Ukr.Biochem.J. 2024; Volume 96, Issue 2, Mar-Apr, pp. 19-26

doi: https://doi.org/10.15407/ubj96.02.019

Determination of thrombin and plasmin activity using the turbidimetric analysis of clot formation and dissolution in human blood plasma

16.04.2024   Uncategorized   No comments

A. Udovenko*, Ye. Makogonenko, O. Hornytska,
G. Gogolinska, O. Yusova, V. Chernyshenko

Palladin Institute of Biochemistry, National Academy of Sciences of Ukraine, Kyiv;
*e-mail: nudovenko26@gmail.com

Received: 05 February 2024; Revised: 17 March 2024;
Accepted: 17 March 2024; Available on-line: 30 April 2024

Based on the turbidimetric curve of formation and dissolution of a blood plasma clot initiated by the activated partial thromboplastin time reagent, a method for determining the coagulation component of thrombin activity and fibrinolytic activity of plasmin is proposed. The activity of thrombin was calculated by the value of the lag period, and plasmin by its amidase activity at the moment of complete dissolution of the clot. At the end of the lag period, about 0.45% of the available prothrombin was activated, and at the moment of complete dissolution of the clot 1.05% of the available plasminogen was activated. This method makes it possible to determine the ratio of the thrombin generation rate to that of plasmin, the time of clot formation to the time of its dissolution, as well as the overall hemostasis potential and coagulation and fibrinolytic components and their ratio.

Keywords: , , , ,

References:

  1. He S, Zhu K, Skeppholm M, Vedin J, Svensson J, Egberg N, Blombäck M, Wallen H. A global assay of haemostasis which uses recombinant tissue factor and tissue-type plasminogen activator to measure the rate of fibrin formation and fibrin degradation in plasma. Thromb Haemost. 2007;98(4):871-882. PubMed, CrossRef
  2. Matsumoto T, Nogami K, Shima M. Simultaneous measurement of thrombin and plasmin generation to assess the interplay between coagulation and fibrinolysis. Thromb Haemost. 2013;110(4):761-768. PubMed, CrossRef
  3. Crawley JTB, Zanardelli S, Chion CKNK, Lane DA. The central role of thrombin in hemostasis. J Thromb Haemost. 2007;5(Suppl 1):95-101. PubMed, CrossRef
  4. Verhagen MJA, Valke LLFG, Schols SEM. Thrombin generation for monitoring hemostatic therapy in hemophilia A: A narrative review. J Thromb Haemost. 2022;20(4):794-805. PubMed, PubMedCentral, CrossRef
  5. Hemker HC, Giesen PL, Ramjee M, Wagenvoord R, Béguin S. The thrombogram: monitoring thrombin generation in platelet-rich plasma. Thromb Haemost. 2000;83(4):589-591. PubMed, CrossRef
  6. Hemker HC, Giesen P, Al Dieri R, Regnault V, de Smedt E, Wagenvoord R, Lecompte T, Béguin S. Calibrated automated thrombin generation measurement in clotting plasma. Pathophysiol Haemost Thromb. 2003;33(1):4-15. PubMed, CrossRef
  7. Tripodi A. Thrombin Generation Assay and Its Application in the Clinical Laboratory. Clin Chem. 2016;62(5):699-707. PubMed, CrossRef
  8. Hulshof AM, Hemker HC, Spronk HMH, Henskens YMC, Ten Cate H. Thrombin-Fibrin(ogen) Interactions, Host Defense and Risk of Thrombosis. Int J Mol Sci. 2021;22(5):2590. PubMed, PubMedCentral, CrossRef
  9. Depasse F, Binder NB, Mueller J, Wissel T, Schwers S, Germer M, Hermes B, Turecek PL. 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. PubMed, PubMedCentral, CrossRef
  10. Storozhuk BG, Pyrogova LV, Chernyshenko TM, Kostiuchenko OP, Kolesnikova IM, Platonova TM, Storozhuk OB, Storozhuk LO, Bereznitsky GK, Tsap PYu, Masenko OO, Makogonenko EM, Lugovskoy EV. Overall hemostasis potential of the blood plasma and its relation to some molecular markers of the hemostasis system in patients with chronic renal disease of stage VD. Ukr Biochem J. 2018;90(5):60-70. CrossRef
  11. Deutsch DG, Mertz ET. Plasminogen: purification from human plasma by affinity chromatography. Science. 1970;170(3962):1095-1096. PubMed, CrossRef
  12. Urano T, Chibber BA, Castellino FJ. The reciprocal effects of epsilon-aminohexanoic acid and chloride ion on the activation of human [Glu1]plasminogen by human urokinase. Proc Natl Acad Sci USA. 1987;84(12):4031-4034. PubMed, PubMedCentral, CrossRef
  13. Varetskaia TV. Microgeterogenety of fibrinogen. Cryofibrinogen. Ukr Biokhim Zhurn. 1960;32(1):13-24.
  14. La Corte ALC, Philippou H, Ärience RAS. Role of fibrin structure in thrombosis and vascular disease. Adv Protein Chem Struct Biol. 2011;83:75-127. PubMed, CrossRef
  15. Udovenko A, Makogonenko Y, Korolova D, Druzhyna N, Chernyshenko V, Komisarenko S. Formation and elimination of soluble fibrin and D-dimer in the bloodstream. Croat Med J. 2023;64(6):421-429. PubMed, PubMedCentral, CrossRef
  16. Belitser VA. Fibrinogen B (soluble fibrinogen), its nature and methods of determination. Lab Delo. 1980:(9):529-532. (In Russian). PubMed
  17. Storozhuk NV, Pyrogova LV, Chernyshenko ТМ, Kostyuchenko OP, Platonova TM, Storozhuk OB, Storozhuk BG, Marunich RYu, Bereznytsky GK, Makogonenko EM. Overall hemostasis potential of blood plasma and its connection to molecular markers of the hemostasis system in patients with stenosis of coronary artery. Ukr Biochem J. 2021;93(5):31-42. CrossRef
  18. Belitser VA, Varetskaia TV. Fibrinogen and fibrin: molecular structure, self-assembly of fibers. Usp Sovrem Biol. 1975;80(1):5-21. (In Russian). PubMed
  19. Makogonenko EM, Kirpa, SA, Lugovskoy EV, Nazarenko NA, Kudinov SA. Kinetics of glu- and lys-plasminogen activation by the tissue activator in a fibrin clot. Biokhimiia. 1987;52(10):1746-1752. (In Russian). PubMed
  20. Wiman B, Collen D. On the kinetics of the reaction between human antiplasmin and plasmin. Eur J Biochem. 1978;84(2):573-578. PubMed, CrossRef
  21. Sottrup-Jensen L. Alpha-macroglobulins: structure, shape, and mechanism of proteinase complex formation. J Biol Chem. 1989;264(20):11539-11542. PubMed, CrossRef
  22. Suenson E, Petersen LC. Fibrin and plasminogen structures essential to stimulation of plasmin formation by tissue-type plasminogen activator. Biochim Biophys Acta. 1986;870(3):510-519. PubMed, CrossRef
  23. Bauer R, Hansen SL, Jones G, Suenson E, Thorsen S, Ogendal L. Fibrin structures during tissue-type plasminogen activator-mediated fibrinolysis studied by laser light scattering: relation to fibrin enhancement of plasminogen activation. Eur Biophys J. 1994;23(4):239-252. PubMed, CrossRef
  24. Miszta A, Huskens D, Donkervoort D, Roberts MJM, Wolberg AS, de Laat B. Assessing plasmin generation in health and disease. Int J Mol Sci. 2021 9;22(5):2758. PubMed, PubMedCentral, CrossRef
  25. Longstaff C. Measuring fibrinolysis: from research to routine diagnostic assays. J Thromb Haemost. 2018;16(4):652-662. PubMed, PubMedCentral, CrossRef
  26. Korolova DS, Stohnii YM, Gryshchuk VI, Zhuk SI, Us IV, Chernyshenko TM, Kostiuchenko OP, Klymenko KP, Platonov OM, Ivashchenko OI, Chernyshenko VO. Thromboelastographic study of fibrin clot and molecular basis of maximum clot firmness. Ukr Biochem J. 2021;93(2):62-70. CrossRef
  27. Van Geffen M, Loof A, Lap P, Boezeman J, Laros-van Gorkom BA, Brons P, Verbruggen B, van Kraaij M, van Heerde WL. A novel hemostasis assay for the simultaneous measurement of coagulation and fibrinolysis. Hematology. 2011;16(6):327-336. PubMed, CrossRef
  28. Matsumoto T, Wada H, Shiraki K, Suzuki K, Yamashita Y, Tawara I, Shimpo H, Shimaoka M. The Evaluation of Clot Waveform Analyses for Assessing Hypercoagulability in Patients Treated with Factor VIII Concentrate. J Clin Med. 2023;12(19):6320. PubMed, PubMedCentral, CrossRef
  29. Wada H, Shiraki K, Matsumoto T, Shimpo H, Shimaoka M. Clot Waveform Analysis for Hemostatic Abnormalities. Ann Lab Med. 2023;43(6):531-538. PubMed, PubMedCentral, CrossRef
  30. Milos M, Coen Herak D, Mahmoud Hourani Soutari N, Pavic J, Zupancic-Salek S, Zadro R, Antovic JP. Overall hemostasis potential and aPTT-clot waveform analysis as powerful laboratory diagnostic tools for identification of hemophilia A patients with unexpected bleeding phenotype. Int J Lab Hematol. 2021;43(2):273-280. PubMed, CrossRef
Creative CommonsThis work is licensed under a Creative Commons Attribution 4.0 International License.