Photometric Assay of Factor VIIIC With a Chromogenic Substrate

Abstract

By the aid of chromogenic substrates, highly specific assays of some serine proteases can be carried out. The substrate used for factor VIIIC-assays was Bz-Ile-Glu-Gly-Arg-pNA [S-2222 KABI) which measures factor Xa. When all components necessary for factor Xa activation except factor VIIIC are kept at constant levels, the resulting Xa-activity is in direct relation to the concentration of factor VIIIC. The substrate plasma was a mixture of hemophilia A plasma with factor VIII inhibitor plasma with a remaining inhibitor activity of between 0.1 and 0.5 units per ml. This substrate was defibrinated by ancrod. For assays of factor VIIIC, this plasma was mixed with the diluted test plasma, cepheloplastin, and calcium chloride at 37°C After a constant activation time, the chromogenic substrate was added and the difference in OD/min was measured at 405 nm. The calibration curve was linear between 1% and over 200% factor VIIIC activity, and the average CV was 7.9%. This method was compared to a standard one-stage method for factor VIIIC. Identical results were obtained in plasma samples of normal individuals, in samples of high factor VIIIC, activity, in plasma from hemophilia A patients, and in factor VIII concentrates. The advantages of this method over the clotting method are: independence of the results from variations of factors V,II, and I in the reaction mixture, stability of the reagents, a better discrimination of factor VIIIC levels in the range between 30% and over 100%, and the possibility of automatization of the method.