Measurement of active coagulation factors in Autoplex-T with colorimetric active site-specific assay technology.
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.
Lundblad
R L
RL
Hyland/Immuno Division of Baxter Healthcare, Duarte, CA 91010-2923, USA. lundblr@baxter.com
Bergstrom
J
J
De Vreker
R
R
Bray
G
G
Gomperts
E
E
Baker
D
D
Kingdon
H S
HS
Mann
K G
KG
Hartshorn
J
J
Jenny
R J
RJ
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
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