The technique of measuring thrombin generation with fluorescent substrates: 4. The H-transform, a mathematical procedure to obtain thrombin concentrations without external calibration
ABSTRACT In fluorogenic thrombin generation (TG) experiments, thrombin concentrations cannot be easily calculated from the rate of the fluorescent signal increase, because the calibration coefficient increases during the experiment, due to substrate consumption and quenching of the fluorescent signal by the product. Continuous, external calibration via an in a parallel sample therefore was hitherto required for an accurate calculation of the TG curve. A technique is presented that allows mathematical transformation of experimental fluorescence intensities into "ideal" data, i.e. in the data that would have been obtained if substrate consumption and quenching by the product would not play a role. The method applies to fluorescence intensities up to 90% of the maximal fluorescent signal corresponding to total substrate conversion and thereby covers the entire region of interest encountered in practice. The first derivative of the transformed signal can then be converted into thrombin concentrations via a conventional, fixed calibration factor. This calibration factor can be obtained from a separate experiment but also by measuring the amidolytic activity of the alpha(2)macroglobulin-thrombin complex present in the reaction mixture ("serum") after thrombin generation is over. This method halves the amount of sample required per experiment.
- American Journal of Clinical Pathology 01/2010; 133(1):163; author reply 164. DOI:10.1309/AJCPX63EFKZSYOQI · 3.01 Impact Factor
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ABSTRACT: Thrombin generation (TG) in plasma can be monitored continuously with a fluorogenic thrombin substrate using calibrated automated thrombinography (CAT). In the presence of low concentrations of a reversible direct thrombin inhibitor (DTI), CAT shows an unexpected effect: the endogenous thrombin potential (ETP) increases at low concentrations of the inhibitor to subsequently decrease concentration dependently at higher concentrations (> approximately 100 nm). To find an explanation for this phenomenon, we measured the concentrations of free thrombin and alpha(2)-macroglobulin-thrombin complex (alpha(2)MT) with a sub-sampling technique in the presence of AR-H067637, a selective DTI. At all concentrations of the DTI there was a gradual dose-dependent decrease in the concentration of free, not-inhibited thrombin but a transient increase in free alpha(2)MT due to competition of thrombin and alpha(2)MT for the inhibitor. Because the CAT technique uses an algorithm to subtract alpha(2)MT activity from the total amidolytic activity, this transient increase in alpha(2)MT activity is not subtracted and erroneously attributed to thrombin itself. This study explains the spurious increase in ETP observed at low DTI concentrations. The results obtained in plasma were corroborated by observations in a thrombin generating system reconstituted with purified factors. In practise, the effect of DTIs on TG can be reliably evaluated from the area under the curve till time-to-peak.Journal of Thrombosis and Haemostasis 02/2010; 8(6):1281-9. DOI:10.1111/j.1538-7836.2010.03822.x · 5.55 Impact Factor
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ABSTRACT: Calibrated automated thrombin generation assay was adapted to measure thrombin generation in platelet rich plasma from mice. Vena cava phlebotomy appeared the best technique for blood sampling. The concentration-effect curves of tissue factor and platelet count have been determined. Corn trypsin inhibitor 2μM inhibits contact activation effectively. Recombinant human thrombomodulin does not inhibit thrombin generation in mouse plasma but activated protein C (20nM) does. Thrombin generation was dose dependently diminished by low molecular weight heparin and increased by high concentrations of exogenous factor VIII i.e. the assay can detect both hypo- and hypercoagulability.Thrombosis Research 11/2010; 126(5):436-41. DOI:10.1016/j.thromres.2010.08.007 · 2.43 Impact Factor