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.

Prediction of bleeding risk in patients taking vitamin K antagonists using thrombin generation testing

“…The calibrator, α2-macroglobulin-thrombin complex, was prepared as described previously [15]. Hepes buffers containing 5 mg/ml or 60 mg/ml bovine serum albumin were prepared as described by Hemker et al [16]. …”

The paradoxical stimulation by a reversible thrombin inhibitor of thrombin generation in plasma measured with thrombinography is caused by α2‐macroglobulin‐thrombin

“…For this the H-transform was used [21]. The initial slope of the calibrator curve was used to convert F ideal /dt into enzymatic units (nMAEmin).…”

Actions of thrombin in the interstitium

“…3 Direct activation of prothrombin by fibrinogen-like protein 2 (Fgl2)/fibroleukin may also occur [36,37]. The presence of leukocytes of various types in the interstitium in nearly every type of pathology [38][39][40][41] suggests that the cellular Fgl2 pathway could be important in interstitial thrombin generation. Fgl2 activates prothrombin in a manner similar to FXa, in that optimal activity requires calcium ions, FVa, and phospholipid vesicles or platelets [36].…”