Thrombin Generation (PPP, PRP, WB)

Thrombin generation is a global hemostatic assay, which describes the formation and inactivation of thrombin in clotting plasma or whole blood. Synapse Research Institute is the inventor and patent holder of the Calibrated Automated Thrombinography (CAT) method, which allows quantitative assessment of thrombin generation. It is based on the use of a fluorogenic thrombin substrate and a thrombin calibrator, which corrects for inner filter effect, donor-to-donor variability in transparency of plasma, substrate depletion and instrumental differences (http://www.thrombinoscope.com/). The Thrombinograph™ (a 96-well plate fluorimeter available from ThermoFisher) is used to measure raw thrombin generation curves and dedicated software from Thrombinoscope™ calculates thrombin generation parameters from these curves. The main parameters are the lag time, the endogenous thrombin potential (ETP; area under the thrombin generation curve) and the peak height.

The lag time is the time needed to form the first traces of thrombin, the ETP represents the total amount of active thrombin formed during thrombin generation and the peak height the maximal amount of thrombin formed.
Numerous articles have shown that an increased thrombin generation is associated with thrombosis, whereas a decrease in thrombin generation is associated with bleeding.

Thrombin generation by means of the CAT assay can be used for several applications, including:

To monitor the use of antithrombotic therapy, e.g. treatment with direct oral anticoagulants (e.g. Dabigatran, Rivaroxaban), vitamin K antagonists (e.g. warfarin), heparins (e.g. unfractionated heparin, low molecular weight heparin) and antiplatelet drugs (e.g. Clopidogrel)
To support the development of antithrombotic drugs, such direct inhibitors of thrombin or FXa
To determine the coagulation state of patients at increased risk of thrombosis or bleeding
To monitor patients with hemophilia, such as treatment with FVIII concentrates or treatment with FVIIa (NovoSeven)
To measure genetic and acquired thrombotic disorders, e.g. deficiency in antithrombin (AT)
To determine thrombogenic capacity of materials
To quantify the levels of coagulation factors in low sample volumes
To elucidate coagulation mechanisms

Thrombin generation triggered with tissue factor can be measured in platelet poor plasma (PPP), platelet rich plasma (PRP) and whole blood. The conditions of the thrombin generation assay can be modified to better accommodate the question at hand. For example, the addition of thrombomodulin to the trigger allows to include the contribution of the protein C pathway to thrombin generation.

References:
Bloemen S, Huskens D, Konings J, Kremers R, Miszta A, de Laat B, Kelchtermans H. Interindividual variability and normal ranges of whole blood and plasma thrombin generation. JALM. 2017 Sept; 2 (2): 150 -164.

Huskens D, Sang Y, Konings J, van der Vorm L, de Laat B, Kelchtermans H, Roest M. Standardization and reference ranges for whole blood platelet function measurements using a flow cytometric platelet activation test. PLoS One. 2018 Feb 1;13(2):e0192079.