Recombinant hirudin for unstable angina pectoris. A multicenter, randomized angiographic trial.

Topol, Eric J.; Fuster, Valentín; Harrington, Robert A.; Califf, Robert M.; Kleiman, Neal S.; Kereiakes, Dean J.; Cohen, Marc; Chapekis, Anthony T.; Gold, Herman K.; Tannenbaum, Mark

doi:10.1161/01.cir.89.4.1557

Cited by 164 publications

(41 citation statements)

References 29 publications

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“…Although differences in the promoters used in these two vector systems preclude firm conclusions, the superior performance of the adenoviral vector is likely due to the ability of this vector to transfer multiple transgene copies to target cells, 46 whereas our current protocols for retroviral gene transfer achieve delivery of only approximately one transgene copy per cell. 47 This limitation of retroviral gene transfer might be overcome by use of newer generation retroviral vectors, which can be concentrated to higher titers and can effect delivery of multiple transgene copies per cell (up to [10][11][12][13][14][15][16][17][18][19][20] even in the absence of antibiotic selection, (Ref. 48, and Dr D Ory, personal communication).…”

Section: Discussionmentioning

confidence: 99%

“…Although some shortterm benefits of hirudin infusion have been reported, systemic hirudin administration has failed to prevent restenosis and has been associated in some settings with unacceptable bleeding complications. [11][12][13][14] The hemorrhagic complications associated with systemic hirudin administration may preclude the use of doses that are sufficient to achieve local thrombin inhibition at sites of vascular disease.…”

Section: Introductionmentioning

confidence: 99%

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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%

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 makes an irreversible tight bond to thrombin. The complex formed by hirudin and thrombin is very close and extends to a large area around the active site (27). The last 10 amino acid residues of the C-terminal region (residues 55-65) react with the anionic site of thrombin, an important region for fibrinogen binding.…”

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confidence: 99%

Theromin, a Novel Leech Thrombin Inhibitor

Salzet¹,

Chopin²,

Baert³

et al. 2000

Journal of Biological Chemistry

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We purified the most potent thrombin inhibitor described to date from the rhynchobdellid leech Theromyzon tessulatum. Designated theromin, it was purified to apparent homogeneity by gel permeation and anion exchange chromatography followed by two reverse-phase steps of high performance liquid chromatography. The primary sequence of theromin (a homodimer of 67 amino acid residues including 16 cysteine residues) was determined by a combination of reduction and s-␤-pyridylethylation, Edman degradation, trypsin enzymatic digestion, and matrix-assisted laser desorption mass spectrometry measurement. Theromin exhibits no sequence homology with any other thrombin inhibitors. Furthermore, theromin significantly diminishes, in a dose-dependent manner, the level of human granulocyte and monocyte activation induced by lipopolysaccharides. In summary, this potent thrombin inhibitor promises to have high biomedical significance.

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“…2,3 Furthermore, thrombin inhibitors potentiate fibrinolysis induced by plasminogen activators. 4 Several animal experiments demonstrated that hirudin is more effective than heparin in preventing platelet-dependent arterial thrombosis, rethrombosis after reperfusion, and thrombus growth.…”

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confidence: 99%

Construction and Functional Evaluation of a Single-Chain Antibody Fusion Protein With Fibrin Targeting and Thrombin Inhibition After Activation by Factor Xa

et al. 2000

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Background-Recombinant technology was used to produce a new anticoagulant that is preferentially localized and active at the site of the clot. Methods and Results-The variable regions of the heavy and light chains of a fibrin-specific antibody were amplified by polymerase chain reaction (PCR) with hybridoma cDNA. To obtain a functional single-chain antibody (scFv), a linker region consisting of (Gly 4 Ser) 3 was introduced by overlap PCR. After the scFv clones were ligated with DNA encoding the pIII protein of the M13 phage, high-affinity clones were selected by 10 rounds of panning on the B␤15-22 peptide of fibrin (␤-peptide). Hirudin was genetically fused to the C-terminus of the variable region of the light chain. To release the functionally essential N-terminus of hirudin at the site of a blood clot, a factor Xa recognition site was introduced between scFv 59D8 and hirudin. The fusion protein was characterized by its size on SDS-PAGE (36 kDa), by Western blotting, by its cleavage into a 29-kDa (single chain alone) and 7-kDa (hirudin) fragment, by its binding to ␤-peptide, and by thrombin inhibition after Xa cleavage. Finally, the fusion protein inhibited appositional growth of whole blood clots in vitro more efficiently than native hirudin. Conclusions-A fusion protein was constructed that binds to a fibrin-specific epitope and inhibits thrombin after its activation by factor Xa. This recombinant anticoagulant effectively inhibits appositional clot growth in vitro. Its efficient and fast production at low cost should facilitate a large-scale evaluation to determine whether an effective localized antithrombin activity can be achieved without systemic bleeding complications.

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Recombinant hirudin for unstable angina pectoris. A multicenter, randomized angiographic trial.

Abstract: Recombinant hirudin appears to be a promising antithrombotic intervention compared with heparin for inhibition of coronary artery thrombus. Large-scale comparative trials are warranted.

Cited by 164 publications

References 29 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

Theromin, a Novel Leech Thrombin Inhibitor

Construction and Functional Evaluation of a Single-Chain Antibody Fusion Protein With Fibrin Targeting and Thrombin Inhibition After Activation by Factor Xa

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