Effect of substrate and fibrin polymerization inhibitor on determination of plasma thrombin generation in vitro

BACKGROUND: Thrombin generation potential, a critical haemostatic measure, can be determined by continuous detection of total thrombin or direct subsampling. However, differences between methods exist in area under the curve or peak thrombin calculated. Also, impact of anticoagulants on thrombin generation may vary depending on mode of analysis. OBJECTIVE: We studied the effect of components on thrombin generation in the presence or absence of anticoagulants. METHODS: The continuous method was conducted with plasma +/- fibrin(ogen) +/- fibrin polymerization inhibitor. Plasma contained slow-reacting TG5134 substrate at 37 degrees C and reaction was started with dilute thromboplastin in CaCl(2)/Tris buffer. Absorbance (405 nm) was recorded over time and free thrombin calculated from total thrombin activity. For the subsampling method, similar plasma mixtures +/- TG5134 were reacted and free thrombin measured directly as the difference in activity against S2238 substrate of timed subsamples taken into EDTA or EDTA + antithrombin + heparin. RESULTS: Slow-reacting substrate in the continuous method acted as a competitor for thrombin, giving delayed but greater free thrombin than direct subsampling. These differences persisted to varying extents with all anticoagulants tested. In either method, presence of polymerization inhibitor increased the amount of free thrombin. Continuous method detection of alpha(2)macroglobulin complexes was hampered by sensitivity limits leading to inordinate free thrombin calculations. Especially with hirudin, although free thrombin remained at the end of the subsampling method, continuous method calculations assumed no residual free thrombin. CONCLUSION: In vitro plasma thrombin generation is delayed and increased by slow-acting substrate and fibrin polymerization inhibitor.