Thrombin Generation Assays

The TGA is a global test of coagulation systems, with the potential to contribute to the current arsenal of clinical prognostication and drug monitoring tools. By varying the conditions under which it is applied, the test can be made sensitive to virtually every known component of the clotting cascade. The current iterations of the test utilize a fluorogenic substrate to monitor thrombin generation beyond the time point when the fibrin clot is formed. Standardization of testing conditions and defining testing indications for TGA is likely needed before the test enters into widespread clinical use.

Thrombin is a multifunctional enzyme that catalyzes the conversion of fibrinogen to fibrin, promotes additional thrombin formation by activation of coagulation factors (F) XI, V, and VIII, stabilizes a clot via activation of antifibrinolytic factors, and activates platelets and endothelial surfaces. When combined with thrombomodulin (TM) it can activate the anticoagulant protein C pathway, leading to dampening of the coagulation cascade.

Fibrin clots form relatively early in the process of thrombin generation, when 95% of thrombin is yet to form. Thus, traditional coagulation tests that terminate with clot formation do not measure the full thrombin generation potential of the plasma sample. Because thrombin formation is controlled by numerous pro- and anticoagulant factors, antigenic prothrombin level is only weakly reflective of the amount of thrombin that can be generated in plasma samples.

The total amount of thrombin that plasma is capable of generating is potentially quite important. Numerous studies have shown that this quantity may serve as a predictor of a patient’s bleeding or thrombosis risk. By varying test conditions, TGA measurements can be made sensitive to virtually all known risk factors for venous thrombosis, allowing assessment of their interaction in individual patients. The test can also be made sensitive to clotting factor abnormalities and factor replacement therapy. In the recent years it has been shown that elevated thrombin generation in vivo was predictive of recurrent venous thrombosis risk and of a first thrombotic event in high-risk subjects. It also could stratify severe FXI-, VIII-, and FIX-deficient patients into high- and low-risk groups for hemorrhage better than measuring individual factors in isolation.

TGAs have been performed in the research setting for many years but were cumbersome and suffered from numerous technical limitations. Recent advances in the development of fluorogenic substrates, software, and instrumentation have made the assays less laborious, potentially more reproducible outside of highly specialized settings and have allowed for automation of large portions of the test workflow. These developments portend well for adoption of TGA into general clinical practice.