Validation of human recombinant tissue factor–activated thromboelastography on citrated whole blood from clinically healthy dogs

Abstract

Telomeres are specialized DNA-protein complexes that cap the ends of linear chromosomes and protect them from degradation and end to end fusions. Due to the ''end replication problem,'' telomeres undergo progressive shortening with each cell division. Shortening to a critical length triggers cell growth arrest and cellular senescence pathways, thereby acting as a means for regulating cellular lifespan. The enzyme telomerase is a ribonucleoprotein complex that adds nucleotides to the 3 end of telomeres, thus maintaining telomere length and bypassing cellular senescence. Telomerase activity is absent from most adult so-matic cells, with expression limited to activated lymphocytes, germ cells and stem cells. In contrast, up to 90% of cancers possess telomerase activity, suggesting that telomerase activity within cells represents the acquisition of an immortal phenotype. Telomerase is composed of an RNA component, TR, a reverse transcriptase catalytic subunit, TERT, and associated proteins. Whilst TR is present in some somatic cells, the finding that TERT is only present in those cells possessing telomerase activity and that telomerase activity can be induced in telomerase-negative cells through the addition of TERT alone suggests that tel-omerase activity is regulated primarily through regulation of the TERT catalytic subunit. Our group has recently sequenced and characterized the canine TERT gene and identified approximately 5Kb of the upstream regulatory region. The purpose of this study was to sequence the promoter of the canine TERT gene and to identify the core region of the promoter essential for activity. PCR amplification and cloning of selected regions of the canine TERT promoter followed by luciferase assays revealed that core promoter activity is contained within a region extending approximately 300bp upstream of the ATG codon. Transient transfections in telomerase-positive canine cell lines and telo-merase negative fibroblasts showed that the promoter is only active in telomerase positive cell lines. Sequence analysis demonstrated that the 5 regulatory region is GC-rich and contains no TATA or CAAT box, similar to the human TERT promoter. Motif searches revealed the presence of multiple transcription factor binding sites common to both the human and canine TERT promoters, including a single E-box, Sp1, AP1, MZF-2 and ER/Sp1 binding sites. These findings suggest that the canine TERT gene shares similar transcriptional control to the human TERT gene. Identifying the core promoter necessary for activity will enable the development of telomerase-targeted therapies in canine cancer patients similar to those investigated in human patients. 401 2005 ACVIM Abstracts acterization of the Mik-red cell antigen. Screening feline blood donors and patients for the presence of this apparently common red cell antigen and corresponding alloantibody may prove necessary in clinical practice. Thromboelastography (TEG) enables global assessment of hemostatic function in whole blood with evaluation of both plasma and cellular...

Successful treatment of canine hemophilia by continuous expression of canine FVIIa

“…We observed a greatly improved clot dynamics profile (data not shown) and a sustained, near-normalization of the reaction time (time to initial fibrin formation 26 …”

Evaluation of Hemostatic Abnormalities in Canine Spirocercosis and Its Association with Systemic Inflammation

“…The sodium citrate tube was filled to ensure the correct anticoagulant‐to‐blood ratio (1 : 9). Citrated‐blood was left to stabilize for 30 minutes after collection before TEG measurements, a fixed time point after sampling was chosen to minimize interassay variation 20. TEG was performed using a computerized thromboelastograph1 according to a previously described method using human recombinant tissue factor2 as the activator 20.…”

“…Reaction time (R) represents the latency period from the start of the assay until a preset fibrin formation is reached. K (clotting time) is a measurement of the rapidity of clot development 20. The angle (α) represents the rapidity of fibrin buildup and cross linking (clot formation).…”

Comparing citrated native, kaolin-activated, and tissue factor–activated samples and determining intraindividual variability for feline thromboelastography

“…However, recombinant tissue factor must be diluted from stock solution, potentially introducing a source of variability in TEG analysis. 4,5,16 The R and κ values are measures of the time to thrombus initiation. The R-value reflects the time it takes for the clot to start forming, while κ indicates the time to reach a defined clot strength.…”