Large scale preparation of hirudin

Bagdy, D; Barabás, Éva; Gráf, László

doi:10.1016/0049-3848(73)90086-8

Cited by 27 publications

(9 citation statements)

References 15 publications

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“…9 The pure substance obtained in this way is a one-chain carbohydrate-free polypeptide containing three intramolecular disulfide bridges and a sulfated tyrosine residue. [10][11][12][13][14] Preliminary establishment of the primary structure showed that the polypeptide chain contains 65 amino acids from which a molecular weight of about 7000 was calculated. 4,15,16 The amino acid composition of hirudin is characterized by a remarkably high content of dicarboxylic amino acids as well as by the absence of arginine, methionine, and tryptophan.…”

Section: Biochemistrymentioning

confidence: 99%

Development of Hirudin as an Antithrombotic Agent

Markwardt¹

1989

Semin Thromb Hemost

122

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Within the framework of ours studies on blood clotting inhibitors from blood sucking animals, 1 we succeeded in isolating the anticoagulant agent from medicinal leeches (Hirudo medicinalis) in the late 1950s. [2][3][4] It was characterized as a specific thrombin inhibitor with polypeptide structure and was named hirudin. 5,6 Since our successful isolation and analysis of the naturally occurring thrombin inhibitor, hirudin preparations have been employed for diagnostic and scientific use in hemostaseology. 7,8 Pharmacologic studies have shown that due to its pronounced and specific anticoagulant effect, hirudin is an antithrombotic agent of high quality. 9 But the clinical use of hirudin has remained limited, since this substance is not available in adequate amounts for therapeutic purposes.Today, there is renewed interest in the naturally occurring thrombin inhibitor. Advanced methods of peptide isolation and genetic engineering are now able to provide sufficient quantities of the anticoagulant as a recombinant-type hirudin. The latter is currently being profiled in clinical settings to evaluate its efficacy. This development prompted us to resume our investigation of native hirudin and to present new experimental and clinical pharmacologic studies on recombinant hirudin, revealing the true potential of this anticoagulant agent. NATIVE HIRUDINRecombinant hirudin was developed on the basis of our knowledge of native hirudin. The biochemical and pharmacologic characteristics of native hirudin are outlined below. BiochemistryHirudin is produced by the peripharyngeal glands of medicinal leeches. The substance is extracted from the homogenized head parts of the animal and enriched by precipitation procedures followed by ion exchange chromatography and gel filtration. Recently, affinity chromatography on matrix-bound thrombin was used to obtain highly purified hirudin. 9 The pure substance obtained in this way is a one-chain carbohydrate-free polypeptide containing three intramolecular disulfide bridges and a sulfated tyrosine residue. 10-14 Preliminary establishment of the primary structure showed that the polypeptide chain contains 65 amino acids from which a molecular weight of about 7000 was calculated. 4,15,16 The amino acid composition of hirudin is characterized by a remarkably high content of dicarboxylic amino acids as well as by the absence of arginine, methionine, and tryptophan. In studies of the amino acid sequence of highly purified hirudin preparations, variants (isoinhibitor forms) were found with different N-terminal amino acids (Val-Val or Ile-Thr-hirudin) and certain differences in the amino acid sequence 16-21 (Fig. 1). Conflicting reports on the amino acid composition, which were reviewed by us, 8 can now be explained as being due to isolation of multiple forms of hirudin. The analysis of the anticoagulant effect

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Section: Biochemistrymentioning

confidence: 99%

Development of Hirudin as an Antithrombotic Agent

Markwardt¹

1989

Semin Thromb Hemost

122

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“…An early update was given in 1970. [10][11][12][13][14][15][16] In the time that followed, the secondary and tertiary structures of hirudin and of isoinhibitors were established. [17][18][19][20][21] Hirudin has an unusual asymmetry of structural elements.…”

Section: Isolation and Biochemical Characterizationmentioning

confidence: 99%

Hirudin As Alternative Anticoagulant- A Historical Review

Markwardt¹

2002

Semin Thromb Hemost

105

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Advances in separation techniques and biotechnology have contributed to the development of anticoagulant agents from hematophagous animals. The most potent known natural thrombin inhibitor from blood-sucking leeches ( Hirudo medicinalis), hirudin has served as a standard for designing the natural coagulation inhibitors as an anticoagulant drug. The search for the development of hirudin from leech extract to genetically engineered products as an alternative anticoagulant has been resumed. The pharmacological profiling of the isolated thrombin inhibitor has shown that native hirudin is an antithrombotic agent of high quality. However, its clinical use has remained limited, because the substance has not been available in adequate amounts. The progress in molecular biology has stimulated the interest in the structure and function of hirudin. This development resulted in the production of recombinant hirudins (r-hirudins) through gene technology. The biological properties of hirudin combined with the ready availability of recombinant forms make the specific thrombin inhibitor well-suited for use as an antithrombotic drug. Its use should lead to a decisive progress in the management of thromboembolic diseases of both arterial and venous origin. Clinical trials, especially in diseases in which thrombin plays a crucial role, are in progress.

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“…6 In the subsequent years, several purification procedures were developed on the basis of chromatographic methods. [8][9][10][11] By gel filtration, the bulk of high and low molecular weight contamination could be separated from the thrombin inhibitor. 9 At neutral pH, hirudin was assumed to be eluted as a dimer, at pH 3.0, as a monomer.…”

Section: Isolation Proceduresmentioning

confidence: 99%

Isolation and Characterization of Hirudin fromHirudo medicinalis

Walsmann

1991

Semin Thromb Hemost

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Large scale preparation of hirudin

Cited by 27 publications

References 15 publications

Development of Hirudin as an Antithrombotic Agent

Development of Hirudin as an Antithrombotic Agent

Hirudin As Alternative Anticoagulant- A Historical Review

Isolation and Characterization of Hirudin fromHirudo medicinalis

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