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J Thorac Cardiovasc Surg 1994;108:381
© 1994 Mosby, Inc.

LETTERS TO THE EDITOR

Recombinant desulfatohirudin as a substitute for heparin during cardiopulmonary bypass*

A. Bernabei, MD, A. K. Rao, MD, S. Niewiarowski, MD, PhD, R. W. Colman, MD, L. Sun, MD, L. H. Edmunds, Jr., MD

Department of Surgery
School of Medicine
University of Pennsylvania
Sol Sherry Thrombosis Research Center
Hematology Division
Departments of Medicine and Physiology
Temple University
Philadelphia, PA. 19104

To the Editor:

Despite large doses of heparin sufficient to maintain activated clotting times greater than 400 seconds, thrombin is produced during cardiopulmonary bypass. Both the prothrombin fragment, F1.2, and the fibrinogen fragment, fibrinopeptide A (FPA), increase progressively during bypass.Go Go 1, 2 Thrombin is a powerful protease that converts fibrinogen to fibrin but also activates platelets, induces endothelial cells to release tissue plasminogen activator, and contributes importantly to bleeding and thromboembolic complications associated with cardiac operations.

Hirudin is a rapid, reversible, tight-binding thrombin inhibitor (Ki = 20 fmol) Go 3 found in leeches (Hirudo medicinalis) that directly and stoichiometrically binds thrombin. Go 3 The natural protein contains 65 amino acids (molecular weight 7000)Go 4; recombinant desulfatohirudin (HV1, CGP 39393, Ciba-Geigy Pharmaceuticals, Summit, N.J.) (r-hirudin) differs from the natural peptide by the absence of a sulfate group on tyrosine 63. Go 5 R-hirudin (molecular weight 6963) is a potent and selective inhibitor of thrombin (Ki = 231 fmol). Go 5 We tested the hypothesis that r-hirudin reduces the rise in FPA and F1.2, markers of thrombin activity, and platelet and neutrophil activation during in vitro simulated extracorporeal circulation. Go 6

Methods
Fresh blood (300 ml) from aspirin-abstaining donors was drawn directly into the reservoir bag to which either beef lung heparin (1500µg, The Upjohn Company, Kalamazoo, Mich., n = 10) or r-hirudin (2.25µg, n = 5, or 6.3µg, n = 5) for final concentrations of 5 U/ml, 1.07µmol/L, and 3.0µmol/L was added. The perfusion circuit consisted of a spiral coil membrane oxygenator (model 0400-2A, Avecor Inc., Minneapolis, Minn.) and roller pump as described previously. Go 6 Blood was recirculated at 37° C for 2 hours. Estimated surface area of the system was 0.45 m 2. Samples were drawn before recirculation and 5, 30, 60, and 120 minutes after the pump was started. Samples were prepared and platelet count, platelet aggregation to adenosine diphosphate, plasma ß-thromboglobulin, neutrophil elastase, and complement C -C inhibitor complex were assayed as previously described. Go 6 Plasma samples for FPA and F1.2 were collected into citrate tubes containing heparin (1000 U/ml) and aprotinin (1 mg/ml) and centrifuged at 2000g for 20 minutes and then 27,000g for 5 minutes. Supernatant plasma was harvested and stored at -80° C. FPA and F1.2 concentrations were measured by enzyme-linked immunoassay (obtained from American Bioproducts, Parsipanny, N.J., and Behring Diagnostics, Somerville, N.J.).

Results
Recirculation significantly (p < 0.050) reduced platelet count and platelet response to adenosine diphosphate and significantly increased plasma ß-thromboglobulin, neutrophil elastase, and C -C inhibitor complex within each group. No significant differences were apparent among the three groups for any measurement at any sampling time except for C -C inhibitor complex. Plasma C -C inhibitor complex concentrations were significantly (p < 0.05) lower with high-dose r-hirudin than those observed with heparin at 30, 60, and 120 minutes (1.8 ± 0.4 U/ml versus 3.2 ± 0.3 U/ml [standard error of the mean] at 2 hours).

Fig.1 illustrates FPA concentrations for the three groups at five sampling times. Values greater than 50 ng/ml were not further quantified; for calculations, the figure 50was used. As compared with heparin, FPA concentrations were significantly higher (p < 0.05 or 0.01) at 60 and 120 minutes for r-hirudin concentrations of 3.0µmol/L and at 30, 60, and 120 minutes for r-hirudin concentrations of 1.07µmol/L by Student's unpaired ttest.



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  		Fig. 1. Plasma fibrinopeptide A concentrations (mean ± standard error of the mean) during in vitro recirculation of fresh human blood. Circles, Heparin (n = 10); squares, r-hirudin 1.07µmol/L (n = 5); triangles, r-hirudin 3.0µmol/L (n = 5).

 
Fig. 2 shows plasma F1.2 concentrations for heparin (n = 5) and r-hirudin (1.07µmol/L) (n = 5). F1.2 concentrations were significantly (p < 0.05 or 0.01) higher with r-hirudin at 30, 60, and 120 minutes.



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  		Fig. 2. Plasma F1.2 concentrations (mean± standard error of the mean) for heparin (n = 5) and r-hirudin (1.07µmol/L, n = 5). Stored plasma was not available to measure F1.2 in five heparin samples and in r-hirudin (3.0µmol) samples.

 
DISCUSSION
The doses of r-hirudin used in this study are high, because partial thromboplastin times double at r-hirudin concentrations of 0.1 µmol/L and quadruple at 1.0 µmol/L. Go 5 In patients with stable coronary disease, infusion of r-hirudin in a dosage of 0.3 mg/kg per hour resulted in plasma concentrations of about 0.3 µmol/L and a threefold increase in partial thromboplastin times. Go 7 The fact that r-hirudin is less successful than heparin in controlling thrombin activity in this in vitro system underscores the massive thrombotic stimulus produced by blood circulation through extracorporeal perfusion systems that contain an oxygenator. Lindhout, Blezer, and Hemker Go 8 previously found that the reaction between thrombin and r-hirudin does not prevent trace amounts of thrombin produced via the extrinsic pathway. In dogs, Walenga and associates Go 9 observed more fibrin formation during 60 minutes of cardiopulmonary bypass with r-hirudin anticoagulation than with a reduced dose of heparin.

The difference between heparin and r-hirudin in suppressing FPA formation is not explained by reaction speed. Both drugs bind thrombin very rapidly. The second/order rate constant for native hirudin with thrombin Go 3 is3.25 x 10 7mol/L -1sec -1 and for antithrombin III with thrombin in the presence of heparin, Go 10 3.7 x 10 7mol/L -1 sec -1. The superior inhibitory effect of heparin-catalyzed antithrombin III on thrombin formation is explained by the ability to also inhibit factor Xa. Heparin–antithrombin III complex strongly inhibits thrombin and factor Xa and weakly inhibits other soluble coagulation proteins Go 11; r-hirudin only binds thrombin. In this system, F1.2, which is produced when prothrombin is cleaved to form thrombin by factor Xa, increases in the presence of r-hirudin but not with heparin. Heparin, therefore, remains the best anticoagulant for cardiopulmonary bypass.

References

  1. Boisclair MD, Lane DA, Philippou H, Sheikh S, Hunt B. Thrombin production, inactivation and expression during open heart surgery measured by assays for activation fragments including a new ELISA for prothrombin Fragment F1 + 2. Thromb Haemost 1993;70:253-8.[Medline]
  2. Brister SJ, Ofosu FA, Buchanan MR. Thrombin generation during cardiac surgery: Is heparin the ideal anticoagulant? Thromb Haemost 1993;70:259-62.[Medline]
  3. Stone SR, Hofsteenge J. Kinetics of the inhibition of thrombin by hirudin. Biochem 1986;25:4622-8.[Medline]
  4. Talbot M. Biology of recombinant hirudin (CGP 39393): a new prospect in the treatment of thrombosis. Sem Thromb Hemost 1989;15:293-301.[Medline]
  5. Marki WE, Wallis RB. The anticoagulant and antithrombotic properties of hirudins. Thromb Haemost 1990;61:344-8.
  6. Wachtfogel Y, Kucich U, Hack CE, Niewarowski S, Colman RW, Edmunds LH Jr. Aprotinin inhibits the contact, neutrophil, and platelet activation systems during simulated extracorporal perfusion. J THORAC CARDIOVASC SURG 1993;106:1-10.[Abstract]
  7. Zoldhelyi P, Webster MWI, Fuster V, et al. Recombinant hirudin in patients with chronic stable coronary artery disease. Circulation 1993;88:2015-22.[Abstract/Free Full Text]
  8. Lindhout T, Blezer R, Hemker HC. The anticoagulant mechanism of action of recombinant hirudin (CGP 39393) in plasma. Thromb Haemost 1990;64:464-8.[Medline]
  9. Walenga JM, Bakhos M, Messmore HL, Fareed J, Pifarré R. Potential use of recombinant hirudin as an anticoagulant in a cardiopulmonary bypass model. Ann Thorac Surg 1991;51:271-7.[Abstract/Free Full Text]
  10. Olson ST, Björk I. Predominant contribution of surface approximation to the mechanism of heparin acceleration of the antithrombin-thrombin reaction. J Biol Chem 1991;266:6353-62.[Abstract/Free Full Text]
  11. Rosenberg RD, Bauer KA. The heparin-antithrombin system: a natural anticoagulant mechanism. In: Colman RW, Hirsh J, Marder VJ, Salzman EW. Hemostasis and thrombosis, basic principles and clinical practice. R.W. Colman, J. Philadelphia: JB Lippincott, 1994:837-60.



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