Mutational analysis of antistasin, an inhibitor of blood coagulation factor xa derived from the mexican leech Haementeria officinalis

Theunissen, Henri J.M.; Dijkema, R.; Swinkels, Joop C.; Poorter, T. L. De; Vink, Paul; Dinther, Theo G. van

doi:10.1016/0049-3848(94)90138-4

Cited by 20 publications

(25 citation statements)

References 22 publications

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“…contains a sequence that is homologous to the reactive site in domain I, as shown in Figure 7 (Dunwiddie et al, 1992). In antistasin domain 11. changing Lys 90(P;) into Val(P; in antistasin domain I ) does not result in the generation of a second reactive site (Theunissen et al, 1994). By analogy with antistasin, the following hirustasin residues comprise the putative reactive site loop: Glu 26(P5).…”

Section: Discussionmentioning

confidence: 99%

Recombinant hirustasin: Production in yeast, crystallization, and interaction with serine proteases

et al. 1997

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A synthetic gene coding for the 55-amino acid protein hirustasin, a novel tissue kallikrein inhibitor from the leech Hirudo medicinalis, was generated by polymerase chain reaction using overlapping oligonucleotides, fused to the yeast a-factor leader sequence and expressed in Saccharomyces cerevisiae. Recombinant hirustasin was secreted mainly as incompletely processed fusion protein, but could be processed in vitro using a soluble variant of the yeast yscF protease. The processed hirustasin was purified to better than 97% purity. N-terminal sequence analysis and electrospray ionization mass spectrometry confirmed a correctly processed N-terminus and the expected amino acid sequence and molecular mass. The biological activity of recombinant hirustasin was identical to that of the authentic leech protein.Crystallized hirustasin alone and in complex with tissue kallikrein diffracted beyond 1.4 A and 2.4 A, respectively. In order to define the reactive site of the inhibitor, the interaction of hirustasin with kallikrein, chymotrypsin, and trypsin was investigated by monitoring complex formation in solution as well as proteolytic cleavage of the inhibitor. During incubation with high, nearly equimolar concentration of tissue kallikrein, hirustasin was cleaved mainly at the peptide bond between Arg 30 and Ile 3 1, the putative reactive site, to yield a modified inhibitor. In the corresponding complex with chymotrypsin, mainly uncleaved hirustasin was found and cleaved hirustasin species accumulated only slowly. Incubation with trypsin led to several proteolytic cleavages in hirustasin with the primary scissile peptide bond located between Arg 30 and Ile 3 1. Hirustasin appears to fall into the class of protease inhibitors displaying temporary inhibition.

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Section: Discussionmentioning

confidence: 99%

Recombinant hirustasin: Production in yeast, crystallization, and interaction with serine proteases

et al. 1997

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“…This protein is a potent inhibitor of coagulation factor Xa (FXa) and was also found to exert an antimetastatic effect in animal tumor models [36,37]. An FXa inhibitor was also identified in the saliva of H. medicinalis.…”

Section: Bioactive Compounds In Leech Salivamentioning

confidence: 98%

An overview of leech and its therapeutic applications

2015

J Coast Life Med

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“…Antistasin is a protein consisting of 119 amino acid residues distributed in three distinct domains [I, II, III] [21,22]. Antistasin is a competitive, slow-tight binding inhibitor of FXa with a long half-life and superior efficacy to heparin in different animal thrombosis models.…”

Section: Antistasinmentioning

confidence: 99%

Heterogeneity of Synthetic Factor Xa Inhibitors

Gerotziafas

Samama²

2005

CPD

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Heparins and vitamin K antagonists are the landmarks of antithrombotic treatment. Both of them were discovered by serendipity; they are multi-targeted drugs and share several limitations. New molecules have been designed in order to be both more selective concerning their biological target and more homogeneous in their biochemical structure aiming at an improved benefit/risk ratio in the treatment of thrombotic disease. In this article, we will review the pharmacological characteristics of the new synthetic direct or antithrombin dependent inhibitors of FXa in the light of the modern concept of blood coagulation process. We will also present the most recent data from the clinical trials with synthetic inhibitors of FXa. Among them, the synthetic pentasaccharide fondaparinux is the first synthetic and specific FXa inhibitor, which has been approved by health authorities in Europe and in the USA for the prophylaxis of venous thromboembolism in major orthopaedic surgery and is being approved for the treatment of pulmonary embolism and DVT as a single daily subcutaneous injection. The phase II dose-finding trial of the "meta-pentasaccharide" idraparinux administered subcutaneously once weekly in the secondary prevention of VTE has been completed. DX-9065a is the first direct synthetic inhibitor which has been studied in patients with coronary disease. Razaxaban, BAY59-7939, ZK-807834 and JTV-803 are orally active direct FXa inhibitors, which have been studied in phase II trials. Several other synthetic direct inhibitors of FXa (such as FXV673, YM60828, KFA-1411) are in a pre-clinical stage of research. From a clinical point of view, the results of recent trials with the synthetic specific FXa inhibitors clearly show that the inhibition of FXa is a critical point in the antithrombotic strategy.

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Mutational analysis of antistasin, an inhibitor of blood coagulation factor xa derived from the mexican leech Haementeria officinalis

Cited by 20 publications

References 22 publications

Recombinant hirustasin: Production in yeast, crystallization, and interaction with serine proteases

Recombinant hirustasin: Production in yeast, crystallization, and interaction with serine proteases

An overview of leech and its therapeutic applications

Heterogeneity of Synthetic Factor Xa Inhibitors

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