Download PDF
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)

    Permissions and Reprints

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
 

  • References

  • 1 Dargaud Y, Spronk HM, Leenders P. et al. Monitoring platelet dependent thrombin generation in mice. Thromb Res 2010; 126: 436-441.
    CrossrefPubMedGoogle Scholar
  • 2 Hogan KA, Weiler H, Lord ST. Mouse models in coagulation. Thromb Haemost 2002; 87: 563-574.
    Article in Thieme ConnectPubMedGoogle Scholar
  • 3 Mackman N. Mouse models in haemostasis and thrombosis. Thromb Haemost 2004; 92: 440-443.
    Article in Thieme ConnectPubMedGoogle Scholar
  • 4 Tchaikovski SN, BJ VANV, Rosing J. et al. Development of a calibrated automated thrombography based thrombin generation test in mouse plasma. J Thromb Haemost 2007; 05: 2079-2086.
    CrossrefPubMedGoogle Scholar
  • 5 Tsakiris DA, Scudder L, Hodivala-Dilke K. et al. Haemostasis in the mouse (Mus musculus): a review. Thromb Haemost 1999; 81: 177-188.
    Article in Thieme ConnectPubMedGoogle Scholar
  • 6 Bunker JP, Goldstein R. Coagulation during hypothermia in man. Proc Soc Exp Biol Med 1958; 97: 199-202.
    CrossrefPubMedGoogle Scholar
  • 7 Martini WZ, Cortez DS, Dubick MA. et al. Thrombelastography is better than PT, aPTT, and activated clotting time in detecting clinically relevant clotting abnormalities after hypothermia, haemorrhagic shock and resuscitation in pigs. J Trauma 2008; 65: 535-543.
    CrossrefPubMedGoogle Scholar
  • 8 Reed 2nd RL, Johnson TD, Hudson JD. et al. The disparity between hypothermic coagulopathy and clotting studies. J Trauma 1992; 33: 465-470.
    CrossrefPubMedGoogle Scholar
  • 9 Rohrer MJ, Natale AM. Effect of hypothermia on the coagulation cascade. Crit Care Med 1992; 20: 1402-1405.
    CrossrefPubMedGoogle Scholar
  • 10 De Smedt E, Al Dieri R, Spronk HM. et al. The technique of measuring thrombin generation with fluorogenic substrates: 1. Necessity of adequate calibration. Thromb Haemost 2008; 100: 343-349.
    Article in Thieme ConnectPubMedGoogle Scholar
  • 11 De Smedt E, Wagenvoord R, Coen Hemker H. The technique of measuring thrombin generation with fluorogenic substrates: 3. The effects of sample dilution. Thromb Haemost 2009; 101: 165-170.
    Article in Thieme ConnectPubMedGoogle Scholar
  • 12 Hemker HC, Al Dieri R, De Smedt E. et al. Thrombin generation, a function test of the haemostatic-thrombotic system. Thromb Haemost 2006; 96: 553-561.
    Article in Thieme ConnectPubMedGoogle Scholar
  • 13 Hemker HC, Giesen P, Al Dieri R. et al. Calibrated automated thrombin generation measurement in clotting plasma. Pathophysiol Haemost Thromb 2003; 33: 4-15.
    CrossrefPubMedGoogle Scholar
  • 14 Hemker HC, Giesen P, Al Dieri R. et al. The calibrated automated thrombogram (CAT): a universal routine test for hyper- and hypocoagulability. Pathophysiol Haemost Thromb 2002; 32: 249-253.
    CrossrefPubMedGoogle Scholar
  • 15 Al Dieri R, Hemker CH. Thrombin generation in whole blood. Br J Haematol 2008; 141: 895 author reply 896-897.
    CrossrefPubMedGoogle Scholar
  • 16 Tappenden KA, Gallimore MJ, Evans G. et al. Thrombin generation: a comparison of assays using platelet-poor and -rich plasma and whole blood samples from healthy controls and patients with a history of venous thromboembolism. Br J Haematol 2007; 139: 106-112.
    CrossrefPubMedGoogle Scholar
  • 17 Ninivaggi M, Apitz-Castro R, Dargaud Y. et al. Whole-blood thrombin generation monitored with a calibrated automated thrombogram-based assay. Clin Chem 2012; 58: 1252-1259.
    CrossrefPubMedGoogle Scholar
  • 18 Tripodi A. The long-awaited whole-blood thrombin generation test. Clin Chem 2012; 58: 1173-1175.
    CrossrefPubMedGoogle Scholar
  • 19 Hemmeryckx B, Himmelreich U, Hoylaerts MF. et al. Impact of clock gene Bmal1 deficiency on nutritionally induced obesity in mice. Obesity 2011; 19: 659-661.
    CrossrefPubMedGoogle Scholar
  • 20 Kondratov RV, Kondratova AA, Gorbacheva VY. et al. Early aging and age-related pathologies in mice deficient in BMAL1, the core componentof the circadian clock. Genes Dev 2006; 20: 1868-1873.
    CrossrefPubMedGoogle Scholar
  • 21 Somanath PR, Podrez EA, Chen J. et al. Deficiency in core circadian protein Bmal1 is associated with a prothrombotic and vascular phenotype. J Cell Physiol 2011; 226: 132-140.
    CrossrefPubMedGoogle Scholar
  • 22 Hemmeryckx B, Hoylaerts MF, Lijnen HR. Effect of premature aging on murine adipose tissue. Exp Gerontol 2012; 47: 256-262.
    CrossrefPubMedGoogle Scholar
  • 23 Hemmeryckx B, Van Hove CE, Fransen P. et al. Progression of the prothrombotic state in aging Bmal1-deficient mice. Arterioscler Thromb Vasc Biol 2011; 31: 2552-2559.
    CrossrefPubMedGoogle Scholar
  • 24 Wolberg AS. Thrombin generation and fibrin clot structure. Blood Rev 2007; 21: 131-142.
    CrossrefPubMedGoogle Scholar
  • 25 Wolberg AS, Campbell RA. Thrombin generation, fibrin clot formation and haemostasis. Transfus Apher Sci 2008; 38: 15-23.
    CrossrefPubMedGoogle Scholar
  • 26 Wolberg AS, Monroe DM, Roberts HR. et al. Elevated prothrombin results in clots with an altered fiber structure: a possible mechanism of the increased thrombotic risk. Blood 2003; 101: 3008-3013.
    CrossrefPubMedGoogle Scholar
  • 27 Whelihan MF, Mann KG. The role of the red cell membrane in thrombin generation. Thromb Res 2013; 131: 377-382.
    CrossrefPubMedGoogle Scholar
  • 28 Whelihan MF, Mooberry MJ, Zachary V. et al. The contribution of red blood cells to thrombin generation in sickle cell disease: meizothrombin generation on sickled red blood cells. J Thromb Haemost 2013; 11: 2187-2189.
    CrossrefPubMedGoogle Scholar
  • 29 Whelihan MF, Zachary V, Orfeo T. et al. Prothrombin activation in blood coagulation: the erythrocyte contribution to thrombin generation. Blood 2012; 120: 3837-3845.
    CrossrefPubMedGoogle Scholar