Chemical Synthesis and Expression of a Gene Coding for Hirudin, the Thrombin-Specific Inhibitor from the Leech<i>Hirudo medicinalis</i>

Bergmann, Cornelia C.; Dodt, Johannes; Köhler, Stefanie; Fink, E; Gassen, Hans Günter

doi:10.1515/bchm3.1986.367.2.731

Cited by 72 publications

(25 citation statements)

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“…Recombinant hirudin has been expressed previously in prokaryotes and lower eukaryotes, including E. coli, yeast and insect cells (using a baculovirus vector). [31][32][33] In each of these systems, hirudin secretion was directed by heterologous signal peptides already known to function efficiently in these species. Because our goal was to express hirudin in a mammalian cell and because there is evidence that use of host cell species-specific signal peptides can result in enhanced secretion and more reliable post-translational processing, 34,35 we initially used the human t-PA signal peptide to direct hirudin secretion from human endothelial cells.…”

Section: Discussionmentioning

confidence: 99%

Retroviral vector-mediated expression of hirudin by human vascular endothelial cells: implications for the design of retroviral vectors expressing biologically active proteins

et al. 1999

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We constructed a hirudin cDNA cassette, HV-1.1, that and mass spectroscopic analysis revealed the presence of encodes mature hirudin variant-1 fused to the signal pepan extra N-terminal serine residue, indicating aberrant tide of human tissue-type plasminogen activator (t-PA).cleavage of the t-PA signal peptide and likely accounting The cassette was subcloned into retroviral vectors and for the diminished activity. We therefore constructed a used to transduce human vascular endothelial cells in vitro.second cDNA cassette, HV-1.2, in which hirudin secretion Hirudin antigen and activity were measured by ELISA and was directed by the signal peptide of human growth horthrombin inhibition assays, respectively. Transduced cells mone. Hirudin expressed from the HV-1.2 cassette had a secreted up to 35 ± 2 ng/10 6 cells/24 h of biologically active specific activity of 13.5 ± 0.2 ATU/ g. Protein sequencing hirudin; expression was stable for at least 7 weeks.and mass spectroscopic analysis demonstrated proper Recombinant hirudin, expressed from the HV-1.1 cassette, cleavage of the growth hormone signal peptide. Thus, we had a specific activity of 7.1 ± 0.2 antithrombin units per achieved high level retrovirus-mediated secretion of biomicrogram (ATU/ g), compared with specific activities of logically active hirudin from endothelial cells in vitro. Use approximately 12 ATU/ g for both native leech hirudin and of these vectors may permit sustained local antagonism of recombinant hirudin produced in yeast. Protein sequencing thrombin activity in vivo.

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Section: Discussionmentioning

confidence: 99%

Retroviral vector-mediated expression of hirudin by human vascular endothelial cells: implications for the design of retroviral vectors expressing biologically active proteins

et al. 1999

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“…Concerning thrombin specificity we decided to generate hirudin mutants with single amino acid substitutions: in order to obtain definite results positions of basic amino acid residues should be replaced by apolar, uncharged and polar, negatively charged amino acid residues, respectively. According to the codons created by chemical gene synthesis [16] the AAA codons for Lys 27, Lys 36 and Lys 47 were changed to ATA for Ile and AGA for Glu; the CAG codon for His 51 was altered to CTC for Leu and GAC for Asp by the method of oligonucleotide-directed site-specific ,mutagenesis via the gapped-duplex (gd) DNA approach. 50-80°70 of examined clones showed the desired point mutation as could be verified by dot-blot hybridization of phages and dideoxy sequencing of positive clones.…”

Section: Construction and Expression Of Hirudin Variantsmentioning

confidence: 99%

Interaction of site specific hirudin variants with α‐thrombin

Dodt

Köhler

Baici³

1988

FEBS Letters

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105

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The kinetics of complex formation between recombinant hirudin or recombinant hirudin mutants with thrombin were analyzed. In order to elucidate the inhibitor's reactive site peptide bond predetermined amino acid substitutions were introduced at positions of basic amino acid residues by means of site-directed mutagenesis of a hirudin gene. In comparison to recombinant hirudin (Ki = 19 pM) only those mutant inhibitors which were modified at amino acid position Lys 47 showed a higher K~ value for their complexes with thrombin. The observed effects are mainly due to increased kofr rate constants.

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“…20 This problem was resolved when recombinant hirudin was developed in 1986. 2,4 Recombinant hirudin differs from native hirudin in that the Tyr residue at position 63 is not sulfated. Although this difference marginally reduces its affinity for thrombin, recombinant hirudin remains a potent and highly specific inhibitor of thrombin.…”

Section: Hirudinmentioning

confidence: 99%

“…1 This finding subsequently led to the development of hirudin, which was initially extracted from leeches and later obtained via rDNA technology. [2][3][4][5] The second observation, made in 1956 by Bettelheim, was that fibrinopeptide A, a small peptide released from fibrinogen by the action of thrombin, inhibited thrombin activity. 6 The third key event in the development of DTIs was the elucidation of the 3-dimensional structure of thrombin, first through molecular modeling and then by x-ray crystallographic analysis of thrombin and the hirudin/thrombin complex in 1989 and 1990, respectively.…”

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Translational Success Stories

Coppens

Eikelboom

Gustafsson

et al. 2012

Circulation Research

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ReviewTranslational Success Stories highlight how basic discoveries have led to clinical advances (such as the use of new drugs or diagnostic modalities in patients). This initiative reflects the renewed emphasis of our journal on translational research. It is hoped that these articles will stimulate efforts to translate basic insights into clinical practice. Translational Success Stories Development of Direct Thrombin InhibitorsMichiel Coppens, John W. Eikelboom, David Gustafsson, Jeffrey I. Weitz, Jack Hirsh Abstract: Anticoagulants are the cornerstone of therapy for conditions associated with arterial and venous thrombosis.Direct thrombin inhibitors (DTIs) are anticoagulants that bind to thrombin and block its enzymatic activity. The bivalent parenteral DTIs hirudin and bivalirudin were based on the observation that the salivary extracts of medicinal leeches prevented blood from clotting. Key events that facilitated the subsequent development of small molecule active site inhibitors, such as argatroban, were the observation that fibrinopeptide A had antithrombotic properties and determination of the crystal structure of thrombin. Hirudin and argatroban have found their niche for the treatment of patients with heparin-induced thrombocytopenia, whereas bivalirudin is approved as an alternative to heparin for patients undergoing percutaneous coronary intervention. The development of orally active direct thrombin inhibitors was challenging because of the need to convert water-soluble, poorly absorbable, active site inhibitors into fat-soluble prodrugs that were then transformed back to the active drug after intestinal absorption. Dabigatran etexilate was the first new oral anticoagulant to be approved for long-term anticoagulant treatment in 6 decades. This Review highlights the development of DTIs as a translational success story; an example in which the combination of scientific ingenuity, structure-based design, and rigorous clinical trials has created a new class of anticoagulants that has improved patient care. (Circ Res. 2012;111:920-929.) Key Words: thrombin Ⅲ anticoagulant Ⅲ drug development D irect thrombin inhibitors (DTIs) are anticoagulants that bind directly to thrombin and block its activity. Prior to their development, the injectable anticoagulants in clinical use were heparin and low-molecular-weight heparins (LMWHs). Both are classified as indirect inhibitors because they have no intrinsic activity; instead, they produce their anticoagulant effect by activating antithrombin. The only class of oral anticoagulants that was available at that time was the vitamin K antagonists (VKAs), such as warfarin.Several key events led to the development of DTIs. The first was the observation in 1884 that salivary secretions of the medicinal leech, Hirudo medicinalis, prevented blood from clotting. 1 This finding subsequently led to the development of hirudin, which was initially extracted from leeches and later obtained via rDNA technology. [2][3][4][5] The second observation, made in 1956 by Bettelheim, was th...

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Chemical Synthesis and Expression of a Gene Coding for Hirudin, the Thrombin-Specific Inhibitor from the LeechHirudo medicinalis

Cited by 72 publications

References 20 publications

Retroviral vector-mediated expression of hirudin by human vascular endothelial cells: implications for the design of retroviral vectors expressing biologically active proteins

Retroviral vector-mediated expression of hirudin by human vascular endothelial cells: implications for the design of retroviral vectors expressing biologically active proteins

Interaction of site specific hirudin variants with α‐thrombin

Translational Success Stories

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