Thromb Haemost 2009; 101(05): 867-877
DOI: 10.1160/TH08-08-0535
Blood Coagulation, Fibrinolysis and Cellular Haemostasis
Schattauer GmbH

A long-lasting, plasmin-activatable thrombin inhibitor aids clot lysis in vitro and does not promote bleeding in vivo

William P. Sheffield
1   Department of Pathology and Molecular Medicine, McMaster University, Hamilton, Ontario, Canada
2   Canadian Blood Services, Research and Development, Hamilton, Ontario, Canada
,
Louise J. Eltringham-Smith
1   Department of Pathology and Molecular Medicine, McMaster University, Hamilton, Ontario, Canada
,
Sharon Gataiance
1   Department of Pathology and Molecular Medicine, McMaster University, Hamilton, Ontario, Canada
,
Varsha Bhakta
2   Canadian Blood Services, Research and Development, Hamilton, Ontario, Canada
› Institutsangaben
Financial support: This work was made possible by Grant-In-Aid T5827 from the Heart and Stroke Foundation of Ontario to WPS.
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Publikationsverlauf

Received: 20. August 2008

Accepted after minor revision: 28. März 2008

Publikationsdatum:
24. November 2017 (online)

Summary

The leech protein hirudin is a potent inhibitor of thrombin, but clinical use of recombinant hirudin is restricted by haemorrhagic risks, and complicated by hirudin’s rapid clearance from the circulation. We previously employed albumin fusion to slow hirudin variant 3 (HV3) clearance. In this study, we hypothesized that reconfiguration of the chimera, appending human serum albumin (HSA) to the N-terminus of HV3, with an intervening plasmin cleavage site, would create a slowly cleared, plasmin-activatable HV3. Potential plasmin cleavage sites were screened by expression in Escherichia coli, interposed between glutathione sulfotransferase and HV3 domains. The most reactive sequence (GSGIYR-ITY) was recreated in C-terminally His-tagged albumin fusion protein HSACHV3, expressed in Pichia pastoris yeast and purified by nickel-chelate affinity chromatography. HSACHV3 showed no thrombin inhibitory activity in the absence of plasmin, but liberated active HV3 in a time- and concentration-dependent manner in its presence. In a discontinuous clot assay involving clot-bound thrombin, HSACHV3 assisted clot lysis by limiting clot extension in a tPA- and concentration-dependent manner. Similar results were obtained in plasma at higher concentrations of HSACHV3. The chimeric protein exhibited much slower clearance in mice than unfused HV3, and indistinguishable pharmacokinetics from unfused recombinant HSA. In a mouse tail transection bleeding model, doses of HSACHV3 identical to those of HV3 that elicited a four-fold increase in the volume of shed blood were without effect. Our results suggest that HSACHV3 is a fully latent, plasmin activatable, long-lasting hirudin, of potential benefit in thrombotic disorders resistant to natural or pharmacological clot lysis.

 
  • References

  • 1 Colman RW. Are hemostasis and thrombosis two sides of the same coin?. J Exp Med 2006; 203: 493-495.
  • 2 Gettins PG. Serpin structure, mechanism, and function. Chem. Rev. 2002; 102: 4751-4804.
  • 3 Dodt J, Machleidt W, Seemuller U. et al. Isolation and characterization of hirudin isoinhibitors and sequence analysis of hirudin PA. Biol Chem Hoppe Seyler 1986; 367: 803-811.
  • 4 Stone SR, Hofsteenge J. Kinetics of the inhibition of thrombin by hirudin. Biochemistry 1986; 25: 4622-4628.
  • 5 Anonymous.. Hirudins: return of the leech?. Lancet 1992; 340: 579-580.
  • 6 Hirsh J, Heddle N, Kelton JG. Treatment of heparin-induced thrombocytopenia: a critical review. Arch Intern Med 2004; 164: 361-369.
  • 7 von Dobschuetz E, Hoffmann T, Messmer K. Inhibition of neutrophil proteinases by recombinant serpin Lex032 reduces capillary no-reflow in ischemia/ reperfusion-induced acute pancreatitis. J Pharmacol Exp Ther 1999; 290: 782-788.
  • 8 Hirsh J. Current anticoagulant therapy--unmet clinical needs. Thromb Res 2003; 109 (Suppl. 19) S1-8.
  • 9 Markwardt F, Nowak G, Sturzebecher J. Clinical pharmacology of recombinant hirudin. Haemostasis 1991; 21 (Suppl. 01) 133-136.
  • 10 Markwardt F, Nowak G, Sturzebecher U. et al. Studies on the pharmacokinetics of hirudin. Biomed Biochim Acta 1987; 46: 237-244.
  • 11 Syed S, Schuyler PD, Kulczycky M. et al. Potent antithrombin activity and delayed clearance from the circulation characterize recombinant hirudin genetically fused to albumin. Blood 1997; 89: 3243-3252.
  • 12 Sheffield WP, Gataiance S, Eltringham-Smith LJ. Combined administration of barbourin--albumin and hirudin--albumin fusion proteins limits fibrin(ogen) deposition on the rabbit balloon-injured aorta. Thromb Res 2007; 119: 195-207.
  • 13 Lazar JB, Winant RC, Johnson PH. Hirudin: amino-terminal residues play a major role in the interaction with thrombin. J Biol Chem 1991; 266: 685-688.
  • 14 Peter K, Graeber J, Kipriyanov S. et al. Construction and functional evaluation of a single-chain antibody fusion protein with fibrin targeting and thrombin inhibition after activation by factor Xa. Circulation 2000; 101: 1158-1164.
  • 15 Zhang C, Yu A, Yuan B. et al. Construction and functional evaluation of hirudin derivatives with low bleeding risk. Thromb Haemost 2008; 99: 324-330.
  • 16 Weitz JI, Hudoba M, Massel D. et al. Clot-bound thrombin is protected from inhibition by heparin-anti-thrombin III but is susceptible to inactivation by anti-thrombin III-independent inhibitors. J Clin Invest 1990; 86: 385-391.
  • 17 Fischer KG. The role of recombinant hirudins in the management of thrombotic disorders. BioDrugs 2004; 18: 235-268.
  • 18 Loscalzo J. Thrombin inhibitors in fibrinolysis. A Hobson’s choice of alternatives. Circulation 1996; 94: 863-865.
  • 19 Mann KG, Butenas S, Brummel K. The dynamics of thrombin formation. Arterioscler Thromb Vasc Biol 2003; 23: 17-25.
  • 20 Aoki N. The past, present and future of plasmin inhibitor. Thromb Res 2005; 116: 455-464.
  • 21 Kolev K, Longstaff C, Machovich R. Fibrinolysis at the fluid-solid interface of thrombi. Curr Med Chem Cardiovasc Hematol Agents 2005; 03: 341-355.
  • 22 Sheffield WP, Smith IJ, Syed S. et al. Prolonged in vivo anticoagulant activity of a hirudin-albumin fusion protein secreted from Pichia pastoris. Blood Coagul Fibrinolysis 2001; 12: 433-443.
  • 23 Cunningham MA, Bhakta V, Sheffield WP. Altering heparin cofactor II at VAL439 (P6) either impairs inhibition of thrombin or confers elastase resistance. Thromb Haemost 2002; 88: 89-97.
  • 24 Sheffield WP, Smith IJ, Syed S. et al. Prolonged in vivo anticoagulant activity of a hirudin-albumin fusion protein secreted from Pichia pastoris. Blood Coagul Fibrinolysis 2001; 12: 433-443.
  • 25 Sheffield WP, Wilson B, Eltringham-Smith LJ. et al. Recombinant albumins containing additional peptide sequences smaller than barbourin retain the ability of barbourin-albumin to inhibit platelet aggregation. Thromb Haemost 2005; 93: 914-921.
  • 26 Sheffield WP, McCurdy TR, Bhakta V. Fusion to albumin as a means to slow the clearance of small therapeutic proteins using the Pichia pastoris expression system: a case study. Methods Mol Biol 2005; 308: 145-154.
  • 27 Sheffield WP, Mamdani A, Hortelano G. et al. Effects of genetic fusion of factor IX to albumin on in vivo clearance in mice and rabbits. Br J Haematol 2004; 126: 565-573.
  • 28 Begbie ME, Mamdani A, Gataiance S. et al. An important role for the activation peptide domain in controlling factor IX levels in the blood of haemophilia B mice. Thromb Haemost 2005; 94: 1138-1147.
  • 29 Fortkamp E, Rieger M, Heisterberg-Moutses G. et al. Cloning and expression in Escherichia coli of a synthetic DNA for hirudin, the blood coagulation inhibitor in the leech. DNA 1986; 05: 511-517.
  • 30 Wallace A, Dennis S, Hofsteenge J. et al. Contribution of the N-terminal region of hirudin to its interaction with thrombin. Biochemistry 1989; 28: 10079-10084.
  • 31 Schechter I, Berger A. On the size of the active site in proteases. I. Papain. Biochem Biophys Res Commun 1967; 27: 157-162.
  • 32 Hervio LS, Coombs GS, Bergstrom RC. et al. Negative selectivity and the evolution of protease cascades: the specificity of plasmin for peptide and protein substrates. Chem Biol 2000; 07: 443-453.
  • 33 Marques JA, George JK, Smith IJ. et al. A barbour-in-albumin fusion protein that is slowly cleared in vivo retains the ability to inhibit platelet aggregation in vitro. Thromb Haemost 2001; 86: 902-908.
  • 34 Komatsu Y, Misawa S, Sukesada A. et al. CX-397, a novel recombinant hirudin analog having a hybrid sequence of hirudin variants-1 and –3. Biochem Biophys Res Commun 1993; 196: 773-779.
  • 35 Peters Jr. T. Serum albumin. Adv Protein Chem 1985; 37: 161-245.
  • 36 Collet JF, Bardwell JC. Oxidative protein folding in bacteria. Mol Microbiol 2002; 44: 1-8.
  • 37 Fenton 2nd JW, Villanueva GB, Ofosu FA. et al. Thrombin inhibition by hirudin: how hirudin inhibits thrombin. Haemostasis 1991; 21 (Suppl. 01) 27-31.
  • 38 Weitz JI, Leslie B, Hudoba M. Thrombin binds to soluble fibrin degradation products where it is protected from inhibition by heparin-antithrombin but susceptible to inactivation by antithrombin-independent inhibitors. Circulation 1998; 97: 544-552.
  • 39 Haskel EJ, Eisenberg PR, Abendschein DR. Inhibition of concomitant thrombin-mediated fibrin formation enhances clot lysis in whole blood. Blood Coagul Fibrinolysis 1993; 04: 7-13.
  • 40 Markwardt F. Hirudin and derivatives as anticoagulant agents. Thromb Haemost 1991; 66: 141-152.
  • 41 Eichler P, Friesen HJ, Lubenow N. et al. Antihirudin antibodies in patients with heparin-induced thrombocytopenia treated with lepirudin: incidence, effects on aPTT, and clinical relevance. Blood 2000; 96: 2373-2378.
  • 42 Fischer KG, Liebe V, Hudek R. et al. Anti-hirudin antibodies alter pharmacokinetics and pharmacodynamics of recombinant hirudin. Thromb Haemost 2003; 89: 973-982.
  • 43 Curtis LD, Brown A, Comer MB. et al. Pharmacokinetics and pharmacodynamics of BB-10153, a thrombin-activatable plasminogen, in healthy volunteers. J Thromb Haemost 2005; 03: 1180-1186.