Increased Thrombin Generation and Fibrin Formation In Premature Aging BMAL1 Deficient Mice
Authors: Ninivaggi Marisa , Kelchtermans Hilde , Kuipers Marijke , Hemmeryckx Bianca , Heemskerk Johan , Lindhout Theo , Hoylaerts Marc F. , De Laat Bas 
Published: October 2019

Abstract

Introduction
The occurrence of venous thromboembolism is strongly age-dependent and has a substantial morbidity and mortality. While the incidence is<1 per 1000 per year up to an age of 50, it thereafter increases rapidly end exponentially. The cause of this increased risk is still unclear, however immobility, decreased muscular tone, aging of the veins and the presence of other or more risk factors and diseases than young individuals all contribute to this increased risk. Whether age-induced circulatory changes and/or increased levels of blood coagulation levels are responsible for the increased thrombogenicity remains unclear. We recently developed a method in which these problems were overcome enabling the measurement of thrombin generation (TG) in a small aliquot of blood. The advantage of the use of this whole blood assay is that the small volume enables us to apply this assay to mice and the use of whole blood enables us to include the effect of blood cells on TG.

Objectives
By modifying the classic plasma Calibrated Automated Thrombogram (CAT) assay, we were able of measuring TG in whole blood in mice. The objective was to validate this assay in mice blood and to examine the rate and extent TG in a mouse model of premature aging.

Methods
TG was assayed in 20- to 28-week-old brain and muscle ARNT-like protein-1 (Bmal1)-deficient (knockout, KO) mice and wild-type (WT) littermates. The Bmal1-KO mice have an impaired circadian behavior and demonstrate loss of rhythmicity in the expression of their target genes. They are known to have a reduced lifespan and display symptoms of premature aging. Mice blood samples were taken from the orbital sinus using a capillary anticoagulated with citrate (3.8%). Coagulation and therefore TG was initiated with a solution containing calcium, tissue factor (TF) and a thrombin specific substrate. Varying the TF concentration revealed an optimal of 0.5 pM. At the end of the TG assay, the samples were fixated, prepared for and analysed by scanning electron microscopy (SEM).

Results
The intra-assay variations (CV%) in mice blood of the endogenous thrombin potential (ETP), peak height, lagtime, time-to-peak and velocity index were 10% or less (n=24). The whole blood TG test showed that Bmal1-KO mice have a significantly (p<0.001) higher ETP (437±7 nM.min; mean±SD, n=5) when compared with wild type mice (ETP=220±45 nM.min; mean±SD, n=5). The peak heights also differed significantly (p=0.027). However, differences in lagtime, TTP and velocity index were not significantly different (p-values were respectively 0.45, 0.15 and 0.74). Applying SEM we found that Bmal1 deficient mice display a more dense fibrin network with smaller pores compared to WT-mice.

Conclusions
The whole blood TG assay in mice blood revealed to be reproducible and we demonstrated that aging Bmal1-KO mice have a hypercoagulable state when compared with WT mice indicating that a higher thrombotic risk in elderly is partially due to a more procoagulation state.

Disclosures:
No relevant conflicts of interest to declare.