Thrombin generation as an intermediate phenotype for venous thrombosis

Summary

In vitro thrombin generation, which reflects an individual’s plasma coagulation potential and has been shown to correlate with the risk of venous thromboembolism (VTE), might represent a useful intermediate phenotype for the genetic dissection of VTE. As a proof of principle, we have investigated whether the thrombin generation assay can detect changes in the haemostatic balance associated with common genetic variation affecting the level or function of coagulation factors and inhibitors. The study population consisted of 140 healthy individuals. Plasma levels of coagulation factors and inhibitors and thrombin generation parameters determined at low tissue factor (TF) ± thrombomodulin (TM) and at high TF ± activated protein C (APC) were available from a previous study. All individuals were genotyped for F5 Leiden, F2 G20210A and 19 additional single nucleotide polymorphisms (SNPs) in haemostasis-related genes. The association of each SNP with plasma levels of the corresponding proteins and with thrombin generation parameters (lag time, peak height and endogenous thrombin potential [ETP]) was evaluated by statistical analysis. Not only F5 Leiden and F2 G20210A, but also several other common SNPs, significantly affected thrombin generation parameters. In particular, FGA A1069G (Thr312Ala) decreased the ETP_-APC, F2 A19911G increased the ETP_-APC, F10 IVS2 C+517G decreased the ETP_+APC, F12 C-46T decreased peak height at low TF, and TFPI T-287C and TFPI IVS7 T-33C decreased the ETP_+APC. These results indicate that the thrombin generation assay is sensitive to genetic variation in haemostasis-related genes, which makes it a promising tool to identify novel genetic risk factors of VTE.

Keywords

VTE - thrombin generation - SNP - intermediate phenotype

• References

• 1 Souto JC. et al. Genetic susceptibility to thrombosis and its relationship to physiological risk factors: the GAIT study. Genetic Analysis of Idiopathic Thrombophilia. Am J Hum Genet 2000; 67: 1452-1459.
• 2 Reitsma PH, Rosendaal FR. Past and future of genetic research in thrombosis. J Thromb Haemost 2007; 05 (Suppl. 01) 264-269.
• 3 Smith NL. et al. Association of genetic variations with nonfatal venous thrombosis in postmenopausal women. J Am Med Assoc 2007; 297: 489-498.
• 4 Bezemer ID. et al. Gene variants associated with deep vein thrombosis. J Am Med Assoc 2008; 299: 1306-1314.
• 5 Soria JM, Fontcuberta J. New approaches and future prospects for evaluating genetic risk of thrombosis. Haematologica 2005; 90: 1212-1222.
• 6 Wichers IM. et al. Assessment of coagulation and fibrinolysis in families with unexplained thrombophilia. Thromb Haemost 2009; 101: 465-470.
• 7 Hemker HC. et al. The calibrated automated thrombogram (CAT): a universal routine test for hyper- and hypocoagulability. Pathophysiol Haemost Thromb 2002; 32: 249-253.
• 8 Dielis AW. et al. Coagulation factors and the protein C system as determinants of thrombin generation in a normal population. J Thromb Haemost 2008; 06: 125-131.
• 9 Brandts A. et al. The risk of venous thrombosis associated with a high endogenous thrombin potential in the absence and presence of activated protein C. J Thromb Haemost 2007; 05: 416-418.
• 10 Dargaud Y. et al. Use of calibrated automated thrombinography +/- thrombomodulin to recognise the prothrombotic phenotype. Thromb Haemost 2006; 96: 562-567.
• 11 van Hylckama Vlieg A. et al. Elevated endogenous thrombin potential is associated with an increased risk of a first deep venous thrombosis but not with the risk of recurrence. Br J Haematol 2007; 138: 769-774.
• 12 Tripodi A. et al. The endogenous thrombin potential and the risk of venous thromboembolism. Thromb Res 2007; 121: 353-359.
• 13 ten Cate-Hoek AJ. et al. Thrombin generation in patients after acute deep-vein thrombosis. Thromb Haemost 2008; 100: 240-245.
• 14 Hron G. et al. Identification of patients at low risk for recurrent venous thromboembolism by measuring thrombin generation. J Am Med Assoc 2006; 296: 397-402.
• 15 Eichinger S. et al. Prediction of recurrent venous thromboembolism by endogenous thrombin potential and D-dimer. Clin Chem 2008; 54: 2042-2048.
• 16 Besser M. et al. High rate of unprovoked recurrent venous thrombosis is associated with a high thrombin generating potential in a prospective cohort study. J Thromb Haemost 2008; 06: 1720-1725.
• 17 Tripodi A. et al. High thrombin generation measured in the presence of thrombomodulin is associated with an increased risk of recurrent venous thromboembolism. J Thromb Haemost 2008; 06: 1327-1333.
• 18 Lutsey PL. et al. Peak thrombin generation and subsequent venous thromboembolism: the longitudinal investigation of thromboembolism etiology (LITE). J Thromb Haemost 2009; 07: 1639-1648.
• 19 Bertina RM. et al. Mutation in blood coagulation factor V associated with resistance to activated protein C. Nature 1994; 369: 64-67.
• 20 Poort SR. et al. A common genetic variation in the 3‘-untranslated region of the prothrombin gene is associated with elevated plasma prothrombin levels and an increase in venous thrombosis. Blood 1996; 88: 3698-3703.
• 21 Duchemin J. et al. Influence of coagulation factors and tissue factor concentration on the thrombin generation test in plasma. Thromb Haemost 2008; 99: 767-773.
• 22 Standeven KF. et al. Functional analysis of the fibrinogen Aalpha Thr312Ala polymorphism: effects on fibrin structure and function. Circulation 2003; 107: 2326-2330.
• 23 Carter AM. et al. Alpha-fibrinogen Thr312Ala polymorphism and venous thromboembolism. Blood 2000; 96: 1177-1179.
• 24 Uitte de Willige S. et al. Genetic variation in the fibrinogen gamma gene increases the risk for deep venous thrombosis by reducing plasma fibrinogen gamma’ levels. Blood 2005; 106: 4176-4183.
• 25 Martinelli I. et al. Prothrombin A19911G polymorphism and the risk of venous thromboembolism. J Thromb Haemost 2006; 04: 2582-2586.
• 26 Chinthammitr Y. et al. The association of prothrombin A19911G polymorphism with plasma prothrombin activity and venous thrombosis: results of the MEGA study, a large population-based case-control study. J Thromb Haemost 2006; 04: 2587-2592.
• 27 Yamazaki T. et al. Molecular basis of quantitative factor V deficiency associated with factor V R2 haplotype. Blood 2002; 100: 2515-2521.
• 28 Scanavini D. et al. Modulation of factor V levels in plasma by polymorphisms in the C2 domain. Arterioscler Thromb Vasc Biol 2004; 24: 200-206.
• 29 Siegert G. et al. Method-dependent influence of certain polymorphisms in the factor V B-domain on the response to activated protein C. Blood Coagul Fibrinolysis 2000; 11: 519-527.
• 30 Green F. et al. A common genetic polymorphism associated with lower coagulation factor VII levels in healthy individuals. Arterioscler Thromb 1991; 11: 540-546.
• 31 Marchetti G. et al. A polymorphism in the 5’ region of coagulation factor VII gene (F7) caused by an inserted decanucleotide. Hum Genet 1993; 90: 575-576.
• 32 Kanaji T. et al. A common genetic polymorphism (46 C to T substitution) in the 5‘-untranslated region of the coagulation factor XII gene is associated with low translation efficiency and decrease in plasma factor XII level. Blood 1998; 91: 2010-2014.
• 33 Tirado I. et al. Association after linkage analysis indicates that homozygosity for the 46C-->T polymorphism in the F12 gene is a genetic risk factor for venous thrombosis. Thromb Haemost 2004; 91: 899-904.
• 34 Bertina RM. et al. The 46C-->T polymorphism in the factor XII gene (F12) and the risk of venous thrombosis. J Thromb Haemost 2005; 03: 597-599.
• 35 Antón AI. et al. Functional consequences of the prothrombotic SERPINC1 rs2227589 polymorphism on antithrombin levels. Haematologica 2009; 94: 589-592.
• 36 Spek CA. et al. Genotypic variation in the promoter region of the protein C gene is associated with plasma protein C levels and thrombotic risk. Arterioscler Thromb Vasc Biol 1995; 15: 214-218.
• 37 Aiach M. et al. Complex association of protein C gene promoter polymorphism with circulating protein C levels and thrombotic risk. Arterioscler Thromb Vasc Biol 1999; 19: 1573-1576.
• 38 Leroy-Matheron C. et al. Genetic modulation of plasma protein S levels by two frequent dimorphisms in the PROS1 gene. Thromb Haemost 1999; 82: 1088-1092.
• 39 Moatti D. et al. A new T-287C polymorphism in the 5’ regulatory region of the tissue factor pathway inhibitor gene. Association study of the T-287C and C-399T polymorphisms with coronary artery disease and plasma TFPI levels. Thromb Haemost 2000; 84: 244-249.
• 40 Ameziane N. et al. The –33T-->C polymorphism in intron 7 of the TFPI gene influences the risk of venous thromboembolism, independently of the factor V Leiden and prothrombin mutations. Thromb Haemost 2002; 88: 195-199.
• 41 Bernardi F. et al. A factor V genetic component differing from factor V R506Q contributes to the activated protein C resistance phenotype. Blood 1997; 90: 1552-1557.
• 42 Soria JM. et al. A new locus on chromosome 18 that influences normal variation in activated protein C resistance phenotype and factor VIII activity and its relation to thrombosis susceptibility. Blood 2003; 101: 163-167.
• 43 van Veen JJ. et al. Thrombin generation testing in routine clinical practice: are we there yet?. Br J Haematol 2008; 142: 889-903.
• 44 Baumann RE, Henschen AH. Human fibrinogen polymorphic site analysis by restriction endonuclease digestion and allele-specific polymerase chain reaction amplification: identification of polymorphisms at positions A alpha 312 and B beta 448. Blood 1993; 82: 2117-2124.
• 45 Ceelie H. et al. Polymorphisms in the prothrombin gene and their association with plasma prothrombin levels. Thromb Haemost 2001; 85: 1066-1070.
• 46 Mingozzi F. et al. A FV multiallelic marker detects genetic components of APC resistance contributing to venous thromboembolism in FV Leiden carriers. Thromb Haemost 2003; 89: 983-989.
• 47 Mustafa S. et al. Two new frequent dimorphisms in the protein S (PROS1) gene. Thromb Haemost 1996; 76: 393-396.
• 48 Diepstraten CM. et al. A CCA/CCG neutral dimorphism in the codon for Pro 626 of the human protein S gene PS alpha (PROS1). Nucleic Acids Res 1991; 19: 5091.
• 49 Miyata T. et al. C-399T polymorphism in the promoter region of human tissue factor pathway inhibitor (TFPI) gene does not change the plasma TFPI antigen level and does not cause venous thrombosis. Thromb Haemost 1998; 80: 345-346.
• 50 Moatti D. et al. Polymorphisms of the tissue factor pathway inhibitor (TFPI) gene in patients with acute coronary syndromes and in healthy subjects: impact of the V264M substitution on plasma levels of TFPI. Arterioscler Thromb Vasc Biol 1999; 19: 862-869.