MEASUREMENT OF ACTIVE COAGULATION-FACTORS IN AUTOPLEX(R)-T WITH COLORIMETRIC ACTIVE SITE-SPECIFIC ASSAY TECHNOLOGY

Autoplex(R)-T is a partially activated prothrombin complex concentrate used primarily for the treatment of patients expressing factor VIII i nhibitors. While Autoplex(R)-T has a demonstrated record of clinical e ffectiveness, the procoagulant composition of this material has not be en reported. This absence of composition data is a reflection of the l ack of techniques appropriate for accurately measuring an individual p rotease such as factor IXa in complex mixtures of similar proteases. T he development of Colorimetric Active Site-Specific ImmunoAssay techno logy (CASSIA) has permitted the accurate analysis of the coagulant enz ymes present in Autoplex(R)-T. Ten lots of Autoplex(R)-T were reacted with both biotinylated phenylalanylprolylarginine chloromethylketone a nd biotinylated glutamylglycylarginine chloromethylketone. Only activa ted forms of the clotting factors present in Autoplex(R)-T react with the peptide chloromethylketones and were thus separated from the other proteins present in Autoplex(R)-T by adsorption onto streptavidin. Th e individual proteins bound to streptavidin were then detected with sp ecific antibodies. Mean results from the analysis of ten lots of Autop lex(R)-T (mean values) are as follows: factor XIa, 5.9 nM or 0.8 mu g/ ml; factor Xa, 46.5 nM or 2.1 mu g/ml; factor IXa, 177.8 nM or 11.7 mu g/ml; factor VIIa, 68.6 nM or 3.3 mu g/ml and factor IIa, 5.3 nM or 0 .2 mu g/ml. These results are discussed with respect to the mechanism of action of Autoplex(R)-T in the treatment of factor VIII inhibitor p atients.