A screening procedure to evaluate the anticoagulant activity and the kinetic behaviour of direct thrombin inhibitors

• Published in: Thrombosis research Vol. 78; no. 3; pp. 217 - 225
• Main Authors: Rupin, Alain, Mennecier, Philippe, de Nanteuil, Guillaume, Laubie, Michel, Verbeuren, Tony J.
• Format: Journal Article
• Language: English
• Published: New York, NY Elsevier Ltd 01.05.1995; Elsevier Science
• Subjects: Amino Acid Sequence; anticoagulant; Anticoagulants - pharmacology; Antithrombins - pharmacology; Binding Sites - drug effects; Biological and medical sciences; Blood Coagulation Tests; Blood. Blood coagulation. Reticuloendothelial system; Boron Compounds - pharmacology; Dipeptides - pharmacology; Drug Evaluation, Preclinical - methods; Fibrin - biosynthesis; Fibrinogen - metabolism; Hirudins - pharmacology; Humans; kinetic; Kinetics; Medical sciences; Microchemistry; Molecular Sequence Data; Oligopeptides - pharmacology; Peptide Fragments - pharmacology; Pharmacology. Drug treatments; Piperidines - pharmacology; Protein Structure, Tertiary; Recombinant Proteins - pharmacology; Thrombin - antagonists & inhibitors; Thrombin - chemistry; Thrombin inhibitor; Thrombin inhibitor; kinetic; anticoagulant; Human; Blood coagulation; Serine endopeptidases; Enzyme; Hydrolases; Thrombin; Anticoagulant; Kinetic parameter; Proteinases; Antithrombin; Biological activity
• ISSN: 0049-3848; 1879-2472
• DOI: 10.1016/0049-3848(95)00051-R

The development of a fibrin clot microassay to define both the kinetic behaviour and the anticoagulant activity of direct thrombin inhibitors targeting various domains of thrombin (catalytic site, anion binding exosite or both) is described. Since classical kinetics studies are difficult to perform in a fibrin-clot assay, methodological conditions were selected in order to obtain a linear relationship between fibrin formation and the thrombin concentration i.e. 0.67 nM thrombin, 6 μM fibrinogen, 5 minutes reaction. Under those conditions, the concentration of the complex thrombin-inhibitor can easily be calculated from a standard curve performed with increasing concentrations of thrombin and fitted versus the total inhibitor concentration using adapted equations. To detect the slow establishment of the thrombin inhibition, results obtained with a protocol in which the inhibitor is pre-incubated with thrombin before the addition of fibrinogen is compared to a protocol in which the inhibitor is pre-incubated with fibrinogen before thrombin is added. Our assay which is validated using different types of thrombin inhibitors (classical competitive: NAPA) and hirudin 55–65; tight binding: r-hirudin; slow tight binding: DUP-714), provides a rapid screening protocol allowing to evaluate the biochemical and anticoagulant properties of any direct thrombin inhibitor.