Download PDF
Thromb Haemost 2005; 93(03): 430-436
DOI: 10.1160/TH04-08-0480
Blood Coagulation, Fibrinolysis and Cellular Haemostasis
Schattauer GmbH

Improvement of the specificity of dipetarudin by site directed mutagenesis

Mercedes López
1   Instituto Venezolano de Investigaciones Científicas, Laboratorio de Trombosis Experimental, Centro de Biofísica y Bioquímica, Caracas, Venezuela
,
Katrin Mende
2   Friedrich Schiller University Jena, Medical Faculty, Research Unit “Pharmacological Haemostaseology“, Jena, Germany
,
Goetz Nowak
2   Friedrich Schiller University Jena, Medical Faculty, Research Unit “Pharmacological Haemostaseology“, Jena, Germany
› Author AffiliationsFinancial support: This work was partly supported by Fresenius Medical Care GmbH, Germany.
Further Information

Publication History

Received 05 August 2004

Accepted after resubmission 07 February 2004

Publication Date:
14 December 2017 (online)

Also available at
    Permissions and Reprints

Summary

Protease specificity is crucial to the design of thrombin inhibitors as inhibition of other physiologically relevant serine-proteases can compromise their clinical use. Dipetarudin, a potent thrombin inhibitor, also inhibits trypsin and plasmin. Due to the specificity of an inhibitor being influenced by the amino acid residue at the P1 position, we replaced the Arg10 at P1 position of dipetarudin by a histidine, which is the P1 residue of rhodniin, a very specific thrombin inhibitor. The amino acid replacement was carried out by site directed mutagenesis. The mutant, dipetarudin R10H, showed a loss of plasmin and trypsin inhibitory activities present in its wild-type counterpart and a 3-fold higher dissociation constant for thrombin than dipetarudin. However, compared to dipetarudin and r-hirudin, dipetarudin R10H showed similar activity in coagulation screening assays such as activated partial thromboplastin time (aPTT), prothrombin time (PT), ecarin clotting time (ECT) and ecarin chromogenic assay (ECA).

Keywords

Dipetarudin - specificity - P1 position - site directed mutagenesis - thrombin
 

  • References

  • 1 Markwardt F. Die Isolierung und chemische Charakterisierung des Hirudins. Z Physiol Chem 1957; 312: 85-9.
    PubMedGoogle Scholar
  • 2 Stone S, Hofsteenge J. Kinetics of the inhibition of thrombin by hirudin. Biochemistry 1986; 25: 4622-8.
    CrossrefPubMedGoogle Scholar
  • 3 Braun P, Dennis S, Hofsteenge J. et al. Use of sitedirected mutagenesis to investigate the basis for the specificity of hirudin. Biochemistry 1988; 27: 6517-22.
    CrossrefPubMedGoogle Scholar
  • 4 Dodt J, Koehler S, Baici A. Interaction of site specific hirudin variants with a-thrombin. FEBS Lett 1988; 229: 87-90.
    CrossrefPubMedGoogle Scholar
  • 5 Friedrich T, Kroeger B, Bialojan S. et al. A Kazal-type inhibitor with thrombin specificity from Rhodnius prolixus. J Biol Chem 1993; 268: 16216-22.
    PubMedGoogle Scholar
  • 6 van de Locht A, Lamba D, Bauer M. et al. Two heads are better than one: crystal structure of the insect derived double domain Kazal inhibitor rhodniin in complex with thrombin. EMBO J 1995; 14: 5149-57.
    PubMedGoogle Scholar
  • 7 Dodt J, Otte M, Strube K. et al. Thrombin inhibitors of bloodsucking animals. Semin Thromb Hemost 1996; 22: 203-8.
    Article in Thieme ConnectPubMedGoogle Scholar
  • 8 Lange U, Keilholz W, Schaub G. et al. Biochemical characterization of a thrombin inhibitor from the bloodsucking bug Dipetalogaster maximus. Haemostasis 1999; 29: 204-11.
    PubMedGoogle Scholar
  • 9 Mende K, Petoukhova O, Koulitchkova V. et al. Dipetalogastin, a potent thrombin inhibitor from the blood-sucking insect Dipetalogaster maximus. Eur J Biochem 1999; 266: 583-90.
    CrossrefPubMedGoogle Scholar
  • 10 López M, Mende K, Steinmetzer T. et al. Cloning, purification and biochemical characterization of dipetarudin, a new chimeric thrombin inhibitor. J Chrom B 2003; 786: 73-80.
    CrossrefPubMedGoogle Scholar
  • 11 Dang Q, Di Cera E. A simple activity assay for thrombin and hirudin. J Protein Chem 1994; 13: 367-73.
    CrossrefPubMedGoogle Scholar
  • 12 Morrison J, Stone S. Approaches to the study and analysis of the inhibition of enzymes by slow and tight binding inhibitors. Comm Mol Cell Biophys 1995; 2: 347-68.
    PubMedGoogle Scholar
  • 13 Dixon M. Determination of enzyme inhibitor constants. Biochem J 1953; 55: 170-1.
    CrossrefPubMedGoogle Scholar
  • 14 Segel IH. Behaviour and analysis of rapid equilibrium and steady-state enzyme systems. Enzyme kinetics, John Wiley and Sons, Inc; New York: 1975: 170-6.
    Google Scholar
  • 15 Nowak G, Bucha E. Quantitative determination of hirudin in blood and body fluids. Semin Thromb Hemost 1996; 22: 197-202.
    Article in Thieme ConnectPubMedGoogle Scholar
  • 16 Copley AL, Niewiarowski S, Marechel J. A micro method of euglobulin fibrinolysis in plasma of human subjects and small laboratory animals. J Lab Med 1959; 53: 468-73.
    PubMedGoogle Scholar
  • 17 Moser M, Auerswald E, Mentele R. et al. Bdellastasin, a serine protease inhibitor of the antistasin family from the medical leech ( Hirudo medicinalis) primary structure, expression in yeast, and characterization of native and recombinant inhibitor. Eur J Biochem 1998; 253: 212-20.
    CrossrefPubMedGoogle Scholar
  • 18 Wen L, Lee I, Chen G. et al. Changing the inhibitory specificity and function of Cucurbita maxima trypsin inhibitor-V by site-directed mutagenesis. Biochem Biophys Res Commun 1995; 207: 897-902.
    CrossrefPubMedGoogle Scholar
  • 19 Heinz D, Hyberts S, Peng J. et al. Changing the inhibitory specificity and function of the proteinase inhibitor eglin c by site-directed mutagenesis: functional and structural investigation. Biochemistry 1992; 31: 8755-66.
    CrossrefPubMedGoogle Scholar
  • 20 Longstaff C, Campbell A, Fersht A. Recombinant chymotrypsin inhibitor 2: expression, kinetic analysis of inhibition with alpha-chymotrypsin and wild-type and mutant subtilisin BPN', and protein engineering to investigate inhibitory specificity and mechanism. Biochemistry 1990; 29: 7339-47.
    CrossrefPubMedGoogle Scholar
  • 21 Stone S, Braun P, Hofsteenge J. Identification of regions of alpha-thrombin involved in its interaction with hirudin. Biochemistry 1987; 26: 4617-24.
    CrossrefPubMedGoogle Scholar
  • 22 Stone S, Dennis S, Hofsteenge J. Quantitative evaluation of the contribution of ionic interactions to the formation of the thrombin-hirudin complex. Biochemistry 1989; 28: 6857-63.
    CrossrefPubMedGoogle Scholar
  • 23 Betz A, Hofsteenge J, Stone S. Interaction of the N-terminal region of hirudin with the active-site cleft of thrombin. Biochemistry 1992; 31: 4557-62.
    CrossrefPubMedGoogle Scholar
  • 24 de Cristofaro R, Rocca B, Bizzi B. et al. The linkage between binding of the C-terminal domain of hirudin and amidase activity in human alpha-thrombin. Biochem J 1993; 289: 475-80.
    CrossrefPubMedGoogle Scholar
  • 25 Nowak G. Clinical monitoring of hirudin and direct thrombin inhibitors. Semin Thromb Hemost 2001; 27: 537-42.
    Article in Thieme ConnectPubMedGoogle Scholar
  • 26 Marbet GA, Verstraete M, Kienast J. et al. Clinical pharmacology of intravenously administered recombinant desulfatohirudin (CGP 39393) in healthy volunteers. J Cardiovasc Pharmacol 1993; 22: 364-72.
    CrossrefPubMedGoogle Scholar
  • 27 Grutter MG, Priestle JP, Rahuel J. et al. Crystal structure of the thrombin-hirudin complex: a novel mode of serine protease inhibition. EMBO J 1990; 9: 2361-5.
    PubMedGoogle Scholar
  • 28 Rydel TJ, Ravichandran KG, Tulinsky A. et al. The structure of a complex of recombinant hirudin and human alpha-thrombin. Science 1990; 249: 277-80.
    CrossrefPubMedGoogle Scholar
  • 29 Rydel TJ, Tulinsky A, Bode W. et al. Refined structure of the hirudin-thrombin complex. J Mol Biol 1991; 221: 583-601.
    CrossrefPubMedGoogle Scholar
  • 30 Bode W, Huber R. Natural protein proteinase inhibitors and their interaction with proteinases. Eur J Biochem 1992; 204: 433-51.
    CrossrefPubMedGoogle Scholar
  • 31 Krishnaswamy S, Mann K, Nesheim M. The prothrombinase- catalyzed activation of prothrombin proceeds through the intermediate meizothrombin in an ordered, sequential reaction. J Biol Chem 1986; 261: 8977-84.
    PubMedGoogle Scholar
  • 32 Rosing J, Zwaal R, Tans G. Formation of meizothrombin as intermediate in factor Xa-catalyzed prothrombin activation. J Biol Chem 1986; 261: 4224-8.
    PubMedGoogle Scholar
  • 33 Rosing J, Tans G. Meizothrombin, a major product of factor Xa-catalyzed prothrombin activation. Thromb Haemost 1988; 60: 355-60.
    Article in Thieme ConnectPubMedGoogle Scholar
  • 34 Boskovic D, Bajzar L, Nesheim M. Channeling during prothrombin activation. J Biol Chem 2001; 276: 28686-93.
    CrossrefPubMedGoogle Scholar