An improved method for two-stage prothrombin determination by the addition of a new factor V reagent using chromogenic peptide substrates was described. Two hundred and forty pl of citrated plasma (1 in 120), diluted with tris buffer, pH 8.4, 10.15 was activated with 100 pl of a commercial thromboplastin and calcium in the presence of 20 pl of the F. V reagent. After 4 minutes incubation at 37C 75 pl of a 1 mM solution of the chromogenic substrate Bz-Phe-Val-Arg-p-nitroanilide was added and the

more »

... increase in absorbance were recorded in a reaction rate analyzer (LKB 8600). An approximate linear relationship was found between JA/min and dilutions of normal plasma in the buffer. The influence of heparin was eliminated by the adding a minute amount of polybrene to the buffer. The new method was well correlated with conventional clotting methods in normal individuals and warf arin treated patients. However, approximately 40 % elevation of the prothrombin level was obtained in plasmas of warf arin treated patients, when echis carinatus venom was used as a prothrombin activator instead of the thromboplastin reagent. Detection of the dissociation of prothrombin level by methods with thromboplastin and with echis carinatus venom and/or by immunological methods is useful for the elucidation of abnormal prothrombin states.