Thrombin Generation Assays: Possibilities and Limitations

Abstract

Thrombin generation assays (TGA) provide a dynamic and integrative assessment of thrombin generation in clotting plasma ex vivo. The method is characterized by triggering coagulation, typically via the extrinsic pathway, and continuously measuring thrombin activity using a fluorogenic peptide substrate to derive key parameters such as peak thrombin and the endogenous thrombin potential. Several assay platforms are currently available, with the original Calibrated Automated Thrombogram (CAT) still being widely used in clinical and research settings, not least due to its flexibility and ability to analyze both platelet-poor as well as platelet-rich plasma. Thrombin generation assays have the potential to support the evaluation and monitoring of treatment of bleeding disorders, including hemophilia A and B and other inherited or acquired coagulation factor deficiencies. They may contribute to risk stratification in thrombotic disorders, and support the assessment of anticoagulant therapies. However, besides ongoing developments and optimization of trigger reagents, inter-assay variability and susceptibility to pre- and analytical variables challenge assay standardization and inter-laboratory comparability. Continued refinement, harmonization, and prospective clinical validation will be essential to unlock the full diagnostic potential of TGA.

Keywords

thrombin generation - diagnostic tool - hemophilia - thrombosis risk - anticoagulation

Authors' Contributions

J.M.: wrote the manuscript; B.P. and J.O.: provided additional scientific/clinical data. All authors reviewed, revised, and finally agreed on the final version of the manuscript.

Publication History

Received: 19 November 2025

Accepted: 23 November 2025

Article published online:
17 February 2026

• References

• 1 Winter WE, Flax SD, Harris NS. Coagulation testing in the core laboratory. Lab Med 2017; 48 (04) 295-313
• 2 Reda S, Morimont L, Douxfils J, Rühl H. Can we measure the individual prothrombotic or prohemorrhagic tendency by global coagulation tests?. Hamostaseologie 2020; 40 (03) 364-378
• 3 Depasse F, Binder NB, Mueller J. et al. Thrombin generation assays are versatile tools in blood coagulation analysis: a review of technical features, and applications from research to laboratory routine. J Thromb Haemost 2021; 19 (12) 2907-2917
• 4 Binder NB, Depasse F, Mueller J. et al. Clinical use of thrombin generation assays. J Thromb Haemost 2021; 19 (12) 2918-2929
• 5 de Laat-Kremers RMW, Ninivaggi M, Devreese KMJ, de Laat B. Towards standardization of thrombin generation assays: inventory of thrombin generation methods based on results of an International Society of Thrombosis and Haemostasis Scientific Standardization Committee survey. J Thromb Haemost 2020; 18 (08) 1893-1899
• 6 Ninivaggi M, de Laat-Kremers R, Tripodi A. et al. Recommendations for the measurement of thrombin generation: communication from the ISTH SSC Subcommittee on Lupus Anticoagulant/Antiphospholipid Antibodies. J Thromb Haemost 2021; 19 (05) 1372-1378
• 7 Park S, Park JK. Back to basics: the coagulation pathway. Blood Res 2024; 59 (01) 35
• 8 Esmon CT. The protein C pathway. Chest 2003; 124 (03) 26S-32S
• 9 Huntington JA. Natural inhibitors of thrombin. Thromb Haemost 2014; 111 (04) 583-589
• 10 Sidonio Jr RF, Hoffman M, Kenet G, Dargaud Y. Thrombin generation and implications for hemophilia therapies: a narrative review. Res Pract Thromb Haemost 2022; 7 (01) 100018
• 11 Hemker HC, Giesen P, AlDieri R. et al. The calibrated automated thrombogram (CAT): a universal routine test for hyper- and hypocoagulability. Pathophysiol Haemost Thromb 2002; 32 (5-6): 249-253
• 12 Kintigh J, Monagle P, Ignjatovic V. A review of commercially available thrombin generation assays. Res Pract Thromb Haemost 2017; 2 (01) 42-48
• 13 Jackson JW, Longstaff C, Woodle SA, Chang WC, Ovanesov MV. Sources of bias and limitations of thrombinography: inner filter effect and substrate depletion at the edge of failure algorithm. Thromb J 2023; 21 (01) 104
• 14 Talon L, Feriel J, Sinegre T, Lebreton A, Lecomte M, Hemar C. Multicenter analytical performance evaluation of the ST Genesia thrombin generation system. Ann Biol Clin (Paris) 2025; 83 (01) 35-48
• 15 Pasi KJ, Rangarajan S, Georgiev P. et al. Targeting of antithrombin in hemophilia A or B with RNAi therapy. N Engl J Med 2017; 377 (09) 819-828
• 16 Shapiro AD, Angchaisuksiri P, Astermark J. et al. Subcutaneous concizumab prophylaxis in hemophilia A and hemophilia A/B with inhibitors: phase 2 trial results. Blood 2019; 134 (22) 1973-1982
• 17 Mahlangu JN, Lamas JL, Morales JC. et al. A phase 1b/2 clinical study of marstacimab, targeting human tissue factor pathway inhibitor, in haemophilia. Br J Haematol 2023; 200 (02) 229-239
• 18 Haisma B, Schols SEM, van Oerle RGM. et al. Comparative analysis of thrombin generation platforms for patients with coagulation factor deficiencies: a comprehensive assessment. Thromb Res 2024; 240: 109045
• 19 Spronk HM, Dielis AW, Panova-Noeva M. et al. Monitoring thrombin generation: is addition of corn trypsin inhibitor needed?. Thromb Haemost 2009; 101 (06) 1156-1162
• 20 Luddington R, Baglin T. Clinical measurement of thrombin generation by calibrated automated thrombography requires contact factor inhibition. J Thromb Haemost 2004; 2 (11) 1954-1959
• 21 Rodgers SE, Wong A, Gopal RD, Dale BJ, Duncan EM, McRae SJ. Evaluation of pre-analytical variables in a commercial thrombin generation assay. Thromb Res 2014; 134 (01) 160-164
• 22 Touw CE, de Jong Y, van Hylckama Vlieg A. The influence of corn trypsin inhibitor on the contribution of coagulation determinants to the Technoclone Thrombin Generation Assay (TGA) and the Calibrated Automated Thrombogram (CAT). PLoS One 2022; 17 (02) e0263960
• 23 Mohammed BM, Martin EJ, Salinas V, Carmona R, Young G, Brophy DF. Failure of corn trypsin inhibitor to affect the thrombin generation assay in plasma from severe hemophiliacs. J Thromb Haemost 2014; 12 (09) 1558-1561
• 24 Valke LLFG, Rijpma S, Meijer D, Schols SEM, van Heerde WL. Thrombin generation assays to personalize treatment in bleeding and thrombotic diseases. Front Cardiovasc Med 2022; 9: 1033416
• 25 Mingot-Castellano ME, Rodríguez-Martorell FJ, Nuñez-Vázquez RJ, Marco P. Acquired haemophilia A: a review of what we know. J Blood Med 2022; 13: 691-710
• 26 Dreier T, Mehic D, Gebhart J. Usefulness of global coagulation tests, thrombin generation and viscoelastic tests for assessing the bleeding phenotype in rare coagulation factor deficiencies. Hamostaseologie 2025; 45 (05) 419-430
• 27 Josset L, Rezigue H, Dargaud Y. Thrombin generation assay to support hematologists in the era of new hemophilia therapies. Int J Lab Hematol 2025; 47 (02) 212-220
• 28 Müller J, Büchsel M, Oleshko O. et al. Laboratory monitoring in patients receiving emicizumab. Hamostaseologie 2025; . Epub ahead of print.
• 29 van der Zwet K, Roest M, Huskens D. et al. No correlation between thrombin generation and emicizumab levels: implications for monitoring emicizumab therapy. Res Pract Thromb Haemost 2024; 9 (01) 102658
• 30 Dargaud Y, Nougier C, Desage S. et al. Utilising thrombin generation assay to guide co-administration of factor therapies with fitusiran. Haemophilia 2025; 31 (06) 1336-1339
• 31 Peraza MA, Rakhe S, Hurst S, Sirivelu M, Brenneman K, Pittman DD. Marstacimab, an antitissue factor pathway inhibitor, combined with bypassing agents: effects on thrombin generation and in hemostatically normal rats. Res Pract Thromb Haemost 2025; 9 (07) 103180
• 32 Dubut J, Goin V, Derray C, Huguenin Y, Fiore M. Targeting tissue factor pathway inhibitor with concizumab to improve hemostasis in patients with Glanzmann thrombasthenia: an in vitro study. J Thromb Haemost 2024; 22 (09) 2589-2600
• 33 Souza MEM, Ferreira LGR, Silva NRO, Lopes LR, das Graças Carvalho M, Rios DRA. Thrombin generation assay in venous thromboembolism: a scoping review. Thromb Res 2025; 252: 109384
• 34 Martin-Fernandez L, Ziyatdinov A, Carrasco M. et al. Genetic determinants of thrombin generation and their relation to venous thrombosis: results from the GAIT-2 Project. PLoS One 2016; 11 (01) e0146922
• 35 Gehlen R, Vandevelde A, de Laat B, Devreese KMJ. Application of the thrombin generation assay in patients with antiphospholipid syndrome: a systematic review of the literature. Front Cardiovasc Med 2023; 10: 1075121
• 36 Ferreira LGR, Figueiredo RC, das Graças Carvalho M, Rios DRA. Thrombin generation assay as a biomarker of cardiovascular outcomes and mortality: a narrative review. Thromb Res 2022; 220: 107-115
• 37 Ay C, Dunkler D, Simanek R. et al. Prediction of venous thromboembolism in patients with cancer by measuring thrombin generation: results from the Vienna Cancer and Thrombosis Study. J Clin Oncol 2011; 29 (15) 2099-2103
• 38 Tripodi A, Capecchi M, Scalambrino E. et al. Comparative investigation of thromboelastometry and thrombin generation for patients receiving direct oral anticoagulants or vitamin K antagonists. Diagnostics (Basel) 2024; 14 (22) 2553
• 39 Shaw JR, Castellucci LA, Siegal D, Carrier M. DOAC-associated bleeding, hemostatic strategies, and thrombin generation assays—a review of the literature. J Thromb Haemost 2023; 21 (03) 433-452
• 40 Pezeshkpoor B, Sereda N, Berkemeier AC. et al. Antidrug antibodies against the polyethylene glycol moiety inhibit the procoagulant activity of therapeutic polyethylene glycolated factor VIII. J Thromb Haemost 2023; 21 (06) 1503-1514
• 41 Rojnik T, Sedlar N, Turk N, Kastrin A, Debeljak M, Božič Mijovski M. Comparison of antithrombin activity assays in detection of patients with heparin binding site antithrombin deficiency: systematic review and meta-analysis. Sci Rep 2023; 13 (01) 16734
• 42 Machlus KR, Colby EA, Wu JR, Koch GG, Key NS, Wolberg AS. Effects of tissue factor, thrombomodulin and elevated clotting factor levels on thrombin generation in the calibrated automated thrombogram. Thromb Haemost 2009; 102 (05) 936-944
• 43 Mairesse A, Bayart JL, Desmet S. et al. Biological variation data and analytical specification goal estimates of the thrombin generation assay with and without thrombomodulin in healthy individuals. Int J Lab Hematol 2021; 43 (03) 450-457
• 44 Sedzro JC, Adam F, Auditeau C. et al. Antithrombotic potential of a single-domain antibody enhancing the activated protein C-cofactor activity of protein S. J Thromb Haemost 2022; 20 (07) 1653-1664
• 45 Nakajima Y, Ogiwara K, Inaba K, Kitazawa T, Nogami K. NXT007-mediated hemostatic potential is suppressed by activated protein C-catalyzed inactivation of activated factor V. Res Pract Thromb Haemost 2023; 8 (01) 102271
• 46 Waters EK, Hilden I, Sørensen BB, Ezban M, Holm PK. Thrombin generation assay using factor XIa to measure factors VIII and IX and their glycoPEGylated derivatives is robust and sensitive. J Thromb Haemost 2015; 13 (11) 2041-2052
• 47 Gribkova IV, Galstyan GM, Polyanskaya TY, Sinauridze EI. Kaolin, used to trigger coagulation in thrombin generation test, increases sensitivity of the method in hemophilia patients. Blood Coagul Fibrinolysis 2020; 31 (03) 193-197
• 48 Ogiwara K, Nogami K, Matsumoto N. et al. A modified thrombin generation assay to evaluate the plasma coagulation potential in the presence of emicizumab, the bispecific antibody to factors IXa/X. Int J Hematol 2020; 112 (05) 621-630
• 49 van de Berg TW, Beckers EAM, Heubel-Moenen FCJI, Henskens YMC, Thomassen MCLGD, Hackeng TM. Sensitive measurement of clinically relevant factor VIII levels in thrombin generation assays requires presence of factor XIa. Thromb Haemost 2023; 123 (11) 1034-1041
• 50 Rudez G, Meijer P, Spronk HM. et al. Biological variation in inflammatory and hemostatic markers. J Thromb Haemost 2009; 7 (08) 1247-1255