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Recombinant desulfatohirudin as a substitute for heparin during cardiopulmonary bypass*Supported by grant HL 19055 from the National Heart, Lung, and Blood Institute, National Institutes of Health, Bethesda, Md

1994; Elsevier BV; Volume: 108; Issue: 2 Linguagem: Inglês

10.1016/s0022-5223(94)70022-2

1097-685X

Alvise Bernabei, A. Koneti Rao, Stefan Niewiarowski, Robert W. Colman, Liyun Sun, L H Edmunds,

Leech Biology and Applications

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.1Boisclair 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: 253-258Crossref PubMed Scopus (143) Google Scholar, 2Brister SJ Ofosu FA Buchanan MR Thrombin generation during cardiac surgery: Is heparin the ideal anticoagulant?.Thromb Haemost. 1993; 70: 259-262Crossref PubMed Scopus (142) Google Scholar 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) 3Stone SR Hofsteenge J Kinetics of the inhibition of thrombin by hirudin.Biochem. 1986; 25: 4622-4628Crossref PubMed Scopus (513) Google Scholar found in leeches (Hirudo medicinalis) that directly and stoichiometrically binds thrombin. 3Stone SR Hofsteenge J Kinetics of the inhibition of thrombin by hirudin.Biochem. 1986; 25: 4622-4628Crossref PubMed Scopus (513) Google Scholar The natural protein contains 65 amino acids (molecular weight 7000)4Talbot M Biology of recombinant hirudin (CGP 39393): a new prospect in the treatment of thrombosis.Sem Thromb Hemost. 1989; 15: 293-301Crossref PubMed Scopus (58) Google Scholar; 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. 5Marki WE Wallis RB The anticoagulant and antithrombotic properties of hirudins.Thromb Haemost. 1990; 61: 344-348Google Scholar R-hirudin (molecular weight 6963) is a potent and selective inhibitor of thrombin (Ki = 231 fmol). 5Marki WE Wallis RB The anticoagulant and antithrombotic properties of hirudins.Thromb Haemost. 1990; 61: 344-348Google Scholar 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. 6Wachtfogel Y Kucich U Hack CE Niewarowski S Colman RW Edmunds Jr, LH Aprotinin inhibits the contact, neutrophil, and platelet activation systems during simulated extracorporal perfusion.J THORAC CARDIOVASC SURG. 1993; 106: 1-10Abstract Full Text PDF PubMed Google Scholar Fresh blood (300 ml) from aspirin-abstaining donors was drawn directly into the reservoir bag to which either beef lung heparin (1500 μg, The Upjohn Company, Kalamazoo, Mich., n = 10) or r-hirudin (2.25 μg, n = 5, or 6.3 μg, n = 5) for final concentrations of 5 U/ml, 1.07 μmol/L, and 3.0 μmol/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. 6Wachtfogel Y Kucich U Hack CE Niewarowski S Colman RW Edmunds Jr, LH Aprotinin inhibits the contact, neutrophil, and platelet activation systems during simulated extracorporal perfusion.J THORAC CARDIOVASC SURG. 1993; 106: 1-10Abstract Full Text PDF PubMed Google Scholar 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 β-thromboglobulin, neutrophil elastase, and complement C -C inhibitor complex were assayed as previously described. 6Wachtfogel Y Kucich U Hack CE Niewarowski S Colman RW Edmunds Jr, LH Aprotinin inhibits the contact, neutrophil, and platelet activation systems during simulated extracorporal perfusion.J THORAC CARDIOVASC SURG. 1993; 106: 1-10Abstract Full Text PDF PubMed Google Scholar 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.). Recirculation significantly (p < 0.050) reduced platelet count and platelet response to adenosine diphosphate and significantly increased plasma β-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 50 was 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 3.0 μmol/L and at 30, 60, and 120 minutes for r-hirudin concentrations of 1.07 μmol/L by Student’s unpaired t test. Fig. 2 shows plasma F1.2 concentrations for heparin (n = 5) and r-hirudin (1.07 μmol/L) (n = 5). F1.2 concentrations were significantly (p < 0.05 or 0.01) higher with r-hirudin at 30, 60, and 120 minutes. 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. 5Marki WE Wallis RB The anticoagulant and antithrombotic properties of hirudins.Thromb Haemost. 1990; 61: 344-348Google Scholar 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. 7Zoldhelyi P Webster MWI Fuster V et al.Recombinant hirudin in patients with chronic stable coronary artery disease.Circulation. 1993; 88: 2015-2022Crossref PubMed Scopus (104) Google Scholar 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 8Lindhout T Blezer R Hemker HC The anticoagulant mechanism of action of recombinant hirudin (CGP 39393) in plasma.Thromb Haemost. 1990; 64: 464-468Crossref PubMed Scopus (47) Google Scholar 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 9Walenga 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: 271-277Abstract Full Text PDF PubMed Scopus (41) Google Scholar 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 3Stone SR Hofsteenge J Kinetics of the inhibition of thrombin by hirudin.Biochem. 1986; 25: 4622-4628Crossref PubMed Scopus (513) Google Scholar is3.25 × 10 7 mol/L -1 sec -1 and for antithrombin III with thrombin in the presence of heparin, 10Olson ST Björk I Predominant contribution of surface approximation to the mechanism of heparin acceleration of the antithrombin-thrombin reaction.J Biol Chem. 1991; 266: 6353-6362PubMed Google Scholar 3.7 × 10 7 mol/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 11Rosenberg RD Bauer KA The heparin-antithrombin system: a natural anticoagulant mechanism.in: Hemostasis and thrombosis, basic principles and clinical practice. R.W. Colman, J. JB Lippincott, Philadelphia1994: 837-860Google Scholar; 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.