The technique of measuring thrombin generation with fluorogenic substrates: 3. The effects of sample dilution

Assessing the clotting function inevitably brings about dilution of plasma. With the existing techniques of thrombin generation (TG) measurement, dilution ranges from 2:3 to 1:8. However, the possibility that dilution alters procoagulant and anticoagulant pathways differently has not been examined. We investigated the effects of dilution on the thrombin generation process and found that the anticoagulant pathways are far more affected by dilution than the procoagulant pathways. That is, when prothrombin and antithrombin concentrations are kept constant, dilution of plasma does not significantly affect tissue factor (TF)-driven thrombin generation. We demonstrate that dilution of plasma slows down the inhibitory activity of tissue factor pathway inhibitor (TFPI) to a greater extent when compared with the down regulation by diluting procoagulant factors. Dilution of plasma has also a negative effect on the participation of the antihaemophiliac factors VIII and IX in TG driven by contact activation or low TF concentration. We also investigated the effect of dilution on the participation of the anticoagulant system that consists of thrombomodulin, protein C and protein S (APC system). We found that plasma dilution causes a loss of sensitivity towards TM and APC. Furthermore, at high dilutions (> 1:12) a second wave of prothrombinase-activity was observed that could be attributed to the suppression of protein S-dependent inhibition. In conclusion, the mechanism of TG is profoundly disturbed by plasma dilution. As a consequence, the less a plasma sample is diluted, the better a TG experiment represents the physiological process.