Effects of oral anticoagulant therapy and haplotype 1 of the endothelial protein C receptor gene on activated protein C levels

Pilar Medina; Elena Bonet; Silvia Navarro; Laura Martos; Amparo Estellés; Fernando Ferrando; Vicente Vicente; Rogier M. Bertina; Francisco España

doi:10.1160/TH11-07-0510

Thrombosis and Haemostasis, Table of Contents

Thromb Haemost 2012; 107(03): 448-457
DOI: 10.1160/TH11-07-0510

Blood Coagulation, Fibrinolysis and Cellular Haemostasis

Schattauer GmbH

Effects of oral anticoagulant therapy and haplotype 1 of the endothelial protein C receptor gene on activated protein C levels

Authors

Pilar Medina

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

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

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

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

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

²Hemostasis and Thrombosis Unit, La Fe University Hospital, Valencia, Spain
Vicente Vicente

³Centro Regional de Hemodonación, Universidad de Murcia, Spain
Rogier M. Bertina

⁴Department of Thrombosis and Haemostasis, Leiden University Medical Centre, Leiden, The Netherlands
Francisco España

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

Abstract

Summary

Oral anticoagulants (OACs) reduce activated protein C (APC) plasma levels less than those of protein C (PC) in lupus erythematosus and cardiac patients. Carriers of the H1 haplotype of the endothelial PC receptor gene (PROCR) have higher APC levels than non-carriers. We aimed to confirm these results in a large group of patients treated with OACs because of venous thromboembolism (VTE) and to assess whether the effect is influenced by the PROCR H1 haplotype. We evaluated APC, PC, and factor (F)II levels in 502 VTE patients (158 with and 344 without OACs) and in 322 healthy individuals. Mean APC, PC and FII levels were significantly lower in OAC patients than in patients not taking OACs. During anticoagulant therapy, the FII/PC ratios were independent of the PC values, whereas APC/FII and APC/PC ratios significantly increased when FII and PC levels decreased. Of the 22 OAC patients carrying the H1H1genotype, 11 (50%) showed APC/PCag ≥2.0 and 10 (45%) APC/ FIIag ratios ≥2.0, whereas for the 49 OAC patients non-carrying the H1 haplotype these figures were 6 (12%) and 4 (8%), respectively (p<0.001). Barium citrate adsorption of plasma from OAC patients showed that most of the circulating free and complexed APC, but only part of PCag, is fully carboxylated. In conclusion, during anticoagulant therapy VT patients have APC levels disproportionately higher than the corresponding PC levels, mainly due to the presence of the PROCR H1 haplotype. Furthermore, a sufficiently carboxylated PC Gla-domain seems to be essential for PC activation in vivo.

Keywords

Activated protein C - endothelial protein C receptor - factor II - oral anti-coagulation - venous thrombosis

Full Text

References

References
1 van Leeuwen Y, Rosendaal FR, Cannegieter SC. Prediction of hemorrhagic and thrombotic events in patients with mechanical heart valve prostheses treated with oral anticoagulants. J Thromb Haemost 2008; 06: 451-456.
2 van der Heijden JF, Rosendaal FR, Vandenbroucke J P. et al. Bleeding complications in oral anticoagulant therapy. An analysis of risk factors. Arch Intern Med 1993; 153: 1557-1562.
3 Suttie JW. Mechanism of action of vitamin K: synthesis of gamma-carboxyglu-tamic acid. Crit Rev Biochem 1980; 08: 191-223.
4 Esmon CT. The role of protein C and thrombomodulin in the regulation of blood coagulation. J Biol Chem 1989; 264: 4743-4746.
5 Stearns-Kurosawa DJ, Kurosawa S, Mollica JS. et al. The endothelial cell protein C receptor augments protein C activation by the thrombin-thrombomodulin complex. Proc Natl Acad Sci USA 1996; 93: 10212-10216.
6 Taylor Jr FB, Peer GT, Lockhart MS. et al. Endothelial cell protein C receptor plays an important role in protein C activation in vivo. Blood 2001; 97: 1685-1688.
7 Centelles MN, Puy C, López-Sagaseta J. et al. Blocking endothelial protein C receptor (EPCR) accelerates thrombus development in vivo. Thromb Haemost 2010; 103: 1239-44. 8. Saposnik B, Reny JL, Gaussem 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: 1311-1318.
8 Medina P, Navarro S, Estellés A. et al. Contribution of polymorphisms in the en-dothelial protein C receptor gene to soluble endothelial protein C receptor and circulating activated protein C levels, and thrombotic risk. Thromb Haemost 2004; 91: 905-911.
9 Uitte de Willige S, Van Marion V, Rosendaal FR. et al. Haplotypes of the EPCR gene, plasma sEPCR levels and the risk of deep venous thrombosis. J Thromb Haemost 2004; 02: 1305-1310.
10 Estellés A, García-Plaza I, Dasí A. et al. Severe inherited “homozygous” protein C deficiency in a newborn infant. Thromb Haemost 1984; 52: 53-56.
11 Walker FJ. Regulation of activated protein C by protein S. The role of phos-pholipids in factor Va inactivation. J Biol Chem 1981; 256: 11128-11131.
12 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: 486-489.
13 España F, Medina P, Navarro S. et al. The multifunctional protein C system. Curr Med Chem-Cardiovasc Hematol Agents 2005; 03: 119-131.
14 Schouten M, van ’t Veer C, Roelofs JJTH. et al. Recombinant activated protein C attenuates coagulopathy and inflammation when administered early in murine pneumococcal pneumonia. Thromb Haemost 2011; 106: 1189-1196.
15 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: 1368-1373.
16 Zorio E, Navarro S, Medina P. et al. Circulating activated protein C is reduced in young survivors of myocardial infarction and inversely correlates with the severity of coronary lesions. J Thromb Haemost 2006; 04: 1530-1536.
17 Medina P, Navarro S, Estellés A. et al. Polymorphisms in the endothelial protein C receptor gene and thrombophilia. Thromb Haemost 2007; 98: 564-569.
18 Pecheniuk NM, Elias DJ, Xu X. et al. Failure to validate association of gene polymorphisms in EPCR, PAR-1, FSAP and protein S Tokushima with venous throm-boembolism among Californians of European ancestry. Thromb Haemost 2008; 99: 453-455.
19 Medina P, Navarro S, Estellés A. et al. Influence of the 4600A/G and 4678G/C polymorphisms in the endothelial protein C receptor (EPCR) gene on the risk of venous thromboembolism in carriers of factor V Leiden. Thromb Haemost 2005; 94: 389-394.
20 Simmelink MJA, de Groot PG, Derksen RHWM. et al. Oral anticoagulation reduces activated protein C less than protein C and other vitamin K-dependent clotting factors. Blood 2002; 100: 4232-4233.
21 España F, Berrettini M, Griffin JH. Purification and characterization of plasma protein C inhibitor. Thromb Res 1989; 55: 369-384.
22 España F, Griffin JH. Determination of functional and antigen protein C inhibitor and its complexes with activated protein C in plasma by ELISA's. Thromb Res 1989; 55: 671-682.
23 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.
24 España F, Gruber AG, Heeb MJ. et al. In vivo and in vitro complexes of activated protein C with two inhibitors in baboon. Blood 1991; 77: 1754-60.
25 Gruber A, Griffin JH. Direct detection of activated protein C in blood from human subjects. Blood 1992; 79: 2340-2348.
26 España F, Estellés A, Aznar J. et al. Assay of protein C in human plasma: comparison of amidolytic, coagulation, and immunochemical assays. Thromb Res 1986; 44: 771-782.
27 Navarro S, Medina P, Mira Y. et al. Haplotypes of the ERCR gene, prothrombin levels, and the risk of venous thrombosis in carriers of the prothrombin G20210A mutation. Haematologica 2008; 93: 885-891.
28 Medina P, Navarro S, Corral J. et al. for the RECAVA thrombo sis groups. Endothelial protein C receptor polymorphisms and risk of myocardial infarction. Haematologica 2008; 93: 1358-1363.
29 Varadi K, Philapitsch A, Santa T. et al. Activation and inactivation of human protein C by plasmin. Thromb Haemost 1994; 71: 615-621.
30 Rezaie AR. Rapid activation of protein C by factor Xa and thrombin in the presence of polyanionic compounds. Blood 1998; 91: 4572-4580.
31 Esmon NL, DeBault E, Esmon CT. Proteolytic formation and properties of ?-car-boxyglutamic acid-domainless protein C. J Biol Chem 1983; 258: 5548-5553.
32 Regan LM, Mollica JS, Rezaie AR. et al. The interaction between the endothelial cell protein C receptor and protein C is dictated by the gamma-carboxyglutamic acid domain of protein C. J Biol Chem 1997; 272: 26279-26284.
33 Taylor Jr FB, Peer GT, Lockhart MS. et al. Endothelial cell protein C receptor plays an important role in protein C activation in vivo. Blood 2001; 97: 1685-1688.
34 Stearns-Kurosawa DJ, Swindle K, D'Angelo A. et al. Plasma levels of endothelial protein C receptor respond to anticoagulant treatment. Blood 2002; 99: 526-530.
35 Gu JM, Katsuura Y, Ferrell GL. et al. Endotoxin and thrombin elevate rodent en-dothelial cell protein C receptor mRNA levels and increase receptor shedding in vivo. Blood 2000; 95: 1687-1693.
36 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: 6704-6710.