Abstract

Introduction Recently, the whole blood calibrated automated thrombogram (CAT) was miniaturized enabling the measurement of thrombin generation (TG) in a limited volume (5 µl) of whole blood. Consequently, this approach may be used to determine TG in small lab animals. Zebrafish are readily available test animals for genetic research. However, their small size has been a hurdle in thrombosis and hemostasis research since most assays require large amounts of plasma. In this study we verified the possibility to measure TG in zebrafish using our newly developed miniaturized whole blood assay.

Methods For TG, 5 µl of whole blood was mixed with 5 µl of buffer containing a rhodamin-based thrombin-sensitive P2Rho substrate (final concentration (fc) 300 µM). 5 µl of this mixture was put on a paper disk and covered with mineral oil to prevent evaporation. Calibration was done as described previously (Ninivaggi et al. Clin Chem 2012) by adding 5 µl of whole blood to 5 µl of a mixture containing P2Rho (fc 300 µM), a2M-thrombin calibrator (fc 100 nM) and citrate (fc 9,8 mM). Fluorescence was detected with a fluorometer (485/538 nm).

Results Due to their limited blood volume, it is impossible to perform both a TG and calibrator measurement on the same fish. Since calibrator measurements performed on blood from different fish demonstrated acceptable variation (CV calibrator slopes < 15%), the average calibrator slope was used for calculations. The calculated TG parameters from 2 independent experiments are depicted in Table 1. TG measured in individual fish showed the same amount of inter-individual variation as in humans. Striking differences with human TG parameters were the short lag time, high peak and high velocity index. Moreover a further analysis of the fibrin network of the clot, by means of scanning electron microscopy (SEM), showed a much denser network composed of thinner fibers compared to humans.

Table 1.

Thrombin generation parameters and their coefficient of variance (CV) in zebrafish in two independent experiments.

 Experiment 1
(n=4) 
Experiment 2
(n=9) 
 Mean CV Mean CV 
Peak ETP (nM.min) 575.68 19.69 689.71 24.91 
Peak (nM) 1573.16 29.58 1668.05 14.46 
Lag time (min) 0.27 0.00 0.49 10.92 
Time to peak (min) 1.08 5.30 1.37 11.49 
Velocity (nM/min) 4002.33 38.21 3826.35 17.04 
 Experiment 1
(n=4) 
Experiment 2
(n=9) 
 Mean CV Mean CV 
Peak ETP (nM.min) 575.68 19.69 689.71 24.91 
Peak (nM) 1573.16 29.58 1668.05 14.46 
Lag time (min) 0.27 0.00 0.49 10.92 
Time to peak (min) 1.08 5.30 1.37 11.49 
Velocity (nM/min) 4002.33 38.21 3826.35 17.04 

Conclusion These results demonstrate the feasibility of measuring TG in whole blood collected from zebrafish. Consequently, zebrafish may be used as a in vivo model to test the effect of (novel) anticoagulant therapeutics on thrombin generation and serve as a model organism for mechanistical research in thrombosis and haemostasis.

Disclosures

No relevant conflicts of interest to declare.

Author notes

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