Axelsson G, Korsan-Bengtsen K, Waldenström J
Thromb Haemost. 1976 Dec 31;36(3):517-24.
A two stage method to determine prothrombin with the chromogenic peptide substrate benzoyl-phe-val-arg-p-nitroanilide has been worked out. Citrated plasma (10 mul) was diluted in 600 mul tris buffer, pH 8.2, ionic strength 0.18 and activated with 250 mul of a commercial rabbit brain-lung thromboplastin. After 325 s incubation at 37 degrees C 200 mul of a 1 mM solution of the chromogenic substrate was added and the increase in absorbance was recorded in a LKB-Beckman 8600 enzyme analyzer. A reading time of 1 minute (including a delay of 20 s) was used which permitted 55 analyses per hour to be carried out. An approximate linear relationship was found between delta A/min and dilutions of normal plasma in prothrombin deficient plasma. The method is insensitive to variations of factors V, VII and X. Less than 10% or normal plasma was needed to "normalize" plasmas deficient in factor V or VII or X. A group of 99 dicoumarol treated patients and 23 normal subjects has been investigated using the present method and compared with a factor II-VII-X determination method. A correlation coefficient of 0.95 was found.
已制定出一种用生色肽底物苯甲酰 - 苯丙 - 缬 - 精 - 对硝基苯胺测定凝血酶原的两阶段方法。将枸橼酸盐血浆（10微升）在600微升pH 8.2、离子强度0.18的Tris缓冲液中稀释，并用250微升市售兔脑肺凝血活酶激活。在37℃孵育325秒后，加入200微升1毫摩尔/升的生色底物溶液，并在LKB - 贝克曼8600酶分析仪中记录吸光度的增加。使用1分钟的读数时间（包括20秒的延迟），每小时可进行55次分析。在凝血酶原缺乏血浆中，发现ΔA/分钟与正常血浆稀释度之间存在近似线性关系。该方法对因子V、VII和X的变化不敏感。“校正”因子V、VII或X缺乏的血浆所需的正常血浆不到10%。已使用本方法对一组99例双香豆素治疗患者和23名正常受试者进行了研究，并与因子II - VII - X测定方法进行了比较。发现相关系数为0.95。
