter, and only clots within 5% of the mean count value were used in the experiments. Experiments were initiated by the addition to the washed clots of 100 mL r-hirudin (or fibrin-targeted hirudin) solution for which antithrombin activity in the S-2238 assay had been determined. The clots were then incubated with 200 mL platelet-free baboon plasma that had been trace labeled with 125I-labeled fibrinogen (250 000 cpm/mL). After incubation times of 60, 120, or 180 minutes, clots were removed from the test tubes and washed three times with 2 mL of 0.15 mol/L NaCl, and radioactivity was measured in a gamma counter.
Inhibition of Thrombosis in Extracorporeal AV Shunts In Vivo
The thrombosis model described below was developed for quantitative and reproducible determination of antithrombotic doses for arterial and venous thrombi. The clinical relevance of this model is based on its defined flow geometry, the reproducibility of thrombus formation, and the similarities between baboon and human hemostatic mechanisms. Six normal male juvenile baboons weighing between 9 and 11.5 kg and bearing chronic AV femoral shunts were used in these studies. Different test agents and doses were studied using random sequence. Some baboons received fibrin-targeted hirudin first, followed by hirudin on a subsequent day, whereas other baboons received r-hirudin first, followed by fibrin-targeted hirudin on a subsequent day. All procedures were approved by the Institutional Animal Care and Use Committee of Emory University in compliance with National Institutes of Health guidelines, Public Health Service policy, the Animal Welfare Act, and related university policies. Before experimentation, all animals were observed to be disease free for at least 3 months.
For surgical procedures, animals were given ketamine hydrochloride (20 mg/kg IM) for induction, 1% halothane through an endotracheal tube for anesthetic maintenance, and buprenorphine analgesic (0.01 mg/kg every 8 hours as needed) postoperatively. For subsequent short-term immobilization when performing experimental procedures postoperatively, ketamine hydrochloride (5 to 20 mg/kg IM) was used.
Chronic exteriorized AV access shunts were surgically placed between the femoral artery and vein to permit interpositioning of thrombogenic devices, drug infusions, and blood sampling. The chronic AV shunts were composed of silicone rubber tubing, 3.0-mm ID (Silastic, Dow Corning Corp). The arterial and venous arms of the shunt were connected with a 1-cm length of polytetrafluoroethylene (Teflon) tubing (2.8-mm ID). All materials were sterilized by autoclaving before surgical placement. These chronic AV shunts do not detectably activate platelets or fibrinogen.18
To measure the effects of r-hirudin and fibrin-targeted hirudin on rates of new thrombus formation, a thrombogenic synthetic polyester textile fiber graft was interposed between the arms of the chronic exteriorized AV shunt system. 111In-labeled platelets were prepared and administered as described previously.19 A boundary infusion port was placed just proximal to the graft so that hirudin, hirudin-antibody conjugate, or saline could be added to the circulating blood. Blood flow was maintained at 40 mL/min by use of a variable-speed peristaltic roller pump (Masterflow model 7016; Cole-Parmer Instrument Co.) interposed between the device and the femoral vein (ie, distal to the device). The concentrations of r-hirudin and conjugate used were equalized in terms of antithrombin activity by use of the S-2238 activity test described above. The total dose of both r-hirudin and fibrin-targeted hirudin used in all experiments was 450 ATU/kg. For a 10-kg baboon, 6 mL of a 750-ATU/mL solution was administered during a 1-hour period. The infusion was weight adjusted to reach a final dose of 450 ATU/kg. Blood samples were taken at 0, 60, and 90 minutes for analysis of hemostatic parameters. Systemic levels of fibrin-targeted hirudin and r-hirudin were undetectable at 60 minutes. Infusions of either r-hirudin or fibrin-targeted hirudin and 111In-labeled platelet imaging were begun as soon as blood flow was established. r-Hirudin or fibrin-targeted hirudin, diluted in saline solution before the experiment, was administered by continuous infusion during the first 60 minutes to maintain a constant systemic drug level throughout the experiment. Saline was infused for the subsequent 60 minutes. Maximal inhibition of thrombus formation was determined at the end of drug infusion.
Images of the vascular graft, including proximal and distal segments of the AV shunts, were acquired with a General Electric 400T MaxiCamera and stored and analyzed with a Medical Data Systems A3 image-processing system (Medtronic) interfaced with the camera. The low-energy peak (172 keV) of 111In was imaged with a 10% energy window. Dynamic images were acquired at 5-minute intervals. Immediately after each dynamic study, standards were imaged, including a syringe containing 5.0 mL whole blood (blood standard) and an identical thrombogenic device filled with static autologous blood (device standard). The imaging routines and isotopic detection protocols for these shunt studies have been reported previously.20 21 Deposited autologous 111In-labeled platelets were counted by scintillation camera imaging. Device patency was assured by Doppler flow analysis through the shunt with the use of a C-clamp–type flow probe interfaced with a Transonic T206 Blood Flow Analyzer. Measurement of parameters of hemostasis was performed as described previously.19
The descriptive data generated from experiments comparing r-hirudin with hirudin-antibody conjugate were reported as mean±SE. Statistical comparisons were calculated with the use of Sigma Stat 1.0 (Jandel) software. For the in vitro studies, the 95% and 99% CIs of the linear regressions of fibrin deposition in response to various doses of the two thrombin inhibitors were contrasted to yield discrimination at the level of P<.01. For the in vivo studies, the dose of thrombin inhibitor that produced maximal inhibition of thrombus formation (Tmax) was determined by the measured platelet deposition after 60 minutes of infusion, and 95% CIs were used to determine a statistical difference between the responses at the level of P<.05.
Inhibition of Thrombus Growth in Baboon Plasma In Vitro
To determine the feasibility of in vivo studies, we first compared the antithrombotic potencies of fibrin-targeted hirudin and r-hirudin in baboon plasma. As indicated in Fig 1⇓, fibrin-targeted hirudin was nine times more effective than r-hirudin in inhibiting fibrin deposition on experimental baboon clot surfaces in baboon plasma (P<.01) when both were administered at doses of 0.8 ATU/mL for 120 minutes. Comparable results were determined over a range of doses (0.8, 8.0, and 80.0 ATU/mL) and times (60, 120, and 180 minutes) as shown in Fig 1A⇓. The dose response at 120 minutes of fibrin deposition demonstrated a statistically significant difference between r-hirudin and hirudin-59D8 at 0.8 and 8.0 ATU/mL (Fig 1B⇓). At the highest dose of 80.0 ATU/mL, r-hirudin achieved a nearly complete effect, so that no difference between r-hirudin and fibrin-targeted hirudin was observed at this dose.
Inhibition of Thrombosis in Baboon Extracorporeal AV Shunts In Vivo
The in vivo potency of fibrin-targeted hirudin was compared with that of r-hirudin in a baboon thrombosis model measuring 111In-labeled platelet deposition on a segment of the synthetic vascular graft placed into an extracorporeal AV shunt. The doses of fibrin-targeted hirudin and r-hirudin were standardized to produce a predictable antithrombin (in ATU) on the basis of the peptide substrate S-2238 assay results.
The antithrombotic potency of fibrin-targeted hirudin was >10-fold that of r-hirudin when identical doses (750 ATU/mL) were delivered into the shunt, as illustrated in Fig 2⇓, on the basis of platelet activation at 60 minutes. The thrombogenic extracorporeal AV shunt contained 0.135±0.019×109 (mean±SE) platelets when 450 ATU/kg fibrin-targeted hirudin was used but 1.853±0.416×109 platelets when the same dose of r-hirudin was used. There was no overlap between the 95% CIs of these means, indicating a significant difference at P<.05. After 60 minutes of infusion, saline was substituted for either r-hirudin or fibrin-targeted hirudin. At this point, both response curves increased in parallel as platelet aggregation and thrombus formation in the synthetic graft were no longer inhibited. There was no significant prolongation of the activated partial thromboplastin time, prothrombin time, or thrombin time in either experimental group (treated with fibrin-targeted hirudin or r-hirudin).
The results of this study demonstrate that fibrin-targeted hirudin is more potent than r-hirudin in preventing platelet deposition and thrombus formation in vivo in a baboon model. This conclusion is based on the observation that in animals treated with fibrin-targeted hirudin, the antithrombin activity was 10-fold less than that in the r-hirudin–treated animals, yet there was less platelet deposition in the fibrin-targeted hirudin–treated animals. This finding is consistent with at least a 10-fold difference in potency between r-hirudin and fibrin-targeted hirudin. This parallels the difference in potency observed in a baboon plasma clot model in the present study and previously demonstrated in a human plasma clot model.11
Although increased potency of fibrin-targeted hirudin was observed in a controlled setting in vitro, it was not clear that this effect would occur in vivo. Because hirudin binds to thrombin with higher affinity (≈10−9 mol/L) than antibody 59D8 binds to fibrin (≈10−7 mol/L), one could argue that no additional effect would be observed after fibrin targeting. In addition, the conditions used for in vivo thrombus formation in the present study favor the formation of platelet-rich thrombi, theoretically lessening the potential therapeutic benefit attainable by targeting hirudin to fibrin. Thus, these studies suggest the following: (1) even though hirudin has a high affinity for thrombin, it is possible to increase local concentrations of hirudin through targeting other molecules present in the thrombus; (2) there is sufficient fibrin present in platelet-rich thrombi to allow targeting by this method; and (3) fibrin targeting increases local concentrations of hirudin, perhaps allowing further interaction of hirudin with both free and fibrin-bound thrombin.
The lack of an effect of either treatment on parameters of hemostasis was likely due to the design of these experiments, which used local delivery of doses too small to have a systemic effect. A study using systemic administration of higher doses will be required to determine whether the increased potency of fibrin-targeted hirudin is accompanied by fewer bleeding complications.
In summary, the data presented herein suggest that fibrin targeting of a thrombin inhibitor represents a viable strategy for decreasing the administered dose of antithrombotic agents and potential for decreasing their systemic effects.
Selected Abbreviations and Acronyms
|chromogenic substrate S-2238||=||H-d-Phenylalanyl-l-pipecolyl-l-arginine-p-nitroaniline dihydrochloride|
This research was supported by grants from the National Institutes of Health (grants HL-48667, HL-02414, HL-44307, HL-41619, HL-31950, HL-31469, and RR-00165).
Drs Bode and Hanson contributed equally to this work.
- Received August 19, 1996.
- Revision received December 12, 1996.
- Accepted December 18, 1996.
- Copyright © 1997 by American Heart Association
Meyer BJ, Fernandez-Ortiz A, Mailhac A, Falk E, Badimon L, Michael AD, Chesebro JH, Fuster V, Badimon JJ. Local delivery of r-hirudin by a double-balloon perfusion catheter prevents mural thrombosis and minimizes platelet deposition after angioplasty. Circulation. 1994;90:2474-2480.
Rubsamen K, Hornberger W, Kirchengast M. Inhibition of arterial thrombus formation in two canine models: comparison of ancrod, a fibrinogen-depleting agent, the thrombin-inhibitor r-hirudin, and the glycoprotein IIb/IIIa-receptor antagonist RO 43-8857. Thromb Haemost. 1995;74:1353-1360.
Topol EJ, Fuster V, Harrington RA, Califf RM, Kleiman NS, Kereiakes DJ, Cohen M, Chapekis A, Gold HK, Tannenbaum MA, Rao AK, Debowey D, Schwartz D, Henis M, Chesebro J. Recombinant hirudin for unstable angina pectoris: a multicenter, randomized angiographic trial. Circulation. 1994;89:1557-1566.
Cannon CP, Braunwald E. Hirudin: initial results in acute myocardial infarction, unstable angina and angioplasty. J Am Coll Cardiol. 1995;25:30S-37S.
Gold HK, Yasuda T, Jang IK, Guerrero JL, Fallon JT, Leinbach RC, Collen D. Animal models for arterial thrombolysis and prevention of reocclusion: erythrocyte-rich versus platelet-rich thrombus. Circulation. 1991;83(suppl IV):IV-26-IV-40.
Haskel EJ, Prager NA, Sobel BE, Abensdschein DR. Relative efficacy of antithrombin compared with antiplatelet agents in accelerating coronary thrombolysis and preventing early reocclusion. Circulation.. 1991;83:1048-1056.
Randomized trial of intravenous heparin versus recombinant hirudin for acute coronary syndromes: the Global Use of Strategies to Open Occluded Coronary Arteries (GUSTO) Investigators. Circulation.. 1994;90:1631-1637.
Antman EM. Hirudin in acute myocardial infarction: Thrombolysis and Thrombin Inhibition in Myocardial Infarction (TIMI) 9B trial. Circulation.. 1996;94:911-921.
Neuhas KL, von Essen R, Tebbe U, Jessel A, Heinrichs H, Maurer W, Doring W, Harmjanz D, Kotter V, Kalhammer E, Simon H, Horacek T. Safety observations from the pilot phase of the randomized r-Hirudin for Improvement of Thrombolysis (HIT-III) study: a study of the Arbeitsgemeinschaft Leitender Kardioogischer Krankenhausartze (ALKK). Circulation.. 1994;90:1638-1642.
Bode C, Hudelmayer M, Mehwald P, Bauer S, Freitag M, Von Hodenberg E, Newell JB, Kubler W, Haber E, Runge MS. Fibrin-targeted recombinant hirudin inhibits fibrin deposition on experimental clots more efficiently than recombinant hirudin. Circulation.. 1994;90:1956-1963.
Runge MS, Bode C, Matsueda GR, Haber E. Antibody-enhanced thrombolysis: targeting of tissue plasminogen activator in vivo. Proc Natl Acad Sci U S A.. 1987;84:7659-7662.
Bode C, Matsueda GR, Hui KY, Haber E. Antibody-directed urokinase: a specific fibrinolytic agent. Science.. 1985;229:765-767.
Bode C, Runge MS, Newell JB, Matsueda GR, Haber E. Characterization of an antibody-urokinase conjugate: a plasminogen activator targeted to fibrin. J Biol Chem.. 1987;262:10819-10823.
Bode C, Meinhardt G, Runge MS, Freitag M, Nordt T, Arens M, Newell JB, Kubler W, Haber E. Platelet-targeted fibrinolysis enhances clot lysis and inhibits platelet aggregation. Circulation.. 1991;84:805-813.
Bode C, Runge MS, Schonermark S, Eberle T, Newell JB, Kubler W, Haber E. Conjugation to antifibrin Fab′ enhances fibrinolytic potency of single-chain urokinase plasminogen activator. Circulation.. 1990;81:1974-1980.
Hui KY, Haber E, Matsueda GR. Monoclonal antibodies to a synthetic fibrin-like peptide bind to human fibrin but not fibrinogen. Science.. 1983;222:1129-1132.
Savage B, McFadden PR, Hanson SR, Harker LA. The relation of platelet density to platelet age: survival of low and high density 111indium-labeled platelets in baboons. Blood.. 1986;68:386-393.
Runge MS, Harker LA, Bode C, Ruef J, Kelly AB, Marzec UM, Allen E, Caban R, Shaw S-Y, Haber E, Hanson SR. Enhanced thrombolytic and antithrombotic potency of a fibrin-targeted plasminogen activator in baboons. Circulation.. 1996;94:1412-1422.