Whole blood thrombin generation in Bmal1-deficient mice

Marisa Ninivaggi; Hilde Kelchtermans; Marijke J. Kuijpers; Bianca Hemmeryckx; Johan W. M. Heemskerk; Theo Lindhout; Marc F. Hoylaerts; Bas de Laat

doi:10.1160/TH13-11-0910

Thrombosis and Haemostasis, Table of Contents

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

¹Synapse BV, CARIM, Maastricht University Medical Centre, Maastricht, The Netherlands

,

Hilde Kelchtermans

¹Synapse BV, CARIM, Maastricht University Medical Centre, Maastricht, The Netherlands

,

Marijke J. Kuijpers

²Biochemistry, CARIM, Maastricht University Medical Centre, Maastricht, The Netherlands

,

Bianca Hemmeryckx

³Center for Molecular and Vascular Biology, KU Leuven, Leuven, Belgium

,

Johan W. M. Heemskerk

²Biochemistry, CARIM, Maastricht University Medical Centre, Maastricht, The Netherlands

,

Theo Lindhout

¹Synapse BV, CARIM, Maastricht University Medical Centre, Maastricht, The Netherlands

,

Marc F. Hoylaerts

³Center for Molecular and Vascular Biology, KU Leuven, Leuven, Belgium

,

Bas de Laat

¹Synapse BV, CARIM, Maastricht University Medical Centre, Maastricht, The Netherlands

²Biochemistry, CARIM, Maastricht University Medical Centre, Maastricht, The Netherlands

› Author Affiliations

Abstract

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

Full Text

References

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