Design and characterization of dipetacompinR10H, a dipetalogastin II-derived, classical competitive thrombin inhibitor

Thomas Bitter; Mercedes López; Goetz Nowak

doi:10.1160/TH06-06-0308

Thrombosis and Haemostasis, Table of Contents

Thromb Haemost 2007; 97(01): 139-145
DOI: 10.1160/TH06-06-0308

New Technologies, Diagnostic Tools and Drugs

Schattauer GmbH

Design and characterization of dipetacompinR10H, a dipetalogastin II-derived, classical competitive thrombin inhibitor

Thomas Bitter

¹Research Unit Pharmacological Haemostaseology, Medical Faculty, Friedrich-Schiller-University, Jena, Germany

,

Mercedes López

²Instituto Venezolano de Investigaciones Científicas, Laboratorio de Trombosis Experimental, Centro de Biofísica y Bioquímica, Caracas, Venezuela

,

Goetz Nowak

¹Research Unit Pharmacological Haemostaseology, Medical Faculty, Friedrich-Schiller-University, Jena, Germany

› Author Affiliations

Abstract

Summary

The design of small chimeric thrombin inhibitors based on the structure of dipetalogastin II has been previously described. These proteins are effective inhibitors of thrombin showing slow binding or slow, tight-binding kinetics. We report here about dipetacompinR10H, a new dipetalogastin II-derived chimeric thrombin inhibitor, which exhibits classical competitive kinetics. The dissociation constant K_i of dipetacompinR10H was determined to be 17.1 ± 0.8 pM. In various coagulation assays it showed a comparable anticoagulant activity like r-hirudin and r-dipetalogastin II. DipetacompinR10H’s inhibition of thrombin was specific, since no inhibition of other serine proteases like factor Xa, plasmin, trypsin or chymotrypsin has been observed.

Keywords

Thrombin inhibitor - Dipetalogastin - rational drug design - chimeric protein - classical competitive kinetics

Full Text

References

References
1 Coughlin SR. Thrombin signalling and proteaseactivated receptors. Nature 2000; 407: 258-64.
2 Huntington JA, Baglin TP. Targeting thrombin--rational drug design from natural mechanisms. Trends Pharmacol Sci 2003; 24: 589-95.
3 Davie EW, Fujikawa K, Kisiel W. The coagulation cascade: initiation, maintenance, and regulation. Biochemistry 1991; 30: 10363-70.
4 Kahn ML, Zheng YW, Huang W. et al A dual thrombin receptor system for platelet activation. Nature 1998; 394: 690-4.
5 Bode W, Turk D, Karshikov A. The refined 1.9-A X-ray crystal structure of D-Phe-Pro-Arg chloromethylketone-inhibited human alpha-thrombin: structure analysis, overall structure, electrostatic properties, detailed active-site geometry, and structure-function relationships. Protein Sci 1992; 1: 426-71.
6 Nutescu EA, Wittkowsky AK. Direct thrombin inhibitors for anticoagulation. Ann Pharmacother 2004; 38: 99-109.
7 Badimon L, Meyer BJ, Badimon JJ. Thrombin in arterial thrombosis. Haemostasis 1994; 24: 69-80.
8 Markwardt F. Die Isolierung und chemische Charakterisierung des Hirudins. Hoppe Seylers Z Physiol Chem 1957; 308: 147-56.
9 Stone SR, Hofsteenge J. Kinetics of the inhibition of thrombin by hirudin. Biochemistry 1986; 25: 4622-8.
10 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.
11 Braun PJ, Dennis S, Hofsteenge J. et al Use of sitedirected mutagenesis to investigate the basis for the specificity of hirudin. Biochemistry 1988; 27: 6517-22.
12 Lange U, Keilholz W, Schaub GA. et al Biochemical characterization of a thrombin inhibitor from the bloodsucking bug Dipetalogaster maximus. Haemostasis 1999; 29: 204-11.
13 Mende K, Petoukhova O, Koulitchkova V. et al Dipetalogastin, a potent thrombin inhibitor from the blood-sucking insect Dipetalogaster maximus. cDNA cloning, expression and characterization. Eur J Biochem 1999; 266: 583-90.
14 Lopez ML, Mende K, Steinmetzer T. et al Cloning, purification and biochemical characterization of dipetarudin, a new chimeric thrombin inhibitor. J Chromatogr B 2003; 786: 73-80.
15 Lopez M, Mende K, Nowak G. Improvement of the specificity of dipetarudin by site directed mutagenesis. Thromb Haemost 2005; 93: 430-6.
16 Nowak G, Bucha E. Quantitative determination of hirudin in blood and body fluids. Semin Thromb Hemost 1996; 22: 197-202.
17 Lineweaver H, Burk D. The determination of enzyme dissociation constants. J Am Chem Soc 1934; 56: 658-66.
18 Dixon M. The determination of enzyme inhibitor constants. Biochem J 1953; 55: 170-1.
19 Nutescu EA, Shapiro NL, Chevalier A. New antico-agulant agents: direct thrombin inhibitors. Clin Geriatr Med 2006; 22: 33-56.
20 Kaplan KL. Direct thrombin inhibitors. Expert Opin Pharmacother 2003; 4: 653-66.
21 Even-Ram SC, Maoz M, Pokroy E. et al Tumor cell invasion is promoted by activation of protease activated receptor-1 in cooperation with the alpha vbeta 5 integrin. J Biol Chem 2001; 276: 10952-62.
22 Karpatkin S. Role of thrombin in tumor angiogenesis, implantation, and metastasis. Pathophysiol Haemost Thromb 2003; 33 (Suppl. 01) 54-5.
23 Nierodzik ML, Chen K, Takeshita K. et al Protease-activated receptor 1 (PAR-1) is required and ratelimiting for thrombin-enhanced experimental pulmonary metastasis. Blood 1998; 92: 3694-700.
24 Lopez ML, Nowak G. Special pharmacokinetics of dipetarudin suggests a potential antitumor activity of this thrombin inhibitor. Anticancer Drugs 2004; 15: 145-9.
25 Schafberg H, Nowak G, Kaufmann R. Thrombin has a bimodal effect on glioma cell growth. Br J Cancer 1997; 76: 1592-5.
26 Maraganore JM, Bourdon P, Jablonski J. et al Design and characterization of hirulogs: a novel class of bivalent peptide inhibitors of thrombin. Biochemistry 1990; 29: 7095-101.
27 Hu L, Lee M, Campbell W. et al Role of endogenous thrombin in tumor implantation, seeding, and spontaneous metastasis. Blood 2004; 104: 2746-51.
28 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: 2373-8.