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Background: Prothrombin (FII) G20210A mutation and elevated plasma prothrombin activity are known risk factors for venous thrombosis. The risk of venous thrombosis among 19911G carriers of the prothrombin A19911G polymorphism has not been extensively investigated., Objectives and Methods: We assessed prothrombin activity, FIIG20210A, and FIIA19911G polymorphisms in a large population-based case-control study, the Multiple Environmental and Genetic Assessment (MEGA) study of risk factors for venous thrombosis. Four thousand three hundred and sixty-five consecutive patients with a first episode of deep vein thrombosis of the leg or pulmonary embolism were included. The control group (n = 4779) consisted of partners of patients or persons gathered using a random-digit dialing method. We studied the effect of FIIA19911G polymorphism on prothrombin activity and thrombosis risk, also in combination with factor V Leiden., Results: Among FII20210-GG control subjects, FII19911-GG carriers had 7.1% [95% confidence interval (CI): 5.7-8.5] higher mean prothrombin activity than FII19911-AA carriers and the risk for GG carriers was 1.43-fold increased compared to AA carriers [odds ratio (OR) 1.43; 95% CI: 1.27-1.61]. Among FII20210-GA control carriers, the mean prothrombin activity in both FII19911-AA and -AG carriers was nearly equivalent [131.7% and 133.4%; mean difference (95% CI) = 1.7% (-7.2-10.7)]. Because of genetic linkage, FII19911-GG carriers were very rare on a FII20210-GA background, as only one FII20210A carrier had FII19911-GG. In FII20210-GA carriers, the OR increased from 3.05 (95% CI: 2.17-4.27) in subjects with FII19911-AA to 3.33 (2.28-4.85) in subjects with FII19911-AG, compared to those with FII20210-GG and FII19911-AA., Conclusions: The FIIA19911G polymorphism is associated with mildly elevated prothrombin activity and is a risk factor for venous thrombosis.

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.

To find genetic causes of high plasma prothrombin levels, an established prothrombotic risk factor. we searched for sequence variations in the prothrombin gene. We selected subjects with the 20210-GG genotype (since the 20210-A allele is already known to be associated with high levels) and elevated prothrombin levels (> or = 130 U/dl) from the Leiden Thrombophilia Study (LETS). No mutations were found in the 1 kb promoter region of the prothrombin gene in seven individuals with an isolated high prothrombin level. Comparison of the allelic frequencies of four different polymorphisms in the prothrombin gene in healthy volunteers and in the control subjects among the selected LETS individuals indicated a higher frequency of the 19911-G allele in the latter group (allele frequency 52 vs. 78%, respectively). Homozygous carriers of the 19911-G allele had 8 U/dl higher plasma prothrombin levels than 19911-AA carriers. This difference in prothrombin levels did not affect the thrombotic risk in 20210-GG carriers. In heterozygous 20210-A carriers the odds ratio increased from 1.6 (95% CI: 0.6-4.3) in subjects with 19911-A to 4.7 (1.6-14.0) in subjects with 19911-G on the other prothrombin allele.

Context: Estrogens are known to be prothrombotic, and findings from the Heart and Estrogen/progestin Replacement Study suggest that in women with clinically recognized heart disease, hormone replacement therapy (HRT) may be associated with early harm and late benefit in terms of coronary events., Objective: To assess whether, as hypothesized, prothrombotic mutations modify the association between HRT use and incidence of first myocardial infarction (MI)., Design and Setting: Population-based, case-control study conducted in a Seattle-based health maintenance organization., Participants: Cases were 232 postmenopausal women aged 30 to 79 years who had their first nonfatal MI between 1995 and 1998. Controls were a stratified random sample of 723 postmenopausal women without MI who were frequency-matched to cases by age, calendar year, and hypertension status., Main Outcome Measure: Risk of first nonfatal MI based on current use of HRT and the presence or absence of coagulation factor V Leiden and prothrombin 20210 G-->A variants among cases and controls, stratified by hypertension., Results: One hundred eight MI cases and 387 controls had hypertension and 124 MI cases and 336 controls did not. Among hypertensive women, the prothrombin variant was a risk factor for MI (odds ratio [OR], 4.32; 95% confidence interval [CI], 1.52-12.1) and, in this stratum, there was also a significant interaction between use of HRT and presence of the prothrombin variant on risk of MI. Compared with nonusers of HRT with wild-type genotype, women who were current users and who had the prothrombin variant (n = 8) had a nearly 11-fold increase in risk of a nonfatal MI (OR, 10.9; 95% CI, 2.15-55.2). The interaction with the prothrombin variant was more pronounced in analyses assuming 100% compliance than in those assuming 80% compliance with HRT. The interaction was absent among nonhypertensive women and was less pronounced if hypertensive and nonhypertensive women were combined into 1 group. No interaction was found for factor V Leiden in either hypertensive or nonhypertensive women. Among hypertensive women, the estimates were affected only in trivial ways by adjustment, and the interaction with the prothrombin variant was specific to HRT., Conclusions: Our results suggest that among postmenopausal hypertensive women, the association between HRT use and MI risk differed between those with and without the prothrombin 20210 G-->A variant. If these findings are confirmed in other studies, screening for the prothrombin variant may permit a better assessment of the risks and benefits associated with HRT in postmenopausal women.

Hypoprothrombinemia is a rare hereditary coagulation defect characterized by low levels of biologically active prothrombin. In this paper we report the laboratory and genetic analysis of a patient with a severe hypoprothrombinemia and some of her relatives. Laboratory analysis showed very low levels of prothrombin antigen. Molecular analysis of the prothrombin genes of the patient resulted in the identification of two novel sequence variations in heterozygous state, a 20079 G to A transition, which predicts a Trp 569-->Stop mutation, and a 1261C-->G change within intron B near the acceptor splice site. A cosegregation of prothrombin deficiency in family members with the two genetic defects was observed.

Background and Purpose: Factor V Leiden and a prothrombin gene variant, G20210A, are mutations associated with a thrombotic risk. The aim of our study was to assess whether these mutations increase the risk of stroke in women under 45 years of age., Methods: We conducted a case-control study in western Washington state. Case patients were women aged 18 to 44 years with a first stroke (n = 106). Control subjects were women without stroke recruited from the same region by use of random-digit telephone dialing (n = 391). All were interviewed and provided blood specimens, which were genotyped for these mutations., Results: Factor V Leiden was found in 0.9% of case patients, a single patient with a subarachnoid hemorrhage, and in 4.1% of control subjects. The odds ratio (OR) for any stroke was 0.2 (95% confidence interval [CI], 0.03 to 1.7). The prothrombin variant was found in 1.9% of case patients, 1 with a venous stroke and 1 with an ischemic stroke, and in 1.6% of control subjects. The OR for any stroke was 1.48 (95% CI, 0.14 to 9.17). ORs for stroke types were also not statistically significant., Conclusions: In this study, neither factor V Leiden nor the prothrombin variant (G20210A) was an important risk factor for stroke in young women. In this setting, screening for these mutations cannot be recommended. Unanswered by this study is whether screening would be useful in select patients, such as those with a strong family history of thrombophilia or those with venous strokes.

Using specimens from a population-based case control study among women ages 18 to 44 years in western Washington, we assessed the relationship between carriership of a genetic clotting factor II variant (20210 G-->A) and myocardial infarction (MI). The factor II variant was previously shown to be present in 1% to 2% of the population, to increase the levels of factor II, and to be associated with venous thrombotic disease. Personal interviews and blood samples were obtained from 79 women with a first myocardial infarction and 381 control women identified through random-digit telephone dialing. Polymerase chain reaction (PCR) method was used to determine the factor II genotypes. The factor II 20210 G to A transition was present more often in women with MI (5.1%) than among control women (1.6%). The age-adjusted odds ratio for MI was 4.0 (95% confidence interval 1.1 to 15.1). The relative risk was high when another major cardiovascular risk factor was also present, such as smoking (odds ratio 43.3, 95% confidence interval 6.7 to 281), and the risk seemed limited to those with other risk factors. These results, in which the effect of major coronary risk factors is enhanced fourfold to sixfold by the prothrombin variant, are similar to those previously reported for another genetic clotting abnormality, factor V Leiden. We conclude that factor II 20210 G to A increases the risk of myocardial infarction in young women, especially in the women with other major risk factors for coronary heart disease.