The paradoxical stimulation by a reversible thrombin inhibitor of thrombin generation in plasma measured with thrombinography is caused by alpha-macroglobulin-thrombin

Thrombin generation (TG) in plasma can be monitored continuously with a fluorogenic thrombin substrate using calibrated automated thrombinography (CAT). In the presence of low concentrations of a reversible direct thrombin inhibitor (DTI), CAT shows an unexpected effect: the endogenous thrombin potential (ETP) increases at low concentrations of the inhibitor to subsequently decrease concentration dependently at higher concentrations (> approximately 100 nm). To find an explanation for this phenomenon, we measured the concentrations of free thrombin and alpha(2)-macroglobulin-thrombin complex (alpha(2)MT) with a sub-sampling technique in the presence of AR-H067637, a selective DTI. At all concentrations of the DTI there was a gradual dose-dependent decrease in the concentration of free, not-inhibited thrombin but a transient increase in free alpha(2)MT due to competition of thrombin and alpha(2)MT for the inhibitor. Because the CAT technique uses an algorithm to subtract alpha(2)MT activity from the total amidolytic activity, this transient increase in alpha(2)MT activity is not subtracted and erroneously attributed to thrombin itself. This study explains the spurious increase in ETP observed at low DTI concentrations. The results obtained in plasma were corroborated by observations in a thrombin generating system reconstituted with purified factors. In practise, the effect of DTIs on TG can be reliably evaluated from the area under the curve till time-to-peak.