title: Measurement of active coagulation factors in Autoplex-T with colorimetric active site-specific assay technology.
abstract: Autoplex-T is a partially activated prothrombin complex concentrate used primarily for the treatment of patients expressing factor VIII inhibitors. While Autoplex-T has a demonstrated record of clinical effectiveness, the procoagulant composition of this material has not been reported. This absence of composition data is a reflection of the lack of techniques appropriate for accurately measuring an individual protease such as factor IXa in complex mixtures of similar proteases. The development of Colorimetric Active Site-Specific ImmunoAssay technology (CASSIA) has permitted the accurate analysis of the coagulant enzymes present in Autoplex-T. Ten lots of Autoplex-T were reacted with both biotinylated phenylalanylprolylarginine chloromethylketone and biotinylated glutamylglycylarginine chloromethylketone. Only activated forms of the clotting factors present in Autoplex-T react with the peptide chloromethylketones and were thus separated from the other proteins present in Autoplex-T by adsorption onto streptavidin. The individual proteins bound to streptavidin were then detected with specific antibodies. Mean results from the analysis of ten lots of Autoplex-T (mean values) are as follows: factor Xla, 5.9 nM or 0.8 microg/ml; factor Xa, 46.5 nM or 2.1 microg/ml; factor IXa, 177.8 nM or 11.7 microg/ml; factor VIIa, 68.6 nM or 3.3 microg/ml and factor IIa, 5.3 nM or 0.2 microg/ml. These results are discussed with respect to the mechanism of action of Autoplex-T in the treatment of factor VIII inhibitor patients.
authors.affiliation: Hyland/Immuno Division of Baxter Healthcare, Duarte, CA 91010-2923, USA. lundblr@baxter.com
authors.forename: R L
authors.initials: RL
authors.lastname: Lundblad
authors.forename: J
authors.initials: J
authors.lastname: Bergstrom
authors.forename: R
authors.initials: R
authors.lastname: De Vreker
authors.forename: G
authors.initials: G
authors.lastname: Bray
authors.forename: E
authors.initials: E
authors.lastname: Gomperts
authors.forename: D
authors.initials: D
authors.lastname: Baker
authors.forename: H S
authors.initials: HS
authors.lastname: Kingdon
authors.forename: K G
authors.initials: KG
authors.lastname: Mann
authors.forename: J
authors.initials: J
authors.lastname: Hartshorn
authors.forename: R J
authors.initials: RJ
authors.lastname: Jenny
mesh_terms: D000590:Amino Acid Chloromethyl Ketones / Q000378:metabolism; D001665:Binding Sites; D001710:Biotin / Q000031:analogs & derivatives / Q000378:metabolism; D019921:Biotinylation; D001779:Blood Coagulation Factors / Q000032:analysis* / Q000737:chemistry*; D003124:Colorimetry / Q000379:methods*; D005069:Evaluation Studies as Topic; D015949:Factor IXa / Q000032:analysis; D015942:Factor VIIa / Q000032:analysis; D015945:Factor XIa / Q000032:analysis; D015951:Factor Xa / Q000032:analysis; D006801:Humans; D011516:Prothrombin / Q000032:analysis; D013379:Substrate Specificity
chemical_list: D000590:Amino Acid Chloromethyl Ketones; D001779:Blood Coagulation Factors; C093995:biotinyldicaproyl-phenylalanyl-prolyl-arginyl-chloromethylketone; C086298:glutamyl-glycyl-arginyl-chloromethyl ketone; C025667:prothrombin complex concentrates; D001710:Biotin; D011516:Prothrombin; C025337:Factor IIa; D015942:Factor VIIa; D015949:Factor IXa; D015945:Factor XIa; D015951:Factor Xa