[69] Hirudin as an inhibitor of thrombin

Markwardt, Fritz

doi:10.1016/0076-6879(70)19082-3

Cited by 341 publications

(198 citation statements)

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“…[2][3][4][5] Hirudin, a 65-amino acid protein secreted by the salivary glands of the medicinal leech, is a potent and specific inhibitor of thrombin. 6 By blocking both the catalytic site and the anion-binding exosite of thrombin, hirudin inhibits the interaction of thrombin both with fibrinogen and with thrombin receptors that are present on the surface of platelets and vascular cells. Recent studies, perfor- med in several animal model systems, have demonstrated that systemically administered hirudin can prevent thrombosis and neointimal proliferation after arterial injury.…”

Section: Introductionmentioning

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: Introductionmentioning

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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“…Hirudin is a thrombin-specific inhibitor originating from leech Hirudo medicinalis (Markwardt, 1990). The inhibitor contains two functional domains (Rydel et al, 1990).…”

Section: Introductionmentioning

confidence: 99%

Controlling the speed of hirudin folding

Chang

1994

Biochemical Journal

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The folding of hirudin undergoes an initial stage of non-specific packing, followed by consolidation (re-organization) of partially packed intermediates to attain the native structure [Chatrenet and Chang (1993) J. Biol. Chem. 268, 20988-20996]. Non-specific packing leads to the formation of scrambled hirudins as folding intermediates. A systematic study was carried out to search for conditions which would selectively control and enhance the processes of packing and consolidation. It is demonstrated here that under optimized conditions, including the use of cystine/cysteine and protein disulphide isomerase, the folding of hirudin in vitro can be achieved quantitatively within 30 s.

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“…Although cu.thrombin is highly specific toward fibrinogen as its natural substrate, its szruccurai homology to other serine proteases may preclude the development of specific and effective active-site directed inhibitors which could be useful to treat thrombotic disorders, Indeed a large number of synthetic or substratemrelated inhibitors have been reported [l], but most of them suffer from vaeious drawbacks such as low potency, poor selectivity or potential toxicity. The most potent inhibitor of Mthrombin known is hirudin [2,3], a '7 kDa family of isoproteins isolated from the glandular extracts of the leech Wirudo medicinalis. I-Iirudin forms a high affinity non-covalent stoichiometric complex with a-thrombin (Ki = 10'" to lo-*' M), but does not inhibit trypsin, kaltikrein or other hcmocoagulant swine proteases (41.…”

Section: Introductio'nmentioning

confidence: 99%

A new class of potent thrombin inhibitors that incorporates a scissile pseudopeptide bond

DiMaio

Gibbs

et al. 1991

FEBS Letters

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INTRODUCTIO'NThrombin is the principal enzyme that acts in concert with other activated factors of the hemocoagulative system to regulate clot formation in response to vascular tissue clamage. Although cu.thrombin is highly specific toward fibrinogen as its natural substrate, its szruccurai homology to other serine proteases may preclude the development of specific and effective active-site directed inhibitors which could be useful to treat thrombotic disorders, Indeed a large number of synthetic or substratemrelated inhibitors have been reported [l], but most of them suffer from vaeious drawbacks such as low potency, poor selectivity or potential toxicity. The most potent inhibitor of Mthrombin known is hirudin [2,3], a '7 kDa family of isoproteins isolated from the glandular extracts of the leech Wirudo medicinalis. I-Iirudin forms a high affinity non-covalent stoichiometric complex with a-thrombin (Ki = 10'" to lo-*' M), but does not inhibit trypsin, kaltikrein or other hcmocoagulant swine proteases (41. It is now known that the high affinity is due to the multiple interactions of hirudin with cu-thrombin while the specificity stems from the interaction with at least one complementary binding site that is unique to CYthrombin [5,6]. This putative 'anion' exosite may also be required for fibrinogen binding. We have previously demonstrated that desulfohirudin'5'65 corresponding to the carboxyl termincrl fragment of recombinant hirudin variant 1 (r-HVl) can inhibit the amidolytic activity of human cr-thrombin towards synthetic substrates [7]. Although the inhibition by this native fragment was weak and the mechanism partially competitive compared to rhirudin, a modification of the NHZ-terminal residues by substituting D-Phe and Arg in positions 45 and 47 respectively, afforded an inhibitor with a Ki = 2.8 ZL 0.9 nM against human a-thrombin. The enhancement of enzyme affinity compared to the corresponding native fragment was attributed to favored interaction with the S1 and S3 subsites of the catalytic center while the G-terminal region comprising residues 55-65 could serve as an anchor that binds to the putative 'anion' exosite.tlbbreviu&ms: NOE, nuclear Qvsrhaussr enhancement; Boc-AOGO, SN-ten-butyloxycarbonyl-4-oxo-8.tosyl guanidinooctanoic acid; TFA, trifluoroacetic acid; AMC, 'I-amino-l-methyl coumarin; APTT, activated partial thromboplastin time In this communication, we report that this model was used to obtain a new class of reversible inhibitors prototyped by P49 (Pig. 1) with high enzyme affinity and whose in vitro plasma antithrombin activity approaches the level of r-hirudin despite the drastically reduced Published by Elsevier Science Publishers B. V.

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[69] Hirudin as an inhibitor of thrombin

Cited by 341 publications

References 11 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

Controlling the speed of hirudin folding

A new class of potent thrombin inhibitors that incorporates a scissile pseudopeptide bond

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