Determination of Activated Factor IX in Factor IX Concentrates with a Chromogenic Substrate

Abstract

In the last years, prothrombin complex concentrates were replaced by factor IX (FIX) concentrates for the treatment of haemophilia B. Although high purity FIX concentrates were proven to be less thrombogenic than prothrombin complex concentrates [ 1 Smith K.J. Immunoaffinity purification of factor IX from commercial concentrates and infusion studies in animals. Blood. 1988; 72: 1269-1277 Crossref PubMed Google Scholar , 2 Hampton K.K. Preston F.E. Lowe G.D. Walker I.D. 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: 279-284 Crossref PubMed Scopus (35) Google Scholar , 3 Thomas D.P. Hampton K.K. Dasani H. Lee C.A. Giangrande P.L. Harman C. Lee M.L. Preston F.E. A cross-over pharmacokinetic and thrombogenicity study of a prothrombin complex concentrate and a purified factor IX concentrate. Br J Haematol. 1994; 87: 782-788 Crossref PubMed Scopus (30) Google Scholar , 4 Philippou H. Adami A. Lane D.A. MacGregor I.R. Tuiddenham E.G.D. Lowe G.D.O. Rumlley A. Ludlam C.A. 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: 23-28 PubMed Google Scholar ] there is still the necessity to test the preparations for the presence of trace amounts of activated clotting factors. Particularly activated FIX (FIXa) has been suggested as one of the potential thrombogenic factors in high purity FIX concentrates [ 5 Hultin M.B. Activated clotting factors in factor IX concentrates. Blood. 1979; 54: 1028-1038 PubMed Google Scholar , 6 Gray E. Tubbs J. Thomas S. Oates A. Boisclair M. Kemball-Cook G. Barrowcliffe T.W. Measurement of activated factor IX in factor IX concentrates with in vivo thrombogenicity. Thromb Haemost. 1995; 73: 675-679 PubMed Google Scholar , 7 Gray E. Walker D. Heath A. Barrowcliffe T.W. Collaborative study on assays of activated FIX (FIXa). Thromb Haemost. 1996; 76: 1114-1117 PubMed Google Scholar ]. Methods for measurement of FIXa are not well established. The method described in the European Pharmacopeia (EP) for evaluating FIX concentrates is based on the measurement of the influence of these concentrates on the nonactivated partial thromboplastin time (NAPTT) of platelet-poor plasma. However, it has been shown that NAPTT results do not correlate well with the thrombogenic potential of the concentrates in animal experiments [ 2 Hampton K.K. Preston F.E. Lowe G.D. Walker I.D. 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: 279-284 Crossref PubMed Scopus (35) Google Scholar ]. Chromogenic methods for determination of FIXa were based on the generation of factor Xa by FIXa in the presence of an excess of purified factor VIII, factor X, phospholipid, and calcium. With this method, trace amounts of FIXa in FIX concentrates were detected, which correlate with the in vivo thrombogenicity of the concentrates [ 6 Gray E. Tubbs J. Thomas S. Oates A. Boisclair M. Kemball-Cook G. Barrowcliffe T.W. Measurement of activated factor IX in factor IX concentrates with in vivo thrombogenicity. Thromb Haemost. 1995; 73: 675-679 PubMed Google Scholar , 8 Hultin M.B. Nemerson Y. Activation of factor X by factors IXa and VIII; a specific assay for factor IXa in the presence of thrombin-activated factor VIII. Blood. 1978; 52: 928-940 Crossref PubMed Google Scholar ]. Up to now, a direct chromogenic determination of FIXa was not possible because of the low amidolytic activity of this clotting enzyme against chromogenic substrates. Recently, it could be shown that certain alcohols, especially ethylene glycol, dramatically increase the catalytic activity of FIXa toward synthetic substrates of the type R-D-Xxx-Gly-Arg-pNA [ 9 Stürzebecher J. Kopetzki E. Bode W. Hopfner K.-P. Dramatic enhancement of the catalytic activity of coagulation factor IXa by alcohols. FEBS Lett. 1997; 412: 295-300 Abstract Full Text Full Text PDF PubMed Scopus (47) Google Scholar ]. This finding opens the way for a direct determination of activated FIX. In the present study, we investigated the possibility to determine trace amounts of activated FIX in FIX concentrates with the chromogenic substrate CH3SO2-D-CHG-Gly-Arg-pNA. The advantages of this new method over the commonly used clotting method, NAPTT, are discussed.