Contribution of polymorphisms in the endothelial protein C receptor gene to soluble endothelial protein C receptor and circulating activated protein C levels, and thrombotic risk

Pilar Medina; Silvia Navarro; Amparo Estellés; Amparo Vayá; Barry Woodhams; Yolanda Mira; Piedad Villa; Martine Migaud-Fressart; Fernando Ferrando; Justo Aznar; Rogier M. Bertina; Francisco España

doi:10.1160/TH03-10-0657

Thrombosis and Haemostasis, Table of Contents

Thromb Haemost 2004; 91(05): 905-911
DOI: 10.1160/TH03-10-0657

Blood Coagulation, Fibrinolysis and Cellular Haemostasis

Schattauer GmbH

Contribution of polymorphisms in the endothelial protein C receptor gene to soluble endothelial protein C receptor and circulating activated protein C levels, and thrombotic risk

Authors

Pilar Medina

¹Research Center, La Fe University Hospital, Valencia, Spain
Silvia Navarro

¹Research Center, La Fe University Hospital, Valencia, Spain
Amparo Estellés

¹Research Center, La Fe University Hospital, Valencia, Spain
Amparo Vayá

²Department of Clinical Pathology, La Fe University Hospital, Valencia, Spain
Barry Woodhams

³Serbio (Groupe Stago), Gennevilliers, France
Yolanda Mira

²Department of Clinical Pathology, La Fe University Hospital, Valencia, Spain
Piedad Villa

²Department of Clinical Pathology, La Fe University Hospital, Valencia, Spain
Martine Migaud-Fressart

⁴Diagnostica Stago, Asnieres, France
Fernando Ferrando

²Department of Clinical Pathology, La Fe University Hospital, Valencia, Spain
Justo Aznar

²Department of Clinical Pathology, La Fe University Hospital, Valencia, Spain
Rogier M. Bertina

⁵Department of Hematology, Leiden University Medical Centre, Leiden, The Netherlands
Francisco España

¹Research Center, La Fe University Hospital, Valencia, Spain

Abstract

Summary

Endothelial cell protein C receptor (EPCR) enhances the generation of activated protein C (APC) by the thrombin-thrombomodulin complex. A soluble form of EPCR (sEPCR), which is generated by metalloprotease activity, is present in plasma. The distribution of sEPCR levels in healthy populations is bimodal. Previously, we described two polymorphisms in exon 4 of the EPCR gene, 4600A/G that encodes the substitution of Ser219 by Gly in the transmembrane region of EPCR and 4678G/C in the 3’-UT region. The aim of this study was to investigate the relationship between these two polymorphisms and plasma sEPCR and APC levels and risk of venous thrombosis. We genotyped 401 healthy controls from the Spanish population and measured their plasma sEPCR and APC levels. Carriers of the 4600AG genotype had significantly higher sEPCR levels than those with the AA genotype, while the 4678CC genotype was associated, to a lesser extent, with elevated APC levels. To assess the effect of these polymorphisms on the risk of thrombosis, we genotyped 405 patients with venous thromboembolism. The frequency of the 4600AG genotype was very similar in patients and controls (p=0.975), whereas the 4678CC genotype was significantly more frequent in controls than in patients (p=0.008). In multivariate analysis, carriers of the 4678CC genotype had a decreased risk of thrombosis (OR=0.61, p=0.009). These data indicate that individuals carrying the 4600AG genotype have high sEPCR levels but do not have an increased risk of thrombosis, whereas individuals carrying the 4678CC genotype have higher APC levels and lower risk of venous thromboembolism.

Keywords

Protein C - endothelial protein C receptor - polymorphism

Full Text

References

References
1 Griffin JH, Evatt B, Zimmerman TS. et al. Deficiency of protein C in congenital thrombotic disease. J Clin Invest 1981; 68: 1370-3.
2 Walker FJ. Regulation of activated protein C by protein S. The role of phospholipids in factor Va inactivation. J Biol Chem 1981; 256c: 11128-31.
3 Fulcher CA, Gardiner JE, Griffin JH. et al. Proteolytic inactivation of human Factor VIII procoagulant protein by activated human protein C and its analogy with Factor V. Blood 1984; 63: 486-9.
4 Bertina RM. Molecular risk factors for thrombosis. Thromb Haemost 1999; 82: 601-9.
5 Laszik Z, Mitro A, Taylor Jr FB. et al. Human protein C receptor is present primarily on endothelial of large vessels: implications in the control of the protein C pathway. Circulation 1987; 96: 3633-40.
6 Stearns-Kurosawa DJ, Kurosawa S, Mollica JS. et al. The endothelial cell protein C receptor augments protein C activation by the thrombinthrombomodulin complex. Proc Natl Acad Sci USA 1996; 93: 10212-6.
7 Esmon CT. The role of protein C and thrombomodulin in the regulation of blood coagulation. J Biol Chem 1989; 264: 4743-6.
8 Kurosawa S, Stearns-Kurosawa DJ, Hidari N. et al. Identification of functional endothelial protein C receptor in human plasma. J Clin Invest 1997; 100: 411-8.
9 Xu J, Qu D, Esmon NL. et al. Metalloproteolytic release of endothelial protein C receptor. J Biol Chem 2000; 275: 6038-44.
10 Liaw PCY, Neuenschwander PF, Smirnov MD. et al. Mechanisms by which soluble endothelial cell protein C receptor modulates protein C and activated protein C function. J Biol Chem 2000; 275: 5447-52.
11 Kurosawa S, Stearns-Kurosawa DJ, Carson CW. et al. Plasma levels of endothelial cell protein C receptor are elevated in patients with sepsis and systemic lupus erythematosus: lack of correlation with thrombomodulin suggests involvement of different pathological processes. Blood 1998; 91: 725-7.
12 Stearns-Kurosawa DJ, Swindle K, D’Angelo A. et al. Plasma levels of endothelial protein C receptor respond to anticoagulant treatment. Blood 2002; 99: 526-30.
13 España F, Medina P, Mira Y. et al. A new polymorphism in the 3’UTR of the endothelial protein C receptor is associated with increased levels of circulating activated protein C and decreased risk of venous thrombosis. Thromb Haemost. 2001 Supplement to XVIII Congress of The International Society on Thrombosis and Haemostasis OC885..
14 Frezzato M, Tosetto A, Rodeghiero E. Validated questionnaire for the identification of previous personal or familial venous thromboembolism. Am J Epidemiol 1996; 143: 1257-65.
15 España F, Zuazu I, Vicente V. et al. Quantification of circulating activated protein C in human plasma by immunoassays. Enzyme levels are proportional to total protein C levels. Thromb Haemost 1996; 75: 56-61.
16 España F, Vayá A, Mira Y. et al. Low level of circulating activated protein C is a risk factor for venous thromboembolism. Thromb Haemost 2001; 86: 1368-73.
17 España F. Complexes of activated protein C with its inhibitors. In: Aznar J, España F. eds. Protein C pathway. Barcelona: Springer-Verlag; 1991: 61-75.
18 Miller SA, Dykes OD, Polesky HF. A simple salting out procedure for extracting DNA from human nucleated cells. Nucleic Acid Res 1988; 16: 1215.
19 Saiki RK, Gelfand DH, Stoffel S. et al. Primerdirected enzymatic amplification of DNA with a thermostable DNA polymerase. Science 1988; 239: 487-90.
20 Simmonds RE, Lane DA. Structural and functional implications of the intron/exon organization of the human endothelial cell protein C/activated protein C receptor (EPCR) gene: comparison with the structure of CD1/major histocompatibility complex α1 and α2 domains. Blood 1999; 94: 632-41.
21 Saposnik B, Reny JL, Gausem P. et al. A haplotype of the EPCR gene is associated with increased plasma levels of sEPCR and is a candidate risk factor for thrombosis. Blood 2004; 103: 1311-18.
22 Gu JM, Katsuura Y, Ferrell GL. et al. Endotoxin and thrombin elevate rodent endothelial cell protein C receptor mRNA levels and increase receptor shedding in vivo. Blood 2000; 95: 1687-93.
23 Xu J, Esmon NL, Esmon CT. Reconstitution of the human endothelial cell protein C receptor with thrombomodulin in phosphatidylcholine vesicles enhances protein C activation. J Biol Chem 1999; 274: 6704-10.
24 Poort SR, Vos HL, Rosendaal FR. et al. The endothelial protein C receptor (EPCR) 23 bp insert mutation and the risk of venous thrombosis. Thromb Haemost 2002; 88: 160-2.
25 Galligan L, Powell C, Livingstone W. et al. The G7763C endothelial protein C receptor (EPCR) gene mutation: prevalence and association with DVT in the Irish population. Thromb Haemost 2002; 88: 163-5.