A Prothrombinase-based Assay for Detection of Resistance to Activated Protein C

Summary

In this paper we present a new method for the detection of resistance to activated protein C (APC) that is based on direct measurement of the effect of APC on the cofactor activity of plasma factor Va. The factor V present in a diluted plasma sample was activated with thrombin and its sensitivity towards APC was subsequently determined by incubation with phospholipids and APC. The loss of factor Va cofactor activity was quantified in a prothrombinase system containing purified prothrombin, factor Xa and phospholipid vesicles and using a chromogen-ic assay for quantitation of thrombin formation. The reaction conditions were optimized in order to distinguish normal, heterozygous and homozygous APC-resistant plasmas. Maximal differences in the response of these plasmas towards APC were observed when factor Va was inactivated by APC in the absence of protein S and when the cofactor activity of factor Va was determined at a low factor Xa concentration (0.3 nM).

Addition of 0.2 nM APC and 20 μM phospholipid vesicles to a 1000-fold diluted sample of thrombin-activated normal plasma resulted in loss of more than 85% of the cofactor activity factor Va within 6 rnin. Under the same conditions, APC inactivated ∼ 60% and ∼20% of the factor Va present in plasma samples from APC-resistant individuals that were heterozygous or homozygous for the mutation Arg⁵⁰⁶⟶Gln in factor V, respectively. Discrimination between the plasma samples from normal and heterozygous and homozygous APC-resistant individuals was facilitated by introduction of the so-called APC-sensitivity ratio (APC-sr). The APC-sr was defined as the ratio of the factor Va cofactor activities determined in thrombin-activated plasma samples after 6 min incubation with or without 0.2 nM APC and was multiplied by 100 to obtain integers (APC-sr = {factor Va_+APC/factor Va_−APC} × 100). Clear differences were observed between the APC-sr of plasmas from normal healthy volunteers (APC-sr: 8-20, n = 33) and from individuals that were heterozygous (APC-sr: 35-50, n = 17) or homozygous APC resistant (APC-sr: 82-88, n = 7). There was no mutual overlap between the APC-sr of normal plasmas and plasmas from heterozygous or homozygous APC resistant individuals (p <0.0001). In all cases our test gave the same result as the DNA-based assay. Since the test is performed on a highly diluted plasma sample there is no interference by conditions that affect APC resistance tests that are based on clotting time determinations (e.g. coagulation factor deficiencies, oral anticoagulation, heparin treatment, the presence of lupus anticoagulants, pregnancy or the use of oral contraceptives). Furthermore, we show that part of the factor Va assay can be performed on an autoanalyzer which increases the number of plasma samples that can be handled simultaneously.

• References

• 1 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 USA 1993; 90: 1004-1008
• 2 Koster T, Rosendaal FR, De Ronde H, Briët E, Vandenbroucke JP, Bertina RM. Venous thrombosis due to a poor anticoagulant response to activated protein C: Leiden Thrombophilia Study. Lancet 1993; 342: 1503-1506
• 3 Griffin JH, Evatt B, Wideman C, Fernandez JA. Anticoagulant protein C pathway defective in a majority of thrombophilic patients. Blood 1993; 82: 1989-1993
• 4 Cadroy Y, Sié P, Boneu B. Frequency of a defective response to activated protein C in patients with a history of venous thrombosis. Blood 1994; 83: 2008-2009
• 5 Svensson PJ, Dahlbäck B. Resistance to activated protein C as a basis for venous thrombosis. N Engl J Med 1994; 330: 517-522
• 6 Halbmayer WM, Haushofer A, Schon R, Fischer M. The prevalence of poor anticoagulant response to activated protein C (APC Resistance) among patients suffering from stroke or venous thrombosis and among healthy subjects. Blood Coagul Fibrinol 1995; 5: 51-57
• 7 Bertina RM, Koeleman BPC, Koster T, Rosendaal FR, Dirven RJ, De Ronde H, Van der Velden PA, Reitsma PH. Mutation in blood coagulation factor V associated with resistance to activated protein C. Nature 1994; 369: 64-67
• 8 Voorberg J, Roelse J, Koopman R, Büller H, Berends F, Ten Cate JW, Mertens K, Van Mourik JA. Association of idiopathic venous thromboembolism with single point-mutation at Arg506 of factor V. Lancet 1994; 343: 1535-1536
• 9 Greengard JS, Sun X, Xu X, Fernandez JA, Griffin JH, Evatt B. Activated protein C resistance caused by Arg506Gln mutation in factor Va. Lancet 1994; 343: 1361-1362
• 10 Alhenc-Gelas M, Gandrille S, Aubry M-L, Emmerich J, Fiessinger J-N, Aiach M. Unexplained thrombosis and factor V Leiden mutation. Lancet 1994; 344: 555-556
• 11 Zöller B, Svensson PJ, He X, Dahlbäck B. Identification of the same factor V gene mutation in 47 out of 50 thrombosis-prone families with inherited resistance to activated protein C. J Clin Invest 1994; 94: 2521-2524
• 12 Kalafatis M, Rand MD, Mann KG. The mechanism of inactivation of human factor V and human factor Va by activated protein C. J Biol Chem 1994; 269: 31869-31880
• 13 Kalafatis M, Bertina RM, Rand MR, Mann KG. Characterization of the molecular defect in factor VR506Q. J Biol Chem 1995; 270: 4053-4057
• 14 Nicolaes GAF, Tans G, Thomassen MCLGD, Hemker HC, Pabinger I, Varadi K, Schwarz HP, Rosing J. Peptide bond cleavages and loss of functional activity during inactivation of factor Va and factor Va^R506Q by activated protein C. J Biol Chem 1995; 270: 21158-21166
• 15 Dahlbäck B. Resistance to activated protein C, the Arg506 to Gin mutation in the factor V gene and venous thrombosis. Thromb Haemost 1995; 73: 739-742
• 16 Gandrille S, Alhenc-Gelas M, Aiach M. A rapid screening method for the factor V Arg506⟶Gln mutation. Blood Coagul Fibrinol 1995; 6: 245-248
• 17 De Ronde H, Bertina RM. Laboratory diagnosis of APC-resistance: a critical evaluation of the test and the development of diagnostic criteria. Thromb Haemost 1994; 72: 880-886
• 18 Rosén S, Johansson K, Lindberg K, dDahlbäck B. Multicenter evaluation of a kit for activated protein C resistance on various coagulation instruments using plasmas from healthy individuals. Thromb Haemost 1994; 72: 255-260
• 19 Nicolaes GAF, Thomassen MCLGD, Béguin S, Tans G, Rosing J, Hemker HC. Effect of activated protein C on thrombin generation and on the thrombin potential in normal and APC resistant individuals. Thromb Haemost, submitted for publication
• 20 Kraus M, Zander N, Fickenscher K. Coagulation assay with improved specificity to factor V mutants insensitive to activated protein C. Thromb Res 1995; 80: 255-264
• 21 Vasse M, Leduc O, Borg J-Y, Chrétien MH, Monconduit M. Resistance to activated protein C: evaluation of three functional assays. Thromb Res 1994; 76: 47-59
• 22 Bokarewa MI, Bremme K, Falk G, Sten-Linder M, Egberg N, Blombäck M. Studies on phospholipid antibodies, APC-resistance and associated mutation in the coagulation factor V gene. Thromb Res 1995; 78: 193-200
• 23 Cadroy Y, Sié P, Alhenc-Gelas M, Aiach M. Evaluation of APC resistance in the plasma of patients with Q506 mutation of factor V (factor V Leiden) and treated by oral anticoagulants. Thromb Haemost 1995; 73: 734-735
• 24 Henkens CMA, Bom VJJ, Seinen AJ, Van der Meer J. Sensitivity to activated protein C; influence of oral contraceptives and sex. Thromb Haemost 1995; 73: 402-404
• 25 Olivieri O, Friso S, Manzato F, Guella A, Bemardi F, Lunghi B, Girelli D, Azzini M, Brocco G, Russo C, Corrocher R. Resistance to activated protein C in healthy women taking oral contraceptives. Br J Haematol 1995; 91: 465-470
• 26 DiScipio RG, Hermodson MA, Yates SG, Davie EW. A comparison of human prothrombin, factor IX (Christmas factor), factor X (Stuart factor), and Protein S. Biochemistry 1977; 16: 698-706
• 27 Bock PE, Craig PA, Olsen ST, Singh P. Isolation of human blood coagulation factor a-factor Xa by soybean trypsin inhibitor-sepharose chromatography and its active site titration with fluorescin mono-p-guanidinobenzoate. Arch Biochem Biophys 1989; 273: 375-388
• 28 Rosing J, Bakker HM, Thomassen MCLGD, Hemker HC, Tans G. Characterization of two forms of human factor Va with different cofactor activities. J Biol Chem 1993; 268: 21130-21136
• 29 Böttcher CJF, van Gent CM, Pries C. A rapid and sensitive sub-micro phosphorus determination. Anal Chim Acta 1961; 24: 203-207
• 30 Sala N, Owen WG, Collen D. A functional assay for protein C in human plasma. Blood 1984; 63: 671-675
• 31 Rosing J, Tans G, Govers-Riemslag JWP, Zwaal RFA, Hemker HC. The role of phospholipids and factor Va in the prothrombinase complex. J Biol Chem 1980; 255: 274-283
• 32 DiScipio RG, Davie EW. Characterization of protein S a gamma-carboxy-glutamic acid containing protein from bovine and human plasma. Biochemistry 1979; 18: 899-904
• 33 Tans G, Rosing J, Thomassen MCLGD, Heeb MJ, Zwaal RFA, Griffin JH. Comparison of anticoagulant and procoagulant activities of stimulated platelets and platelet-derived microparticles. Blood 1991; 77: 2641-2648
• 34 Beauchamp NJ, Daly ME, Hampton KK, Cooper PC, Preston FE, Peake IR. High prevalence of a mutation in the factor V gene within the U.K. population: relationship to activated protein C resistance and familial thrombosis. Br J Haematol 1994; 88: 219-222
• 35 Rosing J, Hoekema L, Nicolaes GAF, Thomassen MCLGD, Hemker HC, Varadi K, Schwarz HP, Tans G. Effects of protein S and factor Xa on peptide bond cleavages during inactivation of factor Va and factor Va^R506Q by activated protein C. J Biol Chem 1995; 270: 27852-27858
• 36 Tracy PB, Eide LL, Bowie EJW, Mann KG. Radioimmunoassay of factor V in human plasma and platelets. Blood 1982; 60: 59-63
• 37 Samaha M, Trossaert M, Conard J, Horellou MH, Elalamy I, Samama MM. Prevalence and patient profile in activated protein C resistance. Am J Clin Pathol 1995; 104: 450-454
• 38 Sun X, Evatt B, Griffin J. Blood coagulation factor Va abnormality associated with resistance to activated protein C in venous thrombophilia. Blood 1994; 11: 3120-3125
• 39 Engel H, Zwang L, van Vliet HHDM, Michiels JJ, Stibbe J, Lindemans J. Phenotyping and genotyping of coagulation factor V Leiden. Thromb Haemost 1996; 75: 267-269
• 40 Le DT, Donnely K, Sevilla B, Rapaport SI. Further experience with the TF-based FV assay for APC resistant FVa (APC-R). Blood 1995; 86: 202a