Cloning and Expression in <i>Escherichia coli</i> of a Synthetic DNA for Hirudin, the Blood Coagulation Inhibitor in the Leech

Fortkamp, E.; Rieger, Michael A.; Heisterberg-Moutses, Gudrun; Schweitzer, Silvia; Sommer, Reinhold

doi:10.1089/dna.1.1986.5.511

Cited by 70 publications

(35 citation statements)

References 25 publications

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“…Substances r-Hirudin, prepared according to Fortkamp et al [14] by Genbiotec, Heidelberg, FRG; specific activity 10 AT-U/pg); bovine thrombin (specific activity 13 NIH-U/pg); heparin (Richter, Budapest, Hungary, specific activity 130 IU/mg); ADP (Reanal, Budapest, Hungary); adrenaline (VEB Jenapharm, Jena, GDR); collagen ('Horm', Hormonchemie, Munich, FRG); l4C-serotonin (5-hydroxy [side chain-2 l4C]-tryptamine creatinine sulphate, specific activity 2.15 GBq/ mmol, Radiochemical Centre, Amersham, England); PAF (semisynthetic PAF acether, l-O-alkyl-2-O-acetyl-sn-glycero-3-phosphocholine, was a generous gift from Prof. Mangold, Münster, FRG).…”

Section: Methodsmentioning

confidence: 99%

Platelet Functions in Recombinant Hirudin-Anticoagulated Blood

Glusa¹,

Markwardt²

1990

Pathophysiol Haemos Thromb

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The influence of genetically engineered recombinant hirudin (r-hirudin) on platelet functions was studied. Depending on the concentration, r-hirudin inhibits the thrombin-induced aggregation and ¹⁴C-serotonin secretion to the same extent as native hirudin. Comparative studies in blood anticoagulated by r-hirudin, heparin or citrate show a significantly lower spontaneous platelet aggregation in r-hirudinized blood. The extent of the ADP-induced aggregation is nearly the same in r-hirudinized, heparinized or citrated plasma. In r-hirudinized plasma, however, aggregation is reversible. In contrast to heparinized or citrated plasma, adrenaline causes only a very slight aggregation in r-hirudinized plasma. ADP- or adrenaline-induced secretion of ¹⁴C-serotonin do not occur in r-hirudinized plasma. The collagen- as well as the PAF-induced aggregation and ¹⁴C-serotonin release in hirudinized plasma do not differ significantly from those in heparinized or citrated plasma. r-Hirudin is a suitable anticoagulant for studying platelet functions because it does not produce any alterations in platelet reactions and does not provoke any changes in the ionized calcium concentration in blood.

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Section: Methodsmentioning

confidence: 99%

Platelet Functions in Recombinant Hirudin-Anticoagulated Blood

Glusa¹,

Markwardt²

1990

Pathophysiol Haemos Thromb

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“…Cloning and sequencing of the hirudin gene [58] allowed large-scale production of recombinant hirudin, e.g. in Escherichia coli [59] and Saccharomyces cerevisiae [60]. Whereas natural hirudins are single-chain polypeptides of approximately 65-66 amino acids containing a sulfated tyrosine, recombinant forms lack the sulfate group on tyrosine 63 [61], and therefore bind less tightly to thrombin.…”

Section: Early Direct Thrombin Inhibitorsmentioning

confidence: 99%

Direct thrombin inhibitors – a survey of recent developments

Schwienhorst¹

2006

Cell. Mol. Life Sci.

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Thrombin is a plasma serine protease that plays a key role in coagulation and hemostasis but also in thromboembolic diseases. Direct thrombin inhibitors could, therefore, be beneficial for future anticoagulant therapy in the prophylaxis of venous and arterial thrombosis as well as myocardial infarction. However, development of direct thrombin inhibitors has brought researchers more heartache than success. The most recent setback came this year when AstraZeneca withdrew Ximelagatran, the first orally bioavailable direct thrombin inhibitor that had received regulatory approval (France, 2003), after reports of serious hepatoxicity in a fraction of patients. This review describes the status of direct thrombin inhibitors, focusing on drug candidates that are at present in clinical trials. In addition, some more recent research strategies in the design of novel direct thrombin inhibitors are discussed, which may very well contribute to future developments of potent anticoagulants.

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“…These recombinant inhibitors showed not only pharmacological properties very similar to those of native hirudin but also an out standing potency as antithrombotic agents that are well tolerated in vivo [5,6], Based on our experience gained in preclinical tests [7][8][9] and after the pharmacotoxicological analysis of r-hirudin [10] (ob tained from Animal Pharma, Erlangen FRG), with the amino acid sequence of na tive hirudin described by Dodt et al [ 11 ] and expressed as a fusion protein in Escheri chia coli by Genbiotec Heidelberg [12], we used this preparation in the present report on clinico-pharmacological investigations.…”

Section: Introductionmentioning

confidence: 99%

Clinical Pharmacology of Recombinant Hirudin

Markwardt¹,

Nowak²,

Stürzebecher³

1991

Pathophysiol Haemos Thromb

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Pharmacological profiling of recombinant hirudin (r-hirudin) has shown that this selective tight-binding thrombin inhibitor is a potent, well-tolerated anticoagulant. Clinical pharmacological studies were performed in human volunteers after single and repeated doses of 0.1–0.5 mg/kg. Generally, administration of r-hirudin was tolerated without side effects. Thrombin time and partial thromboplastin time were prolonged dependent on the r-hirudin level in plasma. Platelet counts, fibrinogen level and fibrinolytic system remained unchanged. Bleeding time was not prolonged. On intravenous injection, r-hirudin was rapidly distributed into the extracellular space and eliminated, with a dose-dependent half-life of 1–2 h (first-order kinetics). After subcutaneous administration, the rH level in blood reached plateau values within 60–120 min. The high recovery of unchanged r-hirudin in the urine identified renal excretion as the predominant route of r-hirudin clearance.

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Cloning and Expression in Escherichia coli of a Synthetic DNA for Hirudin, the Blood Coagulation Inhibitor in the Leech

Cited by 70 publications

References 25 publications

Platelet Functions in Recombinant Hirudin-Anticoagulated Blood

Platelet Functions in Recombinant Hirudin-Anticoagulated Blood

Direct thrombin inhibitors – a survey of recent developments

Clinical Pharmacology of Recombinant Hirudin

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