High Sensitivity Detection of Activated Factor IX: Application to the Analysis of Different Therapeutical Factor IX Concentrates and Prothrombin Complexes

Summary

A very sensitive and highly reliable test system for the detection of activated coagulation factor IX (FIXa) has been established. This assay system is based on the cleavage of a fluorogenic substrate by activated factor X (FXa) which is generated by FIXa. This assay can be used to process a large number of samples at a time and, being based on the convenient microtiter plate format, can easily be adapted to automated processing for routine screening of large sample numbers.

With this assay at hand we determined the FIXa content of different commercially available therapeutic FIX sources, such as high purity FIX (HPFIX) and prothrombin complex concentrates (PCC). Here we demonstrate that PCC from several suppliers do not contain significantly higher levels of FIXa as compared to HPFIX from the same supplier. In fact, there is a tendency for HPFIX to contain more FIXa than PCC. Moreover, HPFIX from certain manufacturers who do not produce PCC are characterized by an exceptionally high content of FIXa. Therefore, the higher thrombogenic potential of PCC which is well documented clinically cannot be explained solely – if at all – by an increased content of FIXa. Rather, it will be necessary to identify other components responsible for this phenomenon.

• References

• 1 Brettler DB, Levine PH. Clinical manifestations and therapy of inherited coagulation factor deficiencies. In: Haemostasis and Thrombosis, 3rd edition. Colman RW, Hirsh J, Marder VJ, Salzman EW. eds. 1994: 169-83.
• 2 Bick RL. Principles of clinical practice. In: Disorders of haemostasis and thrombosis. Georg Thieme Verlag; Stuttgart, New York: 1985
• 3 Scharrer I. The need for highly purified products to treat haemophilia B. Acta haematol 1995; 94 (suppl): 2-7.
• 4 Makris M, Greaves M, Phillips WS, Kitchen S, Rosendaal FR, Preston FE. Emergency oral anticoagulant reversal: The relative efficiency of infusions of fresh frozen plasma and clotting factor concentrate on correction of the coagulopathy. Thromb Haemost 1997; 77: 477-80.
• 5 Fredriksson K, Norrving B, Stromblad LG. Emergency reversal of anticoagulation after intracerebral hemorrhage. Stroke 1992; 23: 972-7.
• 6 Brozovic M. Acquired disorders of coagulation. in: Haemostasis and Thrombosis, 2nd edition. Bloom AL, Duncan PT. eds. 1987: 519-34.
• 7 Ohga S, Saito M, Matsukazi A, Kai T, Ueda K. Disseminated intravascular coagulation in a patient with haemophilia B during factor IX replacement therapy. Br J Haematol 1993; 84: 343-5.
• 8 Gruen DR, Winchester PH, Brill PW, Ramirez E. Magnetic resonance imaging of myocardial infarction during prothrombin complex concentrate therapy of hemophilia A. Pediatric Radiology 1997; 27: 271-2.
• 9 Chistolini A, Mazzucconi MG, Tirindelli MC, La Verde G, Ferrari A, Mandelli F. Disseminated intravascular coagulation and myocardial infarction in a haemophilia B patient during therapy with prothrombin complex concentrates. Acta haematologica 1990; 83: 163-5.
• 10 Kingdon HS, Lundblad RL, Veltkamp JJ, Aronson DL. Potentially thrombogenic materials in factor IX concentrates. Thromb Diath Haemorrh 1975; 33: 617-31.
• 11 Hultin MB. Activated clotting factors in factor IX concentrates. Blood 1979; 54: 1028-38.
• 12 Seligsohn U, Kasper CK, Osterud B, Rapaport SI. Activated factor VII: Presence in factor IX concentrates and persistence in the circulation after infusion. Blood 1979; 53: 828-37.
• 13 Giles AR, Nesheim ME, Hoogendorn H, Tracy PB, Mann KG. The coagulant active phospholipid content is a major determinant of in vivo thrombogenicity of prothrombin complex (factor IX) concentrates in rabbits. Blood 1982; 59: 401-7.
• 14 Berntorp E. Why prescribe highly purified factor VIII and IX concentrates?. Vox Sanguinis 1996; 70: 61-8.
• 15 Roberts HR, Eberst ME. Current management of haemophilia B. Hematology Oncology Clinics of North America 1993; 7: 1269-80.
• 16 Hampton KK, Preston FE, Lowe GDO, Walker ID, Sampson B. Reduced coagulation activation following infusion of a highly purified factor IX concentrate compared to a prothrombin complex concentrate. Br J Haematol 1993; 84: 279-84.
• 17 Mannucci PM, Bauer KA, Gringeri A, Barzegar S, Santagostino E, Tradati FC, Rosenberg RD. No activation of the common pathway of the coagulation cascade after a highly purified factor IX concentrate. Br J Haematol 1991; 79: 606-11.
• 18 Harrison J, Abildgaard C, Lazerson J, Culbertson R, Anderson G. Assessment of thrombogenicity of prothrombin complex concentrates in a porcine model. Thromb Res 1985; 38: 173-88.
• 19 Smith KJ. Immunoaffinity purification of factor IX from commercial concentrates and infusion studies in animals. Blood 1988; 72: 1269-77.
• 20 Philippou H, Adami A, Lane DA, MacGregor IR, Tuddenham EGD, Lowe GDO, Rumley A, Ludlam CA. High purity factor IX and prothrombin complex concentrate (PCC): pharmacokinetics and evidence that factor IXa is the thrombogenic trigger in PCC. Thromb Haemost 1996; 76: 23-8.
• 21 Gray E, Tubbs J, Oates TA, Boisclair M, Kemball-Cook G, Barrowcliffe TW. Measurement of activated factor IX in factor IX concentrates: Correlation with in vivo thrombogenicity. Thromb Haemost 1995; 73: 675-79.
• 22 Kawabata S, Miura T, Morita T, Kato H, Fujikawa K, Iwanaga S, Takada K, Kimura T, Sakakibara S. Highly sensitive peptide-4-methylcoumaryl 7-amide substrates for blood clotting proteases and trypsin. Eur J Biochem 1988; 172: 17-25.
• 23 Williams EB, Krishnaswamy S, Mann KG. Zymogen/enzyme discrimination using peptide chloromethyl ketones. J Biol Chem 1989; 264: 7536-44.
• 24 Hashimoto N, Morita T, Iwanaga S. A method for systematic purification from bovine plasma of six vitamin K dependent coagulation factors: Prothrombin, factor X, factor IX, protein S, protein C, and protein Z. J Biochem 1985; 97: 1347-55.
• 25 Furie BC, Furie B. Coagulant protein of Russel’s viper venom. Methods in enzymology Lorand L. (ed.). New York, Academic press 1976; 45: 191.
• 26 Marsman FR. Parallel line assay: Calculation of potency and 95% confidence limits, version 94-1; in: Manual of laboratory methods for testing the potency of final vaccines used in the WHO expanded programme on immunization. WHO BLG/95.1.
• 27 Laemmli UK. Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature 1970; 227: 680-5.
• 28 Towbin H, Staehelin T, Gordon J. Electrophoretic transfer of proteins from polyacrylamide gels to nitrocellulose sheets: procedure and some applications. Proc Natl Acad Sci 1979; 76: 4350-4.
• 29 Schaffner W, Weissmann C. A rapid, sensitive, and specific method for the determination of protein in dilute solution. Anal Biochem 1973; 56: 502-14.
• 30 Kemkes-Matthes B, Matthes KJ. Protein Z deficiency- a new type of bleeding disorder. Annals of Haematology 1993; 66 (Suppl. 01) A39.