Effects of (Pre-)analytical Variables on Activated Protein C Resistance Determined Via a Thrombin Generation-based Assay

Summary

The normalized activated protein C sensitivity ratio (nAPC-sr) determined with an assay that quantifies the effect of APC on thrombin formation initiated via the extrinsic coagulation pathway identifies hereditary and acquired defects of the protein C system. We investigated the influence of assay conditions (analytical variables) and plasma handling (pre-analytical variables) on nAPC-sr obtained with this APC resistance test. The effect of the analytical variables (CaCl2, phospholipid and APC concentrations and the concentration and source of tissue factor) was determined in pooled normal plasma. Inhibition of thrombin formation by APC was dependent on the APC concentration and was also affected by the tissue factor, Ca2+ and phospholipid concentrations. Thus, strict standardization of reactant concentrations is required to obtain reproducible nAPC-sr. Three different tissue factor preparations were compared by determining nAPCsr in plasma samples obtained from 90 healthy individuals. nAPC-sr were similar for all three tissue factor preparations although, compared with the noncommercially available tissue factor used in earlier studies, values determined with commercial tissue factor preparations showed larger variation. Pre-analytical variables, investigated in plasma of nine volunteers (3 normal individuals and 6 individuals with an APCresistant phenotype) were: concentration of anticoagulant (3.2% vs. 3.8% trisodiumcitrate), time before processing of blood (0, 4 and 24 h), centrifugation speed, storage temperature of plasma (–20° C vs. –80° C) and sample thawing. Multiple linear regression analysis showed that only the citrate concentration affected the nAPC-sr, which was higher in samples collected in 3.2% trisodiumcitrate than in samples collected in 3.8% trisodiumcitrate.

Keywords

APC Resistance - Factor V_Leiden

• References

• 1 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: 64-7.
• 2 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: 1361-2.
• 3 Voorberg J, Roelse J, Koopman R, Buller 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: 1535-6.
• 4 Zöller B, Dahlbäck B. Linkage between inherited resistance to activated protein C and factor V gene mutation in venous thrombosis. Lancet 1994; 343: 1536-8.
• 5 Kalafatis M, Bertina RM, Rand MD, Mann KG. Characterization of the molecular defect in factor VR506Q. J Biol Chem 1995; 270: 4053-7.
• 6 Heeb MJ, Kojima Y, Greengard JS, Griffin JH. Activated protein C resistance: molecular mechanisms based on studies using purified Gln506factor V. Blood 1995; 85: 3405-11.
• 7 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 VaR506Q by activated protein C. J Biol Chem 1995; 270: 27852-8.
• 8 Varadi K, Rosing J, Tans G, Pabinger I, Keil B, Schwarz HP. Factor V enhances the cofactor function of protein S in the APC-mediated inactivation of factor VIII: influence of the Factor VR506Q mutation. Thromb Haemost 1996; 76 (02) 208-14.
• 9 Thorelli E, Kaufman RJ, Dahlbäck B. The C-terminal region of the factor V B-domain is crucial for the anticoagulant activity of factor V. J Biol Chem 1998; 273: 16140-5.
• 10 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. Proceedings of the National Academy of Science of the United States of America 1993; 90: 1004-8.
• 11 Koster T, Rosendaal FR, de Ronde H, Briet E, Vandenbroucke JP, Bertina RM. Venous thrombosis due to poor anticoagulant response to activated protein C: Leiden Thrombophilia Study. Lancet 1993; 342: 1503-6.
• 12 de Visser MC, 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: 1271-6.
• 13 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: 643-50.
• 14 Salomon O, Steinberg DM, Zivelin A, Gitel S, Dardik R, Rosenberg N, Berliner S, Inbal A, Many A, Lubetsky A, Varon D, Martinowitz U, Seligsohn U. Single and combined prothrombotic factors in patients with idiopathic venous thromboembolism: prevalence and risk assessment. Arterioscler Thromb Vasc Biol 1999; 19: 511-8.
• 15 Olivieri O, Friso S, Manzato F, Guella A, Bernardi 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: 465-70.
• 16 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: 402-4.
• 17 Rosing J, Tans G, Nicolaes GAF, Thomassen MCLGD, vanOerle R, vanderPloeg PMEN, Heijnen P, Hamulyak K, Hemker HC. Oral contraceptives and venous thrombosis: Different sensitivities to activated protein C in women using secondand third-generation oral contraceptives. British Journal of Haematology 1997; 97: 233-8.
• 18 Meinardi JR, Henkens CMA, Heringa MP, vanderMeer J. Acquired APC resistance related to oral contraceptives and pregnancy and its possible implications for clinical practice. Blood Coagulation and Fibrinolysis 1997; 08: 152-4.
• 19 Nicolaes GAF, Thomassen MCLGD, 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 Coagulation and Fibrinolysis 1997; 08: 28-38.
• 20 Curvers J, Thomassen MCLGD, Nicolaes GAF, van Oerle R, Hamulyak K, Hemker HC, Tans G, Rosing J. Acquired APC resistance and oral contraceptives: differences between two functional tests. British Journal of Haematology 1999; 105: 88-94.
• 21 Rosing J, Middeldorp S, Curvers J, Thomassen MCLGD, Nicolaes GA, Meijers JC, Bouma BN, Buller HR, Prins MH, Tans G. Low-dose oral contraceptives and acquired resistance to activated protein C: a randomised cross-over study. Lancet 1999; 354: 2036-40.
• 22 Rosing J, Tans G. Effects of oral contraceptives on hemostasis and thrombosis. American Journal of Obstetrics and Gynecology 1999; 180: S375-82.
• 23 Rouser G, Fkeischer S, Yamamoto A. Two-dimensional thin layer chromatographic separation of polar lipids and determination of phospholipids by phosphorus analysis of spots. Lipids 1970; 05: 494-6.
• 24 Sala N, Owen WG, Collen D. A functional assay of protein C in human plasma. Blood 1984; 63: 671-5.
• 25 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: 880-6.
• 26 Duchemin J, Pittet JL, Tartary M, Beguin S, Gaussem P, Alhenc-Gelas M, Aiach M. A new assay based on thrombin generation inhibition to detect both protein C and protein S deficiencies in plasma. Thromb Haemost 1994; 71: 331-8.
• 27 Fischer AM, Tapon-Bretaudiere J, Bros A, Josso F. Respective roles of antithrombin III and alpha 2 macroglobulin in thrombin inactivation. Thromb Haemost 1981; 45: 51-4.
• 28 Hemker HC, Willems GM, Beguin S. A computer assisted method to obtain the prothrombin activation velocity in whole plasma independent of thrombin decay processes. Thromb Haemost 1986; 56: 9-17.
• 29 Thomassen MCLGD, Curvers J, Rimmer JE, Preston FE, van Wersch JWJ, Tans G, Rosing J. Influence of hormone replacement therapy, oral contraceptives and pregnancy on APC-resistance. Thromb Haemost 1999; Suppl. 770-1.
• 30 Sugimura M, Kobayashi T, Kanayama N, Terao T. Detection of decreased response to activated protein C during pregnancy by an endogenous thrombin potential-based assay. Semin Thromb Hemostasis 1999; 25: 497-502.
• 31 Sugimura M, Kobayashi T, Kanayama N, Terao T. Detection of marked reduction of sensitivity to activated protein C prior to the onset of thrombosis during puerperium as detected by endogenous thrombin potential-based assay. Thromb Haemost 1999; 82: 1364-5.
• 32 Bakker HM, Tans G, Janssen TClaessen, Thomassen MCLGD, Hemker HC, Griffin JH, Rosing J. The effect of phospholipids, calcium ions and protein S on rate constants of human factor Va inactivation by activated human protein C. Eur J Biochem 1992; 208: 171-8.
• 33 Nelsestuen GL, Kisiel W, Di Scipio RG. Interaction of vitamin K dependent proteins with membranes. Biochemistry 1978; 17: 2134-8.
• 34 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: 564-6.
• 35 Duncan EM, Casey CR, Duncan BM, Lloyd JV. Effect of concentration of trisodium citrate anticoagulant on calculation of the international normalised ratio and the international sensitivity index of thromboplastin. Thromb Haemost 1994; 72 (01) 84-8.