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Thromb Haemost 2014; 112(02): 271-275
DOI: 10.1160/TH13-11-0910
Blood Coagulation, Fibrinolysis and Cellular Haemostasis
Schattauer GmbH

Whole blood thrombin generation in Bmal1-deficient mice

Marisa Ninivaggi
1   Synapse BV, CARIM, Maastricht University Medical Centre, Maastricht, The Netherlands
,
Hilde Kelchtermans
1   Synapse BV, CARIM, Maastricht University Medical Centre, Maastricht, The Netherlands
,
Marijke J. Kuijpers
2   Biochemistry, CARIM, Maastricht University Medical Centre, Maastricht, The Netherlands
,
Bianca Hemmeryckx
3   Center for Molecular and Vascular Biology, KU Leuven, Leuven, Belgium
,
Johan W. M. Heemskerk
2   Biochemistry, CARIM, Maastricht University Medical Centre, Maastricht, The Netherlands
,
Theo Lindhout
1   Synapse BV, CARIM, Maastricht University Medical Centre, Maastricht, The Netherlands
,
Marc F. Hoylaerts
3   Center for Molecular and Vascular Biology, KU Leuven, Leuven, Belgium
,
Bas de Laat
1   Synapse BV, CARIM, Maastricht University Medical Centre, Maastricht, The Netherlands
2   Biochemistry, CARIM, Maastricht University Medical Centre, Maastricht, The Netherlands
› Author Affiliations Financial support: This project was funded by an unrestricted grant from Center for Translational Molecular Medicine (CTMM) and the Dutch Hart Foundation (NHS 2006T053).
Further Information

Publication History

Received: 05 November 2013

Accepted after major revision: 12 March 2014

Publication Date:
04 December 2017 (online)

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Summary

The Calibrated Automated Thrombogram (CAT) assay that measures thrombin generation (TG) in platelet-poor and -rich plasma, is increasingly being recognised as a more sensitive tool to determine the overall function of the haemostatic system. We developed a method enabling the measurement of TG in a small aliquot of blood. The objective was to validate this assay in mouse blood and to examine the rate and extent of TG in a mouse model of premature aging. TG was assayed in blood from 20– to 28-week-old brain and muscle ARNT-like protein-1 (Bmal1)-deficient (knockout, KO) mice and wild-type (WT) littermates. Bmal1-KO mice are known to display symptoms of premature aging. TG was initiated by adding calcium, tissue factor and a thrombin specific substrate. After TG, the samples were prepared for scanning electron microscopy (SEM). The intra-assay variations (%) in mouse blood of the endogenous thrombin potential (ETP), peak height, lag time, time-to-peak and velocity index were 10% or less (n=24). We found that Bmal1-KO mice have a significantly (p<0.001) higher ETP (437 ± 7 nM.min; mean ± SD, n=7) when compared with WT mice (ETP=220 ± 45 nM.min; mean ± SD, n=5). The peak heights also differed significantly (p=0.027). By applying SEM we found that Bmal1 deficient mice display a denser fibrin network with smaller pores compared to WT mice. In conclusion, the whole blood TG assay in mice revealed to be reproducible. As a proof-of-principle we have shown that the whole blood TG assay is capable of detecting a prothrombotic phenotype in Bmal1-KO mice.

Keywords

Thrombin generation - Bmal1-KO mice - aging
 

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