HEPARIN COFACTOR II: A SIMPLE ASSAY METHOD

A photometric assay method for heparin cofactor II (HC II) is described. In a first step antithrombin III (AT III) in plasma is blocked by an anti human AT III immunoglobuline from goats. After dilution of this plasma with Tris buffer pH 8.4 containing 3 IU/ml heparin and addition of thrombin the remaining thrombin activity is measured by use of the chromogenic substrate S-2238 Kabi. The following preliminary experiments were carried out: Variation of the amount of anti-AT III added to plasma resulted in complete inactivation of 1.25 units AT III by 1.0 ml of the inhibitor. Incubation of 1 ml anti AT III with 1 ml purified AT III ( 1 U/ml} or with 1 ml normal plasma completely abolished AT III activity within 60 sec. Incubation of the reaction mixture with thrombin resulted in maximum inactivation after 180 sec. This is in contrast to AT III activated by heparin which immediately inactivates thrombin. Anti-Xa activity after depletion of AT III was assayed in a similar way by addition of factor Xa to the reaction mixture and measuring the remaining Xa activity by the substrate S-2222. In these tests no anti Xa-activity was found after AT III depletion. From these experiments there was assumed that the anti thrombin activity measured under the following conditions was due to the action of HC II:Plasma ( 50 μl) was mixed with anti AT III (50 μl) and was incubated for 60 sec. Tris buffer with heparin pH 8.4 (900 μl) was added. From this mixture 200 μl was pipetted into a cuvette at 37°C followed by 200 μl thrombin ( 2 IU/ml). After an incubation time of 180 sec 200 μl S-2238 ( 2 mmol/1) was added and the difference in OD/min was determined at 405 nm. A calibration curve was made by series of dilutions of normal AT III depleted plasma from 20 healthy individuals. The following preliminary results ofrHC II activity as a percentage were obtained: