Use of chromogenic peptide substrates in the determination of clotting factors II, VII, IX and X in normal plasma and in plasma of patients treated with oral anticoagulants

Spectrophotometric methods were used to assay the clotting factors II, VII, IX and X in plasma of 33 subjectively healthy human donors and in plasma of 98 patients receiving long-term oral anticoagulant therapy. In 33 normal subjects the interindividual variations in the plasma activities of the clotting factors II, VII, IX and X are respectively 12.2, 21.4, 11.0 and 15.0%. After correction for the assay variations the remaining biological variations are respectively 11.7, 21.2, 9.7 and 14.8%. Plasma from 98 patients receiving long-term anticoagulant therapy was assayed with ‘Thrombotest’, a clotting test in whole blood introduced by Owren and in these plasmas the activity of each of the vitamin K-dependent factors was assayed with spectrophotometric methods. For the clotting factors IX and VII, novel spectrophotometric methods were applied and the plasma activities thus measured were compared to results obtained with factor IX and VII clotting assays. Chromogenic activities of the different factors were correlated among each other and with 1/Thrombotest values. When the therapeutic range for Thrombotest values is set between 5 and 12.5% the corresponding therapeutic ranges for the activity of the factors II, VII, IX and X are respectively 12.6–36.1, 27.0–52.3, 23.1–49.3 and 18.9–36.2% (expressed as a percentage of the activity in normal pool plasma). The chromogenic assays for the factors II, VII, IX and X provide the same information on the therapeutic state of the patients in respectively 86.7, 78.6, 81.6 and 89.8% of the cases. Finally we discuss the suitability of the different assays to monitor oral anticoagulant therapy

Activation of factor IX by factor XIa:A spectrophotometric assay for factor IX in human plasma

The activation of Factor IX by partially purified Factor XIa was followed by active site titration, gelelectrophoresis and by a spectrophotometric assay. The assay is based on the finding that the rate of Factor X activation in the presence of phospholipid and Ca2+ is linear in time and proportional to the amount of Factor IXa present and can be determined with the chromogenic substrate S2222. Conditions were found that allowed complete activation of Factor IX in human plasma by Factor XIa. The amount of Factor IXa present in the plasma sample can be determined with the spectrophotometric assay and is proportional with the amount of plasma present. In plasma from patients receiving vitamin-K antagonists reduced Factor IX activity is found with the spectrophotometric assay, and the new assay method may be useful in monitoring oral anticoagulant therapy