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Background: The prothrombin G20210A mutation is associated with increased plasma prothrombin levels and risk of thrombosis. The mechanism by which this mutation leads to increased prothrombin expression is as yet unclear and still the subject of debate. Objectives: The aim of this study was to investigate the effect of the G20210A mutation on mRNA and protein expression. Methods: We made a set of constructs containing the prothrombin 5′‐regulatory region, the firefly luciferase reporter gene and the prothrombin 3′‐UTR+ downstream region. The latter element contained either the 20210G or A allele and was inserted either as a single unit (constructs G1 and A1) or in tandem (A1A2, G1G2, A1G2, G1A2). Constructs were transiently expressed in HepG2 cells. Expression was evaluated by luciferase assays and reverse transcriptase‐polymerase chain reaction (RT‐PCR), followed by quantification of the products and determination of the ratio of poly(A)site usage. RT‐PCR sequencing was used for determination of the actual site of polyadenylation in mRNAs from constructs G1 and A1 and from endogenous prothrombin mRNAs from HepG2 cells and human liver tissue. Results: The A1 constructs expressed 1.2‐fold more protein than the G1 constructs. The double constructs expressed 1.4‐fold more protein (A1A2 vs. G1G2). Similar results were found in a set of constructs in which an SV40 promoter replaced the prothrombin 5′‐regulatory region. Ratios of poly(A) site usage (expressed as ratio poly(A) site 1 and 2) for the tandem constructs were similar for constructs with two Gs or As at both poly(A)sites; 2.92 (95% confidence interval 2.39–3.45) and 2.75 (2.55–2.95). pA1/pA2 ratios were 1.46 (1.11–1.81) for G1A2 and 6.29 (5.48–7.10) for A1G2 constructs with different poly(A) sites, indicating that the poly(A)site with the 20210A variant is favored over the normal site. In 20210G mRNAs, the G at 20210 was the last non‐A nucleotide in the majority of mRNAs, whereas in most 20210A mRNAs, the last non‐A nucleotide was the C at 20209. Over 70% of the prothrombin 20210G mRNAs from HepG2 cells and human liver tissue is polyadenylated at position 20210. Conclusions: The 20210A variant has a more effective poly(A) site, leading to increased mRNA and protein expression, irrespective of the promoter and gene. It does not affect the position of poly(A) attachment.

Background: Prothrombin is a key component in blood coagulation. Overexpression of prothrombin leads to an increased risk of venous thrombosis. Therefore, the study of the transcriptional regulation of the prothrombin gene may help to identify mechanisms of overexpression. Objectives: The aim of our study was to localize the regions within the prothrombin enhancer responsible for its activity, to identify the proteins binding to these regions, and to establish their functional importance. Methods: We constructed a set of prothrombin promoter 5′ deletion constructs containing the firefly luciferase reporter gene, which were transiently transfected in HepG2, HuH7 and HeLa cells. Putative transcription factor (TF) binding sites were evaluated by electrophoretic mobility shift assays. The functional importance of each TF binding site was evaluated by site directed mutagenesis and transient transfection of the mutant constructs. Results: We confirmed the major contribution of the enhancer region to the transcriptional activity of the prothrombin promoter. Analysis of this region revealed putative binding sites for hepatocyte nuclear factor HNF4, HNF3-beta and specificity protein(Sp)1. We identified six different TFs binding to three evolutionary conserved sites in the enhancer: HNF4-alpha (site 1), HNF1-alpha, HNF3-beta and an as yet unidentified TF (site 2) and the ubiquitously expressed TFs Sp1 and Sp3 (site 3). Mutagenesis studies showed that loss of binding of HNF3-beta resulted in a considerable decrease of enhancer activity, whereas loss of HNF4-alpha or Sp1/Sp3 resulted in milder reductions. Conclusions: The prothrombin enhancer plays a major role in regulation of prothrombin expression. Six different TFs are able to bind to this region. At least three of these TFs, HNF4-alpha, HNF3-beta and Sp1/Sp3, are important in regulation of prothrombin expression.