Improvement of the specificity of dipetarudin by site directed mutagenesis

Mercedes López; Katrin Mende; Goetz Nowak

doi:10.1160/TH04-08-0480

Thrombosis and Haemostasis, Table of Contents

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

¹Instituto Venezolano de Investigaciones Científicas, Laboratorio de Trombosis Experimental, Centro de Biofísica y Bioquímica, Caracas, Venezuela

,

Katrin Mende

²Friedrich Schiller University Jena, Medical Faculty, Research Unit “Pharmacological Haemostaseology“, Jena, Germany

,

Goetz Nowak

²Friedrich Schiller University Jena, Medical Faculty, Research Unit “Pharmacological Haemostaseology“, Jena, Germany

› Author Affiliations

Abstract

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

Full Text

References

References
1 Markwardt F. Die Isolierung und chemische Charakterisierung des Hirudins. Z Physiol Chem 1957; 312: 85-9.
2 Stone S, Hofsteenge J. Kinetics of the inhibition of thrombin by hirudin. Biochemistry 1986; 25: 4622-8.
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.
4 Dodt J, Koehler S, Baici A. Interaction of site specific hirudin variants with a-thrombin. FEBS Lett 1988; 229: 87-90.
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.
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.
7 Dodt J, Otte M, Strube K. et al. Thrombin inhibitors of bloodsucking animals. Semin Thromb Hemost 1996; 22: 203-8.
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.
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.
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.
11 Dang Q, Di Cera E. A simple activity assay for thrombin and hirudin. J Protein Chem 1994; 13: 367-73.
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.
13 Dixon M. Determination of enzyme inhibitor constants. Biochem J 1953; 55: 170-1.
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.
15 Nowak G, Bucha E. Quantitative determination of hirudin in blood and body fluids. Semin Thromb Hemost 1996; 22: 197-202.
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.
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.
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.
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.
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.
21 Stone S, Braun P, Hofsteenge J. Identification of regions of alpha-thrombin involved in its interaction with hirudin. Biochemistry 1987; 26: 4617-24.
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.
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.
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.
25 Nowak G. Clinical monitoring of hirudin and direct thrombin inhibitors. Semin Thromb Hemost 2001; 27: 537-42.
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.
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.
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.
29 Rydel TJ, Tulinsky A, Bode W. et al. Refined structure of the hirudin-thrombin complex. J Mol Biol 1991; 221: 583-601.
30 Bode W, Huber R. Natural protein proteinase inhibitors and their interaction with proteinases. Eur J Biochem 1992; 204: 433-51.
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.
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.
33 Rosing J, Tans G. Meizothrombin, a major product of factor Xa-catalyzed prothrombin activation. Thromb Haemost 1988; 60: 355-60.
34 Boskovic D, Bajzar L, Nesheim M. Channeling during prothrombin activation. J Biol Chem 2001; 276: 28686-93.