Abstract
In the estimation of anti-thrombin III (AT III) in the plasma, biological or immunological methods have generally been employed so far. However, certain limitations such as the intricate and time-consuming nature of the procedures can be raised against their practical use.
Recently conventional methods using chromogenic peptide substrate have been devised, although inasmuch as the reaction time is extremely short with this method it is inevitable that technical failure occures and the method is not convenient for examining large member of specimens. Also, due to the presence of inhibitors other than AT III in the plasma, the dilution effect should be considered.
The above difficulties have been overcome in the following way. Citrated plasma was diluted 71-folds with Tris-HCl buffer (pH 8.8) containing appropriate amounts of heparin and aprotinin. The diluted specimen (0.05ml) was then added to the same buffer (0.2ml), thrombin solution (0.1ml), and allowed to stand at 37°C for 10min. This was followed by addition of the substrate (Tos-Gly-Pro-Arg-pNA: purchased from Pentapharm) and then further incubation at 37°C for 10min. The reaction was stopped with 2.5ml of acetic acid (10%) then absorbancy at 405nm was measured.
The results of our experiment will be summarized as follows:
1) The reaction rate of AT III for anti-thrombin action is accelerated by heparin.
2) The activity of plasma enzymes having amidase activity such as trypsin and plasmin except thrombin, was inhibited by aprotinin.
3) Anti-thrombin action was not affected by any inhibitor other than AT III.
4) Measurements of AT III activity in a series of diluted specimens showed a linearity up to 125%.
5) A good correlation with the immunological method was observed (r=0.988).
6) The procedure was well suited to the measurement of AT III in relation to the estimation of hemorrahgic diathesis.
