Download PDF
Semin Thromb Hemost 2022; 48(06): 680-689
DOI: 10.1055/s-0042-1753511
Review Article

Laboratory Testing for the Evaluation of Phenotypic Activated Protein C Resistance

Laure Morimont
1   Department of Research, Qualiblood Sa, Namur, Belgium
2   Department of Pharmacy, Faculty of Medicine, Namur Thrombosis and Hemostasis Center (NTHC), Namur Research Institute for Life Sciences (NARILIS), University of Namur, Namur, Belgium
,
Nathalie Donis
1   Department of Research, Qualiblood Sa, Namur, Belgium
,
Céline Bouvy
1   Department of Research, Qualiblood Sa, Namur, Belgium
,
François Mullier
3   Hematology Laboratory, Namur Research Institute for Life Sciences (NARILIS), Namur Thrombosis and Hemostasis Center (NTHC), Université Catholique de Louvain, CHU UCL Namur, Yvoir, Belgium
,
Jean-Michel Dogné
2   Department of Pharmacy, Faculty of Medicine, Namur Thrombosis and Hemostasis Center (NTHC), Namur Research Institute for Life Sciences (NARILIS), University of Namur, Namur, Belgium
,
Jonathan Douxfils
1   Department of Research, Qualiblood Sa, Namur, Belgium
2   Department of Pharmacy, Faculty of Medicine, Namur Thrombosis and Hemostasis Center (NTHC), Namur Research Institute for Life Sciences (NARILIS), University of Namur, Namur, Belgium
› Author Affiliations
› Further Information
    Permissions and Reprints

Abstract

Activated protein C (APC) resistance (APCR) is considered a risk factor of venous thromboembolism (VTE). The most common genetic disorder conferring APCR is a factor (F) V Leiden mutation, but many other factors are also implicated, such as other F5 mutations (e.g., FV Hong-Kong and FV Cambridge), protein S deficiency, elevated factor VIII, exogenous hormone use, pregnancy and postpartum, depending on how APCR is defined. Considering the large population affected, the detection of this phenotype is crucial. Two types of tests are currently available: clotting time-based assays (with several versions) and thrombin generation-based assays with the endogenous thrombin potential (ETP)-based assay. The purpose of this review is therefore to discuss the performances of these tests and the cases in which it would be appropriate to use one over the other. Initially, as APCR was thought to be solely related to the FV Leiden mutation, the objective was to obtain a 100% specific assay. Clotting-time based assays were thus specifically designed to detect this inherited condition. Later on, an APCR condition without a FV Leiden mutation was identified and highlighted as an independent risk factor of VTE. Therefore, the development of a less specific assay was needed and a global coagulation test was proposed, known as the ETP-based APCR assay. In light of the above, these tests should not be used for the same purpose. Clotting time-based assays should only be recommended as a screening test for the detection of FV mutations prior to confirmation by genetic testing. On the other hand, the ETP-based APC resistance assay, in addition to being able to detect any type of APCR, could be proposed as a global screening test as it assesses the entire coagulation process.

Keywords

activated protein C resistance - factor V Leiden - venous thromboembolism - blood coagulation tests - phenotype - thrombin generation - thrombophilia - hypercoagulable state


Publication History

Article published online:
02 September 2022

© 2022. Thieme. All rights reserved.

Thieme Medical Publishers, Inc.
333 Seventh Avenue, 18th Floor, New York, NY 10001, USA

 

  • References

  • 1 Dahlbäck B. The importance of the protein C system in the pathogenesis of venous thrombosis. Hematology 2005; 10 (Suppl. 01) 138-139
    Google Scholar
  • 2 Dahlbäck B, Villoutreix BO. The anticoagulant protein C pathway. FEBS Lett 2005; 579 (15) 3310-3316
    Google Scholar
  • 3 Esmon CT. The protein C pathway. Chest 2003; 124 (Suppl. 03) 26S-32S
    Google Scholar
  • 4 Van de Wouwer M, Collen D, Conway EM. Thrombomodulin-protein C-EPCR system: integrated to regulate coagulation and inflammation. Arterioscler Thromb Vasc Biol 2004; 24 (08) 1374-1383
    Google Scholar
  • 5 Castoldi E, Brugge JM, Nicolaes GA, Girelli D, Tans G, Rosing J. Impaired APC cofactor activity of factor V plays a major role in the APC resistance associated with the factor V Leiden (R506Q) and R2 (H1299R) mutations. Blood 2004; 103 (11) 4173-4179
    Google Scholar
  • 6 Tans G, van Hylckama Vlieg A, Thomassen MC. et al. Activated protein C resistance determined with a thrombin generation-based test predicts for venous thrombosis in men and women. Br J Haematol 2003; 122 (03) 465-470
    Google Scholar
  • 7 Rodeghiero F, Tosetto A. Activated protein C resistance and factor V Leiden mutation are independent risk factors for venous thromboembolism. Ann Intern Med 1999; 130 (08) 643-650
    Google Scholar
  • 8 de Visser MCH, Rosendaal FR, Bertina RM. A reduced sensitivity for activated protein C in the absence of factor V Leiden increases the risk of venous thrombosis. Blood 1999; 93 (04) 1271-1276
    Google Scholar
  • 9 Cramer TJ, Griffin JH, Gale AJ. Factor V is an anticoagulant cofactor for activated protein C during inactivation of factor Va. Pathophysiol Haemost Thromb 2010; 37 (01) 17-23
    Google Scholar
  • 10 Dahlbck B. Resistance to activated protein C caused by the R506Q mutation in the gene for factor V is a common risk factor for venous thrombosis. J Intern Med Suppl 1997; 740: 1-8
    Google Scholar
  • 11 Khan S, Dickerman JD. Hereditary thrombophilia. Thromb J 2006; 4: 15
    Google Scholar
  • 12 Kujovich JL, Factor V. Factor V Leiden thrombophilia. Genet Med 2011; 13 (01) 1-16
    Google Scholar
  • 13 Hotoleanu C. Genetic risk factors in venous thromboembolism. Adv Exp Med Biol 2017; 906: 253-272
    Google Scholar
  • 14 Khor B, Van Cott EM. Laboratory evaluation of hypercoagulability. Clin Lab Med 2009; 29 (02) 339-366
    Google Scholar
  • 15 Germain M, Chasman DI, de Haan H. et al; Cardiogenics Consortium. Meta-analysis of 65,734 individuals identifies TSPAN15 and SLC44A2 as two susceptibility loci for venous thromboembolism. Am J Hum Genet 2015; 96 (04) 532-542
    Google Scholar
  • 16 Pezeshkpoor B, Castoldi E, Mahler A. et al. Identification and functional characterization of a novel F5 mutation (Ala512Val, FVB onn ) associated with activated protein C resistance. J Thromb Haemost 2016; 14 (07) 1353-1363
    Google Scholar
  • 17 Bernardi F. Better or worse than the original. J Thromb Haemost 2016; 14 (07) 1350-1352
    Google Scholar
  • 18 Norstrøm E, Thorelli E, Dahlbäck B. Functional characterization of recombinant FV Hong Kong and FV Cambridge. Blood 2002; 100 (02) 524-530
    Google Scholar
  • 19 Castoldi E, Hézard N, Mourey G. et al. Severe thrombophilia in a factor V-deficient patient homozygous for the Ala2086Asp mutation (FV Besançon). J Thromb Haemost 2021; 19 (05) 1186-1199
    Google Scholar
  • 20 Dahlback B. APC resistance: what have we learned since 1993?. J Lab Med 2004; 28 (01) 21-27
    Google Scholar
  • 21 Steen M, Norstrøm EA, Tholander AL. et al. Functional characterization of factor V-Ile359Thr: a novel mutation associated with thrombosis. Blood 2004; 103 (09) 3381-3387
    Google Scholar
  • 22 Nogami K, Shinozawa K, Ogiwara K. et al. Novel FV mutation (W1920R, FVNara) associated with serious deep vein thrombosis and more potent APC resistance relative to FVLeiden. Blood 2014; 123 (15) 2420-2428
    Google Scholar
  • 23 Bernardi F, Faioni EM, Castoldi E. et al. A factor V genetic component differing from factor V R506Q contributes to the activated protein C resistance phenotype. Blood 1997; 90 (04) 1552-1557
    Google Scholar
  • 24 de Visser MC, Guasch JF, Kamphuisen PW, Vos HL, Rosendaal FR, Bertina RM. The HR2 haplotype of factor V: effects on factor V levels, normalized activated protein C sensitivity ratios and the risk of venous thrombosis. Thromb Haemost 2000; 83 (04) 577-582
    Google Scholar
  • 25 Aleksova A, Di Nucci M, Gobbo M. et al. Factor-V HR2 haplotype and thromboembolic disease. Acta Cardiol 2015; 70 (06) 707-711
    Google Scholar
  • 26 ten Kate MK, van der Meer J. Protein S deficiency: a clinical perspective. Haemophilia 2008; 14 (06) 1222-1228
    Google Scholar
  • 27 Yap ES, Timp JF, Flinterman LE. et al. Elevated levels of factor VIII and subsequent risk of all-cause mortality: results from the MEGA follow-up study. J Thromb Haemost 2015; 13 (10) 1833-1842
    Google Scholar
  • 28 Jenkins PV, Rawley O, Smith OP, O'Donnell JS. Elevated factor VIII levels and risk of venous thrombosis. Br J Haematol 2012; 157 (06) 653-663
    Google Scholar
  • 29 de Bastos M, Stegeman BH, Rosendaal FR. et al. Combined oral contraceptives: venous thrombosis. Cochrane Database Syst Rev 2014; (03) CD010813
    Google Scholar
  • 30 Dinger J, Do Minh T, Heinemann K. Impact of estrogen type on cardiovascular safety of combined oral contraceptives. Contraception 2016; 94 (04) 328-339
    Google Scholar
  • 31 Heit JA, Kobbervig CE, James AH, Petterson TM, Bailey KR, Melton III LJ. Trends in the incidence of venous thromboembolism during pregnancy or postpartum: a 30-year population-based study. Ann Intern Med 2005; 143 (10) 697-706
    Google Scholar
  • 32 Jick H, Jick SS, Gurewich V, Myers MW, Vasilakis C. Risk of idiopathic cardiovascular death and nonfatal venous thromboembolism in women using oral contraceptives with differing progestagen components. Lancet 1995; 346 (8990): 1589-1593
    Google Scholar
  • 33 Lidegaard Ø, Løkkegaard E, Svendsen AL, Agger C. Hormonal contraception and risk of venous thromboembolism: national follow-up study. BMJ 2009; 339: b2890
    Google Scholar
  • 34 Spitzer WO, Lewis MA, Heinemann LAJ, Thorogood M, MacRae KD. Transnational Research Group on Oral Contraceptives and the Health of Young Women. Third generation oral contraceptives and risk of venous thromboembolic disorders: an international case-control study. BMJ 1996; 312 (7023): 83-88
    Google Scholar
  • 35 van Hylckama Vlieg A, Helmerhorst FM, Vandenbroucke JP, Doggen CJ, Rosendaal FR. The venous thrombotic risk of oral contraceptives, effects of oestrogen dose and progestogen type: results of the MEGA case-control study. BMJ 2009; 339: b2921
    Google Scholar
  • 36 Rovinski D, Ramos RB, Fighera TM, Casanova GK, Spritzer PM. Risk of venous thromboembolism events in postmenopausal women using oral versus non-oral hormone therapy: a systematic review and meta-analysis. Thromb Res 2018; 168: 83-95
    Google Scholar
  • 37 Douxfils J, Morimont L, Bouvy C. Oral contraceptives and venous thromboembolism: focus on testing that may enable prediction and assessment of the risk. Semin Thromb Hemost 2020; 46 (08) 872-886
    Google Scholar
  • 38 Morimont L, Haguet H, Dogné J-M, Gaspard U, Douxfils J. Combined oral contraceptives and venous thromboembolism: review and perspective to mitigate the risk. Front Endocrinol (Lausanne) 2021; 12: 769187
    Google Scholar
  • 39 Stadel BV, Stadel MD. Oral contraceptives and cardiovascular disease (first of two parts). N Engl J Med 1981; 305 (11) 612-618
    Google Scholar
  • 40 Meade TW, Haines AP, North WRS, Chakrabarti R, Howarth DJ, Stirling Y. Haemostatic, lipid, and blood-pressure profiles of women on oral contraceptives containing 50 microgram or 30 microgram oestrogen. Lancet 1977; 2 (8045): 948-951
    Google Scholar
  • 41 Bonnar J. Coagulation effects of oral contraception. Am J Obstet Gynecol 1987; 157 (4 Pt 2): 1042-1048
    Google Scholar
  • 42 Wessler S, Gitel SN, Wan LS, Pasternack BS. Estrogen-containing oral contraceptive agents. A basis for their thrombogenicity. JAMA 1976; 236 (19) 2179-2182
    Google Scholar
  • 43 Brenner B. Haemostatic changes in pregnancy. Thromb Res 2004; 114 (5-6): 409-414
    Google Scholar
  • 44 Henkens CM, Bom VJ, Seinen AJ, van der Meer J. Sensitivity to activated protein C; influence of oral contraceptives and sex. Thromb Haemost 1995; 73 (03) 402-404
    Google Scholar
  • 45 Olivieri O, Friso S, Manzato F. et al. Resistance to activated protein C in healthy women taking oral contraceptives. Br J Haematol 1995; 91 (02) 465-470
    Google Scholar
  • 46 European Medicines Agency. . Guideline on Clinical investigation of Steroid Contraceptives in Women—EMEA/CPMP/EWP/519/98 Rev 1;. http://www.ema.europa.eu/docs/en_GB/document_library/Scientific_guideline/2009/09/WC500003349.pdf Accessed: 25 Apr 2005
    Google Scholar
  • 47 Dahlbäck B, Carlsson M, Svensson PJ. Familial thrombophilia due to a previously unrecognized mechanism characterized by poor anticoagulant response to activated protein C: prediction of a cofactor to activated protein C. Proc Natl Acad Sci U S A 1993; 90 (03) 1004-1008
    Google Scholar
  • 48 Roshal M, Reyes Gil M. Activated Partial Thromboplastin Time. In: Shaz BH, Gil MR, Hillyer CD. eds. Transfusion Medicine and Hemostasis. Elsevier; 2019: 779-781
    Google Scholar
  • 49 Kadauke S, Khor B, Van Cott EM. Activated protein C resistance testing for factor V Leiden. Am J Hematol 2014; 89 (12) 1147-1150
    Google Scholar
  • 50 Dean E, Favaloro EJ. The changing face of activated protein C resistance testing—a 10-year retrospective. Ann Blood 2020; 5: 6
    Google Scholar
  • 51 External quality Control for Assays and Tests With a focus on Thrombosis and Haemostasis. Thrombophilia - II: APC Resistance (without FV deficient plasma)—Survey 2021–M1 (A). Version 2.0.0. 14 June 2021
    Google Scholar
  • 52 Tripodi A, Chantarangkul V, Negri B, Mannucci PM. Standardization of the APC resistance test. Effects of normalization of results by means of pooled normal plasma. Thromb Haemost 1998; 79 (03) 564-566
    Google Scholar
  • 53 Moore GW, Van Cott EM, Cutler JA, Mitchell MJ, Adcock DM. subcommittee on plasma coagulation inhibitors. Recommendations for clinical laboratory testing of activated protein C resistance; communication from the SSC of the ISTH. J Thromb Haemost 2019; 17 (09) 1555-1561
    Google Scholar
  • 54 Moore GW. Snake venoms in diagnostic hemostasis and thrombosis. Semin Thromb Hemost 2022; 48 (02) 145-160
    Google Scholar
  • 55 Wilmer M, Stocker C, Bühler B, Conell B, Calatzis A. Improved distinction of factor V wild-type and factor V Leiden using a novel prothrombin-based activated protein C resistance assay. Am J Clin Pathol 2004; 122 (06) 836-842
    Google Scholar
  • 56 Gessoni G, Valverde S. Clinical evaluation of a functional prothrombin time-based assay for identification of factor V Leiden carriers in a group of Italian patients with venous thrombosis. Blood Coagul Fibrinolysis 2007; 18 (07) 603-610
    Google Scholar
  • 57 Schöni R, Quehenberger P, Wu JR, Wilmer M. Clinical evaluation of a new functional test for detection of activated protein C resistance (Pefakit APC-R Factor V Leiden) at two centers in Europe and the USA. Thromb Res 2007; 119 (01) 17-26
    Google Scholar
  • 58 Jilma-Stohlawetz P, Lysy K, Sunder-Plassmann R. et al. Limitations of a calibrated, quantitative APC-R assay under routine conditions. Int J Lab Hematol 2021; 43 (02) 318-323
    Google Scholar
  • 59 Castoldi E, Rosing J. APC resistance: biological basis and acquired influences. J Thromb Haemost 2010; 8 (03) 445-453
    Google Scholar
  • 60 Graf LL, Welsh CH, Qamar Z, Marlar RA. Activated protein C resistance assay detects thrombotic risk factors other than factor V Leiden. Am J Clin Pathol 2003; 119 (01) 52-60
    Google Scholar
  • 61 de Visser MC, van Hylckama Vlieg A, Tans G. et al. Determinants of the aPTT- and ETP-based APC sensitivity tests. J Thromb Haemost 2005; 3 (07) 1488-1494
    Google Scholar
  • 62 Nicolaes GA, Thomassen MC, Tans G, Rosing J, Hemker HC. Effect of activated protein C on thrombin generation and on the thrombin potential in plasma of normal and APC-resistant individuals. Blood Coagul Fibrinolysis 1997; 8 (01) 28-38
    Google Scholar
  • 63 Curvers J, Thomassen MC, Rimmer J. et al. Effects of hereditary and acquired risk factors of venous thrombosis on a thrombin generation-based APC resistance test. Thromb Haemost 2002; 88 (01) 5-11
    Google Scholar
  • 64 Douxfils J, Morimont L, Delvigne AS. et al. Validation and standardization of the ETP-based activated protein C resistance test for the clinical investigation of steroid contraceptives in women: an unmet clinical and regulatory need. Clin Chem Lab Med 2020; 58 (02) 294-305
    Google Scholar
  • 65 Tchaikovski SN, van Vliet HA, Thomassen MC. et al. Effect of oral contraceptives on thrombin generation measured via calibrated automated thrombography. Thromb Haemost 2007; 98 (06) 1350-1356
    Google Scholar
  • 66 Hemker HC, Giesen P, Al Dieri R. et al. Calibrated automated thrombin generation measurement in clotting plasma. Pathophysiol Haemost Thromb 2003; 33 (01) 4-15
    Google Scholar
  • 67 Brugge JM, Tans G, Rosing J, Castoldi E. Protein S levels modulate the activated protein C resistance phenotype induced by elevated prothrombin levels. Thromb Haemost 2006; 95 (02) 236-242
    Google Scholar
  • 68 Dargaud Y, Luddington R, Gray E. et al. Standardisation of thrombin generation test–which reference plasma for TGT? An international multicentre study. Thromb Res 2010; 125 (04) 353-356
    Google Scholar
  • 69 Dargaud Y, Luddington R, Gray E. et al. Effect of standardization and normalization on imprecision of calibrated automated thrombography: an international multicentre study. Br J Haematol 2007; 139 (02) 303-309
    Google Scholar
  • 70 Loeffen R, Kleinegris MC, Loubele ST. et al. Preanalytic variables of thrombin generation: towards a standard procedure and validation of the method. J Thromb Haemost 2012; 10 (12) 2544-2554
    Google Scholar
  • 71 Perrin J, Depasse F, Lecompte T. French-speaking CAT group and under the aegis of GEHT, French-speaking CAT group (all in France unless otherwise stated), French-speaking CAT group all in France unless otherwise stated. Large external quality assessment survey on thrombin generation with CAT: further evidence for the usefulness of normalisation with an external reference plasma. Thromb Res 2015; 136 (01) 125-130
    Google Scholar
  • 72 Morimont L, Bouvy C, Delvigne AS, Dogné JM, Douxfils J. Proof of concept of a new scale for the harmonization and the standardization of the ETP-based APC resistance. J Thromb Haemost 2020; 18 (04) 895-904
    Google Scholar
  • 73 Food and Drug Administration. Bioanalytical Method Validation–Guidance for Industry. https://www.fda.gov/downloads/drugs/guidances/ucm070107.pdf Accessed: 6 Feb 2018
    Google Scholar
  • 74 European Medicines Agency. ICH Topic Q2(R1) - Validation of Analytical Procedures: Text and Methodology (CPMP/ICH/381/95). http://www.ema.europa.eu/docs/en_GB/document_library/Scientific_guideline/2009/09/WC500002662.pdf Accessed: 1995. 1995
    Google Scholar
  • 75 Morimont L, Didembourg M, Haguet H. et al. Interlaboratory variability of activated protein C resistance using the ETP-based APC resistance assay. Res Pract Thromb Haemost 2021; 5 (07) e12612
    Google Scholar
  • 76 Hardy M, Douxfils J, Morimont L. et al. Study of in vitro thrombin generation after neutralization of heparin. Int J Lab Hematol 2022; Feb; 44 (01) 168-176
    Google Scholar
  • 77 Monteyne T, De Kesel P, Devreese KMJ. Interference of DOAC stop and DOAC remove in the thrombin generation assay and coagulation assays. Thromb Res 2020; 192: 96-99
    Google Scholar
  • 78 Kopatz WF, Brinkman HJM, Meijers JCM. Use of DOAC Stop for elimination of anticoagulants in the thrombin generation assay. Thromb Res 2018; 170: 97-101
    Google Scholar
  • 79 Morimont L, Leclercq C, Didembourg M. et al. Analytical performance of the endogenous thrombin potential-based activated protein C resistance assay on the automated ST Genesia system. Res Pract Thromb Haemost 2022; 6 (03) e12684
    Google Scholar
  • 80 Baglin T, Gray E, Greaves M. et al; British Committee for Standards in Haematology. Clinical guidelines for testing for heritable thrombophilia. Br J Haematol 2010; 149 (02) 209-220
    Google Scholar
  • 81 Morimont L, Dogné J-M, Douxfils J. Letter to the Editors-in-Chief in response to the article of Abou-Ismail, et al. entitled “Estrogen and thrombosis: a bench to bedside review” (Thrombosis Research 192 (2020) 40-51). Thromb Res 2020; 193: 221-223
    Google Scholar
  • 82 United Nations. , Department of Economic and SocialAffairs, PopulationDivision (2019). Contraceptive Use by Method 2019: Data Booklet (ST/ESA/SER.A/435) 2019
    Google Scholar
  • 83 McDaid A, Logette E, Buchillier V. et al. Risk prediction of developing venous thrombosis in combined oral contraceptive users. PLoS One 2017; 12 (07) e0182041
    Google Scholar
  • 84 Cushman M, Kuller LH, Prentice R. et al; Women's Health Initiative Investigators. Estrogen plus progestin and risk of venous thrombosis. JAMA 2004; 292 (13) 1573-1580
    Google Scholar
  • 85 Curb JD, Prentice RL, Bray PF. et al. Venous thrombosis and conjugated equine estrogen in women without a uterus. Arch Intern Med 2006; 166 (07) 772-780
    Google Scholar
  • 86 Cosmi B, Legnani C, Bernardi F, Coccheri S, Palareti G. Role of family history in identifying women with thrombophilia and higher risk of venous thromboembolism during oral contraception. Arch Intern Med 2003; 163 (09) 1105-1109
    Google Scholar
  • 87 Lavigne-Lissalde G, Sanchez C, Castelli C. et al. Prothrombin G20210A carriers the genetic mutation and a history of venous thrombosis contributes to thrombin generation independently of factor II plasma levels. J Thromb Haemost 2010; 8 (05) 942-949
    Google Scholar
  • 88 Marco A, Brocal C, Martirena F, Lucas J, Marco P. Clinical and biological factors that contribute to thrombin generation in prothrombin G20210A carriers: a case-control study in a single Thrombophilia Center. Thromb Res 2012; 129 (05) e266-e268
    Google Scholar
  • 89 Duchemin J, Pan-Petesch B, Arnaud B, Blouch MT, Abgrall JF. Influence of coagulation factors and tissue factor concentration on the thrombin generation test in plasma. Thromb Haemost 2008; 99 (04) 767-773
    Google Scholar
  • 90 Szlam F, Sreeram G, Solomon C, Levy JH, Molinaro RJ, Tanaka KA. Elevated factor VIII enhances thrombin generation in the presence of factor VIII-deficiency, factor XI-deficiency or fondaparinux. Thromb Res 2011; 127 (02) 135-140
    Google Scholar
  • 91 Duarte RCF, Ferreira CN, Rios DRA, Reis HJD, Carvalho MDG. Thrombin generation assays for global evaluation of the hemostatic system: perspectives and limitations. Rev Bras Hematol Hemoter 2017; 39 (03) 259-265
    Google Scholar