title: Whole blood thrombin generation in Bmal1-deficient mice.
abstract: 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.
authors.forename: M
authors.initials: M
authors.lastname: Ninivaggi
authors.forename: H
authors.initials: H
authors.lastname: Kelchtermans
authors.forename: M J
authors.initials: MJ
authors.lastname: Kuijpers
authors.forename: B
authors.initials: B
authors.lastname: Hemmeryckx
authors.forename: J W M
authors.initials: JW
authors.lastname: Heemskerk
authors.forename: T
authors.initials: T
authors.lastname: Lindhout
authors.forename: M F
authors.initials: MF
authors.lastname: Hoylaerts
authors.affiliation: B. de Laat, PhD, Synapse bv, Oxfordlaan 70, 6229 EV Maastricht, The Netherlands, Tel.: +31 43 3885895, Fax: +31 43 3884159, E-mail: b.delaat@thrombin.com.
authors.forename: B
authors.initials: B
authors.lastname: de Laat
mesh_terms: D056930:ARNTL Transcription Factors / Q000172:deficiency* / Q000235:genetics; D019588:Aging, Premature / Q000097:blood / Q000235:genetics; D000818:Animals; D001777:Blood Coagulation / Q000235:genetics; D001780:Blood Coagulation Tests; D004195:Disease Models, Animal; D005337:Fibrin / Q000378:metabolism / Q000648:ultrastructure; D020022:Genetic Predisposition to Disease; D008297:Male; D008810:Mice, Inbred C57BL; D018345:Mice, Knockout; D008855:Microscopy, Electron, Scanning; D010641:Phenotype; D011237:Predictive Value of Tests; D015203:Reproducibility of Results; D013917:Thrombin / Q000378:metabolism*; D013927:Thrombosis / Q000097:blood / Q000175:diagnosis* / Q000235:genetics
chemical_list: D056930:ARNTL Transcription Factors; C538961:Bmal1 protein, mouse; D005337:Fibrin; D013917:Thrombin
keywords: Bmal1-KO mice; Thrombin generation; aging