Polymorphisms in the 5′‐UTR of the tissue factor gene are associated with altered expression in human endothelial cells

Abstract

Summary. Background: Enhanced tissue factor (TF) expression mediates many disease processes. Recently, four completely concordant polymorphisms were detected in the 5′‐UTR of the TF gene. Three were single base changes and one was an 18‐bp insertion/deletion at −1208. Objectives: This study was undertaken to determine if the I‐allele or the D‐allele would associate with elevated TF expression in human umbilical vein endothelial cells (HUVEC). Methods: HUVEC were genotyped by polymerase chain reaction for 18‐bp insert status. TF expression was induced by interleukin (IL)‐1 or phorbol 12‐myristate 13‐acetate (PMA). Total TF activity was determined by a one‐stage clotting assay and surface TF activity by a chromogenic assay. Protein binding differences between the I‐ and D‐alleles were examined by gel shift assays. Results: IL‐1‐ or PMA‐induced total TF activity in D‐allele HUVEC was increased 2.0–2.5‐fold above that seen in II HUVEC. Surface clotting activity in D‐allele cells was 1.3–1.7‐fold greater than in II‐allele cultures. Experiments with consensus site mutation oligos suggested that the 18‐bp insert creates GATA and CCAAT‐enhancer binding protein (C/EBP) transcription factor recognition sites. Conclusions: The D‐allele is associated with enhanced TF activity in HUVEC. The differences in TF expression between the alleles may be due to variant transcription factor binding in the −1208 region. Further studies are warranted to investigate whether the D‐allele is associated with increased incidence of pathological processes that involve TF.

Tissue factor gene promoter haplotype associates with carotid intima-media thickness in subjects in cardiovascular risk prevention

“…This hypothesis is supported by in vitro studies by Terry et al showing an association of the TF promoter A haplotype (-1208Del) with higher TF mRNA levels and higher surface TF procoagulant activity in endothelial cells [17]. Moreover, although evaluation in silico in another study did not indicate effects of the 4 investigated polymorphisms on transcription factor binding [11], gel shift experiments by Terry et al indeed demonstrated such effects of the Del-1208Ins polymorphism, providing a possible explanation for the genotypic differences in TF expression and activity [17].…”

“…This hypothesis is supported by in vitro studies by Terry et al showing an association of the TF promoter A haplotype (-1208Del) with higher TF mRNA levels and higher surface TF procoagulant activity in endothelial cells [17]. Moreover, although evaluation in silico in another study did not indicate effects of the 4 investigated polymorphisms on transcription factor binding [11], gel shift experiments by Terry et al indeed demonstrated such effects of the Del-1208Ins polymorphism, providing a possible explanation for the genotypic differences in TF expression and activity [17]. An influence on local TF activity in the vessel wall may in turn modulate processes involved in atherosclerosis such as inflammation and VSMC migration and proliferation [2,9,10,[25][26][27][28][29][30], which could result in measurable differences in C-IMT as observed in this study.…”

“…The involvement of TF in pathological processes may be modulated by genetic factors influencing TF expression and activity [11][12][13][14][15][16][17]. Common variants of the TF gene have been described, such as two haplotypes formed by the four completely concordant promoter polymorphisms C-1812T (rs958587), C-1322T (rs3761955), Del-1208Ins (rs ID not available), and A-603G (rs1361600), as reported by Arnaud [11].…”

“…Common variants of the TF gene have been described, such as two haplotypes formed by the four completely concordant promoter polymorphisms C-1812T (rs958587), C-1322T (rs3761955), Del-1208Ins (rs ID not available), and A-603G (rs1361600), as reported by Arnaud [11]. Although not in a fully conclusive manner, these promoter haplotypes have been associated with TF expression levels and/or activity as well as to thrombotic risk in several studies [11][12][13][14][15][16][17]. However, to our knowledge, no studies of possible associations between functionally relevant variants of the TF gene and measures of atherosclerosis were reported so far.…”

“…In order to confirm their previously reported concordance with A-603G, the C-1812T (rs958587), C-1322T (rs3761955), and Del-1208Ins (rs ID not available) polymorphisms were analysed in a set of 249 consecutive patients. C-1812T and C-1322T were analysed with TaqMan SNP Genotyping Assays C 8384819 10 and C 27497686 10, respectively (Applied Biosystems), and the Del-1208Ins polymorphism was analysed by PCR using 2.5 pmol each of previously described oligonucleotide primers [17] as follows; PCR thermocycling was performed with an initial 5 min denaturation at 94 • C, a 40-cycle amplification (45 s at 94 • C, 45 s 53 • C, 45 s 72 • C) and a final 5-min extension at 72 • C using 0.5 units of Platinum Taq polymerase (Invitrogen) in 2.0 mM MgCl 2 in a final volume of 20 l containing 5% dimethylsulfoxide. PCR products were resolved on 3% agarose gels stained with ethidium bromide (BioRad).…”