Fig. 5

An attempt to study substrate consumption in plasma samples supplemented with two substrates, ZGGR-AMC and ZGGR-AFC. FVIII-DP was supplemented with 1 IU/mL FVIII to normalize hemophilic plasma, or not, and was subsequently premixed with the indicated concentrations of two substrates, ZGGR-AMC and ZGGR-AFC, such that the ratio of AMC:AFC equaled to a concentration of 800 µM, prior to initiating coagulation with Ca²⁺. Raw data was produced by the Biotek microplate reader and analyzed in several different ways: (A, B) raw AMC fluorescence in relative fluorescent units (RFU), (C, D) internally calibrated TG curves via a thrombin calibration coefficient (see Materials and Methods), (E, F) Normalized-Uncalibrated curves, (G, H) Calibrated TG curves (via OR software), and (I, J) calibrated TG curves (via SH software). Uncalibrated curve data were produced by differentiating the AMC curves observed in (A, B). CAT calibrated curves were produced using our in-house OR and SH software apps, which use published algorithms similar to CAT calibration. Normalized-uncalibrated curves were produced by normalizing each uncalibrated curve pairing of hemophilic and normalized sample (hemophilic plasma supplemented with FVIII) at each pre-spiked AMC concentration against the TPH value of the normalized plasma sample in each pairing. TG was recorded for 40–60 min. An artifact resembling TG signal in early minutes in Fig. 5 is only seen with ZGGR-AFC experiments, suggesting that it is caused by either the AFC fluorophore itself (similar to Fig. 2 and Fig. S1 discussed above) or background fluorescence signal of un-cleaved ZGG-AFC substrate. Assay conditions: 78 µL of FVIII-DP, 2 µL of FVIII (1 IU/mL), 20 µL of PPP trigger, and 20 µL of custom FluCa mixtures (substrates ZGGR-AMC and ZGGR-AFC at indicated concentrations with calcium chloride buffer)