Background: The improvement of the thrombin generation (TG) assay, since its development in the 1950s, have highlighted its potential to assess the hemostatic capacity in a variety of plasma samples.  Indeed, the use of current fluorogenic thrombin substrates, such as Z-Gly-Gly-Arg 7-amino-4-methylcoumarin (ZGGR-AMC), have alleviated the turbidity issues encountered by its chromogenic predecessor, allowing for larger sample variety, such as platelet rich plasma, for testing. The commercial system, Calibrated Automated Thrombinography (CAT; Stago Diagnostics) performs measurements at an emission of 390 nm with an excitation of 460 nm. Several reports have demonstrated the measurement of TG curves using additional substrates and emission/excitation spectra.

Aims: We sought to investigate the optimal spectra parameters for TG measurement, and the natural fluorescence properties of the thrombin substrate, ZGGR-AMC.

Methods: ZGGR-AMC and free AMC fluorescence was measured in a range of excitations and emission of 450 nm.

Results: The AMC fluorophore has the highest fluorescence at an excitation of 340 nm and emission of ~450 nm. The thrombin substrate, ZGGR-AMC, possesses fluorescence properties, albeit shifted to the left of the excitation and emission spectra of free AMC molecule. The fluorescence of ZGGR-AMC interferes with the maximal AMC fluorescence when excited at 340 nm and emitted at 450 nm, which is completely abrogated if the excitation is set at 380 nm and emission at ~450nm.  

Conclusions: These data provide evidence and corroborates the measurements of TG at an excitation ~ 380 nm, but not lower than 360 nm, providing the maximum AMC fluorescence for the assay readout while effectively denying the contamination of ZGGR-AMC substrate fluorescence. Measurements for TG performed at the optimal wavelength for AMC fluorescence (i.e., 340 nm) runs the risk of detecting an overlapping signal from the thrombin substrate, potentially providing inaccurate results.

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ISTH Congress Abstracts - https://abstracts.isth.org/abstract/natural-fluorescence-of-thrombin-substrate-z-gly-gly-arg-7-amino-4-methylcoumarin-considerations-for-wavelength-choice/