Genetic regulation of fibrin structure and function: complex gene-environment interactions may modulate vascular risk

MECHANISMS OF DISEASE

Mechanisms of disease

Genetic regulation of fibrin structure and function: complex geneenvironment interactions may modulate vascular risk Bernard C B Lim, Robert A S Ariëns, Angela M Carter, John W Weisel, Peter J Grant

Summary Background Polymorphisms in the fibrinogen and factor XIII genes are associated with atherothrombotic risk, but clinical studies have produced inconsistent results and laboratory studies have not explained these findings. We aimed to investigate interactions between polymorphisms in the factor XIII and fibrinogen genes, fibrinogen concentrations, and other cardiovascular risk factors in relation to fibrin structure and function. Methods We used permeation analysis and electron microscopy to investigate interactions between fibrin structure, factor XIII Val34Leu, fibrinogen A
Thr312Ala, fibrinogen B Arg448Lys, and fibrinogen concentrations in plasma and purified systems. Findings Increased fibrinogen concentrations were associated with decreases in permeability, with tighter clot structures in the presence of factor XIII 34Val alleles compared with those in the presence of 34Leu alleles. Findings were confirmed by scanning electron microscopy of fibrin. Similar changes in permeability were noted for A
fibrinogen 312Ala compared with that for 312Thr. Interpretation Our results show interactions between coding polymorphisms in fibrinogen and factor XIII and fibrinogen concentrations that modify fibrin and explain the apparent paradox between epidemiological studies of factor XIII 34Leu and reported in-vitro effects on fibrin structure and function. We suggest a potential complexity of gene-gene and geneenvironment interactions in determining cardiovascular risk. Lancet 2003; 361: 1424–31

Academic Unit of Molecular Vascular Medicine, University of Leeds, Leeds, UK (B C B Lim MB, R A S Ariëns PhD, A M Carter PhD, Prof P J Grant MD); and Department of Cell and Developmental Biology, University of Pennsylvania School of Medicine, Pennsylvania, PA, USA (B C B Lim, J W Weisel PhD) Correspondence to: Prof P J Grant, Academic Unit of Molecular Vascular Medicine, G Floor, Martin Wing, Leeds General Infirmary, Leeds LS1 3EX, UK (e-mail: [email protected])

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Introduction The study of the molecular genetics of atherothrombotic disorders has generated a confusing array of associations with clinical disease. Much of this confusion is due to poor understanding of environmental effects on the molecular mechanisms that regulate THROMBOSIS. Fibrin is the major protein constituent of the blood clot, and through FACTOR XIII CROSS LINKING, on activation by THROMBIN, covalently cross links fibrin to increase clot stability. Findings from epidemiological studies suggest a role for the FIBRINOGEN and factor XIII genes in determining clinical outcome. Study results have, however, been inconsistent and studies of the expressed phenotypes, such as protein concentrations or fibrin structure and function have not been able to convincingly support the findings. A common coding polymorphism in the factor XIII gene, which causes an aminoacid change at codon 34—Val34Leu—is reported to protect against thrombotic disease,1–3 but some studies do not confirm this finding.4,5 Factor XIII Leu34 is activated rapidly by thrombin and produces a fibrin meshwork with thin fibres and small pores,6 which may be contrary to the clinical observation of cardioprotection. In the large ECTIM study,7 polymorphisms in the B chain of fibrinogen were associated with severe coronary artery disease. Fibrinogen Α
Thr312Αla substitution has been associated with post-stroke mortality among patients who have atrial fibrillation.8 In large studies, including the Northwick Park9 and Gothenburg10 studies, fibrinogen is reported as an independent predictor of atherosclerotic disease. Fatah and colleagues11,12 noted that fibrin clot structure was associated with premature coronary artery disease and myocardial infarction. High fibrinogen concentrations lead to the formation of a fibrin clot with thin and tightly packed fibres that has high thrombogenicity, perhaps because the small pore size restricts access of fibrinolytic enzymes.13 Raised fibrinogen concentrations are related to smoking, physical inactivity, and features of the insulin resistance syndrome. As intermediaries for environmental effects, fibrinogen concentrations are an important candidate for interacting with fibrinogen and factor XIII coding polymorphisms to alter vascular risk. We aimed to investigate interactions between polymorphisms in the factor XIII and fibrinogen genes, fibrinogen concentrations and other cardiovascular risk factors in relation to fibrin structure and function.

Participants and methods Participants We recruited 125 white patients who had a clinical diagnosis of acute stroke, from four hospitals in Leeds, UK.14,15 Patients’ clinical characteristics are shown in the table. Patients gave informed consent according to a protocol approved by the Leeds Teaching Hospitals Research Ethics Committee. We chose to measure clot 1424

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Fibrinogen was analysed by the method of Clauss, with use of a KC10 coagulometer (Amelung, Lemgo, Germany).14

GLOSSARY FACTOR XIII CROSS-LINKING

Factor XIII is a transglutaminase that catalyses the formation of covalent bonds between the  chains and the
chains of fibrinogen. Crosslinking imparts tensile strength to the fibrin clot, which may be antithetical to survival in myocardial infarction. FIBRINOGEN

A plasma protein that is the building block of a fibrin clot. FIBRINOLYSIS

The process of clot dissolution. The efficiency with which this occurs can determine clinical outcome in acute myocardial infarction. LIQUID PERMEATION

An experimental method that measures the flow-rates through a fibrin clot. The clot is suspended in a reservoir and physiological buffer allowed to flow through the clot. The speed of flow will generally give an indication of the thickness and density of the fibrin fibres as well as the pore size of the clot. SCANNING ELECTRON MICROSCOPY

A powerful imaging technique that gives nanometre resolution of the ultrastructural features of a fibrin clot. THROMBIN

A coagulation factor (IIa) that cleaves off peptide sequences from fibrinogen to initiate the process of fibrin clot formation. THROMBOSIS

The process of fibrin clot formation, which is initiated by thrombin and includes polymerisation of the fibrin monomers, lateral aggregation of the fibrin protofibrils, and cross-linking of the clot by factor XIII to give a mechanically strong clot.

permeability in this group because they have a wide range of fibrinogen concentrations,14 ranging from 4·0 to 25·6 mol/L (conversion factor to mg/mL 0·34). Procedures Analysis of circulating factors We took venous blood samples within 10 days of acute stroke into 4·3 mmol/L EDTA (edetic acid) and stored for extraction of genomic DNA. Samples for measurement of circulating fibrinogen were taken into 0·1 mol/L trisodium citrate, nine parts to one part sodium citrate, centrifuged at 2500 g at room temperature and stored at –40ºC until analysed. Number (frequency)* Characteristics Mean (SD) age (years) Smoking history Ever smoked Never smoked Sex Male Female Factor XIII Val34Leu Val/Val Val/Leu Leu/Leu A
Thr312Ala Thr/Thr Thr/Ala Ala/Ala B Arg448Lys Arg/Arg Arg/Lys Lys/Lys Mean (SD) fibrinogen (mol/L) Mean (SD) permeability coefficient

69 (12·8) 66 (0·53) 59 (0·47) 65 (0·52) 60 (0·48) 69 (0·58) 43 (0·36) 8 (0·06) 63 (0·52) 46 (0·38) 12 (0·10) 86 (0·74) 29 (0·25) 1 (0·01) 11·6 (4·1) 4·76 (1·97)

*Except where otherwise stated.

Clinical characteristics of patients

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Assessment of factor XIII Val34Leu, B Arg448Lys, A
Thr312Ala genotype We extracted genomic DNA with the BACC3 DNA extraction kit (Nucleon Biosciences, Glasgow, UK) from 10 mL anticoagulated venous blood. We used PCR and single-stranded conformational polymorphism analysis16 to determine factor XIII Val34Leu polymorphism, and we used restriction fragment length polymorphism PCR for the A
Thr312Ala and B Arg448Lys polymorphisms.7,17 Clot permeation We used LIQUID PERMEATION studies to measure flow rates through fibrin clots, since permeability is a sensitive measure of fibrin clot structure. Plasma samples (110 L) were incubated with 1 U/mL human thrombin (Sigma, St Louis, MO, USA) and 20 mmol/L calcium in open tubes for 2 h at room temperature in a wet chamber to allow clot formation. The tubes containing the clots were connected via plastic tubing to a reservoir containing 0·05 mol/L Tris-HCl, 0·15 mol/L sodium chloride, pH 7·5 with a pressure drop of 4 cm. After washing the clots, we measured flow rates of buffer through the fibrin gels by timing six drops for each tube and weighing each drop for exact volume. The permeability or Darcy constant (Ks), which represents the surface of the gel allowing flow through a network and thus provides information on pore structure, was calculated according to the formula: Ks=(QL)/(TAP), where Q is the volume of liquid (mL) with the viscosity  (10–2 poise) flowing through a clot with length L (1·3 cm) and a cross-sectional area A in time t (s) under pressure P (dyne/cm).18 The unit of permeability is cm2, which gives an indication of the size of the pores in the fibrin clot and indirectly gives an estimation of the thickness of the fibrin fibres. Generally, lower permeability indicates a thinner calibre of the fibrin fibres being formed. Relation between factor XIII Val34Leu, permeability, and fibrinogen concentration We selected plasma samples (three each) that were homozygous for the Val/Val or the Leu/Leu genotype. To avoid possible effects from the A
Thr312Ala and B Arg448Lys polymorphisms we chose plasma samples that were homozygous for the most common allele for each. Samples were selected to have similar starting fibrinogen concentrations. We added purified fibrinogen sequentially to a maximum fibrinogen concentration of 28·2 mol/L. Fibrin clots formed and we measured permeability. Purification of factor XIII variants We prepared factor XIII Val34 and Leu34 variants from platelet-poor plasma with a method adapted from previous descriptions.6 Plasma was subjected to repeated precipitations with ammonium sulphate: 20% saturation at room temperature, pH 7·0; 16% saturation at 4ºC, pH 5·4; 16% saturation at 4ºC, pH 7·0; and 36% saturation at 4ºC, pH 7·5. The precipitate was resuspended in one per 1000 plasma volume of 0·05 mol/L Tris-HCl, pH 7·5, 1·0 mmol/L EDTA, dialysed against the same buffer and further purified by gel filtration on a Sepharose 6B (Sigma, St Louis, MO, USA) home-made column (2·640·0 cm), equilibrated and developed with 0·05 mol/L Tris-HCl, pH 7·5, 1·0 mmol/L EDTA. We

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different concentrations. Six replicates of each variant and fibrinogen concentration were prepared for SCANNING ELECTRON MICROSCOPY. We measured fibre diameter with ImageJ (version 1,23y). 100 fibres from each micrograph were measured by an operator, who was unaware of genotype to exclude bias.

Permeability coefficient (10 9 cm2) Ks

12 10 8 6 4 2 0 –0·2

0

0·4

0·2

0·6

0·8

1·0

Log10 fibrinogen Figure 1: Relation between permeability and log-transformed fibrinogen concentration

pooled and concentrated fractions containing factor XIII A subunit and factor XIII B subunit, confirmed by specific ELISA. Purity and activity of the preparations were measured with sodium dodecyl sulphate polyacrylamide (8%, 1:37·5) gel electrophoresis, Asubunit and B-subunit ELISA, and 5-(biotinamido) pentylamine incorporation assay. Electron microscopy After permeation experiments, fibrin clots were washed with sodium cacodylate buffer and fixed overnight in 2% glutaraldehyde solution. We further processed recovered clots by dehydration, with use of a stepwise ethanol gradient, critical-point drying, and sputter coating with gold palladium.19 Plasma clots from four participants homozygous for factor XIII 34Leu and four for factor XIII 34Val were examined and photographed in ten different areas, on a scanning electron microscope.6 In the experiments that involved the purified components, we added purified factor XIII Leu and Val variants to purified fibrinogen free from factor XIII (American Diagnostica, Greenwich, CT, USA) at

Role of the funding source The funding sources had no involvement in the study design, collection, analysis, and interpretation of data, the writing of the report, or in the decision to submit the paper for publication.

12

Val/Val Val/Leu

12

8

Leu/Leu

Permeability coefficient (10 9 cm2)

Permeability coefficient (10 9 cm2) Ks

Factor XIII 10

Statistical analysis From our preliminary study of the factor XIII A subunit Val34Leu polymorphism, the difference in Ks was 4·110–9 (absolute values 8·710–9 for Val and 3·610–9 for Leu) obtained for clots formed from purified fibrinogen in the presence of purified Val 34 factor XIII compared with Leu34.6 On the basis of the results of a pilot study in 49 apparently healthy participants (mean Ks=5·0110–9 [SD 1·7810–9], minimum 2·6210–9, maximum 10·8510–9), the sample size of 125 participants was deemed sufficient to detect a difference of 1·010–9 between Val/Val and Val/Leu plus Leu/Leu groups for Val34Leu, 0·9510–9 between Thr/Thr and Thr/Ala plus Ala/Ala for Thr312Ala, and 1·110–9 between Arg/Arg and Arg/Lys plus Lys/Lys for Arg448Lys at the 5% significance level and with 80% power. We analysed results with SPSS for windows (version 9.0). To assess goodness of fit for permeability, we used the Kolmogorov-Smirnov test. For positively skewed distribution of fibrinogen concentrations, we used log transformation to normalise the distribution and allow analysis by parametric tests. Results are expressed as mean or geometric mean with 95% CI. Between-group differences were investigated by ANOVA and unpaired Student’s t test. We calculated Pearson’s correlation coefficients in a bivariate analysis. Multiple regression analyses were used to assess the contributions of fibrinogen, age, and each polymorphism to permeability, and to identify any interactions between the genotypes and other covariates. We created interaction terms in the linear regression models between genotype and fibrinogen concentrations to assess their effect on permeability. To investigate significant interactions further, we analysed the association of permeability with fibrinogen concentrations according to genotype.

6

4

2

Val allele Leu allele

10 8 6 4 2 0 0·88

0

1·0

1·35

2·3

2·72

Fibrinogen concentration (mol/L) 0·2

0·3

0·4

0·5

0·6

0·7

0·8

Log fibrinogen Figure 2: Interaction between factor XIII Val34Leu polymorphism, fibrinogen, and permeability

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0·91

0·9

1·0 Figure 3: In-vitro permeation experiments investigating relation between factor XIII 34Leu allele, fibrinogen concentration, and permeability Cross-over fibrinogen concentration is 10·0 mol/L. Data are mean (SD) of three replicate experiments.

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Figure 4: Scanning electron micrographs of fibrin clots prepared from plasma samples homozygous for factor XIII 34Val and 34Leu at fibrinogen concentration 22·6 mol/L Magnification is 9400 for each micrograph. Electron micrographs are representative of four samples of each genotype scanned in ten different areas, showing similar results.

Results Permeability Permeability was normally distributed and was strongly inversely correlated with fibrinogen (r= 0·585, p<0·0001; figure 1) and with age (r=–0·239, p=0·007). There was no association between permeability and categorical variables such as sex, myocardial infarction, and smoking (p=0·67, p=0·11, and p=0·43, respectively). The B Arg448Lys polymorphism was significantly associated with permeability; participants who had the Lys448 allele had lower permeability (mean 4·08 cm2 [95% CI 2·52–5·6410–9]) than did those homozygous for the Arg448 allele (5·05 cm2 [4·21–5·8910–9], p=0·02). There was no association between B Arg448Lys and fibrinogen concentrations (p=0·07). There was no association of average permeability with the factor XIII Val34Leu (p=0·62) or the A
Thr312Ala (p=0·07) polymorphisms, nor was there a significant association of either polymorphism with fibrinogen concentrations (p=0·13 and p=0·23 for Val34Leu and Thr312Ala, respectively).

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Multiple linear regression and interactions In the multiple linear regression analyses, only fibrinogen and age were independently related to permeability. In a linear regression model for permeability with age and log-transformed fibrinogen, a 1 g/L increase in fibrinogen led to a fall in permeability of 0·7510 9cm2 (95% CI 0·54–0·9710–9, p<0·0001). There was a significant interaction between fibrinogen concentrations and the factor XIII Val34Leu polymorphism on permeability (p=0·026) and between the A
Thr312Ala polymorphism (p=0·019). There was no significant interaction between the B Arg448Lys polymorphism and fibrinogen concentrations (p=0·34). Factor XIII Val34Leu and fibrinogen The association of permeability with fibrinogen was modulated by Val34Leu genotype (figure 2). In individuals possessing the Val/Val genotype, permeability decreased with increasing fibrinogen concentrations (r=–0·67, p<0·0001). For individuals heterozygous for the Leu allele, the same trend was noted, but permeability changed less as fibrinogen concentrations increased

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Figure 5: Scanning electron micrographs of fibrin clots prepared from purified fibrinogen free from factor XIII and factor XIII Val and Leu variants at different fibrinogen concentrations Magnification 9750. Electron micrographs are representative of six samples of each variant scanned in ten different areas, showing similar results.

(r=–0·40, p=0·01). Individuals homozygous for the Leu allele had permeability that did not change significantly with increasing fibrinogen concentrations (r=–0·17, p=0·69). Thus the rate of change of permeability with increasing fibrinogen concentrations decreased stepwise in the presence of increasing numbers of Leu34 alleles. In-vitro permeation experiments and scanning electron microscopy For patients who had the Leu/Leu genotype, fibrin clots were formed with lower permeability at low fibrinogen concentrations, and with higher permeability at higher fibrinogen concentrations than among those who had the Val/Val genotype. These findings were confirmed by invitro experimentation of the addition of purified fibrinogen to plasma samples from homozygous Val/Val and Leu/Leu people (figure 3). The cross-over point obtained in the invitro experiments was similar to that from the ex-vivo experiments. Scanning electron microscopy of the fibrin

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clots prepared from plasma samples homozygous for the factor XIII 34Leu at 22·6 mol/L fibrinogen concentration showed a fibrin meshwork with thicker fibres (mean 217 nm [SD 35]) and large pores, whereas those formed from plasma samples homozygous for the factor XIII 34Val showed a finer meshwork with thinner fibres (91 nm [12], p<0·0001) and reduced space between the fibrin strands (figure 4). Scanning electron microscopy of clots formed from purified fibrinogen, and factor XIII Leu and Val variants at 2·9, 8·8, and 14·7 mol/L fibrinogen concentrations confirmed the results from the plasma experiments. At low fibrinogen concentrations, fibrin clot structures in samples with the Leu variants had fibres that were thinner (96 nm [14]) and more tightly packed than those in the samples with Val variants (227 nm [28], p<0·0001; figure 5). At intermediate fibrinogen concentrations, the fibrin-fibre diameters approached similar values in the Val/Val and the Leu/Leu samples (165 nm [28] and 152 nm [27],

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respectively), although the differences remained significant (p<0·0001). At high fibrinogen concentrations, the Leu/Leu clots had thicker and more loosely packed fibres than did those in the samples with Val variants (286 [49] vs 93 nm [17], p<0·0001). Fibrinogen polymorphism There was an interaction between fibrinogen A
Thr312Ala genotype, fibrinogen concentration, and fibrin structure; the rate of change of permeability rose with increasing fibrinogen concentrations and increasing numbers of Ala312 alleles. Among individuals who had the Thr/Thr genotype, permeability decreased with increasing fibrinogen concentrations (r=–0·44, p<0·0001). The rate at which permeability decreased with increasing fibrinogen concentrations was greater in individuals heterozygous (r=–0·69, p<0·0001) and homozygous (r=–0·68, p=0·015) for the Ala312 allele than in other participants. There was no significant interaction between the B Arg448Lys genotype and fibrinogen concentrations, although permeability was consistently lower in the B Arg448Lys genotype.

Discussion Our results confirm previous findings that the factor XIII Val34Leu polymorphism affects fibrin clot structure and function at low fibrinogen concentrations. However, we now find that this effect is subject to complex gene (factor XIII, fibrinogen)-environment (fibrinogen concentration) interactions. At high concentrations of fibrinogen, plasma samples homozygous for the Leu allele form clots with increased permeability and looser structures with thicker fibres than do clots formed from plasma samples homozygous for the Val allele. Therefore, a protective effect of factor XIII 34Leu emerges in the presence of fibrinogen concentrations associated with increased vascular risk. In the past 10–15 years, many gene-association studies have been done in coronary artery disease, with mostly inconsistent results.20 Evidence of associations between environmental effects, such as smoking and diet, and interactions with the human genome have helped in the understanding of atherothrombotic processes, but the conflicting evidence suggests a complex relation between the environment and genes. Our findings support the idea that complex genegene and protein-protein interactions with the environment modulate vascular risk. The mechanisms regulating fibrin structure and function might be important in the pathogenesis of thrombotic risk, and evidence suggests that polymorphisms in the genes coding for factor XIII (Val34Leu) and fibrinogen (A
Thr312Ala, B Arg448Lys), and circulating concentrations of fibrinogen all have roles in vascular disorders. Factor XIII Val34Leu has been reported in several studies as being protective against myocardial infarction, ischaemic cerebrovascular disease, and venous thrombosis,1–3 although not all studies confirm these findings.4,5 Additionally, uncertainty exists as to the nature of this relation because invitro studies consistently report the protective 34Leu variant as paradoxically increasing fibrin cross-linking activity and producing a fibrin clot that is less susceptible to FIBRINOLYSIS at low normal fibrinogen concentrations than with the Val variants.6 Fibrinogen A
Thr312Ala has been associated with venous thrombosis and death after stroke,8,21 although the effects of this variant on fibrin structure and function are unclear. The B Arg448Lys polymorphism has been associated with cerebrovascular disease;14 in our unpublished data, we noted an association between the 448Lys allele and a tighter fibrin structure that has thinner fibres, than with other alleles.

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The role of circulating fibrinogen concentrations in vascular disease, and the effects of fibrinogen on fibrin structure and function are important in the understanding of disease mechanisms. In clinical studies, there have been many reports of associations between raised fibrinogen concentrations and risk of atherothrombotic disorders.9,10,22 In the US physicians study, for example, Ma and colleagues22 reported that fibrinogen concentrations higher than 10·0 mol/L resulted in a two-fold increase in risk of developing myocardial infarction.22 In laboratory studies, fibrinogen has an important role in improving platelet aggregation by acting as a ligand for platelet glycoprotein IIb/IIIa receptors,23 and increasing fibrinogen concentrations are associated with the development of a denser fibrin structure with thinner fibres that is resistant to fibrinolysis.11,12 These observations provide mechanisms by which raised fibrinogen concentrations may contribute to vascular disease, but the relation between fibrinogen concentrations and genetic variation in the genes that regulate fibrin structure and function has not been investigated. In previous studies, fibrin clots with low permeability have been implicated in the pathogenesis of atherothrombotic disease. Such clots generally have a fibrin meshwork with decreased fibre thickness and smaller pore size.13 Fatah and colleagues11,12 noted that clots with low permeability were associated with premature coronary artery disease. We show that raised fibrinogen concentrations affect fibrin structure to decrease permeability. Blomback and colleagues24 reported that at concentrations higher than 5·8 mo/L, fibrinogen did not significantly affect the permeability.24 However, we show that fibrinogen affects permeability over a wide range of concentrations. In addition, most previous in-vitro studies, including our own, have been done at low fibrinogen concentrations of 5·8–8·8 mol/L,3–6 which does not take into account the effects of high fibrinogen concentrations on fibrin structure. Evidence suggests that a fibrin clot structure with thick loosely packed fibres has a faster fibrinolysis rate, consistent with 34Leu exerting a protective effect at higher fibrinogen concentrations due to a relative increase in clot lysis.13,25 This effect might be harmful in the presence of a bleeding tendency. In support of this suggestion, Catto and colleagues15 reported that the Leu allele was associated with risk of haemorrhagic stroke in participants with a mean fibrinogen concentration of 12·6 mol/L. Although in several studies of factor XIII genotype associations no fibrinogen concentrations are reported, in one study in which no protective effect of 34Leu was noted, there were fewer smokers among their participants5 than in studies in which a protective effect was shown.2,3,26 Smoking raises fibrinogen concentrations.27–29 Elbaz and colleagues3 reported that the risk of brain infarction increased with the number of pack-years among Val homozygotes, but the trend was not significant among carriers of the Leu allele. Similarly, Franco and colleagues30 reported that the Leu allele attenuated the effect of smoking on the risk of myocardial infarction. Our findings may help to explain those observations. The rise in fibrinogen concentrations related to smoking would unmask the protective effect of the 34Leu allele. The implication of these findings is that hyperfibrinogenaemia is a more potent risk factor for atherothrombotic vascular disorders in people homozygous for the 34Val allele. Since this issue might be important in primary prevention of cardiovascular disease, it needs to be addressed in a prospective clinical study. We report that fibrinogen concentrations directly affect fibrin structure. The implications are that at least part of the well-documented association between fibrinogen

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References

Smoking Physical inactivity Inflammation

Fibrinogen genetics

1

Gene-environment interaction 2

Low fibrinogen

High fibrinogen

3

4

Val34Leu

Leu34

Val34

Gene-environment interaction

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6

Low-risk profile of fibrin structure function

High-risk profile of fibrin structure function

Figure 6: Overview of complex gene-environment interactions and their impact on clinical outcome Environmental effects (smoking, physical inactivity, inflammation) interact with genetic milieu (factor XIII through factor XIII Val34Leu) and fibrinogen concentrations to affect clot structure and clinical outcome.

concentrations and disease may be explained by a direct effect on fibrin structure. Moreover, we have shown genotype affects the association of fibrinogen with fibrin structure and function. Hence, the protective effect of factor XIII Leu34 may be accounted for by the formation of a more permeable fibrin structure at high fibrinogen concentrations when compared with factor XIII Val34, in contrast to observations made at fibrinogen concentrations in the normal range. High fibrinogen concentrations led to a fibrin structure that was less permeable in clots formed from A
Ala312. As well as providing evidence of complex genetic and environmental interactions (figure 6), our data help to explain the apparent paradox of factor XIII 34Leu having a protective effect against thrombotic disorders while being associated with increased activation of factor XIII in the presence of thrombin. The development of a favourable clot profile in 34Leu carriers at fibrinogen concentrations of 10·0–11·2 mol/L and higher suggests a critical range of fibrinogen concentrations above which factor XIII 34Leu has a protective effect compared to Val34 and at which A
Thr312Ala is prothrombotic. The implications of our findings are that merely reporting associations between genetic polymorphisms and cardiovascular disease is oversimplistic. Clinical studies will be required to validate the importance of the current findings in relation to risk of atherothrombotic disease and prevention of these disorders.

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Contributors Bernard Lim did the experimental work. Angela Carter did the statistical analysis. Bernard Lim, Robert Ariëns, John Weisel, and Peter Grant contributed to the overall analysis and design of the study. All investigators contributed to the writing and review of the paper.

Conflict of interest statement None declared.

Acknowledgments The study was funded by the British Heart Foundation (FS/2000023, PG/98104), and supported by grants from the Medical Research Council (G9900904, G0000624) and the National Institutes of Health (HL30954). We thank S MacLennan and E Lee from the Regional Blood Transfusion Centre of Yorkshire, Leeds, UK, for the provision of outdated transfusion plasma and buffy coat, M W Mansfield for statistical advice, and Sekar Nagaswami for technical assistance in the use of the scanning electron microscope.

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Clinical picture ECG artifact due to deep brain stimulation W A Martin, E Camenzind, P R Burkhard A 61-year-old man was referred for evaluation of recentonset anginal symptoms. He was diagnosed in 1985 with Parkinson’s disease and in 1997, bilateral subthalamic neurostimulators (Itrel II, Medtronic) were implanted for the management of severe bilateral tremor, rigidity, and akinesia uncontrolled by dopamine replacement therapy (figure, top). The electrocardiographic tracings (ECG) performed by his referring physician and on the ward (figure, bottom A and B) were uninterpretable because of artifact from the neurostimulators operating in an unipolar configuration at an amplitude of 4 V, frequency 130 Hz, pulse width 90 µsec. An effort test could not be performed and therefore he proceeded directly to diagnostic angiography. To assess the effect on the artifact and any immediate clinical sequelae, the patient’s neurologist deactivated the neurostimulators consecutively using a telemetry programmer (figure, bottom C, D, and E). Severe predominantly left-sided tremor occurred on switching off the right neurostimulator which settled rapidly on reactivation. A satisfactory tracing for ECG monitoring during the procedure was achieved with the left stimulator only turned off. A significant stenosis in the proximal left circumflex artery was found and PTCA with stenting was undertaken with a good result. Ideally the alteration or interruption of deep-brain stimulation therapy should be performed using the appropriate telemetry programmer with the assistance of the patient’s neurologist or neurophysiologist. However this may not be feasible, particularly in emergency situations. Patients may carry their own control magnet and placing this or other strong magnets (similar to those used for pacemaker inhibition) over the implantable pulse generator for <5 s will toggle an on-off switch and allow an electrocardiogram or ECG monitoring to be done without artifact. It is worth noting that deactivation can result in a rapid return of pathological involuntary movements as in our patient which itself may preclude the patient from undergoing any invasive cardiological procedures except under general anaesthesia and neuromuscular blockade. Deep brain and spinal-cord stimulation devices are being used increasingly in the management of advanced Parkinson's disease and chronic pain syndromes refractory to medication. Cardiologists and emergency department physicians will meet patients with these devices more often in the future and need to be aware of the implications and issues when these patients are hospitalised for investigation and management of acute or chronic cardiac ischaemia where initial standard investigations such as electrocardiography and ECG monitoring may be uninterpretable.

Figure (top): Chest radiograph showing bilateral pulse generators implanted subcutaneously in the pectoral area with tunneled leads in the neck; (bottom): Representative complexes from 12 lead ECGs performed: A) by referring physician (digital ECG system, Marquette; frequency filter set at 100Hz); B) on ward (Mac 8 ECG system, Marquette; frequency filter at 150Hz); C) in catheter suite using a mechanical ECG system (Minograf 7, Siemens) with both neurostimulators active; D) right neurostimulator de-activated; E) both neurostimulators de-activated.

Division of Cardiology (W A Martin MRCP, E Camenzind MD) and Division of Neurology, Hopitaux Universitaires de Genève (P R Burkhard MD), CH-1211 Geneva 14, Switzerland

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