Novel genetic risk factors for venous thrombosis; a haplotype-
based candidate gene approach
Uitte de Willige, S.
Citation
Uitte de Willige, S. (2007, May 23). Novel genetic risk factors for venous
thrombosis; a haplotype-based candidate gene approach. Hemostasis and
Thrombosis Research Center, Department of Hematology, Faculty of Medicine,
Leiden University. Retrieved from https://hdl.handle.net/1887/11970
Version: Corrected Publisher’s Version
License: Licence agreement concerning inclusion of doctoral thesis
in the Institutional Repository of the University of Leiden
Downloaded from: https://hdl.handle.net/1887/11970
Note: To cite this publication please use the final published version (if
applicable).
Chapter 3.5
Fibrinogen gamma in ischemic stroke:
a case-control study
Elim Y.L. Cheung, Shirley Uitte de Willige, Hans L. Vos, Frank W.G. Leebeek, Diederik W.J. Dippel, Rogier M. Bertina
and Moniek P.M. de Maat Submitted for publication
Abstract
Background Fibrinogen γ' results from alternative processing of the fibrinogen γ pre- mRNA. Recently, associations between fibrinogen γ' and risk of arterial and deep venous thrombosis have been reported. Furthermore, haplotypes of the fibrinogen gamma (FGG) gene have been associated with fibrinogen γ' levels, the fibrinogen γ'/total fibrinogen ratios and risk of thrombosis.
Objectives: The aim of this study was to determine the role of fibrinogen γ' and FGG haplotypes in ischemic stroke.
Patients/Methods The associations between fibrinogen γ' levels, the fibrinogen γ'/total fibrinogen ratio, and FGG haplotypes and the risk of ischemic stroke were determined in 124 cases and 125 age- and sex- matched population controls.
Results The fibrinogen γ'/total fibrinogen ratio was increased in stroke patients in the acute phase compared with the control subjects. In contrast, in the convalescent phase 3 months after the stroke the ratio was lower than the ratio in the control subjects. FGG haplotype 3 (H3) was associated with a reduced risk of ischemic stroke (odds ratio (OR) 0.61, 95% confidence interval (CI) 0.38-0.95), but not with the fibrinogen γ'/total fibrinogen ratio. In contrast, FGG-H2 was associated with a decreased fibrinogen γ'/total fibrinogen ratio, but not with risk of stroke.
Conclusions The results of our study show an association between fibrinogen γ'/total fibrinogen ratio and ischemic stroke, both in the acute and the convalescent phase of the disease. In addition, carriers of the FGG-H3 haplotype appear to be protected against ischemic stroke.
Introduction
Fibrinogen is a central protein in the haemostatic system. At the final stage of the coagulation cascade, thrombin cleaves fibrinopeptide A and B from the amino termini of the fibrinogen Aα and Bβ chains, converting the soluble fibrinogen into fibrin monomers, which then polymerize to an insoluble fibrin clot. Fibrinogen is a 340-kDa plasma glycoprotein that is synthesized in the liver. It is present in plasma at a concentration of approximately 9 μM (2.6 g/L). The fibrinogen molecule comprises two identical disulphide-linked halves, each consisting of three polypeptide chains termed Aα, Bβ and γ.1 The Aα, Bβ and γ chains are encoded by the fibrinogen alpha (FGA), fibrinogen beta (FGB) and fibrinogen gamma (FGG) genes, respectively. These three genes are located in a 51 kb region on the long arm of chromosome 4.2
A high degree of heterogeneity in the fibrinogen molecule is present in healthy individuals.3,4 Approximately 7-15% of fibrinogen molecules carry an extended γ chain (fibrinogen γ'),5-7 which is the result of alternative processing of the fibrinogen γ pre-mRNA.8 The γ' chain contains 427 amino acids and a unique twenty amino acid sequence (VRPEHPAETEYDSLYPEDDL) replaces the carboxyl terminal four amino
Chapter 3.5
102
acids (AGDV) present in the normal γA chain.9 The γ' chain contains binding sites for thrombin and the factor XIII B subunit,10 but it can no longer bind platelets, because the binding site for platelet integrin αIIbβ3 is disrupted.11 These unique properties of the γ' chain may influence thrombus formation.12 Indeed, a recent study demonstrated an increased risk of deep venous thrombosis for individuals with a reduced fibrinogen γ'/total fibrinogen ratio.13 On the other hand, increased plasma levels of fibrinogen γ' have been reported in patients with coronary artery disease.14 Both associations, although apparently conflicting, indicate that plasma fibrinogen γ' could be an independent risk factor for thrombotic disease.
Polymorphisms in the fibrinogen genes have been associated with fibrinogen levels and functions15-17 and recent studies indicated that haplotypes of the fibrinogen gamma gene are associated with fibrinogen γ' levels, the fibrinogen γ'/total fibrinogen ratio and risk of deep venous thrombosis13 and also with the risk of myocardial infarction.18
Although many studies have confirmed that plasma fibrinogen is an acute phase protein, and high total fibrinogen levels are associated with an increased risk of cardiovascular disease and stroke,19-22 little is known about the role of the fibrinogen γ' variant and genetic variation in FGG in cardiovascular disease. Therefore, the aim of this study was to investigate the association between plasma fibrinogen γ', genetic variations in the FGG gene and ischemic stroke.
Patients, materials and methods Study population
We performed a case-control study with 124 first-ever ischemic stroke or transient ischemic attack (TIA) patients, and 125 controls, aged 18-75 years old. The design of this study has been described in detail previously.23 Briefly, cases were consecutively recruited Caucasian patients with first-ever acute ischemic stroke or TIA. We used population controls, i.e. partners, friends or neighbors of the patients, who were age- and sex-matched, of the same race, without a history of stroke and not related to the patients. Ischemic stroke was defined as the acute onset of focal cerebral dysfunction, because of cerebral ischemia with symptoms lasting more than 24 hours. Patients with TIA (symptoms lasting less than 24 hours) were included if the neurological deficit in the acute phase was observed by a neurologist. All cases were confirmed by a computed tomography of the brain. From both patients and controls, we collected detailed information about cardiovascular risk factors.
Informed consent was obtained from all participants. This study was approved by the local ethics committee and was performed in accordance with the Declaration of Helsinki.
Biochemical analyses
Venipunctures were performed (Vacutainer system, Becton-Dickinson, Plymouth, UK) in the acute phase of the stroke (7-14 days after the event) and in part of the patients also in the convalescent phase of the stroke (three months after the event).
Blood was collected in tubes containing0.129 mol/L sodium citrate in a ratio of 9:1.
After centrifugation at 2000 x g at 4°C for 30 minutes, plasma was stored in aliquots at -70 °C. Genomic DNA was isolated using standard salting-out procedures and stored in aliquots at 4°C. DNA samples were available from 123 patients and 115 control subjects.
Fibrinogen γ' antigen levels were measured by ELISA as described previously.13 Briefly, a mouse anti-human γ' fibrinogen monoclonal antibody (2.G2.H9; Upstate, Charlottesville, VA, USA) was used as catching antibody, and a horseradish peroxidase (HRP)-conjugated rabbit anti-human fibrinogen (DAKO A/S, Glostrup, Denmark) was used as tagging antibody. A human pooled normal plasma (George King Bio-Medical, Overland Park, KS) was used as calibrator, containing 1 U/mL fibrinogen γ' by definition. ELISA measurements were carried out in duplicate.
Plasma samples for measurement of fibrinogen γ' levels were available in 114 patients (samples collected during the acute phase) and in 120 healthy controls. In an unselected group of 48 patients, fibrinogen γ' levels were also measured in the samples collected at 3 months after the event.
Total fibrinogen levels were measured according to von Clauss24 using the Hemosil Fibrinogen-C kit on an automated coagulation analyzer (ACL 300, Instrumentation Laboratory). Missing data were mainly due to unavailability of DNA sample or plasma sample.
Genetic analyses
The SeattleSNPs program for Genomic Applications has identified three haplotype- tagging single nucleotide polymorphisms (SNPs) which tag the total common genetic variation in FGG in Caucasians.25 SNP 8486C>T (rs2066865, 10034C>T by SeattleSNPs) tagged FGG haplotype 2 (H2), SNP 7792T>C (rs1049636, 9340T>C by SeattleSNPs) tagged FGG-H3 and SNP 4288G>A (rs2066860, 5836G>A by SeattleSNPs) tagged FGG-H4. FGG-H1 was assigned to subjects who possessed the common alleles of the three SNPs. Annotation of the SNPs is according to the nomenclature recommendations of the Human Genome Variation Society using GenBank Accession Number AF350254 as reference with nucleotide +1 being the A of the ATG translation initiation codon.26 We determined the three SNPs using 5' nuclease/TaqMan assays as described previously.27 Polymerase chain reactions with fluorescent allele-specific oligonucleotide probes (Assay-by-Design/Assay-on- Demand; Applied Biosystems, Foster City, CA, USA) were performed on a PTC-225 thermal cycler (MJ Research, Cambridge, MA, USA), and fluorescence clustered
Chapter 3.5
104
endpoint reading for allelic discrimination was read on an ABI 7900HT instrument (Applied Biosystems). Primer sequences and probe sequences are available upon request.
Statistical analyses
In order to simplify the interpretation of the fibrinogen γ'/total fibrinogen ratio, the ratio was calculated after converting total fibrinogen levels to units per milliliter (2.80g/L=1 U/mL). Mean values are presented with standard deviation (SD).
Differences between groups were examined by analysis of variance (ANOVA) for patients and controls or paired-samples t-test for acute phase versus convalescent phase in patients. Hardy-Weinberg equilibrium for each SNP was assessed using a Chi-square test in healthy controls. We used the program Haplo.Stats (http://cran.r- project.org/src/contrib/Descriptions/haplo.stats.html)28,29 to determine the association between maximum likelihood estimates of haplotype probability, taking haplotype ambiguity into account. An estimate of the haplotype frequencies was based on the expectation-maximization (EM) algorithm. We determined the association between FGG haplotypes and risk of stroke using the binary family of generalized linear model (GLM) framework. The effect of haplotypes on the fibrinogen γ' level or the fibrinogen γ'/total fibrinogen ratio was computed using the Gaussian family of GLM framework, assuming additivity of the effects of the alleles. A P-value less than 0.05 was considered statistically significant. All statistical analyses were carried out with the SPSS for Windows version 11.0.1 statistical package or Haplo.Stats version 1.2.2.
Results
Table 1 Baseline characteristics of the study participants
Patients Controls
(n=124) (n=125) P
Demographics
Age, years (SD) 56 (±12) 56 (±12) NS
Sex (% female) 58 (47%) 59 (47%) NS
Index event
Stroke : TIA 115 : 9 NA NA
Risk factors
Smoking 61 (49%) 37 (30%) 0.004
Hypertension 60 (48%) 24 (19%) <0.001 Diabetes mellitus 18 (14%) 5 (4%) 0.004 Hypercholesterolemia 78 (63%) 84 (67%) 0.700 Positive family history for
cardiovascular disease 75 (61%) 56 (45%) 0.013 Data for age presented as mean ± SD. Other data are counts and (percentages). SD=Standard Deviation; TIA=Transient Ischemic Attack;
NA=Not Applicable; NS=Not Significant.
Patient population
The baseline characteristics of all patients and controls are shown in Table 1. The mean age of patients and controls was 56 years (SD, 12 years), and 47% of the participants were female. As expected, established risk factors like smoking, hypertension, and diabetes were more frequently present in patients than in controls.
Fibrinogen γ' and stroke
In the acute phase of stroke, the mean plasma fibrinogen γ' level was higher in patients (1.55±0.64 U/mL) than in controls (1.24±0.38 U/mL, P <0.001) (Table 2).
To account for total fibrinogen levels that were also increased in the acute phase of stroke (3.69±1.06 g/L in patients versus 3.41±0.61 g/L in controls), we calculated the fibrinogen γ'/total fibrinogen ratio. This ratio was significantly higher in patients (1.16±0.35) than in controls (1.03±0.30, P =0.002) (Table 2).
Table 2 Fibrinogen γ' levels, fibrinogen γ'/total fibrinogen ratio and total fibrinogen levels in patients and controls
Patients Controls
Acute Convalescent
phase phase P* P† P‡
Fibrinogen γ' 1.55 (0.64) 1.08 (0.33) 1.24 (0.38) <0.001 0.010 <0001 Fibrinogen γ'/total
fibrinogen ratio 1.16 (0.35) 0.91 (0.21) 1.03 (0.30) 0.002 0.010 <0.001 Total fibrinogen 3.69 (1.06) 3.34 (0.76) 3.41 (0.61) 0.006 0.540 0.003 Levels are presented as mean (SD). P*: P-value between controls and patients in acute phase, P†:
P-value between controls and patients in convalescent phase, P‡: P-value between patients in acute and convalescent phase. SD=Standard Deviation.
In the samples collected 3 months after the event, the mean fibrinogen γ' level and fibrinogen γ'/total fibrinogen ratio (1.08±0.33 U/mL and 0.91±0.21, respectively) were significantly lower than the fibrinogen γ' level and fibrinogen γ'/total fibrinogen ratio in the acute phase of stroke, and also compared to controls (Table 2).
FGG haplotypes and risk of ischemic stroke
In the control subjects, the genotypic distributions of all three SNPs were in Hardy- Weinberg equilibrium. The frequency of the FGG-H3 allele was lower in patients (0.24) than in controls (0.33). Carriers of the FGG-H3 allele had a significantly reduced risk of ischemic stroke (OR 0.60, 95%CI 0.38-0.94). A slightly, although not significantly, decreased risk was also seen for FGG-H4 (OR 0.75, 95%CI:0.30- 1.88). For the FGG-H2, no clear association with stroke was observed (Table 3).
Chapter 3.5
106
Table 3 Associations between FGG haplotypes and risk of ischemic stroke Allele Allele
Frequency Frequency Haplotypes
(Patients) (Controls)
OR (95%CI)
H1 (GTC) 0.48 0.39 Reference
H2 (GTT) 0.23 0.23 0.91 (0.58-1.43)
H3 (GCC) 0.24 0.33 0.60 (0.34-0.94)
H4 (ATC) 0.05 0.05 0.75 (0.30-1.88)
Between brackets the nucleotides of the tagging SNPs are given (4288G>A, 7792T>C and 8486C>T, respectively). The minor allele is underlined.
Effect of FGG haplotypes on fibrinogen γ' or total fibrinogen levels
Compared with the reference haplotype FGG-H1, FGG-H2 was associated with a significantly decreased fibrinogen γ'/total fibrinogen ratio in controls and in patients during the acute phase (decrease of 0.093±0.013 and 0.085±0.020, respectively, both P <0.001) or in the convalescent phase (decrease of 0.060±0.015, P <0.001) (Figure 1). No significant relationship was observed between FGG-H3 or FGG-H4 and the fibrinogen γ'/total fibrinogen ratio. Neither did we observe an association between total fibrinogen levels and FGG haplotypes.
Figure 1 Effect of FGG haplotypes on the fibrinogen γ'/total fibrinogen ratio. H1 in controls was used as reference group. * indicates a significant difference from H1 of each subgroup.
Discussion
In the present study, we report that the fibrinogen γ'/total fibrinogen ratio is increased during the acute phase of ischemic stroke, while in the convalescent phase this ratio is decreased and is even lower than the ratio in controls.
Furthermore, FGG-H3 was associated with a reduced risk of ischemic stroke, but not with a change in fibrinogen γ'/total fibrinogen ratio. On the other hand, FGG-H2 carriers had significantly decreased fibrinogen γ'/total fibrinogen ratio, while FGG-H2 was not associated with an increased risk of stroke.
We observed elevated fibrinogen γ' levels and fibrinogen γ'/total fibrinogen ratios in patients with ischemic stroke in the acute phase of their disease. This suggests that the alternative processing mechanism might be affected by the acute phase. For other genes, such an effect has also been suggested.30-32 Another possible mechanism explaining our results may be that the clearance of fibrinogen γA and γ' from the circulation is different during various stages, as suggested by Drouet et al.12 The association between fibrinogen γ'/total fibrinogen ratios and stroke in the convalescent phase was opposite to the association in the acute phase. This interesting observation may be the result of different regulation of alternative processing in the acute and the convalescent phase of stroke.
Fibrinogen γ' may influence the development of thrombosis via several mechanisms.
Fibrinogen γ' contains a unique high-affinity binding site for thrombin and is also known as antithrombin I, because of its inhibitory role on thrombin generation.33,34 High fibrinogen γ' levels are thus expected to have an antithrombotic effect. In addition, the disruption of the binding site for the platelet integrin αIIbβ3 in fibrinogen γ'11 would also predict an antithrombotic effect of increased fibrinogen γ' levels.35 On the other hand, increased fibrinogen γ' levels could also be hypothesized to result in a prothrombotic state, because it results in a fibrin structure with increased resistance to fibrinolysis.36,37 The carboxyl terminus of the γ' chain contains a unique binding site for factor XIIIB,10,38,39 which cross-links fibrin and fibrinogen during polymerization and thereby gives a denser and less permeable fibrin structure such as present in patients with myocardial infarction40,41 and coronary artery disease.42,43
We also studied the fibrinogen γ'/total fibrinogen ratio in the convalescent phase of stroke. The fibrinogen γ'/total fibrinogen ratio in the samples collected 3 months after stroke was significantly lower than the ratio in the samples collected in the acute phase in the same group of patients, and also significantly lower than the ratio in controls. The high fibrinogen γ'/total fibrinogen ratio in the acute phase of the disease may be part of a protective, antithrombotic mechanism of the human body, and studies relating secondary events to fibrinogen γ'/total fibrinogen ratios need to be performed to test this hypothesis. Our observations in the convalescent phase are in line with the results of Uitte de Willige and colleagues,13 who recently demonstrated that reduced fibrinogen γ'/total fibrinogen ratios were associated with an increased risk for deep venous thrombosis. In contrast, two studies have reported that increased fibrinogen γ' levels or fibrinogen γ'/total fibrinogen ratios
Chapter 3.5
108
were associated with risk of arterial thrombotic disease.12,14 The discrepancy between these studies may be explained by differences in endpoint, time after the event of blood sampling, medication and performance of the fibrinogen γ' assay. It may also support that there is a difference in etiology of arterial and venous thrombotic diseases.
An association between genetic variations in the FGG gene and the fibrinogen γ'/total fibrinogen ratio has been recently reported.13 Therefore, we examined the association between FGG haplotypes and fibrinogen γ'/total fibrinogen ratio in ischemic stroke. The fibrinogen γ'/total fibrinogen ratio was indeed strongly reduced in FGG-H2. This effect was seen both in cases (acute phase and convalescent phase) and in controls. The other haplotypes were not significantly associated with the fibrinogen γ'/total fibrinogen ratio in patients nor in controls. It has been observed that the FGG-H2 haplotype gives an improvement of a cleavage stimulatory factor binding site near the γA specific polyadenylation site, which is predicted to result in a relative decrease of the formation of fibrinogen γ' specific mRNA, explaining the reduced fibrinogen γ'/total fibrinogen ratio associated with FGG-H2 (unpublished results).
No association between FGG haplotypes and total fibrinogen levels was observed in our study, which is in accordance with the results of two other studies using Caucasian populations,13,18 but contradicts the results using an European- and African-American population.44
We observed that carriers of FGG-H3 had a reduced risk of ischemic stroke. The underlying mechanism of this association is probably not via an effect on fibrinogen γ', since we did not observe a significant association between the haplotype and fibrinogen γ' levels or the fibrinogen γ'/total fibrinogen ratio, but only a slight increase in all subgroups. Mannila et al observed a similar association between FGG- H3 (called SNP2 in their study) and the risk of myocardial infarction.18 However, Uitte de Willige and colleagues found no significant association between FGG-H3 and the risk of myocardial infarction45 or deep vein thrombosis.13 It is unclear what the underlying mechanisms that influence the development of these diseases might be, since no amino acid variations or known regulatory elements have so far been found to be related to FGG-H3 tagging SNP 7792T>C or SNPs in linkage disequilibrium with it. The results in the literature on the association between γ' and risk of thrombosis are not consistent. A relationship between FGG-H2 and increased risk of venous thrombosis was recently observed,13 whereas Mannila et al and Uitte de Willige et al found no association between FGG-H2 and risk of myocardial infarction.18,45 However, deep venous thrombosis and myocardial infarction may have different mechanisms for the FGG haplotypes and risk of these diseases.
Limitations of our study are the relatively small size of the study population, and the use of a Caucasian population, since it is unclear whether these results can be extrapolated to other populations. Our study only included non-fatal cases of stroke and therefore it is expected that we underestimate the true risk for ischemic stroke if the fatal cases were associated with specific FGG alleles. However, it seems unlikely that the specific FGG alleles play a major role in fatal ischemic stroke only.
Another limitation is that only a small, though unselected, group of patients has been followed-up in the convalescent phase, and clinical outcomes on recurrence or occurrence of other cardiovascular disease were not available, indicating that further studies are needed to investigate the clinical implications.
In conclusion, this study on the association between the fibrinogen γ'/total fibrinogen ratio and ischemic stroke in the acute phase and the convalescent phase shows that the fibrinogen γ'/ total fibrinogen ratio is increased in the acute phase of stroke, which may be an antithrombotic defense mechanism of the human body.
Additionally, carriers of the FGG-H3 haplotype appear to be protected against ischemic stroke.
Acknowledgements
This study was supported by grants from the Dutch program for Tissue Engineering, revolving fund from the Erasmus University Medical Center and grant 912-02-036 from the Netherlands Organization for Scientific Research (NWO).
References
1. Mosesson MW, Siebenlist KR, Meh DA. The structure and biological features of fibrinogen and fibrin. Ann N Y Acad Sci. 2001;936:11-30.
2. Kant JA, Fornace AJ, Jr., Saxe D, Simon MI, McBride OW, Crabtree GR. Evolution and organization of the fibrinogen locus on chromosome 4: gene duplication accompanied by transposition and inversion. Proc Natl Acad Sci U S A. 1985;82:2344-2348.
3. Henschen-Edman AH. Fibrinogen non-inherited heterogeneity and its relationship to function in health and disease. Ann N Y Acad Sci. 2001;936:580-593.
4. de Maat MP, Verschuur M. Fibrinogen heterogeneity: inherited and noninherited. Curr Opin Hematol. 2005;12:377-383.
5. Mosesson MW, Finlayson JS, Umfleet RA. Human fibrinogen heterogeneities. 3.
Identification of chain variants. J Biol Chem. 1972;247:5223-5227.
6. Wolfenstein-Todel C, Mosesson MW. Human plasma fibrinogen heterogeneity: evidence for an extended carboxyl-terminal sequence in a normal gamma chain variant (gamma'). Proc Natl Acad Sci U S A. 1980;77:5069-5073.
7. Francis CW, Marder VJ, Martin SE. Demonstration of a large molecular weight variant of the gamma chain of normal human plasma fibrinogen. J Biol Chem. 1980;255:5599- 5604.
8. Chung DW, Davie EW. Gamma and gamma' chains of human fibrinogen are produced by alternative mRNA processing. Biochemistry. 1984;23:4232-4236.
9. Wolfenstein-Todel C, Mosesson MW. Carboxy-terminal amino acid sequence of a human fibrinogen gamma-chain variant (gamma'). Biochemistry. 1981;20:6146-6149.
10. Mosesson MW. Fibrinogen and fibrin structure and functions. J Thromb Haemost.
2005;3:1894-1904.
11. Farrell DH, Thiagarajan P, Chung DW, Davie EW. Role of fibrinogen alpha and gamma chain sites in platelet aggregation. Proc Natl Acad Sci U S A. 1992;89:10729-10732.
Chapter 3.5
110
12. Drouet L, Paolucci F, Pasqualini N, Laprade M, Ripoll L, Mazoyer E, Bal dit SC, Vanhove N. Plasma gamma'/gamma fibrinogen ratio, a marker of arterial thrombotic activity: a new potential cardiovascular risk factor? Blood Coagul Fibrinolysis. 1999;10 Suppl 1:S35-S39.
13. Uitte de Willige S, de Visser MCH, Houwing-Duistermaat JJ, Rosendaal FR, Vos HL, Bertina RM. Genetic variation in the fibrinogen gamma gene increases the risk of deep venous thrombosis by reducing plasma fibrinogen {gamma}' levels. Blood. 2005;106.
14. Lovely RS, Falls LA, Al Mondhiry HA, Chambers CE, Sexton GJ, Ni H, Farrell DH.
Association of gammaA/gamma' fibrinogen levels and coronary artery disease. Thromb Haemost. 2002;88:26-31.
15. Thomas AE, Green FR, Kelleher CH, Wilkes HC, Brennan PJ, Meade TW, Humphries SE.
Variation in the promoter region of the beta fibrinogen gene is associated with plasma fibrinogen levels in smokers and non-smokers. Thromb Haemost. 1991;65:487-490.
16. Fowkes FG, Connor JM, Smith FB, Wood J, Donnan PT, Lowe GD. Fibrinogen genotype and risk of peripheral atherosclerosis. Lancet. 1992;339:693-696.
17. Green FR. Fibrinogen polymorphisms and atherothrombotic disease. Ann N Y Acad Sci.
2001;936:549-559.
18. Mannila MN, Eriksson P, Lundman P, Samnegard A, Boquist S, Ericsson CG, Tornvall P, Hamsten A, Silveira A. Contribution of haplotypes across the fibrinogen gene cluster to variation in risk of myocardial infarction. Thromb Haemost. 2005;93:570-577.
19. Wilhelmsen L, Svardsudd K, Korsan-Bengtsen K, Larsson B, Welin L, Tibblin G.
Fibrinogen as a risk factor for stroke and myocardial infarction. N Engl J Med.
1984;311:501-505.
20. Maresca G, Di Blasio A, Marchioli R, Di Minno G. Measuring plasma fibrinogen to predict stroke and myocardial infarction: an update. Arterioscler Thromb Vasc Biol.
1999;19:1368-1377.
21. Ernst E, Resch KL. Fibrinogen as a cardiovascular risk factor: a meta-analysis and review of the literature. Ann Intern Med. 1993;118:956-963.
22. van Goor MP, Gomez-Garcia EB, Leebeek FW, Brouwers GJ, Koudstaal PJ, Dippel DW.
The -148 C/T fibrinogen gene polymorphism and fibrinogen levels in ischaemic stroke: a case-control study. J Neurol Neurosurg Psychiatry. 2005;76:121-123.
23. Van Goor ML, Garcia EG, Leebeek F, Brouwers GJ, Koudstaal P, Dippel D. The plasminogen activator inhibitor (PAI-1) 4G/5G promoter polymorphism and PAI-1 levels in ischemic stroke. A case-control study. Thromb Haemost. 2005;93:92-96.
24. Clauss, A. Gerinnungsphysiologische Schnellmethode zur Bestimmung des Fibrinogens.
Acta Haematologica 17, 237-246. 1957.
25. Nickerson, D. SeattleSNPs. NHLBI Program for Genomic Applications, UW-FHCRC, Seattle, WA. http://pga.gs.washington.edu. 2005.
26. den Dunnen JT, Antonarakis SE. Mutation nomenclature extensions and suggestions to describe complex mutations: a discussion. Hum Mutat. 2000;15:7-12.
27. Livak KJ. Allelic discrimination using fluorogenic probes and the 5' nuclease assay.
Genet Anal. 1999;14:143-149.
28. Schaid DJ, Rowland CM, Tines DE, Jacobson RM, Poland GA. Score tests for association between traits and haplotypes when linkage phase is ambiguous. Am J Hum Genet.
2002;70:425-434.
29. Lake SL, Lyon H, Tantisira K, Silverman EK, Weiss ST, Laird NM, Schaid DJ. Estimation and tests of haplotype-environment interaction when linkage phase is ambiguous. Hum Hered. 2003;55:56-65.
30. Bruggemann LW, Drijfhout JW, Reitsma PH, Spek CA. Alternatively spliced tissue factor in mice: induction by Streptococcus pneumoniae. J Thromb Haemost. 2006;4:918-920.
31. Xiong Z, Shaibani A, Li YP, Yan Y, Zhang S, Yang Y, Yang F, Wang H, Yang XF.
Alternative splicing factor ASF/SF2 is down regulated in inflamed muscle. J Clin Pathol.
2006;59:855-861.
32. Mizgerd JP, Spieker MR, Lupa MM. Exon truncation by alternative splicing of murine ICAM-1. Physiol Genomics. 2002;12:47-51.
33. de Bosch NB, Mosesson MW, Ruiz-Saez A, Echenagucia M, Rodriguez-Lemoin A.
Inhibition of thrombin generation in plasma by fibrin formation (Antithrombin I).
Thromb Haemost. 2002;88:253-258.
34. Mosesson MW. Antithrombin I. Inhibition of thrombin generation in plasma by fibrin formation. Thromb Haemost. 2003;89:9-12.
35. Massberg S, Schurzinger K, Lorenz M, Konrad I, Schulz C, Plesnila N, Kennerknecht E, Rudelius M, Sauer S, Braun S, Kremmer E, Emambokus NR, Frampton J, Gawaz M.
Platelet adhesion via glycoprotein IIb integrin is critical for atheroprogression and focal cerebral ischemia: an in vivo study in mice lacking glycoprotein IIb. Circulation.
2005;112:1180-1188.
36. Cooper AV, Standeven KF, Ariëns RA. Fibrinogen gamma-chain splice variant {gamma}' alters fibrin formation and structure. Blood. 2003;102:535-540.
37. Falls LA, Farrell DH. Resistance of gammaA/gamma' fibrin clots to fibrinolysis. J Biol Chem. 1997;272:14251-14256.
38. Mosesson MW, Siebenlist KR, Hainfeld JF, Wall JS. The covalent structure of factor XIIIa crosslinked fibrinogen fibrils. J Struct Biol. 1995;115:88-101.
39. Mosesson MW. Fibrinogen gamma chain functions. J Thromb Haemost. 2003;1:231-238.
40. Fatah K, Hamsten A, Blomback B, Blomback M. Fibrin gel network characteristics and coronary heart disease: relations to plasma fibrinogen concentration, acute phase protein, serum lipoproteins and coronary atherosclerosis. Thromb Haemost.
1992;68:130-135.
41. Fatah K, Beving H, Albage A, Ivert T, Blomback M. Acetylsalicylic acid may protect the patient by increasing fibrin gel porosity. Is withdrawing of treatment harmful to the patient? Eur Heart J. 1996;17:1362-1366.
42. Mills JD, Ariens RA, Mansfield MW, Grant PJ. Altered fibrin clot structure in the healthy relatives of patients with premature coronary artery disease. Circulation.
2002;106:1938-1942.
43. Collet JP, Allali Y, Lesty C, Tanguy ML, Silvain J, Ankri A, Blanchet B, Dumaine R, Gianetti J, Payot L, Weisel JW, Montalescot G. Altered fibrin architecture is associated with hypofibrinolysis and premature coronary atherothrombosis. Arterioscler Thromb Vasc Biol. 2006;26:2567-2573.
44. Reiner AP, Carty CL, Carlson CS, Wan JY, Rieder MJ, Smith JD, Rice K, Fornage M, Jaquish CE, Williams OD, Tracy RP, Lewis CE, Siscovick DS, Boerwinkle E, Nickerson DA.
Association between patterns of nucleotide variation across the three fibrinogen genes and plasma fibrinogen levels: the Coronary Artery Risk Development in Young Adults (CARDIA) study. J Thromb Haemost. 2006;4:1279-1287.
45. Uitte de Willige S, Doggen CJM, de Visser MCH, Bertina RM, Rosendaal FR. Haplotypes of the fibrinogen gamma gene do not affect the risk of myocardial infarction. J Thromb Haemost. 2006;4:474-476.
